WHO TRS 999 Corrigenda Web PDF

999
W H O Te c h n i c a l R e p o r t S e r i e s
999
WHO Expert Committee on Biological Standardization

This report presents the recommendations of a WHO Expert
Committee commissioned to coordinate activities leading to the
adoption of international recommendations for the production
and control of vaccines and other biological substances, and the
establishment of international biological reference materials.
Following a brief introduction, the report summarizes a
number of general issues brought to the attention of the
Committee. The next part of the report, of particular relevance
WHO Expert Committee

to manufacturers and national regulatory authorities, outlines
the discussions held on the development and adoption of
new and revised WHO Recommendations, Guidelines and
on Biological
guidance documents. Following these discussions, a WHO
guidance document on Regulatory assessment of approved
rDNA-derived biotherapeutics was adopted along with WHO
Standardization
Guidelines on the stability evaluation of vaccines for use under
extended controlled temperature conditions and on WHO good
manufacturing practices for biological products. In addition,
revised WHO Recommendations to assure the quality, safety
and efficacy of recombinant human papillomavirus virus-like
particle vaccines were also adopted by the Committee.
Subsequent sections of the report provide information on the
current status and proposed development of international Sixty-sixth report
reference materials in the areas of antibiotics; biotherapeutics
other than blood products; blood products and related
substances; in vitro diagnostic device reagents; and vaccines
WHO Technical Report Series

and related substances.
A series of annexes are then presented which include an
updated list of all WHO Recommendations, Guidelines and
other documents on biological substances used in medicine
(Annex 1). The above four WHO documents adopted on
the advice of the Committee are then published as part
of this report (Annexes 2–5). Finally, all additions and
discontinuations made during the 2015 meeting to the list of
International Standards, Reference Reagents and Reference
Panels for biological substances maintained by WHO are
summarized in Annex 6. The updated full catalogue of
WHO International Reference Preparations is available at:
http://www.who.int/bloodproducts/catalogue/en/.
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on Biological
Standardization
Sixty-sixth report
This report contains the collective views of an international group of experts and
does not necessarily represent the decisions or the stated policy of the World Health Organization
WHO Library Cataloguing-in-Publication Data:
WHO Expert Committee on Biological Standardization, sixty-sixth report.
(WHO technical report series ; no. 999)
1.Biological Products - standards. 2.Vaccines - standards. 3.Blood - standards.
4.Anti-Bacterial Agents - standards. 5.Reference Standards. 6.Diagnostic Test Approval.
I. World Health Organization. II. WHO Expert Committee on Biological Standardization
(2015: Geneva, Switzerland). III. Series.
ISBN 978 92 4 120999 1 (NLM classification: QW 800)
ISBN (PDF) 978 92 4 069563 4
ISSN 0512-3054
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Printed in Italy
Contents
Abbreviations xv
1. Introduction 1
2. General 4
2.1 Current directions 4
2.1.1 Strategic directions in biological standardization: WHO priorities 4
2.1.2 Blood products and related in vitro diagnostics: recent and planned
activities in biological standardization 5
2.1.3 Vaccines and biotherapeutics: recent and planned activities in biological
standardization 7
2.2 Reports 9
2.2.1 Report from the WHO Blood Regulators Network 9
2.2.2 Report of the fifth meeting of WHO collaborating centres to support the
development of WHO biological reference preparations for in vitro
diagnostic devices 10
2.2.3 Report from the network of WHOCCs for the standardization and
evaluation of vaccines 12
2.2.4 Report of a WHO informal consultation on international standards for
biotherapeutic products 13
2.3 Feedback from custodian laboratories 15
2.3.1 Developments and scientific issues highlighted by custodians of
WHO biological reference preparations 15
2.4 Cross-cutting activities of other WHO committees and groups 21
2.4.1 Update on matters arising from the International Nonproprietary Names
Expert Group 21
2.4.2 Collaborative procedure for facilitating national registration of
WHO prequalified medicinal products 22
2.4.3 Proposal to transition from microbiological to physicochemical assays
for antibiotic potency testing 23
2.4.4 Labelling information for influenza vaccines intended for use in
pregnant women 25
2.4.5 Influenza vaccine response during the start of a pandemic 26
3. International Recommendations, Guidelines and other matters related
to the manufacture and quality control of biological substances 28
3.1 General 28
3.1.1 WHO good manufacturing practices for biological products 28
3.1.2 Update on the development of a candidate control material for
adventitious virus detection using deep sequencing 29
3.2 Biotherapeutics other than blood products 30
3.2.1 Regulatory assessment of approved rDNA-derived biotherapeutics 30
3.3 Blood products and related substances 31
3.3.1 Ebola-related issues 31
3.3.2 Local production of blood products 32
3.3.3 Residual risk of virus infections caused by blood products 33
3.3.4 WHO general assessment tool for conducting gap analysis of national
regulatory systems for health products and technologies 34
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3.4 In vitro diagnostic device reagents 35
3.4.1 Calibration of secondary standards for in vitro diagnostic devices 35
3.4.2 WHO prequalification of in vitro diagnostic devices 36
3.5 Vaccines and related substances 37
3.5.1 Recommendations to assure the quality, safety and efficacy of
recombinant human papillomavirus virus-like particle vaccines 37
3.5.2 Guidelines on the stability evaluation of vaccines for use under extended
controlled temperature conditions 38
3.5.3 Vaccine evaluation in public health emergencies – review of regulatory
pathways in selected countries 39
3.5.4 Development of Guidelines on the quality, safety and efficacy of
Ebola vaccines 40
3.5.5 Revision of Guidelines on the safe production and quality control of
inactivated poliomyelitis vaccines manufactured from wild polioviruses 41
3.5.6 International collaborative study to assess the utility of deep sequencing
in the quality control of oral poliomyelitis vaccines and inactivated
poliomyelitis vaccines made from Sabin strains 42
4. International Reference Materials – antibiotics 44
4.1 WHO International Standards and Reference Reagents – antibiotics 44
4.1.1 Fourth WHO International Standard for streptomycin 44
5. International reference materials – biotherapeutics other than
blood products 45
5.1 WHO International Standards and Reference Reagents – biotherapeutics other
than blood products 45
5.1.1 First WHO International Standard for tumour necrosis factor receptor
Fc fusion protein (etanercept) 45
5.1.2 First WHO Reference Panel for antibodies to erythropoietin (human) 46
5.2 Proposed new projects and updates – biotherapeutics other than blood products 47
5.2.1 Proposed First WHO International Standard for darbepoetin 47
5.2.2 Proposed First WHO Reference Panel for in vitro bioassay of
erythropoietin (EPO) 47
5.2.3 Proposed First WHO international standards for pegylated-interferon-α-2a
and pegylated-interferon-α-2b 48
5.2.4 Proposed First WHO Reference Reagent for TGN1412-like functional activity 49
5.2.5 Proposed First WHO Reference Reagent for Pam 3 CSK 4 49
5.2.6 Proposed stability monitoring of the First WHO International Standard for
pegylated granulocyte colony-stimulating factor 50
6. International reference materials – blood products and
related substances 51
6.1 WHO International Standards and Reference Reagents – blood products and
6.1.1 First WHO Reference Reagent for anti-A and anti-B in serum and plasma
for use in haemagglutination assays 51
6.1.2 Fifth WHO International Standard for blood coagulation
factor IX (concentrate) 52
6.2 Proposed new projects and updates – blood products and related substances 53
6.2.1 Proposed First WHO International Standard for activated blood coagulation
factor X 53
iv
6.2.2 Proposed First WHO Reference Panel for procoagulant activity of human
immunoglobulin 54
6.2.3 Proposed Fifth WHO International Standard for thromboplastin
(human, recombinant) 55
6.2.4 Proposed Fifth WHO International Standard for thromboplastin
(rabbit, plain) 56
7. International reference materials – In vitro diagnostic
device reagents 57
7.1 WHO International Standards and Reference Reagents – in vitro diagnostic device
reagents 57
7.1.1 First WHO International Standard for JC virus DNA for NAT-based assays 57
7.1.2 First WHO International Standard for BK virus DNA for NAT-based assays 58
7.1.3 Fifth WHO International Standard for hepatitis C virus RNA for
NAT-based assays 60
7.1.4 Fourth WHO International Standard for anti-Toxoplasma gondii (human) 60
7.1.5 Assignment of a holotranscobalamin value to the First WHO International
Standard for vitamin B12 and folate in human serum 62
7.1.6 First WHO International Standard for human C-peptide 62
7.1.7 First WHO Reference Panel for hepatitis E virus genotypes for
NAT-based assays 63
7.1.8 Extension of the First WHO Repository of platelet transfusion relevant
bacterial strains 65
7.1.9 First WHO Reference Reagent for Ebola virus antibodies 65
7.1.10 First WHO reference reagents for Ebola virus RNA for NAT-based assays 66
7.2 Proposed new projects and updates – in vitro diagnostic device reagents 68
7.2.1 Proposed Fourth WHO International Standard for hepatitis B virus DNA
for NAT-based assays 68
7.2.2 Proposed Fourth WHO International Standard for HIV-1 RNA for
NAT-based assays 68
7.2.3 Proposed First WHO International Standard for varicella zoster virus DNA
7.2.4 Proposed First WHO International Standard for Trypanosoma cruzi DNA
7.2.5 Proposed First WHO International Standard for Plasmodium vivax DNA
7.2.6 Proposed First WHO International Standard for Plasmodium falciparum
antigens 71
7.2.7 Proposed First WHO Reference Panel for KRAS codon 12 and 13 mutations 72
7.2.8 Proposed Fourth WHO International Standard for prolactin (human) 73
7.2.9 Proposed First WHO Reference Panel for hepatitis E virus antibodies 73
7.2.10 Proposed First WHO Reference Panel for HIV-1 p24 antigen 74
7.2.11 Proposed First WHO Reference Panel for Babesia microti antibodies 75
7.2.12 Proposed First WHO Repository of red blood cell transfusion relevant
bacterial strains 76
8. International reference materials – vaccines and related substances 77
8.1 WHO International Standards and Reference Reagents – vaccines and
8.1.1 First WHO International Standard for anti-EV71 serum (human) 77
v
8.1.2 First WHO international standards for meningococcal serogroups A and X
polysaccharide 78
8.1.3 Third WHO International Standard for diphtheria toxoid for use in
flocculation test 79
8.2 Proposed new projects and updates – vaccines and related substances 80
8.2.1 Proposed First WHO international standards for meningococcal serogroups
W and Y polysaccharide 80
8.2.2 Proposed First WHO international standards for Vi polysaccharide 81
8.2.3 Proposed First WHO International Standard for anti-Vi polysaccharide
serum (human) 82
8.2.4 Proposed First WHO Reference Panel for non-typhoidal salmonella
O-antigen polysaccharides 82
8.2.5 Proposed First WHO international standards for antibodies (human) to
Salmonella enterica serovars Typhimurium (O4,5 LPS) and
Enteritidis (O9 LPS) 83
8.2.6 Proposed First WHO International Standard for Salmonella enterica serovar
Paratyphi A O2 polysaccharide 83
8.2.7 Proposed First WHO International Standard for antibodies (human) to
Salmonella enterica Paratyphi A O2 polysaccharide 84
8.2.8 Proposed First WHO Reference Panel for O-antigen polysaccharides for
Shigella strains 84
8.2.9 Proposed First WHO Reference Panel for anti-Shigella O-antigen
polysaccharide serum (human) 85
8.2.10 Proposed First WHO International Standard for anti-Clostridium difficile
serum (human) 86
Annex 1
WHO Recommendations, Guidelines and other documents related to the
manufacture and quality control of biological substances used in medicine 87
Annex 2
WHO good manufacturing practices for biological products 93
Replacement of Annex 1 of WHO Technical Report Series, No. 822 93
Annex 3
Regulatory assessment of approved rDNA-derived biotherapeutics 131
Addendum to Annex 4 of WHO Technical Report Series, No. 987 131
Annex 4
Recommendations to assure the quality, safety and efficacy of recombinant
human papillomavirus virus-like particle vaccines 147
Replacement of Annex 1 of WHO Technical Report Series, No. 962 147
Annex 5
Guidelines on the stability evaluation of vaccines for use under extended controlled
temperature conditions 235
Annex 6
Biological substances: WHO International Standards, Reference Reagents and
Reference Panels 265
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12 to 16 October 2015
Committee members1
Professor K. Cichutek, Paul-Ehrlich-Institut, Langen, Germany
Dr J. Epstein, Center for Biologics Evaluation and Research, Food and Drug Administration,
Silver Spring, MD, United States of America (USA) (also Blood Regulators Network (BRN)
representative)
Dr E. Griffiths, Kingston-upon-Thames, the United Kingdom (Chair)
Dr S. Hindawi, Blood Transfusion Services, Jeddah, Saudi Arabia
Mrs T. Jivapaisarnpong, Institute of Biological Products, Ministry of Public Health,
Nonthaburi, Thailand
Dr H. Klein, National Institutes of Health, Bethesda, MD, the USA (Vice-Chair)
Dr P. Minor, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr J. Petricciani, Palm Springs, CA, the USA (Rapporteur)
Mr V.R. Reddy, South African National Blood Service, Weltevreden Park, South Africa
Dr L.S. Slamet, Technical Adviser and Consultant to the National Agency of Drug and
Food Control, Jakarta Selatan, Indonesia
Dr P. Strengers, Sanquin Plasma Products, Amsterdam, Netherlands
Dr Y. Sohn, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr J. Wang, National Institutes for Food and Drug Control, Beijing, China
Dr K. Zoon, National Institutes of Health, Bethesda, MD, the USA
Representatives of other organizations

Advanced Medical Technology Association
Dr S.R. Binder, Bio-Rad Laboratories, Hercules, CA, the USA
Council of Europe, European Directorate for the Quality of Medicines & HealthCare
Dr K-H. Buchheit, Official Medicines Control Laboratories Network and HealthCare,
Strasbourg, France
The decisions of the Committee were taken in closed session with only members of the Committee
1
present. Each Committee member had completed a Declaration of Interests form prior to the meeting.
These were assessed by the WHO Secretariat and no declared interests were considered to be in conflict
with full meeting participation.
vii
WHO Expert Committee on Biological Standardization Sixty-sixth report
Dr E. Charton, European Pharmacopoeia Department, Strasbourg, France

Developing Country Vaccine Manufacturers Network2
Dr N. Dellepiane, Serum Institute of India, Nyon, Switzerland
Dr C. Gandreuil, BE Vaccines, Nantes, France
Ms T.V. Irviyanti, PT Bio Farma, Bandung, Indonesia
Dr Q.J. Zhao, Innovax, the USA
European Generic Medicines Association
Dr M. Schiestl, Sandoz GmbH, Kundl, Austria
International Alliance of Biological Standardization
Dr J. Bonhoeffer, Brighton Collaboration Foundation, Basel, Switzerland
Dr A. Mire-Sluis, Amgen, Inc., Thousand Oaks, CA, the USA
International Federation of Clinical Chemistry and Laboratory Medicine
Professor P. Gillery, American Memorial Hospital, Reims, France
International Federation of Pharmaceutical Manufacturers & Associations2
D. Colette, GlaxoSmithKline Vaccines, Wavre, Belgium
Dr A. Drapier, GlaxoSmithKline Vaccines, Wavre, Belgium
Dr C. Saillez, GlaxoSmithKline Vaccines, Wavre, Belgium
Dr A. Robertson, Merck & Co., Inc., Kenilworth, NJ, the USA
Dr S. Wong,3 Merck & Co., Inc., Kenilworth, NJ, the USA
Dr A. Saah, Merck Research Laboratories, North Wales, PA, the USA
Dr L. Mallet, Sanofi Pasteur, Marcy L’Etoile, France
International Generic Pharmaceuticals Alliance
Dr S. Kox, European Generic Medicines Association, Brussels, Belgium
WHO Technical Report Series, No. 999, 2016
International Society of Blood Transfusion

Dr C. Bianco, Amsterdam, Netherlands
International Society on Thrombosis and Haemostasis
Dr P. Lenting, Le Kremlin-Bicêtre, France
Pharmaceutical and Medical Device Regulatory Science Society of Japan
Dr T. Murai, Japanese Pharmacopoeia Reference Standards Laboratory, Osaka, Japan
2
A maximum of two representatives of the DCVMN and two representatives of the IFPMA were present in
the meeting room during discussion of any one agenda item.
3
Participated via teleconference.
viii
Plasma Protein Therapeutics Association

Dr D. Misztela, Brussels, Belgium
United States Pharmacopeial Convention
Dr K. Carrick, Science-Global Biologics, Global Science and Standards Division, Rockville,
MD, the USA
Dr T. Morris, Biologics & Biotechnology, Global Science and Standards Division, Rockville,
MD, the USA
Temporary Advisers
Dr A. Chawla, Consultant, Greater Noida, India
Dr C. Conrad, Paul-Ehrlich-Institut, Langen, Germany
Dr P. Krause,4 Center for Biologics Evaluation and Research, Food and Drug Administration,
Bethesda, MD, the USA
Dr M. Lennon, Consultant, Horning, the United Kingdom
Dr V. Maqueda, Biologist, Buenos Aires, Argentina
Dr C. Morris, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom (Rapporteur for the blood products and in vitro diagnostics track)
Dr M. Powell, Medicines and Healthcare Products Regulatory Agency, London, the United
Kingdom
Dr J. Reinhardt, Paul-Ehrlich-Institut, Langen, Germany (Rapporteur for the blood products
and in vitro diagnostics track)
Dr D. Smith, Bacterial and Combination Vaccines Division, Health Canada, Ottawa, Canada
Dr J. Southern, Adviser to the Medicines Control Council of South Africa, Cape Town,
South Africa
Dr Y. Sun, Paul-Ehrlich-Institut, Langen, Germany
Dr E.R. Unger, Centers for Disease Control and Prevention, Atlanta, GA, the USA
Dr A.L. Waddell, Freelance, Stanley, the United Kingdom
Dr T. Wu, Bacterial and Combination Vaccines Division, Health Canada, Ottawa, Canada
Participants
Dr F. Agbanyo, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa,
Canada (also BRN representative)

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Dr N. Almond, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr P. Aprea, National Administration of Drugs, Food and Medical Technology, Buenos
Aires, Argentina
Dr S. Baylis,5 Paul-Ehrlich-Institut, Langen, Germany
Dr A. Bristow, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr P. Bowyer,5 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr J. Boyle,5 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr D. Calam, Pewsey, the United Kingdom (International Nonproprietary Names (INN)
expert)
Dr F. Cano, Agence Nationale de Sécurité du Médicament et des Produits du Santé,
Lyons, France
Mr F. Carazzai Reisdorfer, Agência Nacional de Vigilância Sanitaria, Brasilia, Brazil
Dr H. Chan,5 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr K. Chumakov,5 Center for Biologics Evaluation and Research, Food and Drug
Administration, Silver Spring, MD, the USA
Dr H. Chung, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr L. Coombes,5 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr R. Dominguez Morales, Ministerio de Salud Pública, Havana, Cuba
Professor S. Efstahiou, National Institute for Biological Standards and Control, Potters Bar,
the United Kingdom

Dr L. Elmgren, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa,
Canada (also BRN representative)
Dr S. Fakhrzadeh, Ministry of Health and Medical Education, Tehran, the Islamic Republic
of Iran
Dr I. Feavers, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom

5
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Dr J. Ferguson,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr D.A. Garcia Londono, Assesor del Despacho del Viceministro de Salud, Publica y
Prestación de Servicios, Bogota, Colombia
Professor A. Genazzani, Università del Piemonte Orientale, Novara, Italy (INN expert)
Dr K. Grant, Therapeutic Goods Administration, Woden, ACT, Australia (INN expert)
Dr S. Govind,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr E. Gray,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr M. Gruber,6 Center for Biologics Evaluation and Research, Food and Drug
Administration, Silver Spring, MD, the USA
Dr I. Hamaguchi, National Institute of Infectious Diseases, Tokyo, Japan (also BRN
representative)
Dr K. Haslov, Statens Serum Institute, Copenhagen, Denmark
Dr C.C. Ilonze, National Agency for Food and Drug Administration and Control, Lagos,
Nigeria
Dr S. Inglis, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Mrs W. Jariyapan, Ministry of Public Health, Nonthaburi, Thailand
Dr H. Jia,6 National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom
Ms Juliati, National Agency of Drug and Food Control, Jakarta Pusat, Indonesia
Dr A. Kato, National Institute of Infectious Diseases, Tokyo, Japan
Dr D. Khokal, Health Sciences Authority, Helios, Singapore
Dr S. Kumar,6 Center for Biologics Evaluation and Research, Food and Drug Administration,
Bethesda, MD, the USA
Dr J. Luo, Center for Drug Evaluation, Beijing, China
Dr J. Martin,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr F. Mawas,6 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom

6
xi
Dr E. Mee,7 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr M. Moore,7 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Ms N.A.M. Nur, National Pharmaceutical Control Bureau, Selangor, Malaysia
Dr M. Ochiai, National Institute of Infectious Diseases, Tokyo, Japan
Dr H. Oh, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr M. Ovanesov,7 Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, MD, the USA
Ms O. Panova, Department of Medicines Supply and Regulation of Medical Products,
Ministry of Health, Moscow, Russian Federation
Dr E. Parra, Federal Commission for the Protection against Sanitary Risks, Ministry of
Health, Mexico City, Mexico
Dr M. Pfleiderer, Paul-Ehrlich-Institut, Langen, Germany
Dr S. Rijpkema,7 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr C. Schaerer, Swiss Agency for Therapeutic Products, Bern, Switzerland (also BRN
representative)
Professor R. Seitz, Paul-Ehrlich-Institut, Langen, Germany (also BRN representative)
Dr J.S. Shin, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr G. Smith, Therapeutic Goods Administration, Woden, ACT, Australia (also BRN
representative)
Dr K. Sohn, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr E. Spindler-Raffel,7 Paul-Ehrlich-Institut, Langen, Germany
Dr D. Stahl, Paul-Ehrlich-Institut, Langen, Germany

Dr C. Thelwell,7 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr R. Thorpe, Consultant, Welwyn, the United Kingdom
Dr S. Thorpe,7 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr M. Udell,7 Medicines and Healthcare Products Regulatory Agency, London, the
United Kingdom

7
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Dr A.M.H.P. Van Den Besselaar,8 Leiden University Medical Centre, Leiden, Netherlands
Dr A. Vasheghani Farahani, Food and Drug Organization, Tehran, the Islamic Republic
of Iran
Dr C. Vipond,8 National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr M. Wadhwa, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr J. Weir, Center for Biologics Evaluation and Research, Food and Drug Administration,
Silver Spring, MD, the USA
Dr D. Wilkinson,8 National Institute for Biological Standards and Control, Potters Bar,
the United Kingdom
Dr M. Xu, National Institutes for Food and Drug Control, Beijing, China
WHO Secretariat
Regional Offices
AMRO – Ms M.L. Pombo-Castro
EMRO – Dr H. Langar
SEARO – Mr M. Eisenhawer
WPRO – Dr J. Shin; Dr U. Kim
Headquarters
Mr K. de Joncheere, Director, EMP
Dr J-M Okwo-Bele, Director, IVB and Acting Head, IVR/IVB
Dr L. Rägo, Head, RHT/EMP
Dr D.J. Wood, Coordinator, TSN/EMP (Committee Secretary)
Mr L. Belgharbi, RSS/EMP
Dr J. Fournier-Caruana, PQT/EMP
Dr K. Gao, TSN/EMP
Dr H-N Kang TSN/EMP
Dr I. Knezevic TSN/EMP (Lead for the vaccines and biotherapeutics track)
Dr D. Lei TSN/EMP

8
xiii
Dr M. Nuebling, TSN/EMP (Lead for the blood products and in vitro diagnostics track)
Ms I. Prat, PQT/EMP
Dr M. Refaat, RSS/EMP
Dr C.A. Rodriguez-Hernandez, PQT/EMP
Dr H. Schmidt, TSN/EMP
Dr W. Zhang, PED/GIP
Dr T. Zhou, TSN/EMP
xiv
Abbreviations
AEFI adverse event following immunization
Ag/Ab antigen/antibody
ALIFAR Asociación Latinoamericana de Industrias Farmacéuticas
ATMP advanced therapy medicinal product
BCG bacille Calmette–Guérin
BGTD Biologics and Genetic Therapies Directorate
BQ Biological Qualifier (scheme)
BRN WHO Blood Regulators Network
BSP EDQM biological standardization programme
CBER Center for Biologics Evaluation and Research
CIN cervical intraepithelial neoplasia
CIN2–3 cervical intraepithelial neoplasia grades 2 or 3
CIN2+ cervical intraepithelial neoplasia grade 2 or worse
cLIA competitive Luminex immunoassay
CMI cell-mediated immunity
CTC controlled temperature chain
DCVMN Developing Countries Vaccine Manufacturers Network
DCVRN Developing Country Vaccine Regulators’ Network
DNA deoxyribonucleic acid
ECTC extended controlled temperature conditions
EDQM European Directorate for the Quality of Medicines & HealthCare
EGA European Generic Medicines Association
EIA enzyme immunoassay
ELISA enzyme-linked immunosorbent assay
EMA European Medicines Agency
EMP WHO Department of Essential Medicines and Health Products
EPO erythropoietin
EU ELISA Unit(s)
xv
FIX factor IX
GAPIII WHO Global Action Plan to minimize poliovirus facility-
associated risk after type-specific eradication of wild
polioviruses and sequential cessation of oral polio vaccine use
GCP good clinical practice
GCV geometric coefficient of variation
GMC geometric mean concentration
GMP good manufacturing practice(s)
GMT geometric mean titre
GP glycoprotein
HAV hepatitis B virus
HBsAg hepatitis B surface antigen
HBV hepatitis B virus
HCV hepatitis C virus
HEPA high-efficiency particulate air
HEV hepatitis E virus
HIV human immunodeficiency virus
holoTC holotranscobalamin
HPV human papillomavirus
HVAC heating, ventilation and air conditioning
IARC WHO International Agency for Research on Cancer
ICDRA International Conference of Drug Regulatory Authorities

ICP immune correlate of protection
ICRS International Chemical Reference Substances
IFPMA International Federation of Pharmaceutical Manufacturers
& Associations
IgA immunoglobulin A
IgE immunoglobulin E
IgG immunoglobulin G
IgM immunoglobulin M
xvi
Abbreviations
IGPA International Generic Pharmaceutical Alliance

IIV inactivated influenza vaccine
INN international nonproprietary name(s)
IPV inactivated poliomyelitis vaccine
ISA international standard for antibiotics
ISTH International Society on Thrombosis and Haemostasis
IU International Unit(s)
IVD in vitro diagnostic
LAL limulus amebocyte lysate
LB lower bound
Lf limit of flocculation
LL lower limit
LLOD lower limit of detection
LLOQ lower limit of quantification
LMIC low- and middle-income countries
mAb monoclonal antibody
MAPREC mutant analysis by polymerase chain reaction and restriction
enzyme cleavage
MCB master cell bank
MERS-CoV Middle East respiratory syndrome coronavirus
MFDS Ministry of Food and Drug Safety
MOI multiplicity of infection
MPL monophosphoryl lipid A
MPS massively parallel (deep) sequencing
MRP minimum release potency
MSL master seed lot
MVS master virus seed
NAT nucleic acid amplification technique
NCL national control laboratory
NGS next generation sequencing
xvii
NIBSC National Institute for Biological Standards and Control

NIFDC National Institutes for Food and Drug Control
NIID National Institute of Infectious Diseases
NLT not less than
NMR nuclear magnetic resonance
NMT not more than
NRA national regulatory authority
OPV oral poliomyelitis vaccine
PAGE polyacrylamide gel electrophoresis
PATH Program for Appropriate Technology in Health
PCR polymerase chain reaction
PDL population doubling level
PEI Paul-Ehrlich-Institut
PQR product quality review
PQS pharmaceutical quality system
PS polysaccharide
QRM quality risk management
QM quality management
R&D research and development
RBC red blood cell
RBP reference biotherapeutic product
rDNA recombinant DNA

RNA ribonucleic acid
RRP recurrent respiratory papillomatosis
SAGE WHO Strategic Advisory Group of Experts
SBP similar biotherapeutic product
SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEC–HPLC size-exclusion chromatography – high-performance liquid
chromatography
SoGAT Standardisation of Genome Amplification Techniques (group)
xviii
Abbreviations
SPF specific pathogen free

spp. species
SSC Scientific and Standardization Committee (of ISTH)
TEM transmission electron microscopy
TGA Therapeutic Goods Administration
TNF tumour necrosis factor
TSE transmissible spongiform encephalopathy
USAN United States Adopted Names program
vCJD variant Creutzfeldt-Jakob disease
VLP virus-like particle
VSV vesicular stomatitis virus
WCB working cell bank
WHOCC WHO collaborating centre
WSL working seed lot
WVS working virus seed
xix
1. Introduction
The WHO Expert Committee on Biological Standardization met in Geneva
from 12 to 16 October 2015. The meeting was opened by Mr Kees De Joncheere,
Director of the Department of Essential Medicines and Health Products (EMP).
Mr de Joncheere welcomed the Committee, meeting participants
and observers, and reminded the Committee that it had a mandate to review
developments in the field of biological substances used in human medicine that
included antibiotics, biotherapeutics, blood products, in vitro diagnostic device
reagents and vaccines. During its previous 65 meetings the Committee had
established approximately 70 written standards and around 300 international
biological reference preparations essential for the quality control, regulation and
clinical dosing of biological products.
The development and establishment of such standards, and their
subsequent promotion, are crucially important activities. The inclusive standards
development process implemented by WHO facilitates global consensus on
technical matters and is a very significant factor in promoting convergent
regulatory decision-making between countries. After standards have been
established, proactive technical support from WHO was crucial in obtaining
maximum understanding and impact, and facilitating the consistent application
of standards.
Equitable access to safe, quality, affordable and effective medical products
is one of the cornerstones of universal health care. The development and
adoption of norms and standards to regulate the quality, safety, efficacy and cost-
effective use of medical products is a vital foundation upon which this aspiration
is built. The coordinated efforts of WHO Expert Committees, together with
WHO collaborating centres (WHOCCs) and partner organizations, allow for a
global approach to this core normative work.
Increased levels of international collaboration are now required in the
field of regulatory science. As demands grow for reduced regulatory and policy
burdens, expectations of greater transparency will increase. New norms and
standards will need to reflect these aspirations but not to the point where quality
suffers. As the standards-setting expertise and resources needed to address all
needs are likely to be insufficient in any one Member State, the convergence of
international norms and standards was increasingly being recognized as a key
driver of success. Where appropriate, the use of WHO norms and standards as
global benchmarks represents an opportunity to achieve the desired regulatory
and policy convergence.
The standardization of biological products remains high on the agenda
of Member States and there was now broad recognition of the record of success of
biotherapeutic products in treating many life-threatening and chronic diseases.
However, some of these products were currently very expensive and access
1
to them has been relatively limited, particularly in less-resourced countries.

This situation was likely to change in the near to mid-term as more countries
started to produce biotherapeutics. In addition, the expiry of patents and/or
data protection for several originator biotherapeutics had ushered in an era of
“similar” products. Nevertheless, many countries also recognize that they do not
have the regulatory capacity or expertise to evaluate biotherapeutic products
in general and similar biotherapeutic products (SBPs) in particular. WHO
will therefore enhance its work with countries to strengthen the capacities of
national regulatory authorities (NRAs), quality control laboratories and national
pharmacovigilance centres to regulate the safety, efficacy and quality of medical
products before their authorization for use, and to monitor their subsequent
safety and quality.
Mr de Joncheere reminded the meeting that in May 2014 the World
Health Assembly had adopted two resolutions of particular relevance to the
above areas, namely: (a) Access to biotherapeutic products including similar
biotherapeutic products and ensuring their quality, safety and efficacy; and (b)
Regulatory system strengthening for medical products. The progress made in
implementing the required actions in these areas requested by Member States
will be reported to the Committee, including in the key area of the regulation of
blood therapies.
Mr de Joncheere then drew attention to the need to translate the lessons
learnt from the parallel development, testing, licensure and use of candidate
interventions during the recent response to the complex Ebola epidemic into a
“blueprint” for improving research and development readiness for other highly
infectious diseases. Biological products were among the most promising candidate
interventions for responding to such outbreaks, and the continued strong
engagement of the Committee, and the support of all experts and organizations
represented at this meeting, was of the utmost importance in such efforts.
Mr de Joncheere pointed out the typically full agenda of the Committee
and moved on to the election of meeting officials. In the absence of dissent,

Dr Elwyn Griffiths was elected as Chair and Dr John Petricciani as Rapporteur for
the plenary sessions, and for the track considering vaccines and biotherapeutics.
Dr Harvey Klein was elected as Chair and Dr Clare Morris and Dr Jens Reinhardt
as Rapporteurs for the track considering blood products and in vitro diagnostic
device reagents. Dr Klein was also elected as Vice-Chair for the plenary sessions
of the Committee.
Finally, Mr de Joncheere expressed his thanks on behalf of WHO to the
Committee, to WHOCCs, and to all the experts, institutions and professional
societies working in this area whose efforts provided vital support to WHO
programmes. He concluded by reminding participants that Committee
members acted in their personal capacities as experts and not on behalf of their
organizations or countries.
2
Introduction
The Secretary to the Committee, Dr David Wood, then presented

an overview of WHO Expert Committees and of the important and greatly
valued role they played in providing assistance to Member States. Established
by the World Health Assembly or Executive Board, WHO Expert Committees
acted as official advisory bodies to the Director-General of WHO and were
governed by formal rules and procedures. Expert Committee meetings were
attended by Committee members selected from WHO Expert Advisory Panels;
representatives and observers of other organizations including international
agencies, nongovernmental organizations and professional associations;
Temporary Advisers; and other participants. The reports of the Committee
appear in the WHO Technical Report Series and are presented each year to the
WHO Executive Board.
Dr Wood then introduced the members of the 2015 Expert Committee
and highlighted the new requirement from this meeting onwards that
biographical summaries of the members must be posted for public review and
comment prior to the meeting. All biographical summaries had been posted
and no comments had been received. Dr Wood then outlined the organization
of the meeting and the major issues to be discussed. Declarations of Interests
made by four members of the Committee, one Temporary Adviser and four
participants were then presented to the meeting.1 Following prior evaluation,
WHO had concluded that none of the declarations made constituted a significant
conflict of interest, and that the individuals concerned would be allowed to
participate fully in the meeting.
Following participant introductions, the Committee adopted the
proposed agenda (WHO/BS/2015.2278 Add 2).
Dr E. Griffiths (consulting); Dr P. Minor (public statements and research support); Dr J. Petricciani

1
(consulting and investments); Dr K. Zoon (intellectual property and research support); Dr A. Chawla
(travel support); Professor S. Efstahiou (research support); Dr K. Haslov (consulting); Dr S. Inglis (public
statements); and Dr S. Thorpe (consulting).
3
2. General
2.1 Current directions
2.1.1 Strategic directions in biological standardization: WHO priorities
Dr Wood informed the meeting that in order to facilitate coordination of
overlapping topics and harness synergies, the Expert Committee on Biological
Standardization, the Expert Committee on Specifications for Pharmaceutical
Preparations and the International Nonproprietary Names (INN) Expert
Group were meeting simultaneously. Dr Wood then outlined the three major
categories of outcomes in the area of biological standardization, namely global
written standards, global measurement standards and the provision of support
to regulatory science including through the development of new assays, further
development and refinement of quality control tests, and reviewing of the
scientific basis for the establishment of specifications. In addition, standards-
implementation workshops continue to be conducted and provide extremely
valuable support to participants.
The three global public health priorities of responding to public health
emergencies, promoting access to biotherapeutic products and strengthening
regulatory systems remain key drivers of strategic approaches in this area. In
relation to public health emergencies, an overview from the WHO perspective
was presented on the research and development response to the Ebola outbreak.
The initial challenge had been to compress the usual timeline for unproven
interventions from years to months by working in parallel on product
development, testing, licensure and use. From August 2014, WHO held a series
of consultations with key international experts and stakeholders to identify
potential therapeutic or preventive solutions for Ebola. Based on expert advice,
WHO prioritized a number of products for further investigation through human
testing, namely two candidate vaccines, a shortlist of biotherapeutic products
(including monoclonal antibodies), convalescent whole blood and plasma,

and rapid point-of-care diagnostics. By December 2014, clinical trials were
under way in the affected countries, with efficacy results now either available
or expected for at least one vaccine, six diagnostics and five biotherapeutic
products. Experience with the Ebola epidemic and recognition of the vital need
for research and development (R&D) preparedness led the Director-General of
WHO to conclude that:
...the job now is to harness the lessons from Ebola to create a new R&D
framework that can be used for any epidemic-prone disease, in any
infectious disease emergency. What is needed is an R&D preparedness
plan with clear rules, well-defined platforms for information sharing, and
agreed procedures to expedite development and clinical trials.
4
General
Next steps will include the preparation of a preliminary outline of a

“Blueprint” for R&D preparedness for outbreaks of infectious diseases with
epidemic potential, followed by a broad consultative process and submission
of the outcome to the 2016 World Health Assembly. Five work-streams and
associated deliverables have now been designed to identify the key actions
required to accelerate access to products. In relation to the work-stream on the
identification of research priorities, the four deliverables selected were: (a) a
summary of current status and gaps to be filled to ensure the timely initiation of
human trials and introduction of health technologies; (b) an R&D roadmap for
Middle East respiratory syndrome caused by a novel coronavirus (MERS-CoV);
(c) improved global regulatory preparedness; and (d) a plan for a network of
production platforms for priority health technologies.
Progress in relation to the above global health priority of promoting
access to biotherapeutic products and reviewing WHO reference preparations
in this area is described below in sections 2.1.3 and 2.2.4. In line with the 2014
World Health Assembly, resolution WHA67.21, specific efforts have been made
in the areas of biotherapeutic product standards-setting and implementation,
as well as in the further development of nomenclature by the INN Expert
Group (see section 2.4.1 below). Efforts were also ongoing in the priority
area of strengthening regulatory systems, including through the provision of
WHO assistance in strengthening national blood systems and supporting the
prequalification of in vitro diagnostics (IVDs) (see section 2.1.2 below).
The Committee expressed its support for the focus and priorities outlined
for the work of WHO during the coming year.
2.1.2 Blood products and related in vitro diagnostics: recent

and planned activities in biological standardization
Dr Micha Nuebling presented an overview of activities in a number of
core areas including: (a) strengthening local production of blood products;
(b) prequalification of antivenoms; (c) prequalification of IVDs; (d) regulation of
medical devices; (e) collaborative efforts with WHOCCs; and (f) measurement
standards for consideration by the 2015 Expert Committee.
The WHO Achilles project on improving access to safe blood products
through local production and technology transfer in blood establishments
was then highlighted. This initiative was undertaken in response to resolution
WHA63.12 and aims to increase the supply and safety of blood and blood
products in low- and middle-income countries by raising standards in blood
establishments and reducing the risk of transfusion-transmitted infections.
Following the selection of Indonesia as the pilot country, a broad range of
project activities had been undertaken and an external assessment had now
been made of the extent of implementation of good manufacturing practices
(GMP) in a single blood establishment in Surabaya. In light of the progress
5
made and high level of commitment in this area further activities were now
planned. A brief overview was also presented of blood regulation in Kenya
and of the collaborative efforts now under way between WHO and the Kenyan
National Blood Transfusion Service to improve the safety of the national blood
supply. Following up on recent efforts in the WHO African Region, a workshop
on the development of a regional strategy for blood safety and the establishment
of national regulatory systems for blood and blood products had been held
in Benin in September 2015 involving regulators and blood-establishment
representatives from 13 countries.
Dr Nuebling then drew attention to the pressing need for the assessment
and evaluation of snake antivenoms. With more than 100 000 fatalities
occurring as a result of snake bites each year, and the largely unregulated
nature of potentially therapeutic products of unknown quality, this continued
to be a highly neglected public health issue. As part of its efforts to address
this situation WHO was extending its prequalification programme to cover
antivenoms. Work was now under way to establish a prioritized prequalification
scheme that would be guided by the deliberations of an Expert Review Panel.
The prequalification of IVDs has been an important WHO activity
for many years as part of facilitating access to safe, appropriate and affordable
IVDs of good quality. Through its standards-setting and guidance-development
activities the Committee was requested to further support WHO prequalification
efforts in this area.
In light of resolution WHA67.20 on regulatory system strengthening and
the paramount requirement for all medical products to be safe and effective, the
development of a model regulatory framework for medical devices (including
IVDs) was also highlighted as a WHO EMP priority. Against a backdrop of a
lack of such regulation in many WHO Member States there was a need for an
established process for the approval of medical devices. The development of a
first draft of the model regulatory framework was scheduled for completion in
early 2016. This would then be followed by a round of public consultation prior
to submission of the outcome document for consideration for adoption by the
Expert Committees on Biological Standardization and on Specifications for
Pharmaceutical Preparations in October 2016.
Dr Nuebling then briefly highlighted the main outcomes areas of the
fifth meeting of WHOCCs on the development of WHO biological reference
preparations for in vitro diagnostic devices, which are reported upon more fully
in section 2.2.2 below. Dr Nuebling concluded by summarizing the programme
of measurement standards in the area of blood products and related in vitro
diagnostics proposed for establishment by the Committee in 2016, along with
the new projects to be endorsed.
There was some discussion around the issue of available resources and
expertise to support all the new work areas, particularly the prequalification of
6
General
IVDs. In response, it was noted that although the projected activities in relation
to the prequalification of IVDs were indeed considerable these were longer term
goals with the level of demands likely to build gradually over the coming years,
thus allowing for strengthened collaboration and integration of activities with
other processes.
2.1.3 Vaccines and biotherapeutics: recent and planned

activities in biological standardization
Dr Ivana Knezevic outlined a range of activities and strategic considerations
in relation to the standardization and regulatory evaluation of vaccines and
biotherapeutics. To date, a total of 78 WHO Recommendations, Guidelines
and guidance documents had been published in these areas. As outlined below
in Part 3 of this report, one WHO Recommendations document, two WHO
Guidelines documents and one WHO guidance document had been prepared for
consideration by the Committee in 2015. In addition, five documents were under
development for presentation to the Committee in 2016 with two scheduled
for consideration in 2017. One of the documents scheduled for submission to
the Committee in 2016 was a revised and updated version of the 2001 WHO
Guidelines on clinical evaluation of vaccines: regulatory expectations.2
During the period 2013–2015, eight measurement standards for
vaccines had been established by the Committee with a further three expected
to be established in 2016. Four workshops on the implementation of vaccine
standards had been conducted during 2014–2015, with three planned for 2016
and one for 2017.
In the area of biotherapeutics, six measurement standards had been
established during the period 2013–2015, with three scheduled for consideration
by the Committee in 2016. Two implementation workshops on biotherapeutics
had been conducted in 2014 and two in 2015, with an implementation workshop
on the regulatory assessment of biotherapeutic products scheduled for 2016.
In addition, an informal consultation on the current WHO Guidelines on
SBPs3 had been held in 2015 to assess the need for their revision in view of
recent implementation experience. Participants from 26 countries agreed that
that there was currently no need for revision but that guidance on monoclonal
2
Guidelines on clinical evaluation of vaccines: regulatory expectations. In: WHO Expert Committee on
Biological Standardization: fifty-second report. Geneva: World Health Organization; 2004: Annex 1 (WHO
Technical Report Series, No. 924; www.who.int/biologicals/publications/trs/areas/vaccines/clinical_
evaluation/035-101.pdf?ua=1, accessed 9 February 2016).
3
Guidelines on evaluation of similar biotherapeutic products (SBPs). In: WHO Expert Committee
on Biological Standardization: sixtieth report. Geneva: World Health Organization; 2013: Annex 2
(WHO Technical Report Series, No. 977; http://www.who.int/biologicals/publications/trs/areas/biological_
therapeutics/TRS_977_Annex_2.pdf?ua=1, accessed 9 February 2016).
7
antibody products should be developed as an addendum. An associated

consultation on the regulatory assessment of approved biotherapeutic products
also recommended that an addendum to current WHO Guidelines in this
area 4 be developed and this was one of the written standards scheduled for
consideration by the Committee in 2015 (see section 3.2.1 below).
The importance and potential advantages of using case studies and
publications during implementation workshops was also highlighted. In
addition, the pilot use of interactive e-learning programmes prior to an
implementation workshop for WHO guidelines on biotherapeutic products
and SBPs had proved to be very helpful in facilitating participant involvement
and interaction, with constructive and positive feedback received. Work on the
further development of e-learning tools and approaches, and their application
in standards implementation, was ongoing.
Projects with timelines yet to be defined included the development of
written standards on: (a) respiratory syncytial virus vaccines; (b) meningitis B
and meningitis X vaccines; (c) vector-based vaccines; (d) hepatitis E vaccine;
and (e) cell and other advanced therapies.
Dr Knezevic then highlighted a number of strategic issues, encompassing
the consultation process required for the development of written standards, the
development and implementation of written and measurement standards,
the need for funding, recent World Health Assembly resolutions, establishing
the scope of work and priority setting, improving synergies in vaccine
standardization, and the provision of support to global, regional and inter-
country networks. In relation to scope and priority setting, Dr Knezevic
reported on a proposal for WHO to take the initiative in developing guiding
principles for the scientific regulation of cell-therapy products following
the 2014 WHA resolution on regulatory system strengthening. It was
envisaged that a consultation process may be initiated in 2016 with support
from WHOCCs likely to be a key requirement. A number of opportunities
for regulatory convergence were also highlighted, including through the

collaborative activities of international and regional initiatives, and regulatory
and industry networks.
While the recent expansion of the scope of work in all of the above areas
had been well received by stakeholders, it was also proving to be very demanding.
The Committee agreed that the rate-limiting factor for progress in this area
would be the availability of additional resources, with more-efficient ways of
Guidelines on the quality, safety and efficacy of biotherapeutic protein products prepared by
4
recombinant DNA technology. In: WHO Expert Committee on Biological Standardization: sixty-fourth
report. Geneva: World Health Organization; 2014: Annex 4 (WHO Technical Report Series, No. 987; http://
www.who.int/biologicals/biotherapeutics/TRS_987_Annex4.pdf?ua=1, accessed 9 February 2016).
8
General
achieving the goals now required. It was suggested that in view of the recognized
capabilities of other organizations that the WHO implementation workload
might be reduced through improved collaboration and streamlining of efforts.
2.2 Reports
2.2.1 Report from the WHO Blood Regulators Network
Dr Christian Schaerer reminded the meeting that the objectives of the BRN
were: (a) to identify issues and share expertise and information; (b) to promote
the science-based convergence of regulatory policy, including by fostering the
development of international consensus on regulatory approaches; and (c) to
propose solutions to specific issues, especially emerging public health challenges
such as the vulnerability of countries to communicable disease threats. As
of October 2015 the BRN membership comprised representatives of seven
regulatory agencies from different countries and WHO.
Following an update on membership application decisions and other
organizational issues Dr Schaerer summarized the range of recent BRN
activities. Three teleconferences had taken place in relation to the Ebola
outbreak, with further BRN participation in regular discussions on the situation
and on activities to strengthen blood systems in affected countries. An expert
presentation was given at the Alliance of Blood Operators, and discussions held
on their project on risk-based decision-making for blood safety. Discussions
were also held on national decision-making in relation to donor deferral for men
who have sex with men, and on the potential hazards of transfusion in terms
of the increased risks associated with blood transfusions and the postulated late
sequelae of transfusion.
In late 2014 the BRN proposed that an amendment be made to its
position paper on Collection and use of convalescent plasma or serum as an
element in filovirus outbreak response following its posting on the WHO website
to include a statement on the use of non-immune plasma as a control in studies
with convalescent plasma. In addition, a BRN Letter of support was produced
for a request by the World Federation of Hemophilia to add desmopressin to
the WHO Model List of Essential Medicines. This request was subsequently
accepted at the 2015 meeting of the WHO Expert Committee on the Selection
and Use of Essential Medicines.
Following discussions and recommendations made to WHO at the
16th International Conference of Drug Regulatory Authorities (ICDRA) a draft
guidance document was developed on the implementation of national blood
regulatory systems. BRN representatives also facilitated both the WHO workshop
on the development of a regional strategy for blood safety and the WHO
workshop on the assessment of GMP in a blood establishment in Surabaya,
Indonesia as a part of the WHO Achilles project (see section 2.1.2 above).
9
Dr Schaerer concluded by highlighting the range of topics that had been

identified for discussion by BRN members at its closed meeting during the
current session of the Committee.
2.2.2 Report of the fifth meeting of WHO collaborating centres

to support the development of WHO biological reference
preparations for in vitro diagnostic devices
This meeting was held at the National Institute for Biological Standards and
Control (NIBSC) in July 2015 as a follow-up to the previous meeting held in
2011. The meeting objectives were: (a) to coordinate the needs and priorities of
individual WHOCCs and WHO programmes; (b) to discuss priority projects
in order to avoid the overlap of activities and to strengthen collaboration within
the WHOCC network; and (c) to agree on proposals to put forward to the
Committee for endorsement or establishment.
Meeting topics had included scientific updates on ongoing projects,
an update on relevant WHO guidelines and other guidance documents under
development, general discussions on important topics and specific proposals
for consideration by the Committee. In their overview presentation to the
Committee, Dr Jens Reinhardt and others focused primarily on the scientific
updates that had been provided during the meeting as the above WHO
documents (on residual risk for virus infections in blood products and on
the calibration of secondary standards for IVDs) were dealt with elsewhere in
the agenda (see sections 3.3.3 and 3.3.4 respectively) along with an update of
developments in WHO prequalification of IVDs (see section 3.3.6 below).
Specific scientific updates provided included a report back from a recent
workshop held by the Standardisation of Genome Amplification Techniques
(SoGAT) group. This 20th anniversary meeting was held in London in June
2015 and consisted of four sessions over two days on: (a) understanding the
use of International Standards and encouraging laboratories to calibrate
assays and report results in international units (IU) where an international

standard was available; (b) secondary reference materials, with specific focus
on their production and calibrated by different manufacturers; (c) validation
and calibration of assays in the absence of higher order materials such as
international standards; and (d) the standardization of serological international
standards, which was an area that had not to date been covered by the SoGAT
group. It was agreed that one follow-up activity would be to continue to address
serological standardization with invited experts in this field.
A further update was provided on recent developments in hepatitis E
virus (HEV) testing and associated clinical and regulatory developments in
Europe. The European Pharmacopoeia monograph 1646 had been amended and
a paragraph inserted on HEV, noting the need for HEV nucleic acid amplification
10
General
technique (NAT)-based testing, with such testing then being implemented in

January 2015. This had been preceded by a European Medicines Agency (EMA)
workshop on the viral safety of plasma-derived medicinal products with respect
to HEV in October 2014. Topics discussed included transfusion-associated
infections and related clinical experience, HEV detection and epidemiology
of HEV in blood/plasma donations, and approaches to HEV inactivation or
reduction. One of the meeting outcomes had been a reflection paper on the
viral safety of plasma-derived medicinal products with respect to HEV which
had now undergone public consultation.
An update was also given on two CBER research programmes; one
on global human immunodeficiency virus (HIV) diversity and one on next
generation sequencing (NGS) for pathogens. The first of these projects aimed
to create HIV panels consisting of high-titre virus preparations representative
of worldwide viral diversity. Fully characterized viruses have now been made
available to approved investigators for research purposes and to manufacturers
and developers of test kits to validate their assays for detecting emerging HIV
variants. At present, 216 high-titre samples from 19 HIV subtypes are available
representing the diversity of 29 countries. The use of NSG technology to
detect pathogens has broad potential application, including in full-length HIV
sequencing, new virus discovery, microbiome and virome characterization of
HIV patients and the multiplex detection of bloodborne pathogens.
Significant concerns persist regarding the potential for transfusion-
associated transmission of variant Creutzfeldt-Jakob disease (vCJD) and an
overview was provided of the two current diagnostic approaches, namely
direct detection and amplification-based testing. Both methods were known to
present challenges and the meeting was informed that a review had now been
conducted by the United Kingdom government science and technology select
committee of the progress made in developing a blood test for vCJD. It was
recognized that any test to be used for a blood prevalence study should have a
demonstrated ability to detect markers of vCJD infection in preclinical animal
model samples.
Progress was also outlined in the development of an assay for Ebola
convalescent plasma antibodies using a recombinant construct based on
vesicular stomatitis virus (VSV) in which VSV glycoprotein (GP) was replaced
with Ebola virus GP. Characterization of the resulting construct was described
along with the development of two assays based on the above and similar virus
constructs for the detection of Ebola antibodies that were now undergoing field
validation in West Africa.
An update was then provided on state-of-the-art serological tests for
hepatitis C virus (HCV) and for hepatitis B surface antigen (HBsAg), and on
HIV antigen/antibody (Ag/Ab) combination assays. For HCV core antigen tests
currently on the European market detection limits vary widely. In the area of
11
HCV antibody testing a switch from indirect enzyme-linked immunosorbent

assays (ELISAs) to newer approaches such as “sandwich” ELISA has resulted in
increased sensitivity in the diagnostic window phase. Progress had also been
made in the development of new HCV Ag/Ab combination tests. Comparative
testing of a range of CE-marked HBsAg assays indicated analytical sensitivities
ranging from 0.005 to 0.130 IU/ml. For European Commission Common
Technical Specifications, the required analytical sensitivity of CE-marked
HBsAg screening tests was ≤ 0.130 IU/ml. The development of a new and highly
sensitive HBsAg assay was highlighted indicating the feasibility of sensitivity
improvements for HBsAg assays. However, HBsAg rapid tests still displayed
low sensitivity. For HIV serology, the required analytical sensitivity for p24
antigen detection of at least 2 IU/ml is achieved by the majority of current
HIV Ag/Ab combination assays. However, HIV Ag/Ab rapid tests are still not
commonly available.
General discussion meeting topics included reported stability issues
with established international standards for hepatitis A virus (HAV) RNA, HCV
RNA and hepatitis E virus (HEV) RNA. Standards for HCV RNA and HAV RNA
had displayed reduced stability during shipping at ambient temperatures and an
interim measure had been to send materials on dry ice. Further investigations
were ongoing. Although stool-derived (but not plasma-derived) HEV genotype
1 and genotype 2 are unstable (RNA decay) at elevated temperatures following
lyophilization in plasma matrix, this effect can be prevented by adding stabilizers.
Dr Reinhardt concluded by presenting an update on proposed actions to
be taken with respect to the further development of international standards for
a wide range of infectious markers.
2.2.3 Report from the network of WHOCCs for the

standardization and evaluation of vaccines
The network of WHOCCs for the standardization and evaluation of vaccines
currently consists of eight WHOCCs:

■■ National Institute for Biological Standards and Control (NIBSC),
Medicines and Healthcare Products Regulatory Agency, Potters Bar,
the United Kingdom;
■■ Center for Biologics Evaluation and Research (CBER), Food and
Drug Administration, Silver Spring, MD, the USA;
■■ Department of Bacterial Pathogenesis and Infection Control,
National Institute of Infectious Diseases (NIID), Tokyo, Japan;
■■ Immunobiology and Biochemistry Group, Office of Laboratories
& Scientific Services, Therapeutic Goods Administration (TGA),
Woden, Australia;
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■■ National Institute of Food and Drug Safety Evaluation (NIFDS),

Ministry of Food and Drug Safety (MFDS), Chungcheongbuk-do,
Republic of Korea;
■■ Biologics and Genetic Therapies Directorate (BGTD), Health
Canada, Ottawa, Canada;
■■ Institute for Biological Product Control of the National Institutes for
Food and Drug Control (NIFDC), Beijing, China;
■■ Division of Virology, Paul-Ehrlich-Institut (PEI), Langen, Germany.
Dr Junzhi Wang (NIFDC) reported on two symposia that had
been organized to discuss recent developments in the quality control and
standardization of vaccines. By sharing information on new vaccines, test methods
and reference standards a number of new areas for potential collaboration
were identified along with opportunities for strengthening collaboration within
the network.
An example of the potential benefits of close cooperation between
WHOCCs was provided by NIFDC and NIBSC collaboration in the development
of an international standard for enterovirus 71 neutralizing antibodies. In this
example, leveraging the resources and expertise of two WHOCCs had proved
to be highly beneficial in achieving the goal of providing a new international
standard for WHO endorsement. Dr Wang went on to outline a range of further
successful collaborations between NIFDC and other WHOCCs.
Dr Wang then concluded by describing the key role of regulatory
science in Ebola-related activities in China. This had included an emergency
two-week quality assessment of an SBP for treating infected patients. In addition,
NIFDC had played a central role in Ebola vaccine standardization in China.
A third meeting of the network would be hosted by MFDS in June 2016,
and will include a satellite meeting on the evaluation of vaccines for use in a
public health emergency. The main focus will be on reaching agreement on
priorities, assignment of responsibilities, and the setting of goals and timelines.
The Committee recognized the key importance of collaboration in these
and other areas, welcomed the presentation given and requested that it be kept
updated of the progress and future activities of the network.
2.2.4 Report of a WHO informal consultation on international

standards for biotherapeutic products
Dr Adrian Bristow reported on the above consultation, which had been held
in Geneva in 2015 in order to review a number of key underlying scientific
issues. The consultation had also sought to promote consensus in this area by
examining the results of a previously circulated stakeholder survey on various
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aspects of the standardization of therapeutic monoclonal antibodies, which had

been selected as an exemplar topic for biotherapeutic products in general.
Although the need for such international standards was questioned, the
majority view was that these were valuable in: (a) providing a benchmark for
biological activity; (b) method development and assessment of system suitability;
(c) the calibration of national, pharmacopoieal or in-house reference standards;
(d) assessing the potency of multisource products; (e) facilitating product
surveillance and life-cycle management; and (f) supporting the development of
novel methods.
It was recognized that reference standards are distinct from reference
products or reference medicinal products (for biosimilarity) and are not
interchangeable, having only limited overlap in both form and function. A
reference medicinal product serves to define the quality criteria that a candidate
must meet and is used as a comparator in evaluating the biosimilarity of
candidate preparations – a function that the reference standard does not serve.
Conversely, the reference standard serves to control, define, and calibrate the
performance of the test measurement system – a function that the reference
biological product cannot serve. In assessing biosimilarity, reference standards
thus have a limited but nonetheless essential role. Although such standards do
not serve as comparators for defining the quality of an acceptable product they
are essential in assessing the suitability of assay method and performance.
Biological activity units are considered to be useful during early stages
of SBP development for assay development and qualification purposes but the
specific activity of a reference standard should not be used to define acceptable
specific activity. In addition, unitage was likely to be specific to different assay
classes, and different units for different activities should be assigned. Although
the retrospective establishment of a unit should not in principle affect the
labelling or dosing regimens of existing or future products, a degree of concern
and caution was expressed at the consultation with regard to the unnecessary
retroactive applications of units by regulators. At present, there is some degree

of polarization of views in relation to the optimal extent of standardization
of activity.
In summary, despite recognition of the need for international standards
for biotherapeutic products, different views remain across both the regulatory
and manufacturing sectors with regard to their precise applications. Other
conclusions emerging from the consultation included the need for collaborative
studies to always include the innovator product, ideally as a candidate reference
standard, but at a minimum for comparison purposes. The maintenance of
a global and continuous supply of standards was seen as a key need with
the creation of multiple standards not favoured. Where possible, WHO and
pharmacopoeial standardization should be coordinated and relevant analytical
methods to control method performance developed.
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WHO has proceeded cautiously with a standardization programme

for monoclonal antibodies and other classes of “new” medicines as they gain
marketing authorization through the biosimilar route. There was a need to
recognize the concerns and potential impacts for affected stakeholders, and to
carefully consider the potential use and extent of applicability of these standards.
Consultation participants had therefore urged that WHO consider publicizing
the findings of this consultation.
The Committee noted this report, and reaffirmed its support for
WHO activities in developing international standards in this area, and ensuring
their appropriate use, namely as a benchmark for biological activity, for
method development and system-suitability assessment. The Committee
also recommended that efforts should be undertaken by WHO to address the
polarization of opinion identified in this report and to enhance communication
on the appropriate use of such standards. As a first step, areas of agreement
should be identified and used as the basis for further stakeholder discussion
to further clarify and distinguish scientific and policy issues, and to identify
approaches for their resolution.
2.3 Feedback from custodian laboratories

2.3.1 Developments and scientific issues highlighted by
custodians of WHO biological reference preparations
The Committee was informed of recent developments and issues identified
by the following custodians of WHO biological reference preparations.
National Institute for Biological Standards and Control

(NIBSC), Potters Bar, the United Kingdom
Dr Stephen Inglis presented an overview of the standardization activities
of NIBSC over the past year. While outlining the NIBSC standardization
programme, Dr Inglis highlighted the significant increase in activities and
the unprecedented number of projects being brought to the Committee for
establishment or endorsement in 2015. Such an expanding level of activity
sounded a cautionary note and would necessitate discussion on the specific
ways in which the Committee would deal with such a workload going forward.
In addition, there were other known areas of standardization that were not
yet being addressed by the Committee but which would imminently require
attention, such as the application of NGS and genomic testing.
The overview provided also included specific examples of where the
institute was moving forward and embarking on new developments within the
standardization field. It was noted that the merger with the Medicines and Health
Products Regulatory Agency in recent years had allowed for synergies between
15
the two organizations which had strengthened the position and activities of
NIBSC. After detailing a number of completed projects and new proposals for
consideration by the Committee in 2015, Dr Inglis briefly outlined the response
of NIBSC to the Ebola outbreak, and acknowledged the contribution of all
those who had been involved in developing materials for establishment at this
meeting in a very short time frame.
Among a number of current broader key issues Dr Inglis pointed out
that the expanding field of standardization activities inevitably raised questions
concerning the suitability of the current process in flexibly responding to and
meeting an increasingly complex range of demands. This raised the issue of
whether the current format and associated processes would be sufficiently
responsive and sustainable, particularly in the face of resourcing challenges.
Dr Inglis also drew attention to the increasing difficulty experienced in publishing
research findings in peer-reviewed journal in cases where data had already
been presented in the annual report of the Committee as part of the WHO
Technical Report Series. This was proving to be restrictive in both conveying
standardization messages to the relevant fields and in staff career progression
through publication in recognized journals. Dr Inglis then drew attention to a
number of upcoming events, which included a symposium on regulatory science
in 2016: shaping the future of biological medicines which was being held to mark
the 40th anniversary of NIBSC.
Dr Inglis concluded his presentation by announcing that he would be
retiring from the position of Director at NIBSC in April 2016 and introduced
his successor Dr Christian Schneider.
The Committee joined in thanking Dr Inglis for all his work over the
past 14 years and discussed a number of the key issues raised in his presentation.
In relation to the specific issue of publication there was some discussion around
the use of the journal Biologicals as this peer-reviewed journal was willing to
publish material previously presented in the reports of the Committee.
European Directorate for the Quality of Medicines &

HealthCare (EDQM), Strasbourg, France
Dr Karl-Heinz Buchheit outlined a number of recent EDQM activity areas in
biological standardization, including the European Pharmacopoeia, international
standards for antibiotics (ISAs), the Official Medicines Control Laboratory
network, the European Committee on Blood Transfusion, and in particular its
biological standardization programme (BSP) in which WHO has Observer status.
Dr Buchheit reminded the Committee that EDQM was the custodian
centre for ISAs – a responsibility that it had taken over from NIBSC in 2006.
Since that time, eight ISA replacement batches had been established, with
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another proposed for establishment by the Committee this year. As with other
international standards, ISAs require distribution, replacement as needed
and international collaborative studies. Of the current 23 ISAs eight relate to
antibiotics that are on the WHO Model List of Essential Medicines. Dr Buchheit
reaffirmed the willingness of EDQM to continue in its role as the custodian
centre for ISAs.
BSP goals included the establishment of European Pharmacopoeia
biological reference preparations, the standardization of test methods for
the quality control of biological substances, the elaboration of alternative
methods in support of the 3Rs concept (Replacement, Reduction, Refinement)
to minimize the use of animals in research and the provision of support to
international harmonization efforts, including through collaboration with WHO
and non-European partners. BSP achievements to date included the initiation
or conclusion of 102 projects on reference standards and 41 projects on method
development (including 21 projects on 3R methods).
Dr Buchheit reiterated that the development of alternatives to animal
experiments remained a major commitment of EDQM in line with European
Union directives, and WHO was once again strongly urged to consider
the incorporation of the 3R initiative into its written standards and other
guidance, where appropriate. The inclusion of 3R methods in WHO guidance
was viewed by EDQM as being of paramount importance in promoting their
global acceptance. Dr Buchheit also requested that the Committee evaluate
the possibility of its more proactive and earlier involvement in the validation
of 3R alternatives, such as a replacement for the histamine sensitization test
for pertussis vaccines. EDQM projects of potential interest to the Committee
included the development and evaluation of alternative in vitro tests for both
pertussis toxin and pertussis vaccine.
Dr Buchheit also informed the Committee that one of the main outcomes
of a recent International Alliance for Biological Standardization conference on
3R alternatives was the decision to formally request WHO to initiate steps to
delete the abnormal toxicity test from all WHO Recommendations, Guidelines
and other guidance documents. Dr Buchheit went on to suggest that the
Committee might begin to consider the implications for IUs for vaccines when
in vivo tests are replaced with in vitro tests, and the technical ability to perform
the in vivo test has been lost.
Dr Buchheit concluded by highlighting a number of key harmonization
and other implementation issues for regional standard-setting bodies when no
international standard or other WHO guidance was available. The potential
use of international standards and reagents in the veterinary field was also
highlighted as a specific issue requiring clarification of the most appropriate
lead organization.
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Paul-Ehrlich-lnstitut (PEl), Langen, Germany

Professor Klaus Cichutek presented an update on the recent activities of the
two PEI WHOCCs along with an overview of current scientific issues being
addressed. Activities of the WHOCC for Quality Assurance of Blood Products
and In Vitro Diagnostic Devices had included: (a) development of a WHO
Reference Panel for NAT-based assays for HEV genotypes; (b) development of a
WHO Reference Panel for anti-HEV antibodies; (c) enlargement of the current
WHO repository for transfusion-relevant bacterial strains for platelets; and
(d) establishment of a WHO repository for transfusion-relevant bacterial strains
for red blood cells.
Other PEI activities included regular and active participation in the BRN
and contribution to the development of WHO technical documents on residual
risk for virus infections in blood products and on the calibration of secondary
standards for IVDs (see sections 3.3.3 and 3.3.4 respectively). Collaboration
with the BRN was also undertaken in the important areas of advancing scientific
evaluation of the collection and use of convalescent plasma beyond the Ebola
outbreak, and ensuring the best use of WHO guidance on assessment criteria for
national blood regulatory systems.5
Professor Cichutek then outlined the ongoing PEI support provided to
the WHO Achilles project (see section 2.1.2) which had included participation
in a range of evaluation workshops and other major project activities requiring
the engagement of several PEI experts. PEI was also working to strengthen
the blood regulatory systems in two WHO Member States in the WHO
African Region.
Activities of the PEI WHOCC for the standardization and evaluation
of vaccines during 2014–2015 had included numerous collaborative efforts in
the key areas of Ebola, malaria, influenza and dengue. Other activities had
included identifying novel strategies for vaccination, discussing potential
approaches and requirements for maintaining the emergency availability of

smallpox vaccines, and contributing to the development and establishment
of WHO guidance on novel vaccines and innovative concepts. PEI also
conducted training in the Islamic Republic Iran on the regulation and batch
release of vaccines.
A number of current scientific issues were addressed by PEI in the last
year in relation to the Ebola outbreak 2014–2015, the seasonal influenza situation,
5 Assessment criteria for national blood regulatory systems. In: WHO Expert Committee on Biological
Standardization: sixty-second report. Geneva: World Health Organization; 2013: Annex 7 (WHO Technical
Report Series, No. 979; http://www.who.int/biologicals/expert_committee/TRS_979_62nd_report.pdf?ua=1,
accessed 12 February 2016).
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narcolepsy and MERS-CoV. The Ebola outbreak prompted multiple collaborative

research activities involving PEI, including: (a) support for the development of
the 2014 BRN position paper on Collection and use of convalescent plasma or
serum as an element in filovirus outbreak response; and (b) expert participation
in the development of an ad hoc Working Group guidance document, updated
in April 2015, on the Use of convalescent whole blood or plasma collected from
patients recovered from Ebola virus disease for transfusion, as an empirical
treatment during outbreaks. PEI also collaborated with the German Center for
Infection Research to foster clinical development of the experimental VSV-
ZEBOV vaccine and was the responsible German NRA for assessment of the
Phase I clinical trials in Africa and Europe.
Data on PEI seasonal influenza activities were then presented,
particularly in relation to the now-licensed tetravalent influenza vaccines
and the circulation of influenza B strains in the southern hemisphere, and
the potential implications for the upcoming northern hemisphere influenza
season. An update was also provided on narcolepsy studies in Germany, with
one interesting finding being that the incidence of narcolepsy in the country
had started to rise prior to the initiation of mass vaccination with the H1N1
influenza vaccine. Professor Cichutek then presented data on a measles-based
MERS-CoV vaccine that had shown very promising results in an animal model.
The measles-based system for vaccine development was a platform technology
that could be adapted to different pathogens, potentially allowing for a rapid
response to emerging diseases.
Professor Cichutek concluded by outlining a PEI project on the
standardization of birch pollen. Although the significance of pollen allergies has
been acknowledged for decades little progress has been made in characterizing
the responsible allergens. PEI was investigating the molecular composition of
birch allergens in order to allow for their standardization in diagnostic tests
and desensitization treatments, and to address the currently high degree of
variability in the composition and potency of available products.
Center for Biologics Evaluation and Research (CBER), Silver Spring, MD, the USA
Dr Jay Epstein informed the Committee of a wide range of ongoing and
proposed activities in the further development of potency standards, reference
preparations, international standards, reference panels and reagents.
In response to the Ebola epidemic CBER had expedited the review of
investigational new drugs for Ebola vaccine studies and of an investigational
device exemption for pathogen-reduced plasma for patient care. Information
sharing was facilitated by a Confidentiality Arrangement with WHO, with
frequent information exchange and sharing of updates on Ebola-focused
activities taking place with WHO, EMA, Health Canada and others. CBER also
19
participated in the development of physical standards and methodologies for

antibody and NAT standards, and for assays for total and neutralizing antibodies.
In addition, CBER had participated in African Vaccine Regulatory Forum joint
reviews for Phase I and Phase II/III clinical trial protocols for multiple candidate
Ebola virus vaccines, which had facilitated the initiation of clinical trials.
Dr Epstein also reported on the CBER-WHO Cooperative agreement to
enhance regulatory capacity to support influenza vaccine introduction in low-
and middle-income countries (LMIC). Funding had been provided to support
NRA assessments, training and an international proficiency study of the single
radial immunodiffusion assay. Under another Cooperative agreement, CBER had
provided support for the strengthening of global pharmacovigilance capacities
for vaccines and, in conjunction with the Uppsala Monitoring Centre, training
in the application of pharmacovigilance to regulatory decisions for vaccines.
In 2015, United States Food and Drug Administration approval was given
for the first time to an SBP. In addition, a proposed rule and draft guidance was
issued on nonproprietary naming conventions for biological products. In the
area of blood donation, a final rule on donor eligibility was published in May
2015 that established the requirement to evaluate donors for factors that may
adversely affect the safety, purity and potency of blood components or the health
of the donor. This final rule also provides a flexible framework for responding
to emerging infectious diseases and provides criteria for requiring or removing
testing for transfusion-transmitted infections.
CBER personnel also participated in an international study to assess
the reproducibility of influenza neuraminidase inhibition titres. This study
had been coordinated by the Consortium for the Standardization of Influenza
Seroepidemiology and involved 23 laboratories in 12 countries. Factors
contributing to assay variability were identified and an enzyme-linked lectin
assay protocol will be updated and made available to laboratories working in this
area. The development of reference materials for advanced virus detection using
NGS was another important new area in which CBER had been cooperatively
engaged, and materials should be available by early 2016. Dr Epstein then
outlined CBER participation in a number of international studies to develop and
evaluate vaccine-related standards for respiratory syncytial virus neutralization
assays, human cytomegalovirus immunoglobulin G (IgG), and Ebola antibody.
A number of completed and ongoing standards activities for plasma-derived
coagulation factors were also described, and current and proposed work on the
development of reference preparations for viruses and other pathogens outlined.
Dr Epstein concluded by highlighting a number of recent workshops
sponsored by CBER and noting the value of such workshops in the successful
implementation of regulatory policy, and the importance of regulatory science
research.
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2.4 Cross-cutting activities of other WHO committees and groups

2.4.1 Update on matters arising from the International
Nonproprietary Names Expert Group
The Committee was provided with an overview of the WHO International
Nonproprietary Names (INN) programme and informed that more than 45%
of applications for new INN were for biological products. Areas of particular
interest to the Committee included the challenges and complexities in relation to
the nomenclature used for monoclonal antibodies, recombinant blood products,
peptide and recombinant vaccines, and gene-therapy products.
The Committee was reminded that discussions on an INN Expert
Group proposal for a Biological Qualifier (BQ) scheme for biological products
had been initiated in 2014 with the circulation of a draft proposal for public
comment. Following extensive feedback this proposal had been modified and
further discussion and feedback had taken place in 2015 resulting in further
changes that were to be considered by the INN Expert Group in October 2015.
Although the majority of stakeholders supported the use of the BQ scheme,
some opposition remained. The Committee was informed that the WHO INN
programme will continue to work on modifying the proposal and to propose its
implementation at the appropriate time.
The Committee had also been informed in 2014 that consideration
continued to be given by the INN Expert Group to the establishment of a
nomenclature for cell therapies. The Committee was informed that cell therapies
are now approved in a number of different areas of the world, including the
United States, Europe and Korea, and that many more such products were
reaching the naming stage, with 25 cell therapies in Europe having received
orphan designation. The WHO INN programme had received several formal
requests for the designation of an INN, and the United States Adopted Names
program (USAN) had so far provided over 15 names for cell therapies. Multiple
non-uniform naming schemes have the potential to generate great confusion.
WHO discussions had taken place over the last 5 years on a naming scheme
for cell therapies, with several schemes having been proposed. Difficulties
identified included the use of very long and complex names, which would be
difficult to memorize and at times pronounce. Such complexity might also
lead to transcriptional errors. In addition, as cell-therapy technology rapidly
evolves some schemes may not have considered all the possibilities and risk
been rapidly outdated. In response to these issues, a working group had been
created, stakeholders consulted and a flexible scheme formulated that provided
a compromise between the length of the name and the amount of information
embedded. The proposed scheme was accepted by both USAN and CBER, and
will be discussed during the October 2015 meeting of the INN Expert Group.
21
In relation to the development of a BQ scheme for biological products

the Committee expressed its appreciation for the progress that has been made
since it last considered this issue and asked to be kept informed of future
developments. It was emphasized that implementation of this scheme should
occur soon in order to mitigate the proliferation of national identification
schemes. The Committee also welcomed the progress made in the development of
an agreed-upon nomenclature for cell therapies and asked to be kept informed
of future developments in this area.
2.4.2 Collaborative procedure for facilitating national

registration of WHO prequalified medicinal products
The Committee was provided with a brief overview of the potential advantages
and key features of WHO prequalification of pharmaceutical products and
vaccines. In 2014, the Committee had been informed that a revised procedure
to facilitate national registration of WHO prequalified medicinal products was
under evaluation and that a revised draft had been prepared and made available
for public comment.
Following its endorsement by the Expert Committee on Specifications
for Pharmaceutical Preparations, a number of advocacy workshops and capacity-
building activities were now being planned to facilitate the use of the revised
process. It is intended that a single agreement will be reached with NRAs in
participating countries covering both pharmaceutical products and vaccines. The
importance of early access to data from manufacturers was stressed as a key issue,
particularly in contexts such as pandemic influenza, and the issue of incentives
for manufacturers was identified as an area requiring further consideration.
The revised procedure remains voluntary for both manufacturers and NRAs,
and continues to offer significant potential benefits to both parties. With the
agreement of the manufacturer, the full prequalification dossier and site-audit
report plus initial testing results would be shared with interested NRAs to
facilitate national regulatory decision-making in relation to product registration,
variation or withdrawal. The utility of such a collaborative procedure had already
been demonstrated, including during the facilitated licensing of inactivated
poliomyelitis vaccine (IPV) in several countries.
Discussion was held on a broad range of issues, challenges and
opportunities in this area, including in the areas of national and regional
regulatory capacity-building, the role of regional implementation workshops
and associated initiatives, and the need for manufacturer engagement. The
Committee agreed that the revised document would be useful and looked
forward to being updated on its implementation at a future meeting.
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2.4.3 Proposal to transition from microbiological to

physicochemical assays for antibiotic potency testing
There are currently five International Chemical Reference Substances (ICRS)
established as secondary reference standards for tests according to chapter 3.1
(Microbiological assay of antibiotics) of The International Pharmacopoeia. To
ensure the continuous fitness for purpose of these reference substances, their
assigned potencies have to be monitored regularly in extensive and resource-
consuming collaborative trials. In addition, a total of 21 monographs prescribe
a microbiological assay for antibiotics, but no suitable reference substance has
yet been established.
At its meeting in 2009 the Expert Committee on Specifications for
Pharmaceutical Preparations had decided that in monographs for antibiotics
which specify a microbiological assay, this test should be replaced by a
chromatographic method where possible and appropriate. In view of the above,
the Secretariat of The International Pharmacopoeia proposed to the Expert
Committee on Specifications for Pharmaceutical Preparations and to the
Expert Committee on Biological Standardization to:
1. discontinue the use of five ICRS in microbiological assays of

antibiotics and to delete the potency assignments in the ICRS leaflets;
2. r evise four monographs in order to replace the microbiological
assay with liquid chromatography methods, considering methods
already published in pharmacopoeias;
3. revise five monographs in order to replace the ICRS with WHO
international standards for antibiotics or, preferably, secondary
standards derived from them and established by another
pharmacopoeia for use in microbiological assays, to foster work-
sharing between pharmacopoeias;
4. develop a concept document for the possible transition from
microbiological to physicochemical assays in 14 monographs,
considering in particular chromatographic methods published in
the scientific domain, for discussion and possible endorsement by
the above two Expert Committees; and
5. suppress the monographs for substances containing any of five
active ingredients – medicines containing these substances are no
longer included in the WHO Model List of Essential Medicines
(19th List) or in the relevant invitations for expression of interest
to manufacturers.
23
Both Expert Committees agreed to the proposals described in points

1, 2, 4 and 5 above. With regard to the proposal outlined in point 3, it was
agreed that the experts should be given more time to identify possible reference
standards that could be referred to in each of the monographs. The relevant
ICRS and monographs affected by these decisions are listed in Table 1.
Table 1
Recommendations relating to the use of microbiological assays for antibiotics
1 ICRS no longer to be used for microbiological assays of antibiotics, and potency

assignments to be deleted
1. nystatin (ICRS0369)
2. framycetin sulfate (neomycin B) (ICRS0355)
3. gentamicin sulfate (ICRS0319)
4. spectinomycin hydrochloride (ICRS0415)
5. streptomycin sulfate (ICRS0416)
2 Monographs in which microbiological assay should be replaced by liquid
chromatography methods
1. erythromycin ethylsuccinate
2. erythromycin lactobionate
3. erythromycin stearate
4. tetracycline hydrochloride
3 Monographs for which suitable standards other than ICRS should be identified
1. amphotericin B
2. amphotericin B for injection
3. bleomycin sulfate
4. kanamycin for injection
5. kanamycin monosulfate
4 Monographs for which a concept paper should be developed on the possible
transition from microbiological to physicochemical methods
1. amphotericin B 6. kanamycin acid 11. gentamicin
2. amphotericin B for sulfate sulfate
injection 7. kanamycin for 12. streptomycin
3. bleomycin sulfate injection sulfate
4. erythromycin 8. kanamycin 13. streptomycin for
ethylsuccinate tablets monosulfate injection
5. erythromycin stearate 9. nystatin 14. paromomycin
tablets 10. nystatin tablets sulfate
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Table 1 continued
5 Monographs that should be suppressed
1. bacitracin 4. chlortetracycline 7. oxytetracycline
2. bacitracin zinc hydrochloride dehydrate
3. bleomycin hydrochloride 5. erythromycin (base) 8. oxytetracycline
6. neomycin sulfate hydrochloride
2.4.4 Labelling information for influenza vaccines

intended for use in pregnant women
The Committee was informed that in 2012 WHO had recommended that
pregnant women be vaccinated with trivalent inactivated influenza vaccines
(IIVs) at any stage of pregnancy because of the high risk of severe illness and
even death associated with influenza in pregnant women and children under 6
months of age. Influenza vaccines have a history of safe use in pregnant women,
and the WHO Strategic Advisory Group of Experts (SAGE) on Immunization
had requested that WHO develop a plan and associated process for improving
maternal immunization coverage. In addition, the GAVI Alliance was considering
the inclusion of seasonal influenza vaccination with an emphasis on maternal
immunization into its 2014–2020 Vaccine Investment Strategy.
In response to these initiatives, WHO had established a working group
to develop advice on labelling information for influenza vaccines intended
for use in pregnant women. In July 2014 an informal consultation was held to
assess current approaches and experiences. In some countries the wording used
in product labels allows for the use of influenza vaccines in pregnant women,
as it is not contraindicated. In addition, although precautionary statements
may be required by some regulators to reflect the available safety evidence
these are not intended to be barriers to use. There is however some diversity
among regulators in the use of either cautionary or supportive statements in
the package insert which in the case of the former can be misinterpreted by
other regulatory authorities, pregnant women, vaccine users and immunization
programme managers as a warning against the use of IIVs in pregnant women.
It was concluded at the consultation that information should be presented and
communicated in a way that would be most helpful to vaccine users, policy-
makers, administrators and other relevant parties.
A subsequent survey of Developing Country Vaccine Regulators’ Network
(DCVRN) member countries in October 2014 highlighted inconsistencies in
the wordings used and information provided in package inserts. In addition,
NRAs may have a different interpretation than policy-makers regarding the
precise meaning of the wordings used. It was concluded that WHO guidance on
25
a common policy for package insert wording would improve communication of

the benefits and safety of IIVs administered during pregnancy.
A follow-up working group meeting held in September 2015 had
reviewed current guiding principles in relation to the vaccine labelling to be
used for pregnant women, and had discussed the development and overall
issues to be covered in the proposed WHO guidance. It was agreed that such
guidance could take the form of an addendum to the existing TRS 927 Annex 3
on IIVs, and that its purpose should be to clarify and assist interpretation of the
information provided in the pregnancy subsection of IIV package inserts.
During general discussion a recommendation was made that as
the addendum to TRS 927 is developed, due consideration be given to the
regulatory principle that data must be available to support conclusions about
safety, and that such data are product-specific and usually generated by the
manufacturer. The Committee expressed its interest in this project and asked
to be kept informed of further progress with a view to possible submission of
the addendum document to the Committee in 2016.
2.4.5 Influenza vaccine response during the start of a pandemic

The background context for pandemic influenza preparedness and response
activities is complex and involves a range of international and other initiatives
including the WHO Pandemic Influenza Risk Management framework, the
Pandemic Influenza Preparedness Framework for the sharing of influenza viruses
and access to vaccines and other benefits, and national and regional pandemic
preparedness plans.
As part of ongoing WHO efforts in this area the Committee was
informed that an informal consultation had been held in June 2015 to discuss
and develop specific guidance on initiating an influenza vaccine response
during the start of a pandemic. The consultation had involved 33 participants
from 18 countries, including representatives from WHOCCs, NRAs, individual
manufacturers and manufacturer organizations, academic institutions and

other stakeholders. Discussions also focused on identifying the roles and
responsibilities of all involved entities.
In addition to the progress made towards the development of a draft
operational framework to guide a pandemic vaccine response, key meeting
outcomes had included greatly improved understanding of the currently severe
timeline constraints and production bottlenecks. At present, 3–4 months
appeared to be the minimum time required between the identifying of candidate
vaccine viruses and vaccine availability. Maintaining and strengthening the very
high degree of global coordination required for the current seasonal influenza
vaccine process was also recognized as an essential element in achieving any
future improvements in the vaccine development and production timeline.
26
General
Potential areas for improvement included: (a) reviewing the current WHO
biosafety requirements 6 for manufacturing facilities; (b) reviewing candidate
vaccine virus safety-testing standards and release specifications; (c) improving
the availability of reagents and assays; (d) ensuring clear and effective regulatory
pathways for pandemic vaccines (including for new vaccine platforms and
technologies); and (e) improved understanding of the roles and responsibilities
of stakeholders in terms of communication and interaction.
During discussions, further clarification was given of the vital
importance of promoting and expanding seasonal influenza vaccine use as part
of ensuring that sufficient production capacity would exist for pandemic vaccine
production. The Committee was also informed of the high degree of interest
in moving towards a new paradigm of influenza vaccine virus selection and
development that would allow for the more rapid production of well-matched
seasonal and pandemic influenza vaccines. The Committee expressed its thanks
for the update provided in this important area and asked to be kept informed of
further developments.
WHO biosafety risk assessment and guidelines for the production and quality control of human influenza
6
pandemic vaccines. Update. Geneva: World Health Organization; 2009 (http://www.who.int/biologicals/

areas/vaccines/influenza/CP116_2009-2107_Biosafety_pandemicA_H1N1_flu_vaccines-Addendum-
DRAFTFINAL.pdf, accessed 14 February 2016).
27
3. International Recommendations, Guidelines
and other matters related to the manufacture
and quality control of biological substances
All WHO Recommendations, Guidelines and guidance documents established
at the meeting are listed in Annex 1, which provides an updated listing of all
current WHO Recommendations, Guidelines and other documents related to
the manufacture, evaluation and quality control of biological substances used
in medicine.
3.1 General
3.1.1 WHO good manufacturing practices for biological products
The Committee had been informed in 2014 of the history of WHO good
manufacturing practices (GMP) documents for pharmaceuticals, biological
products and blood establishments. The most recent WHO guidance on GMP
for biological products had been published in 1992 (Annex 2, WHO Technical
Report Series, No. 822). Following the initiation of a review process in 2007, a
consultation had been held in 2014 with the resulting draft revised Guidelines
then undergoing two rounds of public consultation in 2015. The text was
further amended by the guidelines drafting group taking into account the
comments received.
The outcome revised Guidelines document (WHO/BS/2015.2253) was
viewed as complementary to the general recommendations set out in the
current WHO good manufacturing practices for pharmaceutical products: main
principles 7 and in other WHO documents related specifically to the production
and control of biological products. As the revised document does not provide
detailed recommendations for specific classes of biological products, attention
was drawn in the text to the need to consult other relevant WHO documents,
in particular WHO recommendations to assure the quality, safety and efficacy of
specific products.
The proposed revision reflects recent scientific and technological
developments in the manufacture and control of biological products, and in
the application of risk-based approaches to GMP, while recognizing the wide
variability inherent in this evolving class of medicinal products.
WHO good manufacturing practices for pharmaceutical products: main principles. In: WHO Expert
7
Committee on Specifications for Pharmaceutical Preparations: forty-eighth report. Geneva: World Health
Organization; 2014: Annex 2 (WHO Technical Report Series, No. 986; http://www.who.int/medicines/
areas/quality_safety/quality_assurance/TRS986annex2.pdf?ua=1, accessed 14 February 2016).
28
International Recommendations, Guidelines and other matters
The Committee was then provided with a detailed overview of the precise
scope and principal themes covered by the revised Guidelines. The document does
not cover human whole blood, blood components and plasma-derived products
for therapeutic use as separate comprehensive WHO guidance is available and
should be followed.8,9 The characteristically wide variability typical of biological
products and corresponding manufacturing processes, are emphasized in the
revised document, along with specific aspects of the analytical methods and
approaches required. The importance of in-process controls are emphasized and
the quality risk management (QRM) approach recommended as an effective tool
for managing product variability, preventing quality deviations and strengthening
product and process knowledge.
The Committee reviewed the major comments received during the
second round of public consultation and requested a number of clarifications.
After making further changes, the Committee recommended that the revised
WHO Guidelines be adopted and annexed to its report (Annex 2).
3.1.2 Update on the development of a candidate control material

for adventitious virus detection using deep sequencing
The Committee was reminded that in 2010 porcine circovirus-1 DNA had been
detected in two rotavirus vaccines using deep sequencing, thus demonstrating
the potential utility of this technology in improving or replacing existing
adventitious agent tests. However, in order to be useful as a quality control test,
the technology must be optimized to determine the most appropriate assay and
limits of detection, which in turn requires reference materials to enable the
comparison of results and validation of assays.
A reference reagent was formulated by NIBSC (viral multiplex
11/242‑001) comprised of a pool of 25 human viruses produced in cells or eggs,
or obtained from clinical specimens. Post-formulation qPCR results suggested
variable concentrations with six of the viruses being undetectable. The reagent
was distributed to 16 laboratories in nine countries for reagent processing
using their preferred deep sequencing-based detection method. Most of the
laboratories detected at least 20 of the 25 targets with six of the viruses being
detected by every laboratory. Two laboratories detected all 25 viruses.
8
WHO guidelines on good manufacturing practices for blood establishments. In: WHO Expert Committee
on Specifications for Pharmaceutical Preparations: forty-fifth report. Geneva: World Health Organization;
2011: Annex 4 (WHO Technical Report Series, No. 961; http://www.who.int/bloodproducts/publications/
GMP_Bloodestablishments.pdf?ua=1, accessed 2 February 2016).
9
Recommendations for the production, control and regulation of human plasma for fractionation. In: WHO
Expert Committee on Biological Standardization: fifty-sixth report. Geneva: World Health Organization;
2007: Annex 4 (WHO Technical Report Series, No. 941; http://www.who.int/bloodproducts/publications/
TRS941Annex4blood.pdf?ua=1, accessed 2 February 2016).
29
The study demonstrated that current methods differ in their ability

to detect a set of defined viruses. Factors impacting on the ability of a given
method to detect an adventitious virus include variable molecular biology,
bioinformatics methods, databases and criteria/thresholds for defining a “hit”.
The study also underscored the value of a common reagent to enable comparison
of different methods.
The reference reagent will be added to the NIBSC catalogue with about
1200 vials remaining, and may be useful as an interim qualitative reference
material. Discussions are being held on the design of a new reference reagent
containing fewer viruses and at least one prototype from each of the most
relevant viral groups. The source of viruses is also under discussion. Discussions
of sample requirements and study design are also ongoing with regulatory
agencies and other interested parties. It is expected that the new reference
reagent will be available in 12–18 months and that a collaborative study will
begin in about 12 months.
3.2 Biotherapeutics other than blood products

3.2.1 Regulatory assessment of approved rDNA-derived biotherapeutics
The regulatory assessment of biotherapeutic protein products prepared
by recombinant deoxyribonucleic acid (DNA) technology (rDNA-derived
biotherapeutics) which, for various reasons, have been licensed with data
packages that do not follow current international regulatory standards has been
a problematic issue for many countries.
In 2010, ICDRA discussions on such situations were held and WHO
requested to assist in developing approaches for evaluating these already-
licensed products according to current WHO guidance. In 2014, the Sixty-
seventh World Health Assembly adopted two relevant resolutions: one on
promoting access to biotherapeutic products and ensuring their quality, safety
and efficacy, and the other on regulatory systems strengthening in which WHO
was requested to provide guidance, especially on dealing with increasingly

complex biological products.
The purpose of the proposed addendum document10 was to provide
guidance on the regulatory assessment of rDNA-derived biotherapeutics in
cases where their licensing did not follow current international regulatory
standards – including, for example, biotherapeutic products licensed via a
generic pathway or with limited analytical, nonclinical and/or clinical data.
Addendum to: Guidelines on the quality, safety and efficacy of biotherapeutic protein products prepared
10
by recombinant DNA technology. In: WHO Expert Committee on Biological Standardization: sixty-fourth
report. Geneva: World Health Organization; 2014: Annex 4 (WHO Technical Report Series, No. 987; http://
www.who.int/biologicals/biotherapeutics/TRS_987_Annex4.pdf?ua=1, accessed 15 February 2016).
30
Although dealing primarily with rDNA-derived biotherapeutics, some aspects

of the document may also be relevant to other biotherapeutics.
Specific areas addressed included regulatory expectations for rDNA-
derived biotherapeutics (including SBPs), the reviewing of products already
on the market, the points to consider in a stepwise regulatory assessment and
the regulatory actions to be considered following assessment. The stepwise
regulatory assessment approach outlined was designed to offer flexibility and
to promote the accessibility of biotherapeutic products of assured quality, safety
and efficacy.
The outcome document (WHO/BS/2015.2251) had undergone extensive
consultation involving six rounds of regulatory expert review and three rounds
of public consultation. Participation in the consultation process had been broad
and had involved NRAs and national control laboratories (NCLs) from 41
countries in all six WHO regions, EMA and DCVRN, three pharmacopoeias, 11
manufacturer associations and 14 individual manufacturers in nine countries.
The Committee reviewed the document and after proposing a number
of changes to further improve the clarity of the text, recommended that the
proposed addendum be adopted and annexed to its report (Annex 3). The
Committee also emphasized the urgent need for implementation workshops
in this area.
3.3 Blood products and related substances

3.3.1 Ebola-related issues
The Committee was provided with an overview of concerted efforts made by
WHO to prioritize the further investigation of a number of products for the
prevention or treatment of Ebola infection. Three field studies on the use
of convalescent plasma had been conducted in Guinea, Liberia and Sierra
Leone. Differences and similarities in the approaches taken were highlighted
and acknowledgement made of the high level of teamwork demonstrated
by the multiple organizations involved. Final study results from the largest
of these studies, conducted in Guinea and constituting the largest ever trial of
convalescent plasma, were expected in the coming weeks.
In early 2015 a meeting of directors of national blood transfusion
services was held and a range of views expressed in relation to the conducting
of the trials and the challenges faced. Given the extent of pre-existing health
system weaknesses in each of the above affected countries the achievement was
especially notable. One key aim now was to catalyse the short-term support for
research and development efforts in the blood area into medium- and longer-
term sustainable support for the development of the national blood systems. It
was then highlighted to the Committee that while the response to the 2014/2015
Ebola epidemic had progressed well in a short time frame, there was a need for a
31
much more rapid response, and that lessons had been learned that would assist
in defining rapid-response requirements for potential future pathogen outbreaks.
The Committee observed that there had been some degree of criticism
following a perceived slow response from WHO. Nevertheless, it was also
apparent that the scale of work and progress made in a very short space of time
had been considerable.
3.3.2 Local production of blood products

An overview was provided to the Committee of WHO activities conducted in
Indonesia, Kenya and Benin in relation to the strengthening of local production
of blood products. Indonesia had been chosen as a pilot country for the WHO
Achilles project on fostering the use of recovered plasma to produce GMP-
compliant plasma products. This project started in 2013 with a situation
analysis of the national regulatory framework, which was followed in 2014 by
workshops on the evaluation of screening tests and GMP training. In 2015, an
expert assessment was made of GMP in a selected pilot blood establishment
in Surabaya. It was concluded that the establishment had excellent starting
conditions, being a successful blood service with a high donor-recruitment rate
and good infrastructure. Moreover, a quality management (QM) system had been
introduced, albeit to varying degrees in different departments. Recommendations
from the assessment included strengthening of the QM system and liaising with
a blood regulatory system in the geographical region. In continuation of the
project planned activities included: (a) holding a workshop with the Ministry of
Health reference laboratory for blood screening and diagnostic IVDs, covering
the assessment of manufacturer performance data, evaluation algorithms and
cooperation with established reference laboratories; and (b) reassessment of
GMP implementation, potentially again in Surabaya following initiation of the
use of plasma for fractionation.
In Kenya, an assessment of the blood regulatory system was conducted
in 2015. The Committee was provided with a brief overview of the status of
blood regulation in Kenya, the significant challenges still being faced, and the
initiation of collaborative efforts between WHO and the Kenyan National Blood
Transfusion Service to improve the safety of the national blood supply. Following
up on recent efforts in the WHO African Region, a workshop on the development
of a regional strategy for blood safety and the establishment of national regulatory
systems for blood and blood products had been held in Benin in 2015 involving
regulators and blood-establishment representatives from 13 countries, as well as
representatives from WHO, WHO BRN and PEI. This workshop had provided an
opportunity for the sharing of national experiences in blood regulatory activities,
and for the drafting of a regional blood safety strategy for the upcoming 10 years
along with guidelines on the establishing of a regulatory system for blood and
blood products.
32
During discussion, it was clarified that Indonesia intends to initiate

plasma fractionation within the next two to three years. The Committee was
also informed of an upcoming International Society of Blood Transfusion
meeting in November 2015 at which an update on the national blood system in
Indonesia will be presented. The Committee concluded that WHO initiatives
in this area had been successful and should be continued. However, it was also
acknowledged that resourcing and capacity issues would likely arise as levels of
demand for WHO assistance increased.
3.3.3 Residual risk of virus infections caused by blood products

An outline was presented to the Committee of draft WHO Guidelines on
estimating the residual risk of virus infections caused by blood products (such
as cellular blood components or recovered plasma) obtained from whole blood
donations. The development of such guidance had been requested by blood-
donation centres in LMIC during meetings held in the context of the WHO
Achilles project. It had been recognized that currently there is no common and
easy approach for calculating the residual risk because of different screening
assays used in different regions, and a corresponding lack of data on both assay
characteristics and blood-donor epidemiology.
In June 2015, a WHO Working Group meeting had been convened and
draft guidelines prepared to differentiate between screening-test categories
and define the mean diagnostic window phase for each category. Furthermore,
maximal viral loads during the diagnostic windows of different virus infections
(HIV, HBV and HCV) were defined. Combined with epidemiological data
on the donor populations an approach for the calculation of residual risk
was proposed. For repeat-donor subpopulations, residual risk estimation is
primarily based upon the infection rate (incidence) based upon the number
of seroconversions in a one-year observation period, donation frequency and
length of the viraemic window phase. In first-time donor subpopulations
incidence is currently typically determined using more complex approaches
such as modified antibody assays or NAT-only data.
In the draft Guidelines under development it has been proposed that
incidence in this latter subpopulation may be reflected by the incidence in the
respective repeat-donor subpopulation combined with an adjustment factor.
Further guidance includes an adjustment factor for the transient early markers
of HBV infection and a formula for the calculation of maximal numbers of
window-phase donations. Using the proposed approach may allow for greater
consistency in calculation methods, thus allowing for improved comparisons
between different donor populations and different blood centres. It is expected
that the Guidelines will also facilitate decisions on screening-test strategies
based on cost–benefit analyses, and help plasma fractionators to decide upon
the acceptability of recovered plasma.
33
Discussion focused upon the acceptability of using the proposed

extrapolation factor of x3 to estimate incidence in first-time donors based upon
incidence among repeat donors. However, since this adjustment represents
a worst-case scenario and given that further testing would still be possible at
later stages, it was considered acceptable. A further issue raised was the extent
to which the accuracy of IVDs was precisely known and the potential need for
additional cautionary text on this in the proposed document.
3.3.4 WHO general assessment tool for conducting gap analysis of

national regulatory systems for health products and technologies
An overview was presented of a proposed WHO general assessment tool for
conducting gap analysis of national regulatory systems for health products and
technologies. The development of this tool had been agreed upon at the January
2015 WHO International Consultation on Regulatory Systems Strengthening.
The purpose of the general assessment tool was to allow for harmonization
of the broad range of different assessment tools currently used for different
product streams and/or technologies by both WHO and non-WHO agencies,
and to permit the harnessing of synergies between already existing assessment
systems. One such example was the recently adopted WHO assessment criteria
for national blood regulatory systems.11 It was envisaged that use of the general
assessment tool and associated alignment of tools used for individual product
areas would help ensure the compatibility of assessments or other evaluations,
thus avoiding the need to repeat work already done.
The tool had been discussed during the 2015 consultation and developed
by WHO on the basis of the advice received. The current version of the tool was
outlined to the Committee for its information and would be subject to another
round of international consultation in December 2015 with further progress to
be reported to the Committee in 2016.
During discussion, it was pointed out that while such a general assessment
tool may be very useful, and that amendment of the WHO assessment criteria for
national blood regulatory systems would potentially be beneficial in principle, it
would also be important to preserve the details of the different product streams
and/or technologies addressed by individual assessments. It was also reiterated
that the assessments resulting from already existing tools would need to be
accepted after validation to avoid duplication of efforts. It was further pointed
out that the terminology used for the different categories of product needed to
Assessment criteria for national blood regulatory systems. In: WHO Expert Committee on Biological
11
Standardization: sixty-second report. Geneva: World Health Organization; 2013: Annex 7 (WHO
Technical Report Series, No. 979; http://www.who.int/biologicals/expert_committee/TRS_979_62nd_
report.pdf?ua=1, accessed 12 February 2016).
34
be consistent. The Committee recommended that the topic be further discussed

at the next BRN meeting.
3.4 In vitro diagnostic device reagents

3.4.1 Calibration of secondary standards for in vitro diagnostic devices
The development of a WHO guidance document outlining principles for the
calibration of secondary reference materials for IVDs was first discussed in July
2012 and endorsed by the ECBS in October that year. The proposed document
was intended to provide practical guidance on the preparation of secondary
references and their calibration relative to WHO international standards,
facilitate a consistent approach to calibration, improve the availability of
secondary reference materials and contribute to the global harmonization and
quality assurance of IVDs.
The document is intended for use by all manufacturers of secondary
references, and covers both NAT-based and serological infectious disease
assays, the general principles of biological standardization, considerations in
the preparation of secondary references, and the need for collaborative studies,
instructions for use and eventual replacement.
Following collaborative preparation of the first draft by NIBSC and PEI,
the document was presented at the 20th anniversary meeting of the SoGAT
group. Meeting participants provided comments and suggested that a number
of other organizations not present at the meeting should be made aware of the
document. The resulting revised draft combined all comments received to date
and it was now the intention to seek the input of the Committee prior to the
completion of a final draft version for submission to the Committee in 2016.
During discussion, the issue of overlapping WHO guidance was
raised and it was highlighted that the current WHO Guidelines outlining the
development of an international standard already makes general mention
of the need for appropriate calibration of secondary reference materials.
In response, it was suggested that the proposed document was required to
provide more detailed and specific information to address needs in this area.
Discussion then continued in relation to the precise scope and placement of the
proposed document in the context of existing WHO guidance. It was agreed
that such decisions would be driven largely by whether the document was to be
incorporated into the overview guidance or to remain standalone.
Discussion was then held on the envisaged main users of the document.
Although there were commercial manufacturers of such materials the restricted
supply does not meet the needs of assay developers, and the area would likely
benefit from a harmonized approach to the production of these materials. It was
noted that smaller NCLs may also benefit from such a resource, particularly
where they had ready access to patient materials that could serve as a standard.
35
After further consideration it was decided that that a final version of the
document should be presented to the Committee in 2016 prior to a decision
being made on its placement in the context of current guidance.
3.4.2 WHO prequalification of in vitro diagnostic devices

The WHO Prequalification Team is part of WHO/EMP and provides independent
technical information on the safety, quality and performance of IVDs. While
certain IVDs (for example, those used to aid in the diagnosis of HIV infection)
have been assessed by WHO since 1998, a shift occurred in 2008 away from
assessments based solely on performance evaluation and towards a more robust
prequalification assessment which also includes dossier review and manufacturing
site inspection. Future planned developments of the IVD programme were
intended to lead to the development of a set of guidance documents and the
conducting of training sessions to assist IVD manufacturers.
Many of the IVDs used in resource-limited settings were rapid diagnosis
tests, with prequalification dossier reviews indicating that many manufacturers
did not properly use existing reference materials. Independent lot testing at
product delivery to countries was also recommended for such products to
ensure that performance had not been jeopardized during transportation and
storage. It had been concluded that for IVDs used for priority diseases, reference
materials that cover all genotypes and subtypes, as well as common mutants, are
needed. Also, clear regulatory requirements on the use of reference materials
need to be developed, with clear indications that such materials also need to be
commutable and fit for purpose for use outside Europe and the United States.
The Committee congratulated the group on the breadth of this
important work. In the longer term this breadth may raise issues in relation to
the available expertise within current collaborating centres. The Committee was
informed that that the Prequalification Team regularly interacted with other
WHO work programmes to assess priority needs, and it was acknowledged
that linking the prequalification of IVDs to the Committee process would

also assist in the prioritization of standardization projects. However, it was
also acknowledged that sourcing sufficient funding may present a problem
in the continuation of its work. Discussion also took place of the approach of
the Committee in establishing one international standard for each analytical
marker, with subsequent reference materials for that marker comprising a
defined reference panel. Such an approach may prove to be too restrictive for
prequalification needs.
It was noted that the proposed further development of guidance
documents was needed as there was a lack of knowledge regarding available
materials (for example, secondary standards) and of their calibration and correct
use. Data exchange was also discussed and acknowledged to be another area
36
where guidance and development work is needed. After further consideration

it was proposed that the Prequalification Team develop guidance for
manufacturers on the use of international reference materials for the calibration
and characterization of diagnostic assays with a view to presenting this to the
Committee in 2016.
3.5 Vaccines and related substances

3.5.1 Recommendations to assure the quality, safety and efficacy of
recombinant human papillomavirus virus-like particle vaccines
WHO Guidelines to assure the quality, safety and efficacy of recombinant human
papillomavirus (HPV) virus-like particle (VLP) vaccines were first adopted by
the Committee in 2006 12 and were based largely on experience gained from
clinical trials undertaken on the first two licensed HPV vaccines.
Factors that prompted the revision of these Guidelines included the
substantial amount of data accumulated during vaccine implementation, the
development of prophylactic vaccines with extended valency and the use of
other production methods. In addition, the increasing availability and routine
use of HPV VLP vaccines composed of L1 capsid protein and containing at least
types 16 and 18 have important implications for trial designs and end-points for
clinical evaluation of new prophylactic HPV vaccines.
In 2013, a series of meetings was convened by WHO to review new HPV
vaccines under development, and to discuss the evidence and scientific basis for
accepting alternative end-points for evaluating the clinical efficacy of candidate
HPV vaccines. In 2014, a drafting group meeting and informal consultation were
held to draft, review and further develop revised WHO Recommendations in
this area. The informal consultation was attended by experts from around the
world involved in the research, manufacture, licensing/authorization, control-
testing and release of HPV vaccines, with participants drawn from academia,
NRAs, NCLs and industry. Two rounds of public consultation on the outcome
text were then held.
Major issues addressed in the revised WHO Recommendations included:
■■ updating of terminology;
■■ updating of general considerations and other sections to reflect the
current development of HPV vaccines;
Guidelines to assure the quality, safety and efficacy of recombinant human papillomavirus virus-like
12
particle vaccines. In: WHO Expert Committee on Biological Standardization: fifty-seventh report. Geneva:
World Health Organization; 2011: Annex 1 (WHO Technical Report Series, No. 962; http://apps.who.int/
iris/bitstream/10665/44678/1/WHO_TRS_962_eng.pdf, accessed 22 December 2015).
37
■■ updated section on international standards and reference

preparations;
■■ updated Part A in order to include bacteria as a cell substrate;
■■ updated Part B in order to include a new subsection highlighting
tests needed for nonclinical evaluation, and to elaborate regulatory
requirements for both proof-of-concept pharmacology studies and
toxicological testing;
■■ updated Part C in order to reflect appropriate and feasible end-
points in future trials;
■■ updating of appendices.
Additional changes had also been made to bring the revised document
(WHO/BS/2015.2252) into line with other WHO Recommendations, Guidelines
and guidance documents published since the 2006 adoption of the WHO
Guidelines on recombinant HPV VLP vaccines.
The Committee was informed of the key issues addressed and specific
approaches taken in each of the major sections of the revised document, and
provided with a summary of the comments and other inputs received during
the rounds of public consultation. After carefully reviewing the suggested
approaches and comments presented, and making a number of further changes,
the Committee recommended that the revised WHO Recommendations be
adopted and annexed to its report (Annex 4).
3.5.2 Guidelines on the stability evaluation of vaccines for use

under extended controlled temperature conditions
Vaccines are complex biological products and may undergo degradation during
long-term storage under cold chain conditions (for example, 2–8 °C) and this
is typically enhanced at higher temperatures. Consequently, establishing the
stability characteristics of products is a critical element of the overall evaluation

by an NRA to ensure that licensed vaccines remain efficacious at the end of
their shelf-life when stored under the approved conditions.
In response to the stability assessment needs identified by NRAs, WHO
developed guidelines on the stability evaluation of vaccines. While it was well
understood that vaccine quality depends on cold chain storage, it was also
recognized that immunization programmes in certain regions faced substantial
challenges in maintaining cold chains in the field, especially during the final
stage of distribution in remote areas. To address these distribution challenges
and expand immunization programmes into specific regions WHO developed
a “controlled temperature chain” (CTC) programme. This programme currently
requires that a vaccine exhibits a stability profile suitable for a single exposure
to at least 40 °C for a minimum of 3 days just prior to administration, while
38
remaining compliant with the approved vaccine specifications. Additionally, the

programme requires that the CTC provision should be included in the licensure
by the relevant NRA and by WHO prequalification.
The term “extended controlled temperature conditions” (ECTC) had been
introduced to distinguish regulatory requirements from WHO CTC programme
aspects. ECTC assessment establishes the short-term performance of a vaccine
at any temperature above those of a typical cold chain, independent of the
specific programmatic requirements of the current WHO CTC programme.
Vaccines licensed for use under ECTC are required to have sufficient information
regarding the approved conditions (such as maximum temperature and time)
on the package insert.
The proposed WHO Guidelines (WHO/BS/2015.2268) arose from
WHO immunization programme requirements and from discussions held
by an international group of vaccine-stability experts at two WHO-sponsored
consultations – held in Ottawa, Canada in December 2012, and Langen,
Germany in June 2013. The Guidelines are intended to supplement the broader
WHO Guidelines on stability evaluation of vaccines 13 and focus on ECTC-
specific issues not covered in existing guidance with as little overlap as possible.
Key elements include the application of current mathematical modelling and
statistical concepts to the unique short-term requirements that apply to some
cases of vaccine distribution and use. A product-specific ECTC evaluation of
a model monovalent polysaccharide conjugate vaccine is also provided as a
case study.
The Committee reviewed the document and the key issues that had
been raised during the consultations and, after making a number of changes,
recommended that the WHO Guidelines be adopted and annexed to its report
(Annex 5).
3.5.3 Vaccine evaluation in public health emergencies – review

of regulatory pathways in selected countries
The Committee was informed of various WHO initiatives and activities related
to the use of vaccines in public health emergencies. The development and
licensing of vaccines against emerging infections that are declared a public
health emergency involved numerous challenges, including severely contracted
timelines. Of particular importance was the recognition that regulatory
processes needed to be in place to enable the rapid evaluation of submissions
Guidelines on stability evaluation of vaccines. In: WHO Expert Committee on Biological Standardization:
13
fifty-seventh report. Geneva: World Health Organization; 2011: Annex 3 (WHO Technical Report
Series, No. 962; http://who.int/biologicals/vaccines/Annex_3_WHO_TRS_962-3.pdf?ua=1, accessed 11
December 2015).
39
and – following careful risk–benefit assessment – to allow the use of vaccines for
which a full regulatory package may not yet be available.
Following an informal consultation in 2015 and subsequent survey
feedback received from 10 NRAs, WHO had now developed a review document
which aimed to provide an overview of various existing regulatory pathways
in selected countries and to encourage countries to review their state of
regulatory preparedness for public health emergencies. Experience of public
health emergencies since 2000 included the severe acute respiratory syndrome
outbreak in several countries, the H1N1 influenza pandemic (and ongoing risk
of an avian influenza pandemic) and the Ebola epidemic in West Africa. Each of
those events served to reinforce the crucial need for a rapid global response.
It was determined by WHO that a certain level of flexibility already
exists in some countries in relation to the evaluating of vaccines during public
health emergencies. However, there were also countries in which vaccine
licensing could only be done on the basis of a full data package with no
alternative pathway. In such cases, it would be important to initiate discussion at
national and regional (or intercountry) level to explore options for establishing
alternative regulatory pathways as part of overall emergency response efforts.
The most important considerations in evaluating vaccines during public
health emergencies included risk–benefit assessments, the need for a well-
defined and transparent decision-making process, risk-management planning
and pharmacovigilance. It had been further concluded that actual case studies
and lessons learnt from previous public health emergencies could serve as
training materials.
The Committee noted the draft review document and sought
clarifications in a number of areas. While it was agreed that a WHO guidance
document might be of particular value to some NRAs and public health
organizations, there was also recognition of the complexity of emergency
situations. The decision to approve the use of an investigational product in the
event of a public health emergency would ultimately rest on a case-by-case risk–

benefit assessment. The Committee looked forward to reviewing the progress
made in the further development of the document in 2016.
3.5.4 Development of Guidelines on the quality,

safety and efficacy of Ebola vaccines
Following the start of the Ebola epidemic in West Africa in 2014, considerable
efforts have been made in relation to the development, evaluation and licensing
of Ebola vaccines. The Committee was informed that as part of ongoing
WHO measures to support the development of Ebola vaccines a draft concept
paper had been prepared which had outlined the scientific and regulatory
considerations in the evaluation of vaccines intended for emergency use, and
40
which had included related guidance regarding the quality, safety and efficacy
of Ebola vaccines.
Following expert review, the need for a specific Ebola vaccine document
was highlighted. In March 2015, WHO convened an informal consultation
attended by experts, regulatory professionals and other stakeholders involved
in Ebola vaccine development, production, and regulatory evaluation to review
the resulting draft WHO Guidelines and reach consensus on key technical and
regulatory issues.
The draft Guidelines were intended to provide guidance to NRAs and
vaccine manufacturers on the quality, nonclinical and clinical aspects of Ebola
vaccines, particularly those based on viral vectors as these are currently at the
most advanced stage of development.
As there are currently significant knowledge gaps in the scientific
understanding of Ebola disease and Ebola vaccines, and as no Ebola vaccine
has yet been licensed, the document had been developed as WHO Guidelines
rather than Recommendations. It was recognized that subsequent updating of
the Guidelines may be required in the light of recent and future developments.
The Committee noted the comments and suggestions that had been
received by WHO in relation to the draft Guidelines and indicated its agreement
with the proposed content, scope and proposed approach for its further revision.
The Committee also provided a number of additional recommendations,
including the addition of further guidance on post-marketing surveillance and
the need for fluid approaches to updating and posting the current text in this
rapidly evolving area. The Committee expressed its appreciation for the efforts
that had been made in developing such an excellent and comprehensive draft in
such a short period, and looked forward to considering the revised Guidelines
at its next meeting.
3.5.5 Revision of Guidelines on the safe production and quality control of

inactivated poliomyelitis vaccines manufactured from wild polioviruses
The Committee was reminded that the WHO Polio Eradication and Endgame
Strategic Plan 2013–2018 (PEESP) published by the Global Polio Eradication
Initiative sets the goal of achieving a polio-free world by 2018. Towards this goal,
activities include minimizing the risk of facility-associated reintroduction of wild
or attenuated oral poliomyelitis vaccine (OPV, Sabin) polioviruses. In the now
finalized third edition of the WHO Global Action Plan to minimize poliovirus
facility-associated risk after type-specific eradication of wild polioviruses and
sequential cessation of oral polio vaccine use (GAPIII) published in May 2015, the
safe handling and containment of poliovirus infectious and potentially infectious
materials has been aligned with the PEESP. WHO was currently developing a
supplementary document – GAPIII Containment Certification Scheme – to
41
help facilities and oversight bodies better understand and implement GAPIII
requirements as they relate to the certification of poliovirus containment in
essential facilities.
The Committee was informed that current WHO Guidelines for the safe
production and quality control of IPV manufactured from wild polioviruses had
been published as an addendum to the previous WHO Recommendations for
the production and control of poliomyelitis vaccine (inactivated). In light of a
range of associated developments and updates, including in the area of GMP for
biological products, the revision of the current Guidelines had been proposed.
In order to inform the revision process and identify key issues to
be addressed a thorough expert technical review was conducted involving
WHOCCs working in this area. The need for revision of the current guidance
was broadly recognized in order to provide practical and detailed guidance to
poliomyelitis vaccine manufacturers and regulators not fully addressed by any
currently available WHO documents. Ideally the revision would extend the scope
of the current document to cover all poliovirus strains, and provide updated,
detailed, specific and practical guidance on all aspects of poliovirus containment
in vaccine-manufacturing facilities, with an emphasis on issues not addressed
elsewhere. The revision would also take into account the current and anticipated
future global polio eradication situation and be aligned with current resources
and global strategies such as PEESP and GAPIII. Further details and specific
issues will be identified and discussed during consultations with experts from
vaccine manufacturers and NRAs.
The Committee noted the information presented, sought clarifications
in a number of areas and made several specific suggestions. The Committee
agreed with the conclusions and proposals presented, expressed its support
for the development of the revised WHO Guidelines and looked forward to
reviewing progress in 2016.
3.5.6 International collaborative study to assess the utility of deep

sequencing in the quality control of oral poliomyelitis vaccines
and inactivated poliomyelitis vaccines made from Sabin strains
An international collaborative study on the utility of massively parallel (deep)
sequencing (MPS) in monitoring the molecular consistency of OPV had been
endorsed by the Committee in 2013. Preliminary data were subsequently
collected in 2014, and a workshop convened in April 2015 attended by NRAs,
NCLs and poliomyelitis vaccine manufacturers from several countries.
The status of OPV quality control in different countries was reviewed
and the experiences of several groups in using MPS shared. After discussing the
objectives and design of the proposed collaborative study, workshop participants
concluded that Phase 1 of the study should focus on the evaluation of MPS
42
analysis as a potential replacement for the mutant analysis by polymerase chain

reaction and restriction enzyme cleavage (MAPREC) test. Phase 2 of the study
would then concentrate on generating whole-genome sequence polymorphism
profiles for use in assessing the consistency of newly prepared vaccine batches
when compared with previously released vaccine lots.
The Committee was provided with an overview on the objectives, design
and justification for each proposed phase, and updated on the current status
of the study. The Committee noted the proposals made in relation to each of
the two phases, sought clarifications in a number of areas and made several
specific suggestions. The Committee agreed that the study would provide useful
information, and looked forward to reviewing progress in 2016.
43
4. International Reference Materials – antibiotics
All reference materials established at the meeting are listed in Annex 6.
4.1 WHO International Standards and

Reference Reagents – antibiotics
4.1.1 Fourth WHO International Standard for streptomycin
Streptomycin is an aminoglycoside antibiotic used in the treatment of tuberculosis
and of moderate to severe susceptible infections, including plague, tularemia and
bacterial endocarditis. It is also used as second-line therapy for gram-negative
bacillary bacteraemia, meningitis, pneumonia, brucellosis, granuloma inguinale,
chancroid and urinary tract infections. Streptomycin appears on the WHO
Model List of Essential Medicines.
The Third WHO International Standard for streptomycin was established
in 1980 on the basis of an international collaborative study and was assigned
a value of 78 500 IU/ampoule. As stocks of this international standard were
dwindling, appropriate steps were taken by EDQM for its replacement.
An international collaborative study was conducted to establish the
Fourth WHO International Standard for streptomycin. Thirteen laboratories
from 12 countries participated. The biological activity of the candidate material
(EDQM code 55821) was estimated by microbiological assays using sensitive
microorganisms. To ensure continuity between consecutive batches, the current
international standard was used as a reference.
Stability studies demonstrated that the candidate material was stable
at temperatures used for storage (−20 °C). The results of accelerated thermal
degradation studies at 20 °C, 37 °C and 45 °C for 1, 3 and 6 months indicated
that the candidate material was stable for long-term use. Real-time stability
studies were now in progress. The Committee was informed that 993 ampoules
would be available for use as an international standard.

The Committee considered the report of the study (WHO/BS/2015.2277)
and recommended that the candidate material 55821 be established as the
Fourth WHO International Standard for streptomycin with an assigned value
of 76 000 IU/ampoule.
44
5. International reference materials –
biotherapeutics other than blood products
5.1 WHO International Standards and Reference Reagents –

5.1.1 First WHO International Standard for tumour necrosis
factor receptor Fc fusion protein (etanercept)
The recombinant human tumour necrosis factor (TNF) receptor Fc fusion
protein (etanercept) is a large glycoprotein with a molecular weight of
approximately 150 kilodaltons. It acts as a competitive inhibitor of TNF and
prevents it from binding to its cell surface receptors, thereby reducing the
biological activity of TNF. As a result, it has potential application in the treatment
of various autoimmune diseases or disorders associated with increased TNF
and excess inflammation. Current therapeutic indications include rheumatoid
arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis
and plaque psoriasis.
Several versions of etanercept are already approved in some countries
with a number of SBPs now undergoing regulatory evaluation or clinical trials
in various other countries. Etanercept products are dosed in mass units and
current labelling does not provide information on biological activity (that
is, in terms of international units or specific activity of the protein). In vitro
determination of biological activity against proprietary reference materials is
routinely performed by license holders for lot release and stability assessment.
The development of a publically available international standard would facilitate
determination of the biological activity of intended copies or potential SBPs,
thus ensuring patient access to products of consistent quality and effectiveness.
This project had been endorsed by the Committee in 2012.
An international collaborative study was conducted to establish the First
WHO International Standard for tumour necrosis factor receptor Fc fusion
protein (etanercept). Three candidate samples were provided by two different
manufacturers and were analysed by 28 laboratories from 15 countries using
their own in-house bioassay. Within- and between-laboratory repeatability was
generally very high with excellent agreement observed between laboratories
regardless of which candidate was chosen as the standard for determining
sample potency. Two of the candidate samples (13/192 and 13/204) were found
to be suitable for use as an international standard.
Stability studies demonstrated that the above two candidate materials
were stable at temperatures used for storage (−70 °C) and for laboratory
manipulation (4–20 °C). The results of accelerated thermal degradation studies
45
at 14 to 19 months indicated that both candidates would be stable for long-

term use. Real-time stability studies were now in progress. The Committee was
informed that 4700 ampoules would be available for use as an international
standard.
The Committee considered the report of the study (WHO/BS/2015.2257),
made several suggestions on the information to be included in the Instructions
for Use and recommended that candidate material 13/204 be established as the
First WHO International Standard for tumour necrosis factor receptor Fc fusion
protein (etanercept) with an assigned value of 10 000 IU/ampoule.
5.1.2 First WHO Reference Panel for antibodies to erythropoietin (human)

Recombinant human erythropoietin (EPO) has been approved for the treatment
of anaemia with subsequent approval obtained for several other erythropoiesis-
stimulating agents (ESAs) worldwide. Currently, a number of different EPO
products of markedly variable quality are marketed worldwide. Despite
their successful use, EPO products can in rare cases induce a potentially life-
threatening immune response in recipients. Evidence to date has shown that
human EPO induces neutralizing antibodies which not only cross-react with all
EPO products but also neutralize the endogenously produced protein causing
antibody-mediated pure red cell aplasia (PRCA). Isolated PRCA cases have also
been linked to the use of other ESAs.
There are no universally accepted methods or reference reagents for
detecting and measuring EPO antibodies. However, several methods which
differ in the types of antibodies detected and in sensitivity are used to detect
antibodies against EPO. The availability of reference standards/reagents for
the testing of EPO antibodies across different assay platforms would allow
for consistency in the detecting and measuring of EPO antibodies as part of
monitoring the safety and efficacy of ESAs. Because previous attempts to obtain
PRCA sera from patients for the development of reference standards have
proved largely unsuccessful a panel of nine human monoclonal antibodies

(mAbs) against human EPO with defined characteristics was obtained. This
panel is intended to be made available to manufacturers and clinical laboratories
to allow for the selection and validation of appropriate assays, and for the
monitoring of assay performance. The antibody panel is appropriate for use with
all ESAs structurally related to human EPO, and contained a range of mAbs
with different characteristics – non-neutralizing and neutralizing, and spanning
various isotypes and affinities – based on information derived from clinical
samples from non-PRCA and PRCA patients. This project was endorsed by the
Committee in 2010.
An international collaborative study was conducted to establish the First
WHO Reference Panel for antibodies to erythropoietin (human). The panel
46
International reference materials – biotherapeutics other than blood products
was analysed by 18 laboratories from eight countries using their own in-house
assay. Results showed that antibody detection varied between laboratories and
was dependent on antibody characteristics and the method used. Within- and
between-assay repeatability was generally very good with excellent agreement
for most laboratories. Accelerated thermal degradation studies demonstrated
that panel samples stored at 37 °C for 9 months showed no significant loss of
activity. The Committee was informed that 700 vials would be available for use
as an international standard.
and recommended that the proposed panel be established as the First WHO
Reference Panel for antibodies to erythropoietin (human).
5.2 Proposed new projects and updates –

5.2.1 Proposed First WHO International Standard for darbepoetin
Darbepoetin is a recombinant erythropoiesis stimulating protein with a longer
in vivo half-life compared to recombinant erythropoietin (EPO). A number
of darbepoetin BSPs are in development and some have already been licensed.
As non-originator products can vary in quality, efficacy and safety there is now
a need for a WHO international standard for use by manufacturers and control
laboratories in calibrating assays to assess product potency.
A donation of darbepoetin from the originator or from biosimilar
manufacturers will be sought for use in a collaborative study. Potency will be
assigned in mass units to the darbepoetin standard using an in vitro bioassay.
Following discussion and further consideration, the Committee endorsed
the proposal (WHO/BS/2015.2273) to develop a First WHO International
Standard for darbepoetin and agreed that the collaborative study should proceed
with subsequent submission of its outcome to the Committee in 2018.
5.2.2 Proposed First WHO Reference Panel for in vitro

bioassay of erythropoietin (EPO)
The current international standard for EPO, and its recombinant and natural
predecessors, were all value-assigned by in vivo bioassay. The EPO unit, used for
dosing regimens, therefore remains traceable to an in vivo bioassay, which also
remains the pharmacopoeial assay method. Replacement of the in vivo bioassay
with an in vitro method is a high-profile objective. However, although a number
of in vitro assays for EPO have been developed and described, their adoption
as regulatory assays has been hampered by the fact that the two different assay
approaches depend on different rate-limiting parameters of the molecule, and
show opposite correlations with processes such as sialylation. The validation
of in vitro methods is hampered by the absence of system suitability standards
47
which allow the relationships between structure and function for various assays
to be explored.
The proposed panel would span a range of properties, principally pI
(a function of the degree of sialylation), to allow users to define the response of
in vitro and in vivo bioassay systems to variations in sialylation. Globally, there
are numerous EPO manufacturers, pharmacopoeias and control laboratories
who would seek access to such materials.
The Committee considered the document (WHO/BS/2015.2273)
outlining the establishment of a panel of bioassay reference reagents for EPO
but was not convinced of the utility of such a panel in the in vitro bioassay of
EPO and did not endorse the proposal.
5.2.3 Proposed First WHO international standards for pegylated-

interferon-α-2a and pegylated-interferon-α-2b
Despite the availability of new therapeutic options for HCV infection, which
affects 150–180 million people worldwide, the standard therapy for the treatment
of chronic hepatitis C in many countries continues to be pegylated-interferon-α
(peg-IFN-α) in combination with ribavirin. Peg-IFN-α-2a and peg-IFN-α-2b
are licensed in the USA and Europe, and are available globally. WHO added
peg-IFN-α to its Model List of Essential Medicines in 2013. Alternative products
have been marketed or are in development in several counties, and these
products differ in their biological properties both from the originator product
and from the parental unmodified IFN-α-2 forms.
Currently there is no international standard for peg-IFN-α products.
International standards for unmodified IFN-α forms are however available,
and may have been used by some manufacturers to determine the activities of
their products. As potency-determination practices are likely to vary among
manufacturers, this can result in products with discrepant potencies. Therefore,
reference international standards would be very useful for the in vitro assay
of product biological activity. Due to the complexities associated with these

products it was proposed that several different preparations be evaluated,
and assessment made of the feasibility of developing suitable standards for
peg‑IFN‑α-2a and/or peg-IFN-α-2b for use by manufacturers and control
laboratories in calibrating assays for the potency evaluation of such products
in order to ensure their clinical safety and efficacy. The biological activity of
products would be assessed using appropriate bioassays and a collaborative
study organized to assign a unitage to the international standards. Donations
of peg-IFN-α from the originator product manufacturer and from alternative
product manufacturers will be sought.
The Committee considered the proposal (WHO/BS/2015.2273) and
expressed reservations concerning both the feasibility of obtaining candidate
materials for the proposed study and the relevance of in vitro results that had
48
not been shown to correlate with current in vivo test results. The Committee
recommended that an exploratory study be undertaken to compare unmodified
IFN-α with peg-IFN-α products, and that the results be reported to the
Committee in due course.
5.2.4 Proposed First WHO Reference Reagent for

TGN1412-like functional activity
The severe adverse events that occurred during a Phase I clinical trial of the anti-
CD28 superagonist TGN1412 highlighted the need for improved in vitro assays
to better predict the risk of cytokine release syndrome following administration
of monoclonal antibodies to humans. Although a number of cytokine release
assays (CRAs) had since been developed these were of widely varying sensitivity.
One problem faced by many investigators was the difficulty in sourcing a positive
control with TGN1412-like activity. The availability of a TGN1412 analogue
would greatly facilitate assay development and application by manufacturers,
contract research organizations, and regulatory and research laboratories.
A TGN1412 analogue (NIBSC code NIB28SA-G4) had been produced by
NIBSC with subsequent experiments, including a pilot fill, demonstrating no loss
of functional activity in a PBMC/HUVEC co-culture CRA. It was proposed that
production fill material would now be evaluated in a multicentre collaborative
study for its suitability as a positive control for CRAs. Negative control material
would also be provided for the collaborative study.
the proposal (WHO/BS/2015.2273) to develop a First WHO Reference Reagent
for TGN1412-like functional activity, and agreed that the collaborative study
should proceed with subsequent submission of its outcome to the Committee
in 2016.
5.2.5 Proposed First WHO Reference Reagent for Pam 3 CSK 4

The inadvertent contamination of parenteral products by endotoxin and non-
endotoxin pyrogens can potentially lead to life-threatening, uncontrolled
inflammatory events. Assays currently used to identify contamination (bacterial
endotoxin test and rabbit pyrogen test) have a number of inherent limitations
and rely upon animal products and testing. Significant efforts have therefore
been made to establish alternative in vitro methods based upon the use of human
monocytes. This has resulted in the development of a number of monocyte
activation test (MAT) assays of widely varying sensitivity.
Sourcing a reliable positive control for monocyte activation, other than
endotoxin, has proved to be problematic. The availability of an alternative toll-
like receptor ligand would greatly facilitate assay development and application
by manufacturers, contract research organizations, and regulatory and research
laboratories.
49
The toll-like receptor ligand Pam 3CSK 4 reliably activates monocytes

when using two of the most widely used MAT assays. It was proposed that, in
coordination with EDQM, this material be evaluated in an NIBSC multicentre
collaborative study for its suitability as a positive control for MAT assays
employed in assessing pyrogenic contamination of parenteral products.
the proposal (WHO/BS/2015.2271) to develop a First WHO Reference Reagent
for Pam3 CSK4 for use in MAT assays, made a number of recommendations
regarding potential cell sources, and agreed that the collaborative study should
proceed with subsequent submission of its outcome to the Committee in 2017.
5.2.6 Proposed stability monitoring of the First WHO International

Standard for pegylated granulocyte colony-stimulating factor
The First WHO International Standard for pegylated granulocyte colony-
stimulating factor was established by the Committee in 2013 and is the primary
standard for in vitro assays of the biological activity of pegylated granulocyte
colony-stimulating factor preparations.
The Committee was informed that samples of the current standard
stored at elevated temperatures (20 °C, 37 °C and 45 °C) for approximately 2
years had been tested concurrently with samples stored at the recommended
temperature of −20 °C and with baseline samples stored at −70 °C in GNFS-60
bioassays. The calculated potencies of the samples relative to the baseline sample
were used to fit the Arrhenius model for accelerated thermal degradation, and
resulted in a predicted loss in activity of 0.031% per year when stored at −20 °C.
Extended studies after 20 years or upon standard replacement (whichever was
sooner) were now being proposed in order to confirm the appropriate stability
of the current standard.
the proposal (WHO/BS/2015.2260) and looked forward to the subsequent
submission of the stability study outcome to the Committee in due course.
50
6. International reference materials – blood
products and related substances
6.1 WHO International Standards and Reference Reagents –

blood products and related substances
6.1.1 First WHO Reference Reagent for anti-A and anti-B in serum
and plasma for use in haemagglutination assays
Testing for high-titre anti-A and anti-B in serum and plasma using
haemagglutination assays is an important step in minimizing the risk of causing
clinically significant haemolysis when blood components rich in plasma
containing high titre anti-A/B are transfused to patients of blood groups A, B
or AB. Such testing can also facilitate the transplanting of mismatched kidneys
from living donors which can be performed successfully if the recipient has
sufficiently low levels of anti-A and anti-B, and can also identify high-titre anti-
A/B plasma for exclusion from the manufacture of blood products.
A lyophilized serum preparation with high anti-A and anti-B titres
(NIBSC code 14/300) was evaluated in an international collaborative study
for its suitability to serve as a WHO reference reagent for the standardization
and control of haemagglutination titrations for anti-A and anti-B in serum and
plasma. Two plasma-based reserve preparations – NIBSC codes 14/304 (high-
titre anti-A) and 14/208 (high-titre anti-B) – were also included in the study.
Twenty three laboratories in 12 countries tested all three preparations, with
21 laboratories using the recommended methodologies of direct agglutination
at room temperature (DRT) using DiaMed neutral gel cards, and indirect anti-
globulin testing (IAT) using DiaMed anti-IgG and/or LISS/Coombs gel cards.
Up to a 64-fold variation was observed in reported titres per preparation. No
reactivity with group A red blood cells (RBCs) was reported for 14/208 and none
of the preparations reacted with group O RBCs.
Study results indicated that haemagglutination tests can exhibit
wide inter-laboratory variation, even when using a common procedure, and
demonstrated the need for international reference reagents with “nominal”
titres to facilitate inter-laboratory comparisons and allow sample titres to be
reported relative to the reference titres. The establishment of 14/300 as a WHO
reference reagent for high-titre anti-A and anti-B in serum, with assigned
nominal anti-A and anti-B titres, would facilitate the global standardization of
haemagglutination testing in patient samples, and will allow for identification
of more consistent cut-off titres for various applications such as ABO-
incompatible renal transplants.
51

made several suggestions on the information to be included in the Instructions
for Use (including an indication of the range of assay types used in the study and
of the data used for unit assignment), and recommended that candidate material
14/300 be established as the First WHO Reference Reagent for anti-A and anti-B
in serum and plasma for use in haemagglutination assays with nominal anti-A
and anti-B titres of 128 and 256 assigned for DRT and IAT respectively.
6.1.2 Fifth WHO International Standard for blood

coagulation factor IX (concentrate)
Accurate potency labelling against robust and reliable reference standards is
of paramount importance in ensuring the efficacy of clinical products based
on high-purity blood coagulation factor IX (FIX) and prothrombin complex
concentrates used in the treatment of congenital and acquired factor IX
deficiency. Due to depletion of the current WHO international standard a
collaborative study was undertaken with the following three main aims: (a) to
add an FIX antigen value to the Fourth WHO International Standard for
blood coagulation factors II, VII, IX X (plasma); (b) to establish a replacement
international standard for blood coagulation factor IX (concentrate); and
(c) to investigate the suitability of the replacement international standard as a
potency standard for purified full-length recombinant FIX.
For the assignment of an FIX antigen value to the Fourth WHO
International Standard for blood coagulation factors II, VII, IX, X (plasma),
15 laboratories generated 17 sets of data for analysis. Only five of these data
sets exhibited an intra-laboratory geometric coefficient of variation (GCV)
greater than 10%. There was also good inter- laboratory agreement with an
observed GCV of 7.9%. Based on the data analysis, it was recommended
that an FIX antigen value of 0.90 IU/ampoule be added to the label of above
international standard.
For the value assignment of the Fifth WHO International Standard

for blood coagulation factor (concentrate) relative to the current international
standard two plasma-derived candidate materials (NIBSC codes 14/148 and
14/162) were evaluated by 50 laboratories from 18 countries. The laboratories
returned a total of 55 sets of clotting assay results and 15 sets of chromogenic
assay results for analysis. Intra-laboratory variability was reasonably low with
the majority of GCVs below 10%. Lower inter-laboratory agreement was
observed for candidate material 14/148 than for candidate material 14/162.
Although there were no discrepancies between clotting and chromogenic
assays for either sample, a significantly lower potency (approximately −6%) was
obtained for candidate material 14/162 with clotting assays when buffer rather
than FIX‑deficient plasma was used as a pre-diluent. It was recommended
that candidate material 14/148 be established as the Fifth WHO International
52
Standard for blood coagulation factor IX (concentrate) with an assigned potency

of 10.5 IU/ampoule.
The study also investigated the comparability of the plasma-derived
concentrate standard with full-length recombinant FIX products and assessed
the feasibility of establishing an international standard for recombinant FIX. All
three currently licensed full-length recombinant FIX products were represented
in the evaluation of two recombinant candidate materials, and data received
from 49 laboratories. Despite data analysis indicating that a recombinant
standard for recombinant products could minimize assay discrepancies for
some products and improve inter-laboratory agreement, a number of objections
had been raised in relation to potential adverse impacts on the amount of
protein in the final products and the use of multiple international standards for
the same coagulation factor. Following evaluation by experts nominated by the
Scientific and Standardization Committee (SSC) of the International Society
on Thrombosis and Haemotasis (ISTH) and subsequent discussions with the
ISTH/WHO Liaison Group, it had been decided not to request the Committee
to consider the establishment of an international standard for recombinant FIX.
Committee discussions centred on the need for an international standard
for recombinant FIX and on the inherently complex difficulties of establishing
such a standard in light of the different recombinant FIX preparations now
in the development pipeline. Despite the currently unresolved scientific and
practical issues, further work on the possible establishment of an international
standard for recombinant FIX was encouraged given the likely future need to
standardize the potency of such products.
and recommended that candidate material 14/148 be established as the Fifth
WHO International Standard for blood coagulation factor IX (concentrate) with
an assigned value of 10.5 IU/ampoule. The Committee further recommended
that an FIX antigen value of 0.9 IU/ampoule be assigned to the Fourth WHO
International Standard for blood coagulation factors II, VII, IX, X (plasma).
For both these standards the Committee requested that consideration be given
to specifying the post-reconstitution IU/ml value in the Instructions for Use.
6.2 Proposed new projects and updates – blood

products and related substances
6.2.1 Proposed First WHO International Standard for
activated blood coagulation factor X
Activated blood coagulation factor X (FXa) sourced from bovine plasma and
established in 1975 is currently being used as a non-WHO reference material
for the standardized measurement of the FXa content of activated prothrombin
complex concentrate (PCC) used in the treatment of haemophilia. In addition,
53
it is also being used in the investigation of factor X (FX) concentrate, an orphan

drug for the treatment of FX deficiency, currently in clinical trials. The suitability
of bovine FXa as a reference material to meet regulatory requirements in this
area is in question with the material also having being filled externally with no
stability data or information on the uniformity of the fill available. It was therefore
proposed that the material be replaced with a human preparation of FXa.
Potential candidate material for this purpose was identified from three
sources including an internal reference material prepared from human plasma
donated by the manufacturer of the PCC FEIBA. One candidate material (NIBSC
code 15/102) was shown to be active and homogeneous.
In the proposed collaborative study, the activity of 15/102 will be
measured relative to the bovine standard (NIBSC code 75/595) and local FXa
standards using chromogenic and clotting methods. Potency assignment will
be performed relative to 75/595 or an independent arbitrary unit assigned
if this is not possible. The proposed uses of the international standard are to
measure FXa levels in PCCs and assess FXa content as a contaminant of FX
concentrates. Other potential uses include the measurement of FXa inhibitors,
the measurement of FXa in heparin assays and the measurement of truncated
FXa now in development as a reversal agent.
Standard for activated blood coagulation factor X.
6.2.2 Proposed First WHO Reference Panel for procoagulant

activity of human immunoglobulin
In 2012, a proposal had been put to the Committee to endorse the development
of matrix-related reference materials to support assay methods used to
measure procoagulant activity in human immunoglobulin products. Initial
indications were given that the proposed reference materials were not well
defined and the decision was made not to proceed with further discussion at
that point. At the same time, a reference reagent for FXIa was established,
followed in 2013 by the establishment of an international standard for FXIa
to support its measurement in immunoglobulin products. In addition, global
stakeholders continued to work on the development and refinement of assays
for procoagulant activity.
The Committee was informed that the international standard for FXIa
is now helping to harmonize FXIa assay methods, and is also used by regulators
and manufacturers to develop other assay methods such as the Non-Activated
Partial Thromboplastin Time and Thrombin Generation Assay. Because of the
matrix heterogeneity of immunoglobulin products, it was clear that in addition
to the purified FXIa standard, matrix-related reference preparations will also
be required. Such matrix-related preparations may be superior to purified FXIa in
54
standardizing procoagulant activity assays that are not amenable to estimation

in IUs.
Although the measurement of the procoagulant activity of human
immunoglobulins remains an unresolved issue, a degree of commonality
was now apparent between different strategies, and agreement on further
collaboration had been reached. A working group had been convened comprising
representatives of the principal organizations and agencies working in this
area. A large collaborative study was now being proposed to clarify the need to
establish the relevant reference preparations as a WHO reference panel and it
was proposed that the working group report back to the Committee in 2016.
Following discussion of the proposal (WHO/BS/2015.2274), the
Committee noted the intended collaborative study and looked forward to being
updated on its progress in 2016. An indication was also made by the Ministry of
Food and Drug Safety, Republic of Korea of its interest in joining the working
group process.
6.2.3 Proposed Fifth WHO International Standard for

thromboplastin (human, recombinant)
International Standards for thromboplastins are used to: (a) determine the
International Sensitivity Index (ISI) for commercial or local prothrombin
time (PT) test reagents and instruments, which is needed to determine the
international normalized ratio (INR) in patients receiving vitamin K antagonist;
(b) calibrate the INR for whole blood coagulation monitors (point-of-care tests);
and (c) certify frozen or freeze-dried plasma for local INR determination. In
general, any new thromboplastin standard should be calibrated with a similar
reference thromboplastin (like-to-like principle). As stocks of the current WHO
international standard are low, and will be exhausted within a year, a replacement
standard is now required. It was intended that the replacement would be
calibrated against the current international standard.
An international collaborative study has therefore been proposed
to assign ISI values to candidate replacement preparations and to evaluate
their suitability according to predetermined criteria. A candidate for human
recombinant thromboplastin had now been prepared (freeze dried in sealed
ampoules) and assigned the provisional NIBSC code 14/001. Corresponding
reconstitution fluid had also been prepared in sealed ampoules. The study would
involve approximately 20 laboratories. PT determination will be performed by
manual tilt tube technique on 10 different days using blood samples from two
healthy subjects and six patients per day that have been treated with vitamin K
antagonists to yield an INR of 1.5–4.5. Lyophilized control plasma will be tested
in parallel. Accelerated degradation studies will be performed. As long-term
stability studies (at −20 °C) would not be completed within the time frame of
the project future studies should be considered.
55

the proposal (WHO/BS/2015.2274) to develop a Fifth WHO International
Standard for thromboplastin (human, recombinant).
6.2.4 Proposed Fifth WHO International Standard

for thromboplastin (rabbit, plain)
As outlined above, thromboplastins used in the PT test for the laboratory
control of oral anticoagulant treatment must be calibrated against international
standards to determine the ISI necessary to convert PT results into an INR.
Feedback from users has indicated that the calibration of a given thromboplastin
is generally more precise when performed against an international standard
of similar composition, and from the same species. For this reason, WHO
therefore maintains international standards for different species. As stocks of
the current WHO international standard for rabbit thromboplastin are low,
and will be exhausted within a year, a replacement standard is now required.
It was intended that the replacement would be calibrated against the current
As part of the international collaborative study on human prothrombin
outlined above in section 6.2.3 a rabbit thromboplastin candidate material
has also been prepared and assigned the provisional NIBSC code 15/001.
Appropriate reconstitution fluid has again been prepared in sealed ampoules.
During discussion, clarification was sought on why the technical
specifications between the two standards were not identical with the range of
the rabbit preparation being slightly higher. This appeared to be a reflection
of the property of the rabbit brain lysate which needed to be dosed slightly
higher than the human recombinant standard to yield equivalent INR values in
the respective assays.
the proposal (WHO/BS/2015.2274) to develop a Fifth WHO International
Standard for thromboplastin (rabbit, plain).
56
In vitro diagnostic device reagents
7.1 WHO International Standards and Reference

Reagents – in vitro diagnostic device reagents
7.1.1 First WHO International Standard for JC virus DNA for NAT-based assays
JC virus (JCV) is a member of the Polyomaviridae family and is typically
acquired in childhood, resulting in an estimated worldwide seroprevalance of
50–90% by adulthood. Following primary infection, the virus establishes latency
and the vast majority of individuals remain asymptomatic. Under conditions of
immunodeficiency and immune suppression JCV can establish lytic infection in
oligodendrocytes leading to demyelination resulting in progressive multifocal
leukoencephalopathy (PML). PML presents with progressive neurological deficits,
including motor and sensory defects, with a mortality rate of 30–50% within the
first few months of diagnosis. Although the incidence of PML remains highest in
HIV-1 infected individuals (~85% of cases), cases have also recently been detected
among relapsing remitting multiple sclerosis patients undergoing monoclonal
antibody treatment with Natalizumab, an immunomodulatory therapy.
The molecular detection of JCV DNA in cerebrospinal fluid is frequently
used as a confirmatory diagnosis of PML and is also relied upon during post-
treatment monitoring. As highlighted in external quality assessment (EQA)
programmes, the absence of a standardized reference preparation currently
prevents the assured determination of accurate and comparable viral load. It
is envisaged that the proposed international standard will be used by clinical
diagnostics laboratories and IVD manufacturers for the calibration of secondary
reference reagents and working standards.
An international collaborative study was conducted to evaluate a
candidate reference material (NIBSC code 14/114) for use in the standardization
of JCV NAT-based assays. The freeze-dried whole virus preparation JCV 1A
strain, formulated in a universal buffer, was analysed by 23 laboratories in
14 countries using their own routine NAT-based assays for JCV detection.
The freeze-dried candidate was tested alongside its liquid equivalent, as well
as patient samples and spiked clinical samples. Despite a large degree of inter-
laboratory variation in the estimates obtained, overall results demonstrated
the suitability of the candidate material 14/114, with analysis of its dilution in
different matrices indicating no matrix effects, highlighting its potential utility
in the calibration of multiple clinical samples. Furthermore, accelerated thermal
degradation studies performed at 4 and 8 months demonstrated the stability of
57
the candidate material at temperatures used for storage (−20 °C) and laboratory
manipulation (4–20 °C), as well as at the 37–45 °C ambient temperatures
typically encountered during global shipment. Further real-time stability studies
were now in progress to assess the long-term stability of the candidate material.
The Committee was informed that 4103 vials would be available for use as an
Discussion was the held on the potential issues relating to the use of
a single subtype, the possibility of matrix effects and the associated issue of
commutability. The Committee was informed that although the commutability
aspects of the study had been limited, there was nothing to indicate that
proposed material would not be suitable, and the possibility of a wider
commutability study was being discussed with study participants and other
stakeholders. Discussion also centred on the assignment of a unit based on
quantitative data or on combined qualitative and quantitative data. Despite
an observed discrepancy between qualitative and quantitative findings, the
Committee concluded that clinical decision-making for JCV relies primarily on
determining the presence or absence of virus rather than its quantitation and
the use of a combined value would be appropriate.
and recommended that the candidate material 14/114 be established as the First
WHO International Standard for JC virus DNA for NAT-based assays with an
assigned potency of 7.0 log 10 IU/ml.
7.1.2 First WHO International Standard for BK

virus DNA for NAT-based assays
BK virus (BKV) is a member of the Polyomaviridae family and is typically
acquired in childhood, resulting in a seroprevalance of ~90% worldwide by
adulthood. Following primary infection the virus establishes latency and the
vast majority of the population remains asymptomatic. Renal transplantation

and haematopoietic stem cell transplantation (HSCT) patients undergoing
immunosuppression are susceptible to latent BKV reactivation, which can result
in BKV-associated nephropathy (BKVAN) characterized by interstitial nephritis
and/or urinary tract stenosis. Affecting up to 10% of all renal transplant patients,
BKVAN can cause allograft loss in up to 60% of such affected recipients. In HSCT
patients, BKV reactivation can present with haemorrhagic cystitis that can be
associated with significant morbidity and mortality.
In the absence of an effective antiviral to treat BKV reactivation, the
management of renal transplant patients requires rigorous post-transplantation
monitoring of BKV viral load using quantitative polymerase chain reaction
(PCR) analysis of urine and plasma at specified time points. A BKV viral load
58
International reference materials – In vitro diagnostic device reagents
of ≥ 4 log 10 /ml for > 3 weeks is presumed predictive for BKVAN, upon which
a reduction in immunosuppression is recommended. A standardized reference
preparation is therefore needed for accurate and comparable viral load
determination. It is envisaged that such a reference preparation would be used
by clinical diagnostic laboratories and IVD manufacturers for the calibration of
secondary reference reagents and working standards.
An international collaborative study was conducted to evaluate
candidate materials for use in the standardization of BKV NAT-based assays.
Two candidate samples of freeze-dried whole BKV preparations (NIBSC
codes 14/202 and 14/212) formulated in a universal buffer were assessed by
33 laboratories from 15 countries, each using their routine NAT-based assays
for BKV detection. The freeze-dried candidates were tested alongside their
liquid equivalents, two patient samples and a BKV plasmid construct. For both
candidate materials a good degree of assay harmonization was already evident
amongst laboratories, and expression of the data as a relative potency for both
candidate samples suggested that either could serve as a suitable candidate for
an international standard. Of the two candidate samples, 14/212 was considered
to be the most suitable based on its geographical representation of the most
relevant circulating strain in Europe and the United States. Accelerated thermal
degradation stability studies performed at 3 months demonstrated that the
candidate material was stable, and exhibited no loss of titre at temperatures
used for storage (−20 °C), laboratory manipulation (4–20 °C), as well as at the
37–45 °C ambient temperature typically encountered during global shipment.
Further real-time stability studies were now in progress to assess the long-term
stability of the candidate material. The Committee was informed that 4092 vials
would be available for use as an international standard.
Discussion was held on the assignment of the unit with agreement
reached that in light of the minority representation of qualitative assays, the
under quantification typically seen in such assays and the primary requirement
to quantify BKV viral load for the management of renal transplant patients a
unit should be derived solely from the quantitative data obtained. Although
the choice of subtype for the candidate material was also approved, there was
also discussion of the potential value of creating a subtype panel that included
subtype 4 as this was also known to be clinically relevant. It was agreed that
NIBSC would investigate this need with stakeholders.
indicated that the Instructions for Use should clearly specify that assigned unitage
was based on quantitative NAT assays only, and recommended that the candidate
material 14/212 be established as the First WHO International Standard for BK
virus DNA for NAT-based assays with an assigned potency of 7.2 log 10 IU/ml.
59
7.1.3 Fifth WHO International Standard for hepatitis C

virus RNA for NAT-based assays
HCV is a bloodborne virus that causes both acute and chronic liver disease.
HCV is spread by contact with infected body fluids, predominately through
the practice of needle sharing. It is estimated that up to 150 million people
worldwide are infected with HCV with currently no vaccine available.
Although HCV infection can be detected using both serological and molecular
methods the latter are preferred in many settings due to their ability to detect
early infection.
An international standard for HCV RNA for NAT-based assays has
been available since 1996 with the current fourth international standard
having been established in 2011. However, concerns have been raised over the
stability of the current material with users reporting a 0.2 log 10 decrease in titre
when the material is shipped at ambient temperature. In addition, fewer than
50 vials of the current international standard remain. Following a hypothesis
that the presence of antibodies provides some stabilizing effects during the
lyophilization process, a pilot study was undertaken to assess the stability of
HCV products formulated from both antibody positive and negative material.
Following assessment by five laboratories using different commercial HCV
NAT-based assays, no differences in titre were observed. A full international
collaborative study was then conducted, involving 17 laboratories from 11
countries, to assess a candidate replacement material (NIBSC code 14/150)
alongside both the current international standard and the previous second
international standard. Accelerated degradation data for the candidate material
indicated a 0.1 log 10 loss in titre at 20 °C over 6 months relative to the −20 °C
baseline sample. Further real-time stability studies were ongoing.
Following discussion concerning the assignment of a potency value, it
was agreed that this would be based upon the quantitative-only data for relative
potency assessed against the second international standard. Clarification
was also given of the genotype used to prepare the candidate material, and
confirmation provided that this had been shown, via sequencing, to be the
same genotype used in the previous international standards.
and recommended that the candidate material 14/150 be established as the
Fifth WHO International Standard for hepatitis C virus RNA for NAT-based
assays with an assigned value of 5.0 log 10 IU/vial when reconstituted in 1.1 ml
of nuclease-free water.
7.1.4 Fourth WHO International Standard for anti-Toxoplasma gondii (human)

Toxoplasmosis is a disease caused by the parasite Toxoplasma gondii, the
congenital transmission of which remains a considerable burden on global
health. During pregnancy, serological screening programmes are used in
60
some settings to determine maternal toxoplasmosis antibody status as part

of efforts to prevent infection of the fetus. In addition, toxoplasmosis is also a
major cause of mortality among transplant patients. An appropriate antibody
standard is therefore required to validate the serological assays used to diagnose
toxoplasmosis. In 1994, TOXM was established as the Third WHO International
Standard for anti-Toxoplasma serum, human for use by IVD manufacturers,
national reference laboratories and hospital laboratories. Since 2000, stocks of
TOXM have been low and are now exhausted.
An international collaborative study was conducted involving 16
laboratories from 12 countries to assess the suitability of a candidate material
(NIBSC code 13/132) as a replacement international standard for anti-
Toxoplasma immunoglobulin, IgA, IgM and IgG. Each ampoule of the candidate
material contained 0.5 ml of pooled human plasma taken from six donors
who had experienced a recent T. gondii infection. The material was compared
with TOXM using: (a) agglutination assays for IgA, IgM and IgG; (b) enzyme
linked immunosorbent assays (ELISA) and enzyme linked fluorescent assays for
imunoglobulin, IgG, IgM and avidity of IgG; (c) immunofluorescence assays for
IgG and IgM; and (d) immunoblots for IgM and IgG and the Sabin-Feldman dye
tests. For continuity purposes, the first international standard for anti-Toxoplasma
IgG (NIBSC code 01/600) was also included in the collaborative study.
Intra- and inter-laboratory variability was generally found to be low
with the candidate material 13/132 exhibiting high levels of anti-Toxoplasma
IgG and IgM, and a potency intermediate between TOXM and 01/600. The
candidate material was also found to be stable at the temperature used for
storage (−20 °C) with accelerated thermal degradation studies at 15 months
indicating its suitability for long-term use. The Committee was informed that
3695 ampoules would be available for use as an international standard.
Discussion centred on the long-term stability of the IgM based on the
data shown, and on issues arising from the less frequent use of the dye test
and associated potential for problems in using current assays arising from the
reported high IgG titre of the proposed standard. Nevertheless, it was concluded
that the proposed standard would be highly useful in the standardization of
serological testing for toxoplasmosis and in supporting the appropriate clinical
management of this disease.
and recommended that the candidate material 13/132 be established as the
Fourth WHO International Standard for anti-Toxoplasma gondii (human) with
a unitage of 160 IU/ampoule. An IgG unitage of 263 U/ampoule was assigned
and the recommendation made that the high IgM content of the standard be
stated in the Instructions for Use. The Committee further recommended that a
potential replacement standard be developed with IgG and IgM unitages suitable
for use with current analytical methods.
61
7.1.5 Assignment of a holotranscobalamin value to the First WHO

International Standard for vitamin B12 and folate in human serum
Although it is known that vitamin B12 deficiency can lead to anaemia, the
accuracy of current methods for measuring its level in the blood has been
questioned. Approximately 20% of circulating vitamin B12 is bound to the
protein carrier transcobalamin. The resulting complex, holotranscobalamin
(holoTC), is the active portion of B12 available to cells and research indicates
that its measurement is a more reliable marker of early B12 deficiency than
total B12 measurement. In addition, holoTC assays are considered to be more
reliable during pregnancy and in patients taking the oral contraceptive pill as
the complex is not subject to the physiological drops associated with total B12
levels in these groups.
A first WHO international standard (NIBSC code 03/178) covering both
vitamin B12 and folate in human serum had been established in 2005 using
pooled human serum. A consensus B12 value of 480 pg/ml had been assigned
to this standard, along with a folate value based on liquid chromatography and
mass spectrometry methods. The Committee was informed that 2900 ampoules
of this material remained. As commercial assays for holoTC were now available,
with others likely to be developed shortly, a proposal had been endorsed in
2014 to assign a consensus holoTC value to the current international standard
in order to support holoTC measurement approaches.
An international collaborative study involving 12 laboratories in eight
countries had been conducted using a variety of in-house and commercial
methods to evaluate the holoTC content of the current standard 03/178 and of
three patient serum samples. Study results indicated good overall agreement
on the holoTC content of 03/178 with good parallelism also observed with
the patient samples, demonstrating commutability between the standard and
patient materials. Accelerated thermal degradation studies were performed, and
indicated an anticipated potency loss of 0.035% per year for samples stored at
−20 °C, which was considered to be acceptable.

The Committee noted that the proposed unit for this marker was in
pmol/l and not arbitrary WHO IUs. Following discussion it was clarified and
accepted that the proposed assignment in pmol/l would lead to greater industry
adoption and thus improved assay harmonization. The Committee then
considered the report of the study (WHO/BS/2015.2263) and recommended
that a holoTC value of 107 pmol/l be assigned to the WHO First International
Standard for vitamin B12 and folate in human serum.
7.1.6 First WHO International Standard for human C-peptide

Human C-peptide is a single chain peptide of 31 amino acids that is synthesized
in pancreatic beta cells as the connecting peptide for the A and B chains of
62
insulin in the proinsulin molecule. The measurement of C-peptide has a

number of important clinical uses where the monitoring of beta cell function
and endogenous insulin levels are required. Human C-peptide concentrations
are measured by immunoassay using patient serum, plasma or urine and the
first reference reagent used for the standardization of such assays comprised
ampoules of recombinant human C-peptide with a content of 10 μg/ampoule.
Established in 1986, the stocks of this reference reagent were now exhausted and
in 2010 the Committee endorsed a proposal for its replacement.
An international collaborative study involving 24 laboratories in 10
countries was conducted to evaluate the suitability of a candidate material
(NIBSC code 13/146) for use as a first international standard. Following
confirmation of the C-peptide content of a primary calibrant, the candidate
material 13/146 was calibrated accordingly and further characterized.
Estimates obtained from all laboratories were found to be in good agreement,
and indicated a content of 8.64 µg/ampoule. Study results also indicated that
the candidate material exhibited appropriate immunological activity and was
sufficiently stable to serve as an international standard. Assessment of the
commutability of the candidate material using a small cohort of patient samples
also indicated that the candidate material 13/146 was likely to be commutable
with patient serum and urine samples as measured by current immunoassays.
The Committee was informed that 2492 ampoules would be available for use as
an international standard.
Discussion took place on how best to address an apparent discrepancy
in the reported strengths of the formerly used reference reagent and the
proposed international standard, especially during assay revalidation, and on
the acceptability of using SI units instead of IUs.
and recommended that the candidate material 13/146 be established as the First
WHO International Standard for human C-peptide with an assigned value of
8.64 µg/ampoule. A clear indication should also be given in the Instructions for
Use of the known discrepancy in potency against the former reference reagent.
7.1.7 First WHO Reference Panel for hepatitis E virus

genotypes for NAT-based assays
Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. The virus
is transmitted mainly by the faecal-oral route and is associated with waterborne
epidemics in the developing world. Four main genotypes are identified among
the HEV strains capable of human infection with different, albeit partially
overlapping, worldwide distribution. Genotype 1 is predominantly found in
Africa and South-East Asia. Genotype 2 is found in Africa (overlapping in most
areas with genotype 1) and also in Mexico (where genotype 3 is also widespread).
63
Genotype 3 is also found in Australia, Europe, North America, South America

and Northern Asia. Genotype 4 is widespread in South-East Asia (overlapping in
different countries with genotype 1) and is also found in Europe (overlapping with
genotype 3). HEV belonging to genotypes 3 and 4 has been detected in domestic
pigs and in wild animals such as wild boars and deer, with animal contact or
the consumption of under-cooked meat being a likely a source of infection in
humans. Infection with genotype 1 can lead to severe consequences in pregnant
women and in individuals with underlying liver disease. There is also increasing
evidence of persistent infection in immunocompromised patients.
The First WHO International Standard for hepatitis E virus for NAT-
based assays was established in 2011. In the same year, the Committee also
endorsed a proposal to establish a First WHO Reference Panel for hepatitis E
virus genotypes for NAT-based assays comprising all four main genotypes, as
well as important sub-genotypes.
An international collaborative study involving 24 laboratories from
14 countries was conducted to evaluate a candidate panel comprising eight
strains from plasma and three strains from stool, diluted in pooled plasma.
Samples were concurrently evaluated against the current WHO international
standard using a range of different qualitative and quantitative in-house
and commercially available assays. Simultaneously, a biological reference
preparation for HEV genotype 3f was calibrated, initiated by the Council of
Europe and the European Commission in support of the implementation of
HEV NAT-based screening for solvent/detergent-treated plasma in Europe.
Prior to the collaborative study, the stability of the lyophilized samples was
studied with stool-derived HEV genotypes 1 and 2 found to be unstable upon
storage at elevated temperatures. This effect could be prevented by the addition
of stabilizers. Data on the potency of panel members and on the candidate
biological reference preparation were presented.
During discussion it was suggested that information be included on the

stabilization of the stool-derived material and on the nature of the stabilizers
used. The inclusion of the collaborative study results for potency ranges was also
suggested as this might help inform users of the variability of the results for
the included genotypes. Concerns were expressed however that users might
attempt to use the reference panel for quantitative purposes for which it was
not intended.
and recommended that the candidate panel be established as the First WHO
Reference Panel for hepatitis E virus genotypes for NAT-based assays. Although
no unitage would be assigned to the panel members, the geometric means and
ranges for each panel member would be provided in the Instructions for Use for
64
information only, along with information on the stabilizers used for the stool-
derived viruses.
7.1.8 Extension of the First WHO Repository of platelet

transfusion relevant bacterial strains
The bacterial contamination of platelet concentrates remains a persistent problem
in transfusion medicine. For method validation and assessment of blood-safety
measures, the use of a repository of bacterial strains able to proliferate in blood
components under normal storage conditions is crucial. In 2010, the Committee
had established the First WHO Repository of platelet transfusion relevant
bacterial strains, consisting of four bacterial strains. At the same time a proposal
for the future expansion of the repository was endorsed by the Committee.
During an international collaborative study involving 14 laboratories
in 10 countries, 11 new bacterial strains were evaluated for inclusion in
an expanded repository. A total of 15 bacterial strains (the four established
repository strains plus the proposed new candidate strains) were sent to study
participants. During evaluation, nine of the 11 new strains exhibited sufficient
growth properties with seven growing in all laboratories and two growing in 12
of the 14 laboratories. Although two strains did not grow in a sufficient number
of participating laboratories, an alternative strain of one of these (Morganella
morganii; PEI-A-91) was successfully restored to the proposed panel following
successful re-testing in eight laboratories.
Discussion topics included the generation of stability data in relation
to the replacement strain, and the potential methodological benefits arising
from the different growth kinetics of the candidate strains. It was also suggested
that other bacterial strains commonly reported as platelet contaminants might
usefully be added to the repository at a later stage.
and recommended that the First WHO Repository of platelet transfusion relevant
bacterial strains be extended by 10 strains.
7.1.9 First WHO Reference Reagent for Ebola virus antibodies

On 5 October 2015, WHO declared that the 2014/2015 Ebola virus disease
outbreak in West Africa continued to constitute a Public Health Emergency of
International Concern. It was recognized that the availability of internationally
recognized reference materials for antibodies to the Ebola virus would facilitate
the standardization of Ebola antibody assays used in epidemiological studies
to measure past or present Ebola infection and in vaccine studies to measure
antibodies elicited by vaccination in humans. In the absence of such standards,
individual laboratories would apply their own reference standards which would
65
not be harmonized with other laboratories and methods, thus impairing the
reproducibility of results between laboratories.
Recommendations emerging from a Technical Workshop on the
Standardization of Serological and PCR assays for the detection of Ebola virus
held at NIBSC in 2015 included the urgent development of interim Ebola
standards and, in the longer-term, of international standards in accordance with
published WHO guidelines for formal establishment the Committee.
In an international collaborative study involving 17 laboratories in
five countries nine Ebola virus antibody preparations were evaluated for their
suitability as an interim WHO standard for Ebola antibody assays. These nine
blinded-study samples included three preparations of purified anti-Ebola IgG
derived from transchromosomic bovines that had been pre-bled or vaccinated
with experimental vaccines, three solvent-detergent-treated plasma samples
obtained from three patients who had recovered from Ebola virus disease, one
solvent-detergent-treated Ebola-negative plasma and two non-treated plasma
pools obtained from volunteers vaccinated with the monovalent formulation
of the chimpanzee adenovirus 3-vectored vaccine. Results were obtained using
27 assays covering the three general methodological categories of neutralization
of live Ebola virus, neutralization of Ebola pseudotypes or virus-like particles
and enzyme immunoassay.
The Committee recognized the urgent need for such a material and
congratulated the groups involved on their greatly expedited efforts. Discussions
were held on the reasons for a lack of detectable antibodies in vaccinee material
and on the sufficiency of the available data. As the studies were not sufficiently
complete to establish any of the candidate materials as an international standard,
it was decided that the material should be described as a reference reagent with
this designation reflected in the unit terminology used.
The Committee considered the original proposal for the study (WHO/
BS/2015.2275) and the presentation of its expedited results and recommended

that the American Red Cross Ebola virus convalescent sample (sample code 79)
be established as the First WHO Reference Reagent for Ebola virus antibodies
with an assigned unitage of 1 U/ml.
7.1.10 First WHO reference reagents for Ebola virus RNA for NAT-based assays
The calibration and performance assessment of Ebola virus RNA NAT-based
assays requires reference materials that can be used in parallel with clinical
samples to standardize or control for every step of the assay, from extraction
through to detection and quantification.
An international collaborative study was conducted involving 13
laboratories from seven countries using a range of in-house and commercially
66
available qualitative and quantitative Ebola virus RNA NAT-based assays.

To ensure the biosafety of the materials to be provided to end users, candidate
synthetic Ebola virus RNA interim reference materials were developed for
assessment in this study with the longer-term goal being to produce an
inactivated Ebola virus validated as non-infectious and formulated for use as a
fully established international standard. The synthetic reference materials used
in the blinded-panel study were developed using two different approaches. One
approach utilized a lentiviral-based system to produce both candidate reference
materials and in-run controls, with an influenza-like ribonucleoprotein-based
system used to produce additional study materials. The resulting synthetic Ebola
virus RNA candidate materials produced were non-replicating, non-infectious
and suitable for use during the extraction, amplification and detection steps of
NAT-based assays.
Although the commutability of the candidate interim standards for
clinical samples was not fully assessed due to biosafety concerns, the use of a
variety of assay methods and diluents was considered to have addressed some
such aspects. An accelerated thermal degradation study at 1 month indicated
that the freeze-dried candidates were sufficiently stable for storage at −20 °C
and for shipment at ambient temperatures within temperate climate zones. It
was recommended however that any interim reference materials established be
packed in ice packs or dry ice when shipping to hotter climates. As with the
establishment of the First WHO Reference Reagent for Ebola virus antibodies
(see section 7.1.9) it was proposed that the interim materials be established as
reference reagents.
The Committee considered the original proposal for the study (WHO/
BS/2015.2275) and the presentation of its expedited results and recommended
that samples 1003 (Ebola NP-VP35-GP-LVV high for use in assays targeting
the np, vp35 and gp genes) and 1012 (Ebola VP40-L-LVV high for use in assays
targeting the vp40 and l genes) be established the First WHO reference reagents
for Ebola virus RNA for NAT-based assays with assigned unitages of 7.5 log 10
U/ml and 7.7 log 10 U/ml respectively when reconstituted in 1 ml of nuclease-
free water. Although arbitrary these values represent the consensus estimates
obtained across the range of quantitative laboratory assays used in the study. The
Committee also encouraged the conducting of follow-up studies to investigate
commutability with clinical samples. The Committee further recommended that
two lower-titre candidate materials (Ebola NP-VP35-GP-LVV low and Ebola
VP40-L-LVV low) be established as in-run controls with assigned potencies
of 3.5 log 10 U/ml and 3.7 log 10 U/ml respectively when reconstituted in 1 ml of
nuclease-free water. The Committee noted that approximately 200 vials of each
of the interim reference reagents and approximately 4000 ampoules of each of
the in-run controls would be available for distribution.
67

in vitro diagnostic device reagents
7.2.1 Proposed Fourth WHO International Standard for
hepatitis B virus DNA for NAT-based assays
Hepatitis B virus (HBV) is a highly infectious virus transmitted via blood and
bodily fluids and is known to cause cirrhosis of the liver and hepatocellular
carcinoma. Detection of HBV can be performed by serological or molecular
methods. Molecular NAT-based assays are routinely used to manage HBV
infections with a number of countries also mandating the NAT-based HBV
screening of cells, tissues and organs for transplantation. Blood screening using
NAT-based assays is not universally mandated but is widely implemented on a
voluntary basis.
The Third WHO International Standard for hepatitis B virus DNA for
NAT-based assays was established in 2011 and has been used widely for the
calibration of new HBV NAT-based assays and of secondary reference materials.
The Committee was informed that current stocks would be sufficient for
approximately two years and that the development of a replacement standard
would need to commence shortly to maintain an adequate supply.
At the time of preparation of the current international standard the bulk
material was freeze-dried in two batches (NIBSC codes 10/264 and 10/266).
Both the current standard (10/264) and candidate replacement material (10/266)
were evaluated in a collaborative study with mean relative potencies of 5.93
and 5.98 log 10 IU/ml respectively obtained relative to the second international
standard. The Committee was informed that 2533 vials of 10/266 were available
and a collaborative study would be conducted to compare the potencies of
10/264 and 10/266. In addition, HBV-positive plasma samples would be included
to allow for comparison with clinical samples, and calibration of a secondary
reference material for HBV NAT-based assays would also be undertaken.
Following a query regarding the labelling of the secondary reference
reagent and the expected remit of the Committee in its establishment it was
clarified that although the relevant data would be available to the Committee
there would be no requirement for its formal establishment.
the proposal (WHO/BS/2015.2275) to develop a Fourth WHO International
Standard for hepatitis B virus DNA for NAT-based assays.
7.2.2 Proposed Fourth WHO International Standard

for HIV-1 RNA for NAT-based assays
Human immunodeficiency virus (HIV) causes weakening and ultimately failure
of the immune system through infection of lymphocyte cells, predominately CD
4+ helper T cells. The progressive loss of such cells leaves the host susceptible to
68
opportunistic infections, which if left untreated are life threatening. Although

advances in drug therapy have played a major role in slowing the spread of
disease, the accurate detection and monitoring of infection remain essential to
prevention efforts and are key elements in patient care. As the virus is spread
through blood and bodily fluids, NAT-based screening for HIV is also a major
component of blood transfusion screening protocols.
The Third WHO International Standard for HIV-1 RNA for NAT-based
assays was established in 2011. In order to maintain supplies, this standard will
need to be replaced within 2 years. The Committee was informed that high-titre
stocks of the source material used for the current international standard were
still available for use as a replacement standard. Given that the material will be
heat-inactivated, non-infectious and derived from ample tissue culture stocks,
it would be feasible to produce a batch of 5000–6000 vials. It was intended that
a number of HIV-positive plasma donations would also be included in the
proposed collaborative study.
Discussion revolved around the most appropriate subtype to be selected
for the replacement material. Although previous international standards had
been formulated from a stock of subtype B material, it was known that subtype C
was now the most prevalent HIV subtype globally. Nevertheless, it was agreed
that to maintain continuity with the current international standard that the
replacement material would consist of the same subtype B strain. It was also
agreed that, availability permitting, the previous first and second international
standards should also be included in the collaborative study assessment.
Standard for HIV-1 RNA for NAT-based assays.

varicella zoster virus DNA for NAT-based assays
Varicella zoster virus (VZV) is a member of the Herpesviridae family and is
transmitted via the respiratory route. It is known to cause a two-stage infection
with primary infection resulting in chickenpox (varicella) and subsequent
virus reactivation resulting in shingles (herpes zoster). Following primary
infection, often during childhood, lifelong latency is established in the neurons
of the dorsal root ganglia with reactivation often occurring in later in life. As
serious neurological complications can arise from untreated infections, accurate
determination of assay sensitivity is essential for ensuring correct patient
management. Although vaccines are available for both varicella and herpes
zoster the degree of implementation varies between countries.
Molecular detection methods are considered the primary diagnostic tool
with EQA programmes demonstrating considerable variation in assay sensitivity.
69
In addition, a number of tests used by diagnostic laboratories are in-house assays.

It was therefore proposed that an international collaborative study be conducted
to evaluate candidate material for use as a First WHO International Standard for
varicella zoster virus DNA for NAT-based assays. A range of in-house and, where
possible, commercially available assays would be used to test a characterized
cell culture virus supernatant diluted in negative cerebrospinal fluid, along with
clinical cerebrospinal fluid samples and/or cultured virus from skin lesions.
Following discussion and further consideration, the Committee agreed
that there was a need for standardization in this area endorsed the proposal
(WHO/BS/2015.2275) to develop a First WHO International Standard for
varicella zoster virus DNA for NAT-based assays.

Trypanosoma cruzi DNA for NAT-based assays
Trypanosoma cruzi is a protozoan flagellate and is the causative agent of
Chagas disease, the elimination of which is the focus of a WHO programme.
In addition, the World Health Assembly passed a resolution regarding the
need for T. cruzi diagnostics. T. cruzi is transmitted by bites from Triatomine
insects but can also be transmitted via congenital, bloodborne, tissue-borne
and oral routes. An estimated 6–7 million people are infected worldwide, mostly
in the Latin American region, but also, as a result of global travel, in Europe,
Japan, North America and the Western Pacific region.
As no higher-order standard exists for the standardization of T. cruzi
diagnostic assays it was proposed that an international collaborative study be
conducted to evaluate candidate material for use as a First WHO International
Standard for Trypanosoma cruzi DNA for NAT-based assays.
The Committee acknowledged the need for a standard in this area and
requested clarification of the choice of subtype VI material as the main candidate.
As the proposed study also included the development of a panel of materials
of different subtypes it was felt that its outcome may ultimately determine the
correct subtype to be established. In the absence of the immediate resolution
of this issue the Committee requested that full clarification of this point be
provided at its next meeting. Following discussion and further consideration,
the Committee endorsed the proposal (WHO/BS/2015.2275) to develop a First
WHO International Standard for Trypanosoma cruzi DNA for NAT-based assays.

Plasmodium vivax DNA for NAT-based assays
Plasmodium vivax is a protozoan parasite and one of the main causative agents
of malaria with up to 300 million cases of infection annually attributable to this
parasite. P. vivax now causes as many cases of malaria as P. falciparum and in
70
some regions is the most common cause of malaria. Even in low concentrations
P. vivax is capable of causing severe infection and even death, mostly due to
splenomegaly. Addressing P. vivax infection is a recognized priority of the WHO
Malaria Programme, and it is envisaged that the development of a standard in
this area will benefit clinicians, commercial kit manufacturers and researchers.
It was proposed that an international collaborative study be conducted
to evaluate candidate material for use as a First WHO International Standard
for Plasmodium vivax DNA for NAT-based assays. In addition to the candidate
material, the study would also assess clinical samples for commutability purposes.
The Committee recognized the need for this standard while noting that
the study proposal contained only limited information on a number of key
aspects. The Committee suggested that the study should include laboratories
in countries where P. vivax was endemic, and requested that additional study
details be provided at its next meeting. Following discussion and further
consideration, the Committee endorsed the proposal (WHO/BS/2015.2275) to
develop a First WHO International Standard for Plasmodium vivax DNA for
NAT-based assays.

Plasmodium falciparum antigens
Globally, an estimated 3.2 billion people in 97 countries and territories are at
risk of developing malaria with 1.2 billion of these at high risk (greater than a
1 in 1000 chance of developing malaria in a year). According to latest estimates,
198 million malaria cases occurred globally in 2013 (uncertainty range 124–283
million) resulting in 584 000 deaths (uncertainty range 367 000–755 000).
Rapid diagnostic tests (RDTs) are important tools for surveillance and
for malaria case management with approximately 205 million used globally in
2012. The main targets of such products are histidine-rich protein 2 (HRP2),
lactate dehydrogenase and aldolase. Although there are many RDT devices
currently available detecting a variety of antigens, their validation and quality
control are limited as there is currently no international standard for the above
antigens. Current standards used for assessing malaria RDTs are based around
cryopreserved P. falciparum parasites, patient-derived P. falciparum (wild-type),
P. vivax infected blood and a parasite-negative blood panel, housed at the
United States Centers for Disease Control and Prevention, and comprising the
WHO Malaria Specimen Bank. The collection, preparation, characterization
and transport of patient samples for specimen banks are cumbersome and
expensive activities difficult to sustain in the long term.
It is proposed that an international standard be developed for use not
only in the quality control and standardization of RDTs worldwide but also in
monitoring the development of more sensitive diagnostic tests. The proposed
71
standard would consist of a lyophilized lysate of a P. falciparum culture

containing sufficient material for the detection of the three main antigens used
in malaria RDTs. The candidate material would be assessed in an international
collaborative study using capture ELISA and compared against clinical isolates
of P. falciparum from Africa, Asia and South America as well as a recombinant
HRP2 protein.
The Committee agreed that there was a need for this material and
recognized that, although not ideal, the use of recombinant HRP2 to represent
an antigen that naturally displays variation would be an acceptable compromise.
Following discussion and further consideration, the Committee endorsed the
proposal (WHO/BS/2015.2275) to develop a First WHO International Standard
for Plasmodium falciparum antigens.
7.2.7 Proposed First WHO Reference Panel for

KRAS codon 12 and 13 mutations
Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations occur in
approximately 40% of metastatic colorectal cancers and are also frequently
found in lung and pancreatic cancers. About 98% of KRAS mutations are
localized in codons 12 and 13. Patients with KRAS mutations do not respond
to anti-Epidermal Growth Factor Receptor antibody therapies, and KRAS
mutation testing is therefore of clinical importance with > 1 million colorectal
cancer diagnoses made globally each year.
During a proposed international collaborative study eight genomic
DNAs (one wild-type lymphoblastoid and seven heterozygous tumour cell lines)
will be produced and provided as freeze-dried DNA to more than 20 laboratories
for characterization using a broad range of Sanger, next-generation and other
sequencing technologies. Consideration was also been given to the use of a novel
standard format used in CE-marked IVDs (stabilized by anhydrobiosis in the
commercially available GenTegra inorganic matrix and stored in screw-capped

plastic tubes containing 1 µg of material for storage at 20 °C). In any case, the
material would take the form of a reference panel since neither digital PCR nor
sequencing will yield results measurable in IS units.
Discussion centred on potential matrix effects in the different assays
intended to be used in this study arising from the novel formulation. However, no
assays performed to date had exhibited such effects. It was suggested the KRAS
content should be defined as a percentage of the wild-type gene and the product
diluted in wild-type genomic DNA, bearing in mind the potential aneuploidy
of the originator cell lines. Following discussion and further consideration, the
Committee endorsed the proposal (WHO/BS/2015.2275) to develop a First
WHO Reference Panel for KRAS codon 12 and 13 mutations.
72
7.2.8 Proposed Fourth WHO International Standard for prolactin (human)

The measurement of serum prolactin by immunoassay is used to evaluate
pituitary gland function, to diagnose and monitor prolactinomas and to
determine the cause of galactorrhoea, headache, visual disturbance and
infertility. The majority of immunoassays are calibrated with respect to the
current Third WHO International Standard for prolactin, human (NIBSC
code 84/500), stocks of which are now low. The development of a replacement
international standard would require the sourcing of human pituitary prolactin,
which is not readily available. Potential sources include donors who have
previously provided pituitary-derived materials to NIBSC or materials obtained
from commercial organizations.
In the absence of such a bulk donation, it was proposed that an
international collaborative study be conducted of a batch of ampoules (2000
remaining) that was filled using pituitary prolactin donated by a Swedish
manufacturer in the 1980s. Evaluation of this material (NIBSC code 83/573)
had been undertaken during the establishment of the current international
standard in 1986, using bioassays (in vivo and cell-based), immunoassays and
radioreceptor assays. The unitage assigned to 83/573 in that study was 63 IU/
ampoule. The same material had also been included as a pituitary preparation in
a study held in 2000 to establish the First WHO Reference Reagent for prolactin,
human, recombinant. No viral markers have been detected in 83/573 and its
dose-response has been demonstrated to be parallel to 84/500. Study participants
would include immunoassay manufacturers, experts in the measurement of
prolactin and control laboratories, who will be asked to evaluate the current and
candidate international standards (84/500 and 83/573), the First WHO Reference
Reagent for prolactin, human, recombinant and up to 16 human serum samples
containing prolactin within and above the normal range. Accelerated degradation
samples (9 months) were available and would be distributed to participants as
capacity allowed or measured at NIBSC.
Standard for prolactin (human).
7.2.9 Proposed First WHO Reference Panel for hepatitis E virus antibodies
Diagnosis of infection with hepatitis E virus (HEV) requires a variety of tests
including the detection of IgM and (rising) IgG antibodies. The analysis of
IgM antibodies in particular is useful for the confirmation of acute infection.
Anti‑HEV IgG may also be detectable during acute infection. Anti-HEV IgG is
also a marker of past HEV infection and is thus important in seroprevalence
studies of previous HEV exposure in different populations.
73
In 1997, a WHO reference reagent (NIBSC code 95/584) originating

from a single donor and of unknown genotype had been established by the
Committee. Since then there has been growing recognition of the urgent need
for an international validation exercise of all existing HEV serology tests based
on the known lack of sensitivity and/or specificity of many serological assays.
In some cases there appears to have been a significant underestimation of HEV
seroprevalence in blood donors and, particularly in the case of IgM assays,
significant discrepancies in sensitivity and specificity, as well as cross-reactivity
with antibodies to other pathogens.
A project was now being proposed to establish an anti-HEV reference
panel which should ideally represent a wide geographical area. Such a
reference panel would also allow for the re-evaluation of the current WHO
reference reagent if current stock levels justified this, or for the identifying of
possible replacements if not.
Following discussion and further consideration, including the need to
include genotype information for any proposed panel, the Committee endorsed
the proposal (WHO/BS/2015.2275) to develop a First WHO Reference Panel
for hepatitis E virus antibodies.
7.2.10 Proposed First WHO Reference Panel for HIV-1 p24 antigen
The early detection and monitoring of infection with HIV is a key element in
treatment and prevention activities. Although the use of molecular methods
allows for early viral detection their cost is often prohibitive in low-income
countries. In addition, the genetic diversity of HIV presents a challenge to all
assay development with the accurate detection of HIV across all subtypes known
to be problematic.
An HIV-1 p24 antigen reference reagent (NIBSC code 90/636) was first
established by WHO in 1992 using subtype B patient-derived material that had
been inactivated using a solvent detergent treatment. Further development in
this area slowed following the emergence of molecular diagnostic techniques

in the late 1990s.
Following collaboration between NIBSC and the University of Zurich
a panel of samples was developed for use in the assessment of p24 antigen
detection assays. The panel consists of virus-like particle (VLP) constructs
generated in tissue culture from eukaryotic expression vectors. The VLPs contain
subtype-specific structural Gag proteins cloned from patient RNA extracts. The
use of enzymatic proteins (with the exception of PR) from same viral backbone
(subtype B) allows for homogenous quantification and input standardization via
reverse transcriptase activity.
Initial stability studies at NIBSC on lyophilized materials have
demonstrated the potential for the successful production of stable freeze-dried
material. The proposed panel would consist of lyophilized VLPs of the HIV-1
74
subtypes A, B, C, D, F1, G, H, circulating recombinant forms (CRF01_AE, 02_

AG, 12_BF, 20_BG) and group O (plus optional HIV-2). Each VLP would be
diluted in human plasma to physiologically relevant concentrations that could
be traced to the First WHO Reference Reagent for HIV-1 p24 antigen.
The Committee agreed that this was an important project that addressed
a current gap in the standardization of HIV detection. Although of global
importance there may be greater demand in countries with limited resources.
It was therefore suggested that assays (including antigen-only and combination)
that are most commonly used in such settings should be used in the proposed
study, along with the current WHO reference reagent. The participation of
laboratories in the developing world should also be ensured. Committee
members requested that both HIV antibody positive and negative clinical
samples be included in the study. Additionally there should also be broad and
relevant representation of mosaic viruses, parallel testing with seroconversion
panels and inclusion of an HIV-2 VLP.
the proposal (WHO/BS/2015.2275) to develop a First WHO Reference Panel for
HIV-1 p24 antigen.
7.2.11 Proposed First WHO Reference Panel for Babesia microti antibodies
Babesiosis is a tick-borne zoonosis caused by infection with intra-erythrocytic
protozoans of several species of the genus Babesia. Although several Babesia
species are transmitted in the United States, B. microti is the most prevalent.
Endemic B. microti transmission has also been reported in Canada, parts of
Europe and Japan (B. microti-like). The prevalence of such infections in the
United States and other countries is poorly understood with the full extent of
global transmission believed to be underestimated.
In 2014, a total of 1759 babesiosis cases in 21 states were reported to the
national surveillance programme in the United States highlighting the risk of
infection outside the nine known endemic states. In addition, areas of endemic
transmission are reported to be expanding, particularly in states adjoining the
endemic states. Although there is no FDA-licensed test for diagnostic or donor-
screening purposes, some donor testing using investigational tests has been
conducted in the United States.
It was proposed that CBER develop a First WHO Reference Reagent
panel for Babesia microti antibodies. Panel members would be formulated from
defibrinated plasma samples taken from humans who had a current or recent
B. microti infection. In addition, RBCs from DBA/2 mice infected with B. microti
would be used as a source of antigen in immunofluorescence assays. The
reference panel would consist of four serial dilutions of the pooled B. microti
antibody positive samples and one pooled B. microti antibody negative sample.
75
The need for this panel was acknowledged by the Committee with
discussions held in relation to the degree of assay cross-reactivity between
B. microti and other species of Babesia. Although not specifically tested for
in the material proposed for this panel, reports in the literature suggested
that some degree of cross-reactivity existed between species. Following
discussion and further consideration, the Committee endorsed the proposal
(WHO/BS/2015.2275) to develop a First WHO Reference Panel for Babesia
microti antibodies.
7.2.12 Proposed First WHO Repository of red blood cell

transfusion relevant bacterial strains
The initial establishment in 2010 of the First WHO Repository of platelet
transfusion relevant bacterial strains and its extension in 2015 (see section
7.1.8 above) represent the first steps in a strategy to establish repositories of
transfusion-relevant bacterial strains for all blood components. It was now
proposed that a First WHO Repository of red blood cell transfusion relevant
bacterial strains also be established for the validation of both bacterial-screening
methods and pathogen-reduction systems.
Microbial infection of RBC products and resulting infection in recipient
patients is frequently reported, often with fatal outcome. Based on the reported
literature and available haemovigilance data, a number of bacterial strains
had now been chosen for pre-testing for potential inclusion in the proposed
repository based upon their growth properties at 4–6 °C for up to 49 days.
While some strains showed no growth and were not detectable after 49 days,
others were still detectable (and would thus survive in RBC preparations)
and some even grew to high titres in less than 14 days. Bacterial reference
strains with relevance in RBC transfusion include both Gram-positive
bacteria (Bacillus cereus, B. subtilis, Staphylococcus aureus, Micrococcus luteus
and Streptococcus pyogenes) and Gram-negative bacteria (Klebsiella oxytoca,
K. pneumonia, Pseudomonas fluorescens, P. aeruginosa, Yersinia enterocolitica,

Serratia marcescens, S. liquefaciens and Listeria monocytogenes). Since growth
ability may vary by strain an international collaborative study was proposed.
Although spiking experiments to demonstrate bacterial inactivation are
performed by manufacturers, the availability of a bacterial reference panel
would allow standardized comparisons to be made of different technologies.
It was also agreed that the panel should include additional strains that grow
reliably in RBCs and which are relevant to transfusion-induced sepsis.
the proposal (WHO/BS/2015.2275) to establish a First WHO Repository of red
blood cell transfusion relevant bacterial strains.
76
vaccines and related substances
8.1 WHO International Standards and Reference

Reagents – vaccines and related substances
8.1.1 First WHO International Standard for anti-EV71 serum (human)
Vaccines for the effective prevention of hand, foot and mouth disease caused
by enterovirus 71 (EV71) are under development across South-East Asia. The
quantification of anti-EV71 neutralizing antibodies in human sera is an important
marker and diagnostic tool in assessing the immune status of individuals, and is
invaluable in evaluating the immunogenicity of EV71 vaccines.
A proposal to establish a First WHO International Standard for anti-
EV71 serum (human) was endorsed by the Committee in 2012. Candidate
materials were identified among 62 plasma samples provided by the National
Institutes for Food and Drug Control, China. EV71 strains representative of a
wide range of B and C genotypes were used for the initial characterization of
the candidate serum samples. An international collaborative study involving
17 laboratories from 6 countries was then conducted to evaluate the suitability of
two candidate materials (NIBSC codes 14/138 and 14/140) using a wide range
of in-house virus neutralization assays. Both candidate materials exhibited good
levels of neutralizing antibodies against a wide range of EV71 strains of various
genotypes. Furthermore, inter-laboratory variations in neutralization titres were
significantly reduced when values were expressed relative to either of the two
candidate sera. Stability studies demonstrated that both candidate materials were
also stable at temperatures used for storage (−20 °C) and laboratory manipulation
(4–20 °C). Accelerated thermal degradation studies at 3 months further indicated
that both candidates would be stable for long-term use. Real-time stability studies
of samples maintained for 12 months at different temperatures also showed no
significant loss of activity in samples held at 20 °C and below relative to those
stored −20 °C. It was concluded that both candidate materials would be suitable
for use as an international standard, and that in addition a low-titre material
(NIBSC code 13/238) could usefully serve as a WHO reference reagent for the
standardization of EV71 neutralization assays.
and recommended that candidate material 14/140 (5000 ampoules) be
established as the First WHO International Standard for anti-EV71 serum
(human) with an assigned value of 1000 IU/ampoule. Candidate material 14/138
(5000 ampoules) was recommended as a potential replacement international
standard in the future with an assigned value of 1090 IU/ampoule. In addition,
77
material 13/238 was established as the First WHO Reference Reagent for EV71
neutralization assays with an assigned value of 300 IU/ampoule.
8.1.2 First WHO international standards for meningococcal

serogroups A and X polysaccharide
For over a century meningococcal disease caused by meningococcal serogroup
A (MenA) has caused a significant burden to the region of Africa stretching
between Senegal and Ethiopia. In 2010, a conjugate vaccine developed
specifically for that area was introduced by the Meningitis Vaccine Project. As
a result, the incidence of disease caused by MenA has declined dramatically.
However, MenX, a previously rare causative agent of disease, has emerged as a
cause of outbreaks in the region. As a result, initiatives are now under way to
manufacture a conjugate vaccine similar to those used for MenA, C, W and Y
to control disease caused by MenX.
Plain polysaccharide (PS) and conjugate vaccines are primarily
evaluated by physicochemical methods to ensure that batches are consistently
manufactured. As different assays are employed to quantify the PS content as
the potency test of final formulations and bulk intermediates, there is a need for
international standards for both MenA and MenX PS to support calibration of
the internal references used in different laboratories. In 2011, the First WHO
International Standard for meningococcal serogroup C polysaccharide was
established for use in quantification assays for polysaccharide MenC-based
vaccines. MenA and MenX international standards are designed to be utilized
in a similar way, and their proposed development had been endorsed by the
Committee in 2013 and 2014 respectively.
An international collaborative study involving 11 laboratories from
nine countries, each using their own in-house assay, was conducted to establish
a First WHO international standard for meningococcal serogroups A and X
polysaccharide. One candidate material for each serogroup was provided for
analysis by each of two different manufacturers. Intra- and inter-laboratory

repeatability was generally very good with excellent agreement observed
between laboratories when the same methods were used for determining the
potency of the candidate materials. Both candidate materials (NIBSC codes
13/246 and 14/156) were found to be suitable for use as international standards.
Stability studies also demonstrated that the candidate materials were stable at
temperatures used for storage (−20 °C) and laboratory manipulation (4 °C).
During accelerated thermal degradation studies, a significant reduction in
lyophilized plug volume was observed for both candidate materials following
storage at 37 °C and 56 °C for up to 1 year for MenA (13/246) and 6 months
for MenX (14/156). The amount of PS in the ampoules remained constant
under all conditions. Real-time stability, reconstituted and accelerated thermal
degradation studies are ongoing.
78
The Committee considered the report of the studies (WHO/BS/2015.2255

Add 1) and recommended that candidate material 13/246 (3000 ampoules)
be established as the First WHO International Standard for meningococcal
serogroup A polysaccharide with an assigned value of 0.845 ± 0.043 mg/ampoule;
and that candidate material 14/156 (900 ampoules) be established as the First
WHO International Standard for meningococcal serogroup X polysaccharide
with an assigned value of 0.776 ± 0.089 mg/ampoule.
8.1.3 Third WHO International Standard for diphtheria

toxoid for use in flocculation test
Diphtheria is caused by exotoxin-producing strains of the bacterium
Corynebacterium diphtheriae. Active immunization against diphtheria is based
on the use of diphtheria toxoid (DTxd, a chemically detoxified preparation of
diphtheria toxin) to induce protective antibody responses. Diphtheria vaccines
form an essential component of the primary immunization schedule of children
and have been part of the WHO Expanded Programme on Immunization since
its inception in 1974.
DTxd is produced by growing the toxin-producing C. diphtheriae in
liquid media and converting the toxin to inactive toxoid by treatment with
formaldehyde. The antigenic strength and purity of the bulk toxoid is evaluated
by measurement of “limit of flocculation” (Lf) units. Measurement of antigen
content in Lf also serves as a good indicator of the consistency of production,
and testing of the crude toxin prior to inactivation is recommended for
monitoring purposes. Flocculation remains the primary method used by vaccine
manufacturers to evaluate toxin and toxoid concentrations in Lf units.
The Second WHO International Standard for diphtheria toxoid for use
in flocculation test was established in 2007 and defines the Lf unit. In light of
limited remaining stocks of this international standard a project was initiated
to calibrate and establish a replacement material. An international collaborative
study involving 25 laboratories in 15 countries was conducted to establish a
Third WHO International Standard for diphtheria toxoid for use in flocculation
test. Candidate material for the replacement standard was provided by one
manufacturer for formulation and filling prior to freeze-drying (Preparation A).
A similar material from another manufacturer was also provided, and prepared
in the same way, for a proposed new Pharmacopoeial Reference Standard for
diphtheria toxoid (Preparation B). The study was initiated with the primary
aim of calibrating these materials in Lf units using the Ramon flocculation test
standardized against the current international standard. Preparation A was
found to have a unitage of 1874 Lf/ampoule while Preparation B was found to
have a unitage of 714 Lf/ampoule. Intra- and inter-laboratory repeatability was
generally good for both preparations. A secondary aim of the collaborative
study was to assess the suitability of alternative antigen-detection methods
79
for measuring Lf. Comparable results to the original Ramon flocculation test
were obtained when using a laser light-scattering platelet aggregometer as
the detection system for flocculation. Similarly, Lf results returned by ELISA
methods were not significantly different to flocculation, suggesting that ELISA
may be used as a suitable alternative to the flocculation test for measuring the
Lf of diphtheria toxoid samples, subject to validation.
Real-time stability studies were ongoing and had so far demonstrated
that the candidate material (NIBSC code 13/212) was stable at the normal storage
temperature (−20 °C) and suitable for use up to 3 months after reconstitution
when stored at 4 °C. Accelerated thermal degradation study results suggested
that the material would also exhibit suitable long-term stability with a predicted
degradation rate of 0.018% per year when stored at −20 °C.
and recommended that Preparation A (NIBSC code 13/212; 4800 ampoules) be
established as the Third WHO International Standard for diphtheria toxoid for
use in flocculation test with an assigned value of 1870 Lf/ampoule.

vaccines and related substances
8.2.1 Proposed First WHO international standards for
meningococcal serogroups W and Y polysaccharide
Meningococcal disease is found worldwide with severe outbreaks of disease
occasionally occurring. Tetravalent conjugate vaccines to prevent disease
caused by Neisseria meningitidis serogroups ACWY are used globally. Three
ACWY conjugate vaccines are licensed and currently in use. Other conjugate
vaccines with different valencies, containing W and Y polysaccharide are in
development. Quantification of the total and free (unconjugated) saccharide
in conjugate vaccines is of key importance as an indicator of vaccine potency.
With the increased use of multivalent conjugates there is a need to accurately

quantify each different PS in order to achieve the correct balance of each in
the final formulation.
The First WHO International Standard for meningococcal serogroup C
polysaccharide was established in 2011, with WHO international standards for
MenA PS and MenX PS being established in 2015 (see section 8.1.2 above). The
proposed WHO international standards for MenW and MenY PS will allow for
the accurate quantification of PS in both the intermediate PS and bulk conjugate
components and final vaccine, and will complete the set of international standards
used for the quantification of PS in meningococcal conjugate vaccines.
The candidate materials to be used in a proposed international
collaborative study would be manufacturer-donated purified PS of vaccine quality
with low lipopolysaccharide and protein content. Approximately 10 laboratories
80
would be involved in evaluating the suitability of the candidate materials for

determining MenW and MenY PS content in conjugate vaccine samples. In
a departure from previous approaches, it was proposed to use quantitative
nuclear magnetic resonance (NMR) results from a single laboratory to assign
unitages. The proposed international standards would potentially be suitable
for use in three quantitative assays – NMR, high-performance anion‑exchange
chromatography with pulsed amperometric detection and resorcinol assays.
Standard for meningococcal serogroup W polysaccharide, and a First WHO
International Standard for meningococcal serogroup Y polysaccharide, and
agreed that the collaborative study should proceed with subsequent submission
of its outcome to the Committee in 2018.
8.2.2 Proposed First WHO international standards for Vi polysaccharide

Typhoid is a major cause of illness and death among children in developing
countries. In 2008, WHO estimated that typhoid fever causes 216 000–600 000
deaths annually. Most of these deaths occur in developing countries, primarily
in Asia. In such countries, large segments of the population do not have access
to safe water supplies or basic sanitation services, and there are only limited
programmes for detecting carriers and preventing them from handling food.
The vaccination of local populations has now become economically feasible
as vaccine manufacturers in developing countries start to produce Vi PS
conjugate vaccines.
There are minimum requirements for the amount of Vi PS per dose
of plain or conjugate vaccine. The relative ratio of free PS against total PS, the
extent of O-acetylation and molecular size/mass distribution have all been used
as important markers of immunogenicity. As the amount of Vi PS in relation to
the carrier protein is important for assessing vaccine potency an international
standard for Vi PS is required.
Manufacturer-donated GMP-grade Vi PS obtained from Citrobacter
freundii was found to be immunogenic in a Phase I study and immunologically
identical to Salmonella typhi Vi PS. In addition a second GMP-grade Vi PS
preparation purified from S. enterica subspecies Typhi was also being evaluated
as a potential standard.
Following discussion and further consideration, the Committee
endorsed the proposal (WHO/BS/2015.2272) to develop a First WHO
International Standard for Citrobacter freundii Vi polysaccharide and a First
WHO International Standard for Salmonella typhi Vi polysaccharide, and
of its outcome to the Committee.
81

anti-Vi polysaccharide serum (human)
The Committee was reminded that following the submission in 2014 of the
results of an earlier international collaborative study on the development of
an international standard in this area, it had been concluded that further work
was required to determine the cause of the significant inter-laboratory and
inter-assay variation observed. In response to that request, it was determined
that the potential causes of such variation included the technical expertise of
laboratories, poor binding of Vi PS to ELISA plates and the quality of IgG used
in the reference reagents.
A revised collaborative study was therefore being proposed in which
the original candidate material would be analysed by five of the original
participant laboratories. The candidate material would be analysed alongside
nine freeze‑dried preparations of anti-Vi IgG positive sera obtained from
volunteers in a Phase I clinical trial of Vi PS-tetanus conjugate. Following
discussion and further consideration, the Committee endorsed the proposal
(WHO/BS/2015.2272) to continue to develop a First WHO International
Standard for anti-Vi polysaccharide serum (human), and agreed that the
revised collaborative study should proceed after further discussions with
appropriate experts. The results of this study were expected to be submitted to
the Committee in 2017.
8.2.4 Proposed First WHO Reference Panel for non-typhoidal

salmonella O-antigen polysaccharides
Invasive non-typhoidal salmonella disease is caused by Salmonella enterica
serovars Typhimurium (O4,5 LPS) and Enteritidis (O9 LPS), and is a leading
cause of bacteraemia in sub-Saharan Africa, particularly in infants and
immunocompromised individuals. The disease has a high case fatality rate
and multidrug resistance hampers effective treatment. Field studies have shown
that increased serum levels of anti-O IgG in young children reduce the chances
of reinfection with S. enterica serovar Typhimurium. Anti-O IgGs are also
bactericidal in vitro.
Vaccine manufacturers in developing countries have started to produce
bivalent vaccines containing O4,5 and O9 antigens representing these serovars.
As several vaccines targeting the O-antigen PS will be entering clinical
development, a common global standard is now required for the quantification
and testing of quality parameters of O4,5 and O9 PS used in bioassays to measure
the potency of these vaccines and to compare the immunogenicity of various
vaccines. At present, three types of bivalent vaccines (conjugate, attenuated oral
and outer membrane) are in development, and it was therefore proposed that
an international collaborative study be undertaken to establish a First WHO
82
Reference Panel for non-typhoidal salmonella O-antigen polysaccharides. The

candidate materials would be of GMP standard.
for non-typhoidal salmonella O-antigen polysaccharides, and agreed that the
collaborative study should proceed with subsequent submission of its outcome
to the Committee in 2019.
8.2.5 Proposed First WHO international standards for

antibodies (human) to Salmonella enterica serovars
Typhimurium (O4,5 LPS) and Enteritidis (O9 LPS)
As outlined above in section 8.2.4 invasive non-typhoidal salmonella disease is
caused by Salmonella enterica serovars Typhimurium (O4,5 LPS) and Enteritidis
(O9 LPS) and vaccine manufacturers in developing countries have started
producing bivalent vaccines containing O4,5 and O9 antigens. At present,
in-house or commercially available antisera and immunoassays are used to
characterize vaccine immunoreactivity. Given the potential importance of such
vaccines in controlling disease there is now a need to develop international
standards for anti-O4,5 serum (human) and anti-O9 serum (human). It was
proposed that an international collaborative study be undertaken to establish
international standards in this area. It was intended that several vaccine
manufacturers would be approached as potential sources of clinical samples.
Sera from volunteers taking part in Phase I clinical trials would also be collected
and the pooled material screened for IgG reactive with O4,5 or O9 antigen.
Standard for antibodies (human) to Salmonella enterica serovar Typhimurium
(O4,5 LPS) and a First WHO International Standard for antibodies (human)
to Salmonella enterica serovar Enteritidis (O9 LPS), and agreed that the
8.2.6 Proposed First WHO International Standard for Salmonella

enterica serovar Paratyphi A O2 polysaccharide
Enteric fever is endemic in South and South-East Asia and is co-localized
with typhoid. In 2008, WHO estimated that > 27 million cases of enteric fever
occurred annually, and case fatality rates of up to 5% have been reported, mostly
in children < 5 years of age. The incidence of Paratyphi A varies by region but in
general ranges from 15–25% of all enteric fever cases. In a small number of areas
incidences of > 50% have been reported.
83
Several developing country vaccine manufacturers are producing

O2 PS specific for paratyphoid to be combined with typhoid vaccines. These
vaccines are expected to enter clinical trials within two years. A common global
standard is thus required for the quantification and testing of quality parameters
of O2 PS used in bioassays to measure the potency of these vaccines and to
compare the immunogenicity of various vaccine formulations. It was proposed
that an international collaborative study be undertaken to establish the First
WHO International Standard for Salmonella enterica serovar Paratyphi A O2
polysaccharide. Candidate materials would be of GMP standard and suitable for
use in both immunoassays and physicochemical assays.
Standard for Salmonella enterica serovar Paratyphi A O2 polysaccharide, and
of its outcome to the Committee in 2018.
8.2.7 Proposed First WHO International Standard for antibodies

(human) to Salmonella enterica Paratyphi A O2 polysaccharide
As outlined above in section 8.2.6 enteric fever is endemic in South and South-
East Asia, and several developing country vaccine manufacturers are producing
O2 PS specific for paratyphoid to be combined with typhoid vaccines. At present,
in-house or commercially available antisera and immunoassays are used to
characterize the immunoreactivity of O2 PS. Given the potential importance of
such vaccines in controlling enteric fever there is now a need for an international
standard for human antibodies to Salmonella enterica serovar Paratyphi A
O2 PS. It was therefore proposed that an international collaborative study be
undertaken to establish a First WHO International Standard for antibodies
(human) to Salmonella enterica Paratyphi A, O2 polysaccharide. Several vaccine
manufacturers will be approached as potential sources of sera from volunteers
taking part in Phase I clinical trials.

Standard for antibodies (human) to Salmonella enterica Paratyphi A O2
polysaccharide, and agreed that the collaborative study should proceed with
subsequent submission of its outcome to the Committee in 2018.
8.2.8 Proposed First WHO Reference Panel for O-antigen

polysaccharides for Shigella strains
Shigella serotypes are a major global cause of morbidity and mortality. Shigella
flexneri and S. sonnei are responsible for > 125 million cases of moderate to
severe diarrhoea cases per year, mostly in children < 5 years of age. The majority
84
(90%) of infections caused by Shigella occur in sub-Saharan Africa and South

Asia with travellers and deployed members of the military visiting endemic areas
also at risk for shigellosis.
A number of organizations are now developing 4- to 6-valent vaccines to
cover the immunodominant O-antigens of the most epidemiologically relevant
serotypes (S. sonnei and S. flexneri 2a, 3a and 6). Monovalent vaccines for
S. sonnei and S. flexneri 2a are also being developed with some already in early
clinical trials. Given the common targets (the O-PS or repeating unit of LPS) of
many of these vaccines, global standards are required for the quantification and
testing of O-PS used in bioassays to measure the potency of these vaccines and
to compare the immunogenicity of various vaccines. It was therefore proposed
that an international collaborative study be undertaken to establish a First WHO
Reference Panel for O-antigen polysaccharides for Shigella strains. Candidate
materials would be of GMP standard and suitable for use in both immunoassays
and physicochemical assays.
for O-antigen polysaccharides for Shigella strains, and agreed that the
8.2.9 Proposed First WHO Reference Panel for anti-Shigella

O-antigen polysaccharide serum (human)
As outlined above in section 8.2.8, Shigella serotypes are a major global cause
of morbidity and mortality, and the 4- to 6-valent vaccines currently under
development are intended to cover the immunodominant O-antigens of the
most epidemiologically relevant serotypes (S. sonnei, S. flexneri 2a, 3a and 6).
In the case of S. sonnei an O-antigen antibody level has been identified which
is protective. At present, in-house or commercially available antisera and
immunoassays are used to characterize vaccine immunoreactivity. Given the
potential of a multivalent Shigella vaccine in controlling disease, there is now a
need for international standards for human anti-O-antigen PS serum reflecting
the most epidemiologically relevant serotypes. It was therefore proposed that
an international collaborative study be undertaken to establish a First WHO
Reference Panel for anti-Shigella O-antigen polysaccharide serum (human).
Several vaccine manufacturers will be approached as potential sources of sera
obtained from volunteers in Phase I clinical trials.
for anti-Shigella O-antigen polysaccharide serum (human), and agreed that the
85

anti-Clostridium difficile serum (human)
Clostridium difficile infection (CDI) is caused by colonization of the human
intestine by this Gram-positive bacterium, and can result in a high mortality
rate. The risk of recurrence of CDI presents a burden to both patients and the
health-care system with millions of new infections per year and thousands
of deaths. CDI-associated disease constitutes a large majority of nosocomial
diarrhoea cases in developed countries and is mediated by the effects of two
secreted toxins – toxin A and toxin B. These toxins have key toxin-neutralizing
epitopes that are the focus of current vaccine development studies.
Patients who develop strong anti-toxin antibody responses can clear
C. difficile infection and remain disease free, and several vaccines are currently
in various stages of clinical trials. However, there are at present no international
standards or reference reagents available to standardize the immune, functional
or cytotoxic assays necessary for the evaluation of vaccine efficacy and quality.
As a human reference serum with an assigned unitage would be
very useful in calibrating various immunoassays, it was proposed that an
international collaborative study be undertaken to establish a First WHO
International Standard for anti-Clostridium difficile serum (human). Following
testing of several batches of intravenous immunoglobulin, one manufacturer-
donated batch was found to have high titres of antibodies to both toxin A and
toxin B and would serve as a candidate material.
Following discussion and further consideration, the Committee
endorsed the proposal (WHO/BS/2015.2272) to develop a First WHO
International Standard for anti-Clostridium difficile serum (human), and agreed
that the collaborative study should proceed with subsequent submission of its
outcome to the Committee in 2017.
86
Annex 1
WHO Recommendations, Guidelines and other documents
related to the manufacture and quality control of
biological substances used in medicine
WHO Recommendations, Guidelines and other documents are intended to
provide guidance to those responsible for the production of biological substances
as well as to others who may have to decide upon appropriate methods of
assay and control to ensure that products are safe, reliable and potent. WHO
Recommendations (previously called Requirements) and Guidelines are scientific
and advisory in nature but may be adopted by an NRA as national requirements
or used as the basis of such requirements.
Recommendations concerned with biological substances used in
medicine are formulated by international groups of experts and are published
in the WHO Technical Report Series 1 as listed below. A historical list of
Requirements and other sets of Recommendations is available on request from
the World Health Organization, 20 avenue Appia, 1211 Geneva 27, Switzerland.
Reports of the WHO Expert Committee on Biological Standardization
published in the WHO Technical Report Series can be purchased from:
WHO Press
World Health Organization
20 avenue Appia
1211 Geneva 27
Switzerland
Telephone: + 41 22 791 3246
Fax: +41 22 791 4857
Email: [email protected]
Website: www.who.int/bookorders
Individual Recommendations and Guidelines may be obtained free of
charge as offprints by writing to:
Technologies Standards and Norms
Department of Essential Medicines and Health Products
World Health Organization
20 avenue Appia
1211 Geneva 27
Switzerland
Abbreviated in the following pages to “TRS”.

1
87
Recommendations, Guidelines and other Reference

documents
Animal cells, use of, as in vitro substrates for the Revised 2010, TRS 978 (2013)
production of biologicals
BCG vaccines (dried) Revised 2011, TRS 979 (2013)
Biological products: good manufacturing Revised 2015, TRS 999 (2016)
practices
Biological standardization and control: Unpublished document
a scientific review commissioned by the WHO/BLG/97.1
UK National Biological Standards Board (1997)
Biological substances: International Standards Revised 2004, TRS 932 (2006)
and Reference Reagents
Biotherapeutic protein products prepared by Revised 2013, TRS 987 (2014);
recombinant DNA technology Addendum 2015, TRS 999 (2016)
Biotherapeutic products, similar Adopted 2009, TRS 977 (2013)
Blood, blood components and plasma Revised 1992, TRS 840 (1994)
derivatives: collection, processing and quality
control
Blood establishments: good manufacturing Adopted 2010, TRS 961 (2011)
practices
Blood plasma (human) for fractionation Adopted 2005, TRS 941 (2007)
Blood plasma products (human): viral Adopted 2001, TRS 924 (2004)
inactivation and removal procedures
Blood regulatory systems, assessment criteria Adopted 2011, TRS 979 (2013)
for national
WHO Technical Report Series No. 999, 2016
Changes to approved vaccines: procedures Adopted 2014, TRS 993 (2015)

and data requirements
Cholera vaccines (inactivated, oral) Adopted 2001, TRS 924 (2004)
Dengue tetravalent vaccines (live, attenuated) Revised 2011, TRS 979 (2013)
Diphtheria, tetanus, pertussis (whole cell), and Revised 2012, TRS 980 (2014)
combined (DTwP) vaccines
Diphtheria vaccines (adsorbed) Revised 2012, TRS 980 (2014)
DNA vaccines: assuring quality and nonclinical Revised 2005, TRS 941 (2007)
safety
Haemophilus influenzae type b conjugate Revised 1998, TRS 897 (2000)
vaccines
88
Annex 1

documents
Haemorrhagic fever with renal syndrome (HFRS) Adopted 1993, TRS 848 (1994)
vaccines (inactivated)
Hepatitis A vaccines (inactivated) Adopted 1994, TRS 858 (1995)
Hepatitis B vaccines prepared from plasma Revised 1987, TRS 771 (1988)
Hepatitis B vaccines made by recombinant DNA Revised 2010, TRS 978 (2013)
techniques
Human interferons prepared from Adopted 1988, TRS 786 (1989)
lymphoblastoid cells
Influenza, biosafety risk assessment and safe Adopted 2005, TRS 941 (2007)
production and control for (human) pandemic
vaccines
Influenza vaccines (inactivated) Revised 2003, TRS 927 (2005)
Influenza vaccines (live) Revised 2009, TRS 977 (2013)
Influenza vaccines, human, pandemic, Adopted 2007, TRS 963 (2011)
regulatory preparedness
Japanese encephalitis vaccines (inactivated) Revised 2007, TRS 963 (2011)
for human use
Japanese encephalitis vaccines (live, attenuated) Revised 2012, TRS 980 (2014)
for human use
Louse-borne human typhus vaccines (live) Adopted 1982, TRS 687 (1983)
Malaria vaccines (recombinant) Adopted 2012, TRS 980 (2014)
Measles, mumps and rubella vaccines and Adopted 1992, TRS 848 (1994);
combined vaccines (live) Note TRS 848 (1994)
Meningococcal polysaccharide vaccines Adopted 1975, TRS 594 (1976);
Addendum 1980, TRS 658 (1981);
Amendment 1999, TRS 904 (2002)
Meningococcal A conjugate vaccines Adopted 2006, TRS 962 (2011)
Meningococcal C conjugate vaccines Adopted 2001, TRS 924 (2004);
Addendum (revised) 2007,
TRS 963 (2011)
Monoclonal antibodies Adopted 1991, TRS 822 (1992)
Papillomavirus vaccines (human, recombinant, Revised 2015, TRS 999 (2016)
virus-like particle)
89

documents
Pertussis vaccines (acellular) Revised 2011, TRS 979 (2013)
Pertussis vaccines (whole-cell) Revised 2005, TRS 941 (2007)
Pharmaceutical products, storage and transport Adopted 2010, TRS 961 (2011)
of time- and temperature-sensitive
Pneumococcal conjugate vaccines Revised 2009, TRS 977 (2013)
Poliomyelitis vaccines (inactivated) Revised 2014, TRS 993 (2015)
Poliomyelitis vaccines (inactivated): guidelines Adopted 2003, TRS 926 (2004)
for the safe production and quality control of
inactivated poliomyelitis vaccine manufactured
from wild polioviruses
Poliomyelitis vaccines (oral) Revised 2012, TRS 980 (2014)
Quality assurance for biological products, Adopted 1991, TRS 822 (1992)
guidelines for national authorities
Rabies vaccines for human use (inactivated) Revised 2005, TRS 941 (2007)
produced in cell substrates and embryonated
eggs
Regulation and licensing of biological products Adopted 1994, TRS 858 (1995)
in countries with newly developing regulatory
authorities
Regulatory risk evaluation on finding an Adopted 2014, TRS 993 (2015)
adventitious agent in a marketed vaccine:
scientific principles
Rotavirus vaccines (live-attenuated, oral) Adopted 2005, TRS 941 (2007)
Smallpox vaccines Revised 2003, TRS 926 (2004)

Snake antivenom immunoglobulins Adopted 2008, TRS 964 (2012)
Sterility of biological substances Revised 1973, TRS 530 (1973);
Amendment 1995, TRS 872 (1998)
Synthetic peptide vaccines Adopted 1997, TRS 889 (1999)
Tetanus vaccines (adsorbed) Revised 2012, TRS 980 (2014)
Thiomersal for vaccines: regulatory expectations Adopted 2003, TRS 926 (2004)
for elimination, reduction or removal
Thromboplastins and plasma used to control Revised 2011, TRS 979 (2013)
oral anticoagulant therapy
90
Annex 1

documents
Tick-borne encephalitis vaccines (inactivated) Adopted 1997, TRS 889 (1999)
Transmissible spongiform encephalopathies Revised 2005, WHO (2006)
in relation to biological and pharmaceutical http://www.who.int/biologicals/
products, guidelines publications/en/whotse2003.pdf
Tuberculins Revised 1985, TRS 745 (1987)
Typhoid vaccines, conjugated Adopted 2013, TRS 987 (2014)
Typhoid vaccines (live attenuated, Ty21a, oral) Adopted 1983, TRS 700 (1984)
Typhoid vaccines, Vi polysaccharide Adopted 1992, TRS 840 (1994)
Vaccines, clinical evaluation: regulatory Adopted 2001, TRS 924 (2004)
expectations
Vaccines, lot release Adopted 2010, TRS 978 (2013)
Vaccines, nonclinical evaluation Adopted 2003, TRS 926 (2004)
Vaccines, nonclinical evaluation of vaccine Adopted 2013, TRS 987 (2014)
adjuvants and adjuvanted vaccines
Vaccines, prequalification procedure Adopted 2010, TRS 978 (2013)
Vaccines, stability evaluation Adopted 2006, TRS 962 (2011)
Vaccines, stability evaluation for use under Adopted 2015, TRS 999 (2016)
extended controlled temperature conditions
Varicella vaccines (live) Revised 1993, TRS 848 (1994)
Yellow fever vaccines Revised 2010, TRS 978 (2013)
Yellow fever vaccines, laboratories approved Revised 1995, TRS 872 (1998)
by WHO for the production of
Yellow fever virus, production and testing of Adopted 1985, TRS 745 (1987)
WHO primary seed lot 213–77 and reference
batch 168–736
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Annex 2
WHO good manufacturing practices for biological products
Replacement of Annex 1 of WHO Technical Report Series, No. 822
1. Introduction 96
2. Scope 96
3. Terminology 100
4. Principles and general considerations 104
5. Pharmaceutical quality system and quality risk management 106
6. Personnel 106
7. Starting materials 107
8. Seed lots and cell banks 109
9. Premises and equipment 111
10. Containment 113
11. Clean rooms 115
12. Production 116
13. Campaign production 118
14. Labelling 119
15. Validation 119
16. Quality control 121
17. Documentation (batch processing records) 122
18. Use of animals 123
19. Authors and acknowledgements 125
20. References 127
93
Guidelines published by WHO are intended to be scientific and

advisory in nature. Each of the following sections constitutes guidance
for national regulatory authorities (NRAs) and for manufacturers of
biological products. If an NRA so desires, these WHO Guidelines may
be adopted as definitive national requirements, or modifications
may be justified and made by the NRA.
94
Annex 2
Abbreviations
AEFI adverse event following immunization
ATMP advanced therapy medicinal product
BCG bacille Calmette–Guérin
GMP good manufacturing practice(s)
HEPA high-efficiency particulate air
HVAC heating, ventilation and air conditioning
IgE immunoglobulin E
mAb monoclonal antibody
MSL master seed lot
MVS master virus seed
PQR product quality review
PQS pharmaceutical quality system
QRM quality risk management
SPF specific pathogen free
TSE transmissible spongiform encephalopathy
WSL working seed lot
WVS working virus seed
95
1. Introduction
Biological products can be defined according to their source material and
method of manufacture. The source materials and methods employed in the
manufacture of biological products for human use therefore represent critical
factors in shaping their appropriate regulatory control. Biological products are
derived from cells, tissues or microorganisms and reflect the inherent variability
characteristic of living materials. The active substances in biological products
are often too complex to be fully characterized by utilizing physicochemical
testing methods alone and may show a marked heterogeneity from one
preparation and/or batch to the next. Consequently, special considerations
are needed when manufacturing biological products in order to maintain
consistency in product quality.
Good manufacturing practices (GMP) for biological products were
first published by WHO in 1992 (1). This current revision reflects subsequent
developments that have taken place in science and technology, and in the
application of risk-based approaches to GMP (2–14). The content of this
document should be considered complementary to the general recommendations
set out in the current WHO good manufacturing practices for pharmaceutical
products: main principles (2) and in other WHO documents related specifically
to the production and control of biological products.
This document is intended to serve as a basis for establishing national
guidelines for GMP for biological products. If a national regulatory authority
(NRA) so desires, the guidance provided may be adopted as definitive national
requirements, or modifications may be justified and made by the NRA in light
of the risk–benefit balance and legal considerations in each authority. In such
cases, it is recommended that any modification to the principles and technical
specifications set out below should be made only on the condition that the
modifications ensure product quality, safety and efficacy that are at least
equivalent to that recommended in this document.
2. Scope
The guidance provided in this document applies to the manufacture, control
and testing of biological products for human use – from starting materials
and preparations (including seed lots, cell banks and intermediates) to the
finished product.
Manufacturing procedures within the scope of this document include:
■■ growth of strains of microorganisms and eukaryotic cells;
■■ extraction of substances from biological tissues, including human,
animal and plant tissues, and fungi;
96
Annex 2
■■ recombinant DNA (rDNA) techniques;

■■ hybridoma techniques;
■■ propagation of microorganisms in embryos or animals.
Medicinal products of biological origin manufactured by these procedures
include allergens, antigens, vaccines, certain hormones, cytokines, monoclonal
antibodies (mAbs), enzymes, animal immune sera, products of fermentation
(including products derived from rDNA), biological diagnostic reagents for in
vivo use and advanced therapy medicinal products (ATMPs) used for example
in gene therapy and cell therapy.
For human whole blood, blood components and plasma-derived
products for therapeutic use separate comprehensive WHO guidance is available
and should be followed (12, 15).
In some countries certain small-molecule medicinal products (for
example, antibiotics) are not defined as biological products. Nevertheless, where
the manufacturing procedures described in this document are used then the
guidance provided may be followed.
The preparation of investigational medicinal products for use in clinical
trials should follow the basic principles of GMP set out in these and other WHO
GMP guidelines (2, 16) as appropriate. However, certain other requirements
(such as process and analytical method validations) could be completed before
marketing authorization (17–19).
The current document does not provide detailed recommendations
for specific classes of biological products (for example, vaccines). Attention is
therefore directed to other relevant WHO documents, and in particular to WHO
recommendations to assure the quality, safety and efficacy of specific products.1
Table 1 illustrates the typical risk-based application of the current
document (4, 7). It should be noted that this table is illustrative only and is not
intended to describe the precise scope.
See: http://www.who.int/biologicals/en/ (accessed 4 November 2015).

1
97
98
Table 1
Scope of the current document (illustrative)
Type and source Example products Application of this document to steps in manufacture
of material
1. Animal or plant Heparins, insulin, Collection of plant, Cutting, mixing Isolation and Formulation
sources: non- enzymes, proteins, organ, tissue or and/or initial purification and filling
transgenic allergen extract, ATMPs, fluid processing
animal immune sera
2. Virus or bacteria/ Viral or bacterial Establishment Cell culture Inactivation Formulation
fermentation/cell vaccines, enzymes, and and/or when applicable, and filling
culture proteins maintenance of fermentation isolation and
MCB, WCB, MSL/ purification
MVS, WSL/WVS
3. Biotechnology Recombinant products, Establishment Cell culture Isolation, Formulation
fermentation/cell mAbs, allergens, and and/or purification and and filling
culture vaccines, gene therapy maintenance of fermentation modification
(viral and non-viral MCB, WCB, MSL,

vectors, plasmids) WSL
4. Animal sources: Recombinant proteins, Master and Collection, cutting, Isolation, Formulation
transgenic ATMPs working transgenic mixing and/or purification and and filling
bank initial processing modification
5. Plant sources: Recombinant proteins, Master and Growing and/or Initial extraction, Formulation
transgenic vaccines, allergens working transgenic harvesting isolation, and filling
bank purification and
modification
Table 1 continued
Type and source Example products Application of this document to steps in manufacture
of material
6. Human sources Urine-derived enzymes, Collection of fluid Mixing and/or Isolation and Formulation
hormones initial processing purification and filling
7. Human and/or Gene therapy: Donation, Vector Ex vivo genetic Formulation
animal sources genetically modified procurement and manufacture and modification of and filling
cells testing of starting cell purification cells, establish
tissue/cellsa and processing MCB, WCB or
cell stock
Somatic cell therapy Donation, Establishing and Cell isolation, Formulation,
procurement and maintaining MCB, culture purification combination
testing of starting WCB or cell stock and combination and filling
tissue/cellsa with non-cellular
components
Tissue-engineered Donation, Initial processing, Cell isolation, Formulation,
products procurement and isolation and culture, purification combination
testing of starting purification, and combination and filling
tissue/cellsa establishing and with non-cellular
maintaining MCB, components
WCB, primary cell
stock
a
GMP guidelines, as described in this document, are not applied to this step. Other national regulations, requirements, recommendations and/or guidelines may apply as
deemed necessary by the NRA.
MCB = master cell bank; MSL = master seed lot; MVS = master virus seed; WCB = working cell bank; WSL = working seed lot; WVS = working virus seed.
Annex 2
99
3. Terminology
In addition to the terms defined in WHO good manufacturing practices for
pharmaceutical products: main principles (2) and WHO good manufacturing
practices for sterile pharmaceutical products (3), the definitions given below
apply to the terms as used in the current document. These terms may have
different meanings in other contexts.
Active substance: a defined process intermediate containing the active
ingredient, which is subsequently formulated with excipients to produce the drug
product. This may also be referred to as “drug substance” or “active ingredient”
in other documents.
Adventitious agents: contaminating microorganisms of the cell culture
or source materials, including bacteria, fungi, mycoplasmas/spiroplasmas,
mycobacteria, rickettsia, protozoa, parasites, transmissible spongiform
encephalopathy (TSE) agents and viruses that have been unintentionally
introduced into the manufacturing process of a biological product. The source
of these contaminants may be the legacy of the cell line, or the raw materials
used in the culture medium to propagate the cells (in banking, in production or
in their legacy), the environment, personnel, equipment or elsewhere.
Allergen: a molecule capable of inducing an immunoglobulin E (IgE)
response and/or a Type I allergic reaction.
Antibodies: proteins produced naturally by the B-lymphocytes that
bind to specific antigens. Using rDNA technology antibodies are also produced
in other (continuous) cell lines. Antibodies may be divided into two main types
– monoclonal and polyclonal antibodies – based on key differences in their
methods of manufacture. Also called immunoglobulins.
Antigens: substances (for example, toxins, foreign proteins, bacteria,
tissue cells and venoms) capable of inducing specific immune responses.
Axenic: a single organism in culture which is not contaminated with
any other organism.

Bioburden: the level and type (objectionable or not) of microorganisms
present in raw materials, media, biological substances, intermediates or
finished products. Regarded as contamination when the level and/or type
exceed specifications.
Biohazard: any biological material considered to be hazardous to people
and/or the environment.
Biological starting materials: starting materials derived from a biological
source that mark the beginning of the manufacturing process of a drug, as
described in a marketing authorization or licence application, and from which
the active ingredient is derived either directly (for example, plasma derivatives,
ascitic fluid and bovine lung) or indirectly (for example, cell substrates, host/
vector production cells, eggs and viral strains).
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Biosafety risk group: denotes the containment conditions required for

safe handling of organisms associated with different hazards, ranging from Risk
Group 1 (lowest risk, no or low individual and community risk, and unlikely to
cause disease) to Risk Group 4 (highest risk, high individual and community
risk, usually causes severe disease, and which is likely to spread with no
prophylaxis or treatment available) (20).
Campaign manufacture: the manufacture of an uninterrupted sequence
of batches of the same product or intermediate in a given time period, followed
by strict adherence to accepted control measures before switching to another
product or different serotype. The different products are not run at the same
time but may be run on the same equipment.
Cell bank: a collection of appropriate containers whose contents are
of uniform composition and stored under defined conditions. Each container
represents an aliquot of a single pool of cells.
Cell culture: the process by which cells that are no longer organized into
tissues are grown in vitro under defined and controlled conditions. Cell cultures
are operated and processed under axenic conditions to ensure a pure culture
absent of microbial contamination.
Cell stock: primary cells expanded to a given number of cells to be
aliquoted and used as starting material for production of a limited number of
lots of a cell-based medicinal product.
Containment: the concept of using a process, equipment, personnel,
utilities, system and/or facility to contain product, dust or contaminants in one
zone, preventing them from entering into another zone and/or escaping.
Continuous culture: a process by which the growth of cells is maintained
by periodically replacing a portion of the cells and the medium so that there is
no lag or saturation phase.
Control strategy: a planned set of controls derived from current
product and process understanding that assures process performance and
product quality. The controls can include parameters and attributes related
to active substance and finished product materials and components; facility
and equipment operating conditions; in-process controls; finished product
specifications; and the associated methods and frequency of monitoring
and control.
Cross-contamination: contamination of a starting material, intermediate
product or finished product with another starting material or product during
production. In multi-product facilities, cross-contamination can occur throughout
the manufacturing process, from generation of the master cell bank (MCB) and
working cell bank (WCB) to finished product.
Dedicated: facility, personnel, equipment or piece of equipment used
only in the manufacture of a particular product or group of specified products
of similar risk.
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Dedicated area: an area that may be in the same building as another

area but which is separated by a physical barrier and which has, for example,
separate entrances, staff facilities and air-handling systems. Also referred to as
“self-contained facility” in other GMP documents.
Feeder cells: cells used in co-culture to maintain pluripotent stem cells.
For human embryonic stem cell culture, typical feeder layers include mouse
embryonic fibroblasts or human embryonic fibroblasts that have been treated
to prevent them from dividing.
Finished product: a finished dosage form that has undergone all
stages of manufacture, including packaging in its final container and labelling.
Also referred to as “finished dosage form”, “drug product” or “final product” in
other documents.
Fermentation: maintenance or propagation of microbial cells in vitro
(fermenter). Fermentation is operated and progressed under axenic conditions
to ensure a pure culture absent of contaminating microorganisms.
Harvesting: the procedure by which the cells, inclusion bodies or crude
supernatants containing the unpurified active ingredient are recovered.
Hybridoma: an immortalized cell line that secretes desired (monoclonal)
antibodies and which is typically derived by fusing B-lymphocytes with
tumour cells.
Inactivation: removal or reduction to an acceptable limit of infectivity of
microorganisms or detoxification of toxins by chemical or physical modification.
Master cell bank (MCB): a quantity of well-characterized cells of animal
or other origin, derived from a cell seed at a specific population doubling level
(PDL) or passage level, dispensed into multiple containers and stored under
defined conditions. The MCB is prepared from a single homogeneously mixed
pool of cells. In some cases, such as genetically engineered cells, the MCB may
be prepared from a selected cell clone established under defined conditions.
However, the MCB may not be clonal. The MCB is used to derive a working cell
bank (WCB).
Monoclonal antibodies (mAbs): homogenous antibody population
obtained from a single clone of lymphocytes or by recombinant technology
and which bind to a single epitope.
Pharmaceutical quality system (PQS): management system used by a
pharmaceutical company to direct and control its activities with regard to quality.
Polyclonal antibodies: antibodies derived from a range of lymphocyte
clones and produced in humans and animals in response to the epitopes on
most “non-self ” molecules.
Primary containment: a system of containment that prevents the
escape of a biological agent into the immediate working environment. It
involves the use of closed containers or biological safety cabinets along with
secure operating procedures.
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Quality risk management (QRM): a systematic process for the

assessment, control, communication and review of risks to the quality of
pharmaceutical products across the product life-cycle.
Reference sample: a sample of a batch of starting material, packaging
material, intermediate or finished product which is stored for the purpose of
being analysed should the need arise during the shelf-life of the batch concerned.
Retention sample: a sample of a fully packaged unit from a batch
of finished product. It is stored for identification purposes (for example, of
presentation, packaging, labelling, patient information leaflet, batch number and
expiry date) should the need arise during the shelf-life of the batch concerned.
Seed lot: a quantity of live cells or viruses which has been derived from
a single culture (though not necessarily clonal), has a uniform composition and
is aliquoted into appropriate storage containers from which all future products
will be derived, either directly or via a seed lot system. The following derived
terms are used in this document – master seed lot (MSL): a lot or bank of
cells or viruses from which all future vaccine production will be derived. The
MSL represents a well-characterized collection of cells or viruses or bacteria of
uniform composition. Also referred to as “master virus seed” (MVS) for virus
seeds, “master seed bank”, “master seed antigen” or “master transgenic bank” in
other documents; and working seed lot (WSL): a cell or viral or bacterial seed
lot derived by propagation from the MSL under defined conditions and used to
initiate production of vaccines on a lot-by-lot basis. Also referred to as “working
virus seed” (WVS) for virus seeds, “working seed bank”, “working seed antigen”
or “working transgenic bank” in other documents.
Specific pathogen free (SPF): denoting animals or animal materials
(such as chickens, embryos, eggs or cell cultures) derived from groups of animals
(for example, flocks or herds) free from specified pathogens, and used for the
production or quality control of biological products. Such flocks or herds are
defined as animals sharing a common environment and having their own
caretakers who have no contact with non-SPF groups.
Starting materials: any substances of a defined quality used in the
production of a pharmaceutical product, but excluding packaging materials. In
the context of biological products manufacturing, examples of starting materials
may include cryo-protectants, feeder cells, reagents, growth media, buffers,
serum, enzymes, cytokines, growth factors and amino acids.
Transgenic: denoting an organism that contains a foreign gene in its
normal genetic component for the expression of biological pharmaceutical
materials.
Vaccine: a preparation containing antigens capable of inducing an
active immune response for the prevention, amelioration or treatment of
infectious diseases.
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Working cell bank (WCB): a quantity of well-characterized cells of

animal or other origin, derived from an MCB at a specific PDL or passage
level, dispensed into multiple containers and stored under defined conditions.
The WCB is prepared from a single homogeneously mixed pool of cells
(often, this is the MCB). One or more of the WCB containers is used for each
production culture.
4. Principles and general considerations

The manufacture of biological products should be undertaken in accordance with
the basic principles of GMP. The points covered by the current document should,
therefore, be considered as complementary to the general recommendations
set out in the current WHO good manufacturing practices for pharmaceutical
products: main principles (2) and associated specialized guidelines and
recommendations (3, 4, 10, 13, 14) as well as other WHO documents related
specifically to the production and control of biological products established by
the WHO Expert Committee on Biological Standardization.2
The manufacture, control and administration of biological active
substances and finished products require certain specific considerations and
precautions arising from the nature of these products and their processes.
Unlike conventional pharmaceutical products which are manufactured using
chemical and physical techniques capable of a high degree of consistency, the
manufacture of biological active substances and finished products involves
biological processes and materials, such as cultivation of cells or extraction from
living organisms. As these biological processes may display inherent variability,
the range and nature of by-products may also be variable. As a result, quality
risk management (QRM) principles are particularly important for this class of
materials and should be used to develop the control strategy across all stages
of manufacture so as to minimize variability and reduce the opportunity for
contamination and cross-contamination.

Materials and processing conditions used in cultivation processes are
designed to provide conditions for the growth of target cells and microorganisms
– therefore, extraneous microbial contaminants have the opportunity to grow.
Furthermore, many biological products have limited ability to withstand certain
purification techniques, particularly those designed to inactivate or remove
adventitious viral contaminants. The design of the processes, equipment, facilities,
utilities, the conditions of preparation and addition of buffers and reagents,
sampling, and training of the operators are key considerations in minimizing
See: http://www.who.int/biologicals/en/ (accessed 4 November 2015).

2
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such contamination events. Specifications outlined in WHO guidelines and

recommendations will determine whether and to what stage of production
substances and materials can have a defined level of bioburden or need to be
sterile. Similarly, manufacturing should be consistent with other specifications
set out in the product summary files, marketing authorization or clinical trial
approvals (for example, number of generations (expressed as doublings or
passages) between the seed lot or cell bank and the finished product).
Many biological materials (such as live-attenuated bacteria and
viruses) cannot be terminally sterilized by heat, gas or radiation. In addition,
some products, such as certain live and adjuvanted vaccines (for example,
bacille Calmette–Guérin (BCG) or cholera), may not be sterilized by filtration
processes. For these axenic products, processing should be conducted aseptically
to minimize the introduction of contaminants from the point where a potential
contamination cannot be removed from the manufacturing process. Relevant
WHO documents should be consulted on the validation of specific manufacturing
steps such as virus removal or inactivation (21). Robust environmental controls
and monitoring and, wherever feasible, in situ cleaning and sterilization systems,
together with the use of closed systems can significantly reduce the risk of
accidental contamination and cross-contamination.
Control usually involves biological analytical techniques, which typically
have a greater variability than physicochemical determinations. The combination
of variability in starting materials and the potential for subtle changes during
the manufacturing process of biological products also requires an emphasis
on production consistency. This is of particular concern because of the need
to link consistency to original clinical trials documenting the product’s safety
and efficacy. A robust manufacturing process is therefore crucial and in-process
controls take on a particular importance in the manufacture of biological active
substances and medicinal products.
Because of the risks inherent in producing and manipulating pathogenic
and transmissible microorganisms during the production and testing of
biological materials, GMP should prioritize the safety of the recipient to whom
the biological product is administered, the safety of personnel during operation
and the protection of the environment.
Biosafety considerations should follow national guidelines and (if
applicable and available) international guidelines. In most countries, the
regulation of GMP and biosafety are governed by different institutions. In the
context of manufacturing pathogenic biological products of Biosafety Risk Group
3 and 4, close collaboration between such institutions is especially required to
assure that both product contamination and environmental contamination levels
are controlled within acceptable limits. Specific recommendations regarding
containment are outlined below in section 10.
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5. Pharmaceutical quality system and

quality risk management
Biological products, like any pharmaceutical product, should be manufactured
in accordance with the requirements of a pharmaceutical quality system (PQS)
based on a life-cycle approach as defined in WHO good manufacturing practices
for pharmaceutical products: main principles (2). This approach facilitates
innovation and continual improvement, and also strengthens the link between
pharmaceutical development and manufacturing activities.
QRM principles should be used to develop the control strategy across
all manufacturing and control stages – including materials sourcing and
storage, personnel and materials flow, manufacture and packaging, quality
control, quality assurance, storage and distribution activities, as described in
relevant WHO guidelines (14) and other documents (22). Due to the inherent
variability of biological processes and starting materials, ongoing trend analysis
and periodic review are particularly important elements of PQS. Thus, special
attention should be paid to starting material controls, change control, trend
analysis and deviation management in order to ensure production consistency.
Monitoring systems should be designed so as to provide early detection of any
unwanted or unanticipated factors that may affect the quality, safety and efficacy
of the product. The effectiveness of the control strategy in monitoring, reducing
and managing such risks should be regularly reviewed and the systems updated
as required taking into account scientific and technical progress.
6. Personnel
6.1 Personnel responsible for production and control should have an adequate
background in relevant scientific disciplines such as microbiology, biology,
biometry, chemistry, medicine, pharmacy, pharmacology, virology,

immunology, biotechnology and veterinary medicine, together with
sufficient practical experience to enable them to perform their duties.
6.2 The health status of personnel should be taken into consideration as
part of ensuring product safety. Where necessary, personnel engaged
in production, maintenance, testing and animal care (and inspections)
should be vaccinated with appropriate specific vaccines and have regular
health checks. Any changes in the health status of personnel which could
adversely affect the quality of the product should preclude their working
in the production area, and appropriate records kept. The scope and
frequency of health monitoring should be commensurate with the risk to
the product and personnel.
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6.3 Training in cleaning and disinfection procedures, hygiene and microbiology

should emphasize the risk of microbial and adventitious contamination and
the nature of the target microorganisms and growth media routinely used.
6.4 Where required to minimize the opportunity for cross-contamination,
restrictions on the movement of all personnel (including quality control,
maintenance and cleaning staff) should be defined on the basis of QRM
principles. In general, all personnel including those not routinely involved
in the production operation (such as management, engineering staff and
validation staff or auditors) should not pass from areas with exposure to
live microorganisms, genetically modified microorganisms, animal tissue,
toxins, venoms or animals to areas where other products (inactivated or
sterile) or different organisms are handled. If such passage is unavoidable
during a working day, then contamination control measures (for example,
clearly defined decontamination measures such as a complete change of
appropriate clothing and shoes, and showering if applicable) should be
followed by all personnel visiting any such production area unless otherwise
justified on the basis of QRM.
6.5 Because the risks are difficult to manage, personnel working in an animal
facility should be restricted from entering production areas where potential
risks of cross-contamination exist.
6.6 Staff assigned to the production of BCG products should not work with
other infectious agents. In particular, they should not work with virulent
strains of Mycobacterium tuberculosis, nor should they be exposed to
a known risk of tuberculosis infection (23). Additionally, they should
be carefully monitored, with regular health checks that screen for
tuberculosis infection.
6.7 If personnel working in BCG manufacturing and in animal quarters need
to be reassigned to other manufacturing units they should not be allowed
into such units until they pass their health check.
7. Starting materials
7.1 The source, origin and suitability of active substances, starting materials
(for example, cryo-protectants and feeder cells), buffers and media (for
example, reagents, growth media, serum, enzymes, cytokines, growth
factors and amino acids) and other components of the finished product
should be clearly defined and controlled according to the principles set out
in WHO guidance on GMP for pharmaceutical products (2).
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7.2 Manufacturers should retain information describing the source and quality
of the biological materials used for at least 1 year after the expiry date
of the finished products and according to local regulations concerning
biological products. It has been found that documents retained for longer
periods may provide useful information related to adverse events following
immunization (AEFIs) and other investigations.
7.3 All starting material suppliers (that is, manufacturers) should be initially
qualified on the basis of documented criteria and a risk-based approach.
Regular assessments of their status should also be carried out. Particular
attention should be given to the identification and monitoring of any
variability that may affect biological processes. When starting materials
are sourced from brokers who could increase the risk of contamination
by performing repackaging operations under GMP (2, 4) they should be
carefully qualified; an audit may form part of such qualification, as needed.
7.4 An identity test, or equivalent, should be performed on each batch of
received starting materials prior to release. The number of containers
sampled should be justified on the basis of QRM principles and in agreement
with all applicable guidelines (2). The identification of all starting materials
should be in compliance with the requirements appropriate to the stage
of manufacture. The level of testing should be commensurate with the
qualification level of the supplier and the nature of the materials used. In the
case of starting material used to manufacture active substances the number
of samples taken should be based on statistically recognized criteria and
QRM principles (2). However, for starting materials and intermediates used
in the formulation of finished product each container should be sampled
for identity testing in accordance with the main principles of GMP for
pharmaceutical products unless reduced testing has been validated.
7.5 The sampling process should not adversely affect the quality of the
product. Incoming starting materials should be sampled under appropriate

conditions in order to prevent contamination and cross-contamination.
7.6 Where justified (such as the special case of sterile starting materials) it
may be acceptable to reduce the risk of contamination by not performing
sampling at the time of receipt but to perform the testing later on samples
taken at the time of use. In such cases, release of the finished product is
conditional upon satisfactory results of these tests.
7.7 Where the necessary tests for approving starting materials take a
significantly long time, it may be permissible by exception to process
starting materials before the test results are available. The use of these
materials should be clearly justified in a documented manner, and the risks
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should be understood and assessed under the principles of QRM. In such

cases, release of the finished product is conditional upon satisfactory results
from the tests. It must be ensured that this is not standard practice and
occurs only with justification of the risk taken.
7.8 The risk of contamination of starting materials during their passage
along the supply chain should be assessed, with particular emphasis on
adventitious agents such as those causing TSEs (24). Other materials
that come into direct contact with manufacturing equipment and/or
with potential product contact surfaces (such as filter media, growth
media during aseptic process simulations and lubricants) should also be
controlled. A quality risk assessment should be performed to evaluate the
potential for adventitious agents in biological starting materials.
7.9 Where required, the sterilization of starting materials should be carried
out by heat whenever possible. Where necessary, other appropriate
validated methods may also be used for this purpose (such as irradiation
and filtration).
7.10 The controls required for ensuring the quality of sterile starting materials
and of the aseptic manufacturing process should be based on the principles
and guidance contained in the current WHO good manufacturing practices
for sterile pharmaceutical products (3).
7.11 The transport of critical materials, reference materials, active substances,
human tissues and cells to the manufacturing site should be controlled
as part of a written quality agreement between the responsible parties if
they are different commercial entities. Manufacturing sites should have
documentary evidence of adherence to the specified storage and transport
conditions, including cold chain requirements, if required. The required
traceability – starting at tissue establishments through to the recipient(s),
and including the traceability of materials in contact with the cells or tissues
– should be ensured, maintained and documented.
8. Seed lots and cell banks

8.1 The recommendations set out in WHO good manufacturing practices for
active pharmaceutical ingredients (4) should be followed – specifically
section 18 on specific guidance for active pharmaceutical ingredients
manufactured by cell culture/fermentation.
8.2 Where human or animal cells are used as feeder cells in the manufacturing
process, appropriate controls over their sourcing, testing, transport and
storage should be in place.
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8.3 In order to prevent the unwanted drift of genetic properties which

might result from repeated subcultures or multiple generations, the
production of biological products obtained by microbial culture, cell
culture or propagation in embryos and animals should be based on a
system of master and working seed lots and/or cell banks; which is the
beginning of the manufacturing process of certain biological products (for
example, vaccines).
8.4 The number of generations (expressed as passages or doublings) between
the seed lot or cell bank and the finished product, defined as maximum,
should be consistent with the marketing authorization dossier and should
not be exceeded.
8.5 Cell-based medicinal products are often generated from a cell stock
obtained from a limited number of passages. In contrast with the two-
tier system of MCBs and WCBs, the number of production runs from a
cell stock is limited by the number of aliquots obtained after expansion
and does not cover the entire life-cycle of the product. Cell stock changes
should be covered by a validation protocol and communicated to the NRA,
as applicable.
8.6 Establishment and handling of the MCBs and WCBs should be performed
under conditions which are demonstrably appropriate. These should
include an appropriately controlled environment to protect the seed lot and
the cell bank, and the personnel handling them. To establish the minimum
requirements for clean room grade and environmental monitoring in the
case of vaccines see the WHO Environmental monitoring of clean rooms
in vaccine manufacturing facilities: points to consider for manufacturers of
human vaccines (25). During the establishment of the seed lot and cell
bank, no other living or infectious material (such as viruses, cell lines or
microbial strains) should be handled simultaneously in the same area or by

the same persons, as set out in current WHO Recommendations (26).
8.7 Quarantine and release procedures for master and working cell banks/seed
lots should be followed, including adequate characterization and testing for
contaminants. Initially, full characterization testing of the MCB should be
done, including genetic identification. A new MCB (from a previous initial
clone, MCB or WCB) should be subjected to the same established testing as
the original MCB, unless otherwise justified. Thereafter, the viability, purity
and other stability-indicating attributes of seed lots and cell banks should
be checked regularly according to justified criteria. Evidence of the stability
and recovery of the seed lots and banks should be documented and records
should be kept in a manner that permits trend evaluation.
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8.8 Each storage container should be adequately sealed, clearly labelled and
kept at an appropriate temperature. A stock inventory should be kept. The
storage temperature should be recorded continuously and, where applicable,
the liquid nitrogen level should be monitored. Any deviation from the set
limits, and any corrective and preventive action taken, should be recorded.
Temperature deviations should be detected as early as possible (for example,
through the use of an alarm system for temperature and nitrogen levels).
8.9 Seed lots and cell banks should be stored and used in such a way as to
minimize the risks of contamination or alteration (for example, stored
in qualified ultra-low temperature freezers or liquid nitrogen storage
containers). Control measures for the storage of different seeds and/or
cells in the same area or equipment should prevent mix-up and should
take into account the infectious nature of the materials in order to prevent
cross-contamination.
8.10 MSLs, MCBs, and preferably also WSLs and WCBs, should be stored in
two or more controlled separate sites in order to minimize the risk of
total loss due to natural disaster, equipment malfunction or human error.
A contingency plan should be in place.
8.11 The storage and handling conditions for the cell or seed banks should
be defined. Access should be controlled and restricted to authorized
personnel, and appropriate access records maintained. Records of location,
identity and inventory of individual containers should also be kept. Once
containers are removed from the seed lot/cell bank management system
they should not be returned to stock.
9. Premises and equipment

9.1 In general, preparations containing live microorganisms or live viruses
should not be manufactured and containers should not be filled in areas
used for the processing of other pharmaceutical products. However, if the
manufacturer can demonstrate and validate effective containment and
decontamination of the live microorganisms and viruses then the use of
multi-product facilities may be justifiable. In such cases, measures such as
campaign production, closed systems and/or disposable systems should be
considered and should be based on QRM principles (see sections 10 and
13 below on containment and campaign production respectively).
9.2 Documented QRM should be carried out for every additional product
in a biological manufacturing multi-product facility, which may include
a potency and toxicological evaluation based on cross-contamination
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risks. Other factors to be taken into account include facility/equipment

design and use, personnel and material flows, microbiological controls,
physicochemical characteristics of the active substance, process
characteristics, cleaning processes and analytical capabilities relative to
the relevant limits established from product evaluation. The outcome
of the QRM process should be the basis for determining the necessity
for premises and equipment to be dedicated to a particular product or
product family, and the extent to which this should be the case. This
may include dedicating specific product-contact parts. The NRA should
approve the use of a manufacturing facility for the production of multiple
products on case-to-case basis.
9.3 Killed vaccines, antisera and other biological products – including those
made by rDNA techniques, toxoids and bacterial extracts – may, following
inactivation, be manufactured on the same premises provided that
adequate decontamination and cleaning measures are implemented on
the basis of QRM.
9.4 Cleaning and sanitization should take into account the fact that processes
often include the handling of growth media and other growth-promoting
agents. Validation studies should be carried out to ensure the effectiveness
of cleaning, sanitization and disinfection, including elimination of residues
of used agents. Environmental and personnel safety precautions should be
taken during the cleaning and sanitization processes. The use of cleaning
and sanitizing agents should not pose any major risk to the performance
of equipment.
The use of closed systems to improve asepsis and containment
should be considered where practicable. Where open systems are utilized
during processing (for example, during addition of growth supplements,
media, buffers and gases, and during sampling and aseptic manipulations
during the handling of live cells such as in cell-therapy products) control

measures should be put in place to prevent contamination, mix-up and
cross-contamination. Logical and unidirectional flows of personnel,
materials and processes, and the use of clean-in-place and sterilize-in-place
systems, should be considered wherever possible. Where sterile single-use
systems such as bags and connectors are utilized, they should be qualified
with respect to suitability, extractables, leachables and integrity.
9.5 Because of the variability of biological products, and of the corresponding
manufacturing processes, approved starting materials that have to be
measured or weighed for the production process (such as growth media,
solutions and buffers) may be kept in small stocks in the production area
for a specified period of time according to defined criteria – such as for
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the duration of manufacture of the batch or of the campaign. Appropriate

storage conditions and controls should be maintained during such
temporary storage. These materials should not be returned to the general
stock. Materials used to formulate buffers, growth media and so on
should be weighed and made into a solution in a contained area using
local protection (such as a classified weighing booth) and outside the
aseptic processing areas in order to minimize particulate contamination
of the latter.
9.6 In manufacturing facilities, the mix-up of entry and exit of personnel
should be avoided through the use of separate changing rooms or through
procedural controls where Biosafety Risk Group 3 or 4 organisms are
handled (20).
10. Containment
10.1 Airborne dissemination of live microorganisms and viruses used for the
production process, including those from personnel, should be avoided.
10.2 Adequate precautions should be taken to avoid contamination of the
drainage system with dangerous effluents. Drainage systems should be
designed in such a way that effluents can be effectively neutralized or
decontaminated to minimize the risk of cross-contamination. Specific and
validated decontamination systems should be considered for effluents
when infectious and/or potentially infectious materials are used for
production. Local regulations should be complied with in order to
minimize the risk of contamination of the external environment according
to the risk associated with the biohazardous nature of waste materials.
10.3 Dedicated production areas should be used for the handling of live cells
capable of persistence in the manufacturing environment, for pathogenic
organisms of Biosafety Risk Group 3 or 4 and/or for spore-forming
organisms until the inactivation process is accomplished and verified. For
Bacillus anthracis, Clostridium tetani and Clostridium botulinum strictly
dedicated facilities should be utilized for each individual product.
Up‑to-date information on these and other high-risk or “special” agents
should be sought from major information resources (27). Where campaign
manufacture of spore-forming organisms occurs in a facility or suite of
facilities only one product should be processed at any one time.
Use of any pathogenic organism above Biosafety Risk Group 3
may be permitted by the NRA according to the biohazard classification
of the organism, the risk assessment of the biological product and its
emergency demand.
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10.4 Production of BCG-related product should take place in a dedicated

area and by means of dedicated equipment and utilities (such as heating,
ventilation and air conditioning (HVAC) systems) in order to minimize
the hazard of cross-contamination.
10.5 Specific containment requirements apply to poliomyelitis vaccine in
accordance with the WHO global action plan to minimize poliovirus
facility-associated risk (28) and with WHO Guidelines for the safe
production and quality control of inactivated poliomyelitis vaccine
manufactured from wild polioviruses (29). The measures and procedures
necessary for containment (that is, for protecting the environment
and ensuring the safety of the operator) should not conflict with those for
ensuring product quality.
10.6 Air-handling systems should be designed, constructed and maintained to
minimize the risk of cross-contamination between different manufacturing
areas as required. The need for dedicated air-handling units or single-
pass systems should be based on QRM principles, taking into account the
biohazard classification and containment requirements of the relevant
organism, and process and equipment risks. In the case of Biosafety Risk
Group 3 organisms, air should not be recirculated to any other area in the
facility and should be exhausted through high-efficiency particulate air
(HEPA) filters that are regularly checked for performance. A dedicated
non-recirculating ventilation system and HEPA-filtering of exhaust air are
required when handling Biosafety Risk Group 4 organisms (27).
10.7 Primary containment equipment should be designed and initially qualified
for integrity in order to ensure that the escape of biological agents and/
or material into the immediate working area and outside environment is
prevented. Thereafter, in line with relevant guidelines and QRM principles,
periodical tests should be performed to ensure that the equipment is in

proper working condition.
10.8 Activities associated with the handling of live biological agents (such
as centrifugation and blending of products which can lead to aerosol
formation) should be contained in such a way as to prevent contamination
of other products or the egress of live agents into the working and/or
outside environment. The viability of such organisms and their biohazard
classification should be taken into consideration as part of the management
of such risks.
Accidental spillages, especially of live organisms, must be dealt
with quickly and safely. Validated decontamination measures should be
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available for each organism or groups of related organisms. Where different

strains of a single bacteria species or very similar viruses are involved,
the decontamination process may be validated with one representative
strain, unless the strains vary significantly in their resistance to the
decontaminating agent(s) used.
10.9 Areas where Biosafety Risk Group 3 or 4 organisms are handled should
always have a negative air pressure relative to the environment. This will
ensure the containment of the organism in unlikely events such as failure
of the door interlock. Air-lock doors should be interlocked to prevent
them being opened simultaneously. Differential pressure alarms should be
present wherever required, and should be validated and monitored.
10.10 Air-vent filters should be hydrophobic and subject to integrity testing at

intervals determined by a QRM approach.
10.11 Where the filtration of exhaust air is necessary, the safe changing of filters
should be ensured or bag-in-bag-out housings should be employed. Once
removed, filters should be decontaminated and properly destroyed. In
addition to HEPA filtration other inactivation technologies such as heat
inactivation and steam scavenging may be considered for exhaust air to
ensure effective inactivation of pathogenic organisms of Biosafety Risk
Group 3 and/or 4.
11. Clean rooms

11.1 The WHO good manufacturing practices for sterile pharmaceutical
products (3) defines and establishes the required class/grade of clean
areas for the manufacture of sterile products according to the operations
performed, including final aseptic fill. Additionally, in order to address the
specific manufacturing processes involved in the production of biological
products, and particularly vaccines, the WHO Environmental monitoring
of clean rooms in vaccine manufacturing facilities: points to consider for
manufacturers of human vaccines (25) guidance document may be used
to develop the environmental classification requirements for biological
manufacturing processes.
As part of the control strategy, the degree of environmental control
of particulate and microbial contamination of the production premises
should be adapted to the intermediate or finished product, and also to the
production step, taking into account the potential level of contamination of
the starting materials and the risks to the finished product.
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11.2 The environmental monitoring programme should be supplemented with

methods to detect the presence of the specific microorganisms used for
production (for example, recombinant yeast and toxin- or polysaccharide-
producing bacteria). The environmental monitoring programme may also
include detection of the produced organisms and adventitious agents of
production organisms, especially when campaign manufacture is applied
on the basis of QRM principles.
12. Production
12.1 Since cultivation conditions, media and reagents are designed to promote
the growth of cells or microbial organisms, typically in an axenic state,
particular attention should be paid to the control strategy for ensuring that
effective steps are in place for preventing or minimizing the occurrence of
unwanted bioburden, endotoxins, viruses of animal and human origin, and
associated metabolites.
12.2 The QRM process should be the basis for implementing the technical and
organizational measures required to control the risks of contamination
and cross-contamination. These could include, though are not limited to:
■■ carrying out processing and filling in segregated areas;
■■ containing material transfer by means of an airlock and appropriate
type of pass box with validated transfer procedures, clothing change
and effective washing and decontamination of equipment;
■■ recirculation of only treated (HEPA-filtered) air;
■■ acquiring knowledge of the key characteristics (for example,
pathogenicity, detectability, persistence and susceptibility to
inactivation) of all cells, organisms and any adventitious agents within

the same facility;
■■ when considering the acceptability of concurrent work in cases
where production is characterized by multiple small batches from
different starting materials (for example, cell-based products) taking
into account factors such as the health status of donors and the
risk of total loss of a product from or for specific patients during
development of the cross-contamination control strategy;
■■ preventing the risk of live organisms and spores entering non-related
areas or equipment by addressing all potential routes of cross-
contamination (for example, through the HVAC system) through
the use of single-use components and closed systems;
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■■ conducting environmental monitoring specific to the microorganism

being manufactured in adjacent areas while paying attention to
cross-contamination risks arising from the use of certain monitoring
equipment (such as that used for airborne particle monitoring) in
areas handling live and/or spore-forming organisms;
■■ using campaign-based production (see section 13 below).
12.3 When applicable, the inoculum preparation area should be designed so as
to effectively control the risk of contamination, and should be equipped
with a biosafety hood for primary containment.
12.4 If possible, growth media should be sterilized in situ by heat or in-line
microbial-retentive filters. Additionally, in-line microbial-retentive filters
should be used for the routine addition of gases, media, acids, alkalis and
so on to fermenters or bioreactors.
12.5 Data from continuous monitoring of certain production processes (such
as fermentation) should form part of the batch record. Where continuous
culture is used, special consideration should be given to parameters such
as temperature, pH, pO2 , CO2 and the rate of feed or carbon source with
respect to growth of cells.
12.6 In cases where a viral inactivation or removal process is performed,
measures should be taken (for example, in relation to facility layout,
unidirectional flow and equipment) to avoid the risk of recontamination
of treated products by non-treated products.
12.7 A wide variety of equipment and components (for example, resins,
matrices and cassettes) are used for purification purposes. QRM principles
should be applied to devise the control strategy regarding such equipment
and associated components when used in campaign manufacture and
in multi-product facilities. The reuse of components at different stages
of processing of one product is discouraged but, if performed, should
be validated. Acceptance criteria, operating conditions, regeneration
methods, lifespan and sanitization or sterilization methods, cleaning
process, and hold time between the use of reused components should be
defined and validated. The reuse of components for different products is
not acceptable.
12.8 Where adverse donor (human or animal) health information becomes
available after procurement and/or processing, and this information relates
to product quality, then appropriate measures should be taken – including
product recall, if applicable.
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12.9 Antibiotics may be used during the early stages of production to help
prevent inadvertent microbial contamination or to reduce the bioburden
of living tissues and cells. In this case, the use of antibiotics should be well
justified, and they should be cleared from the manufacturing process at
the stage specified in the marketing authorization. Acceptable residual
levels should be defined and validated. Penicillin and other beta-lactam
antibiotics should not be used at any stage of the process.
12.10 A procedure should be in place to address equipment and/or accessories
failure (such as air vent filter failure) which should include a product impact
review. If such failures are discovered following batch release the NRA
should be notified and the need for a batch recall should be considered.
13. Campaign production

13.1 The decision to use a facility or filling line for campaign manufacture
should be justified in a documented manner and should be based on a
systematic risk approach for each product (or strain) taking into account
the containment requirements and the risk of cross-contamination to
the next product. Campaign changeover procedures, including sensitive
techniques used for the determination of residues, should be validated
and proper cleaning acceptance criteria should be defined on a toxicology
basis of product residues from the last campaign, as applicable. Equipment
assigned to continued production or to campaign production of successive
batches of the same intermediate product should be cleaned at appropriate
validated intervals to prevent build-up and carry-over of contaminants
(such as product degradants or objectionable levels of microorganisms).
13.2 For downstream operations of certain products (for example, pertussis
or diphtheria vaccines) campaign production may be acceptable if well
justified. For finishing operations (formulation and filling) the need

for dedicated facilities or the use of campaigns in the same facility will
depend on the specific characteristics of the biological product, on the
characteristics of the other products (including any non-biological
products), on the filling technologies used (such as single-use closed
systems) and on local NRA regulations. Labelling and packaging
operations can be carried out in a multi-product facility.
13.3 Campaign changeover involves intensive decontamination/sterilization
(if required) and cleaning of the equipment and manufacturing area.
Decontamination/sterilization (if required) and cleaning should include
all equipment and accessories used during production, as well as the
facility itself. The following recommendations should be considered:
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■■ waste should be removed from the manufacturing area or sent to the

bio-waste system in a safe manner;
■■ materials should be transferred by a validated procedure;
■■ the Quality Unit should confirm area clearance by inspection, and
review the campaign changeover data (including monitoring results)
prior to releasing the area for the next product.
13.4 When required, the corresponding diluent for the product can be filled in
the same facility in line with the defined campaign production strategy
for finished product.
13.5 When campaign-based manufacturing is considered, the facility layout
and the design of the premises and equipment should permit effective
cleaning and decontamination/sterilization (if required) based on QRM
principles and validated procedures following the production campaign.
In addition, consideration may need to be given at the design stage of
facility layout to the possible need for fumigation.
14. Labelling
14.1 The information provided on the inner label (also called the container
label) and on the outer label (on the packaging) should be readable and
legible, and the content approved by the NRA.
14.2 Minimal key information should be printed on the inner label, and
additional information should be provided on the outer label (for example,
carton) and/or product leaflet.
14.3 The suitability of labels for low and ultra-low storage temperatures should
be verified, if applicable. The label should remain properly attached to the
container under different storage conditions during the shelf-life of the
product. The label and its adhesive should have no adverse effect on the
quality of the product caused by leaching, migration and/or other means.
15. Validation
15.1 Biological processes, handling of live materials and using campaign-based
production, if applicable, are the major aspects of biological product
manufacturing which require process and cleaning validation. The
validation of such processes – given the typical variability of biological
products, the possible use of harmful and toxic materials and the need
for inactivation processes – plays an important role in demonstrating
production consistency and in proving that the critical process parameters
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and product attributes are controlled. Where available, WHO guidance

documents should be consulted on the validation of specific manufacturing
methods (for example, virus removal or inactivation (21)).
15.2 A QRM approach should be used to determine the scope and extent
of validation.
15.3 All critical biological processes (including inoculation, multiplication,
fermentation, cell disruption, inactivation, purification, virus removal,
removal of toxic and harmful additives, filtration, formulation and aseptic
filling) are subject, as applicable, to process validation. Manufacturing
control parameters to be validated may include specific addition sequences,
mixing speeds, time and temperature controls, limits of light exposure
and containment.
15.4 After initial process validation studies have been finalized and routine
production has begun, critical processes should be subject to monitoring
and trending with the objective of assuring consistency and detecting any
unexpected variability. The monitoring strategy should be defined, taking
into consideration factors such as the inherent variability, complexity of
quality attributes and heterogeneity of biological products. A system or
systems for detecting unplanned departures from the process as designed
should be in place to ensure that the process remains in a state of control.
Collection and evaluation of information and data on the performance of
the process will allow for detection of undesired process variability and
will determine whether action should be taken to prevent, anticipate and/or
correct problems so that the process remains under control.
15.5 Cleaning validation should be performed in order to confirm the
effectiveness of cleaning procedures designed to remove biological
substances, growth media, process reagents, cleaning agents, inactivation

agents and so on. Careful consideration should be given to cleaning
validation when campaign-based production is practised.
15.6 Critical processes for inactivation or elimination of potentially harmful
microorganisms of Biosafety Risk Group 2 or above, including genetically
modified ones, are subject to validation.
15.7 Process revalidation may be triggered by a process change as part of
the change-control system. In addition, because of the variability of
processes, products and methods, process revalidation may be conducted
at predetermined regular intervals according to risk considerations.
A detailed review of all changes, trends and deviations occurring within
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a defined time period – for example, 1 year, based on the regular product
quality review (PQR) – may indicate a need for process revalidation.
15.8 The integrity and specified hold times of containers used to store
intermediate products should be validated unless such intermediate
products are freshly prepared and used immediately.
16. Quality control

16.1 As part of quality control sampling and testing procedures for biological
materials and products, special consideration should be given to the
nature of the materials being sampled (for example, the need to avoid
contamination, ensure biocontainment and/or cold chain requirements)
in order to ensure that the testing carried out is representative.
16.2 Samples for post-release use typically fall into one of two categories –
reference samples or retention samples – for the purposes of analytical
testing and identification respectively. For finished products the reference
and retention samples will in many instances be presented identically
as fully packaged units. In such circumstances, reference and retention
samples may be regarded as interchangeable.
Reference samples of biological starting materials should be
retained under the recommended storage conditions for at least 1 year
beyond the expiry date of the corresponding finished product. Reference
samples of other starting materials (other than solvents, gases and
water) as well as intermediates for which critical parameters cannot be
tested in the final product should be retained for at least 2 years after
the release of the product if their stability allows for this storage period.
Certain starting materials such as components of growth media need not
necessarily be retained.
Retention samples of a finished product should be stored in their
final packaging at the recommended storage conditions for at least 1 year
after the expiry date.
16.3 For cell-based products, microbiological tests (for example, sterility tests
or purity checks) should be conducted on cultures of cells or cell banks
free of antibiotics and other inhibitory substances in order to provide
evidence of the absence of bacterial and fungal contamination, and to be
able to detect fastidious organisms where appropriate. Where antibiotics
are used, they should be removed by filtration at the time of testing.
16.4 The traceability, proper use and storage of reference standards should
be ensured, defined and recorded. The stability of reference standards
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should be monitored, and their performance trended. The WHO

Recommendations for the preparation, characterization and establishment
of international and other biological reference standards (30) should
be followed.
16.5 All stability studies – including real-time/real-condition stability, accelerated
stability and stress testing – should be carried out according to relevant
WHO and other guidelines (31) or other recognized documents. Trend
analysis of the test results from the stability monitoring programme should
assure the early detection of any process or assay drift, and this information
should be part of the PQR of biological products.
16.6 For products where ongoing stability monitoring would normally require
testing using animals, and no appropriate alternative or validated
techniques are available, the frequency of testing may take into account a
risk-based approach. The principle of bracketing and matrix designs may
be applied if scientifically justified in the stability protocol.
16.7 All analytical methods used in the quality control and in-process control
of biological products should be well characterized, validated and
documented to a satisfactory standard in order to yield reliable results.
The fundamental parameters of this validation include linearity, accuracy,
precision, selectivity/specificity, sensitivity and reproducibility (32–35).
16.8 For test methods described in relevant pharmacopoeial monographs,
qualification of the laboratory test equipment and personnel should be
performed. In addition, repeat precision and comparability precision should
be shown in the case of animal tests. Repeatability and reproducibility
should also be demonstrated by reviewing retrospective test data.
In addition to the common parameters typically used for validating
assays (such as accuracy and precision) additional measurements (for
example, of the performance of references, critical reagents and/or cell

lines) should be considered during the validation of bioassays based on
the biological nature of the assay and reagents used.
17. Documentation (batch processing records)

17.1 In general, the processing records of regular production batches should
provide a complete account of the manufacturing activities of each
batch of biological product showing that it has been produced, tested and
dispensed into containers in accordance with the approved procedures.
In the case of vaccines, a batch processing record and a summary
protocol should be prepared for each batch for the purpose of lot release
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by the NRA. The information included in the summary protocol should

follow the WHO Guidelines for independent lot release of vaccines by
regulatory authorities (36). The summary protocol and all associated
records should be of a type approved by the NRA.
17.2 Manufacturing batch records should be retained for at least 1 year after
the expiry date of the batch of the biological product and should be readily
retrievable for inspection by the NRA. It has been found that documents
retained for longer periods may provide useful information related to
AEFI and other investigations.
17.3 Starting materials may require additional documentation on source,
origin, supply chain, method of manufacture and controls applied in order
to ensure an appropriate level of control, including of microbiological
quality if applicable.
17.4 Some product types may require a specific definition of what materials
constitute a batch – particularly somatic cells in the context of ATMPs.
For autologous and donor-matched situations, the manufactured product
should be viewed as a batch.
18. Use of animals

18.1 A wide range of animals is used for the manufacture or quality control
of biological products. Special considerations are required when animal
facilities are present at a manufacturing site.
18.2 The presence of live animals in the production area should be avoided
unless otherwise justified. Embryonated eggs are allowed in the production
area, if applicable. If the extraction of tissues or organs from animals is
required then particular care should be taken to prevent contamination
of the production area (for example, appropriate disinfection procedures
should be undertaken).
18.3 Areas used for performing tests involving animals or microorganisms
should be well separated from premises used for the manufacturing
of products and should have completely separate ventilation systems
and separate staff. The separation of different animal species before and
during testing should be considered, as should the necessary animal
acclimatization process, as part of the test requirements.
18.4 In addition to monitoring compliance with TSE regulations (24) other
adventitious agents that are of concern (including those causing zoonotic
diseases and diseases in source animals) should also be monitored and
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recorded in line with specialist advice on establishing such programmes.

Instances of ill health occurring in the source/donor animals should be
investigated with respect to their suitability, and the suitability of in-contact
animals, for continued use (for example, in manufacture, as sources of
starting materials, and for quality control and safety testing). Decisions
should be documented.
18.5 A look-back procedure should be in place in relation to the decision-
making process used to evaluate the continued suitability of the biological
active substance or finished product in which animal-sourced starting
materials have been used or incorporated. This decision-making process
may include the retesting of reference samples from previous collections
from the same donor animal (where applicable) to establish the last
negative donation. The withdrawal period of therapeutic agents used to
treat source/donor animals should be documented and should be taken
into account when considering the removal of those animals from the
programme for defined periods.
18.6 Particular care should be taken to prevent and monitor infections in
source/donor animals. Measures taken should cover aspects such as
sourcing, facilities, husbandry, biosafety procedures, testing regimes,
control of bedding and feed materials, 100% fresh air supply, appropriate
design of the HVAC system, water supply and appropriate temperature
and humidity conditions for the species being handled. This is of special
relevance to SPF animals where pharmacopoeial monograph requirements
should be met. Housing and health monitoring should also be defined for
other categories of animals (for example, healthy flocks or herds).
18.7 For products manufactured from transgenic animals, traceability should
be maintained in the creation of such animals from the source animals.
Note should be taken of national requirements for animal quarters, care

and quarantine.
18.8 For different animal species and lines, key criteria should be defined,
monitored and recorded. These may include the age, sex, weight and health
status of the animals.
18.9 Animals, biological agents and tests carried out should be appropriately
identified to prevent any risk of mix-up and to control all identified hazards.
18.10 The facility layout should ensure a unidirectional and segregated flow of
healthy animals, inoculated animals and waste-decontamination areas.
Personnel and visitors should also follow a defined flow in order to avoid
cross-contamination.
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19. Authors and acknowledgements

The scientific basis for the revision of these WHO Guidelines was discussed at
a working group meeting held in Bangkok, Thailand, 10–13 September 2007
and attended by: Dr M.M.F. Ahmed, Center for Control of Biologicals and
Vaccines, Egypt; Dr H. Alitamsar, PT Bio Farma, Indonesia; Mr P. Angtrakool,
Ministry of Public Health, Thailand; Dr D. Buckley, Consultant, Monash,
Australia; Dr M. Dennehy, The Biovac Institute, South Africa; Ms X. Dong,
Beijing Tiantan Biological Products Co. Ltd, China; Dr H.J.M. van de Donk,
Consultant, Den Haag, Netherlands; Dr M. Gheisarzardeh, Ministry of Health
and Medical Education, the Islamic Republic of Iran; Dr H.T. Hong, National
Institute for Control of Vaccine and Biologicals, Viet Nam; Mrs W. Jariyapan,
WHO Regional Office for South-East Asia, India; Mr M. Javadekar, Serum
Institute of India Ltd, India; Dr D. Jiang, State Food and Drug Administration,
China; Mrs T. Jivapaisarnpong, Ministry of Public Health, Thailand; Dr A.
Khadem, Pasteur Institute of Iran, the Islamic Republic of Iran; Professor S.
Khomvilai, Thai Red Cross Society, Thailand; Dr K-H. Kim, Korean Food and
Drug Administration, Republic of Korea; Dr Kustantinah, National Agency of
Drug and Food Control, Indonesia; Professor C.K. Lee, Advisor to the Korean
Food and Drug Administration, Republic of Korea; Mrs J. Li, Sinovac Biotech
Co. Ltd, China; V.G. Maqueda, Biologist, Buenos Aires, Argentina; Dr K-I.
Min, Korean Food and Drug Administration, Republic of Korea; Mr I. Rees,
Medicines and Healthcare Products Regulatory Agency, the United Kingdom;
Dr C.H. Sia, Health Sciences Authority, Singapore; Dr M. Suhardono, PT Bio
Farma, Indonesia; Ms J. Teo, Centre for Drug Administration, Singapore; Ms P.S.
Thanaphollert, Ministry of Public Health, Thailand; Mr S. Thirapakpoomanunt,
Ministry of Public Health, Thailand; Ms A.R.T. Utami, National Agency of
Drug and Food Control, Indonesia; Dr D.T.H. Van, Institute for Vaccine and
Biologicals, Viet Nam; Mr B. Wibisono, National Agency of Drug and Food
Control, Indonesia; Mr J. Yang, Kunming Institute of Medical Biology, China;
Mr Y. Yu, Kunming Institute of Medical Biology, China; and Dr I. Knezevic
and Dr S. Lambert, World Health Organization, Switzerland – and a WHO
drafting group meeting held in Geneva, Switzerland, 30–31 October 2013 and
attended by: Mr R. Acs, Central Drugs Standard Control Organisation, India;
Mr M. Eisenhawer, WHO Regional Office for South-East Asia, India; Dr S.
Fakhrzadeh, Ministry of Health and Medical Education, the Islamic Republic
of Iran; V.G. Maqueda, Biologist, Buenos Aires, Argentina; Mrs K. Porkaew,
Ministry of Public Health, Thailand; Dr S.O. Rumiano, Consultant, Buenos
Aires, Argentina; Dr Y. Wang, National Institutes for Food and Drug Control,
China; Mr B. Wibisono, National Agency of Drug and Food Control, Indonesia;
and Dr A. Chawla, Dr A.R. Khadem, Dr I. Knezevic, Dr S. Kopp and Dr D. Lei,
World Health Organization, Switzerland.
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Based upon the principles defined in the above working group and
drafting group meetings the first draft of these Guidelines was prepared by Mr R.
Acs, Central Drugs Standard Control Organisation, India; Dr B. Yáñez Chamizo,
Centro para el Control Estatal de Medicamentos, Equipos y Dispositivos Médicos,
Cuba; Dr S. Fakhrzadeh, Ministry of Health and Medical Education, the Islamic
Republic of Iran; Mrs K. Porkaew, Ministry of Public Health, Thailand; Dr S.O.
Rumiano, Consultant, Buenos Aires, Argentina; Dr Y. Wang, National Institutes
for Food and Drug Control, China; Mr B. Wibisono, National Agency of Drug
and Food Control, Indonesia; Mr M. Eisenhawer, WHO Regional Office for
South-East Asia, India; Dr A. Chawla, Consultant, Greater Noida, India; Dr A.R.
Khadem, World Health Organization, Switzerland; V.G. Maqueda, Biologist,
Buenos Aires, Argentina; and Dr D. Lei, World Health Organization, Switzerland.
A second draft was then prepared by V.G. Maqueda, Biologist, Buenos
Aires, Argentina; Dr B. Yáñez Chamizo, Centro para el Control Estatal de
Medicamentos, Equipos y Dispositivos Médicos, Cuba; Dr S. Fakhrzadeh,
Ministry of Health and Medical Education, the Islamic Republic of Iran;
Dr S.O. Rumiano, Consultant, Buenos Aires, Argentina; Dr Y. Wang, National
Institutes for Food and Drug Control, China; Mr B. Wibisono, National Agency
of Drug and Food Control, Indonesia; Mr M. Eisenhawer, WHO Regional
Office for South-East Asia, India; Dr A. Chawla, Consultant, Greater Noida,
India; and Dr A.R. Khadem and Dr D. Lei, World Health Organization,
Switzerland following a consultation held in Tunis, Tunisia, 22–24 July 2014
and attended by: Dr H. Baiao, National Authority for Medicines and Health
Products, Portugal; Mrs R. Bose, Ministry of Health and Family Welfare, India;
Mr C. Cabral, Butantan Institute, Brazil; Dr R. Chaplinsky, GSK Vaccines,
Belgium; Dr A. Chawla, Consultant, Greater Noida, India; Mr M. Diagne,
Direction de la Pharmacie et des Laboratoires, Senegal; Mr M. Eisenhawer,
WHO Regional Office for South-East Asia, India; Dr S. Fakhrzadeh, Ministry
of Health and Medical Education, the Islamic Republic of Iran; Mrs R. Frikha,
Directorate of Pharmacy Inspection, Tunisia; Dr M. Gershman, Pfizer, the USA;
Ms A.R. Cornelio Geyer, Agência Nacional de Vigilância Sanitária, Brazil; Dr E.
Griffiths, Consultant, Kingston-upon-Thames, the United Kingdom; Dr N.
Harjee, Consultant, Ontario, Canada; Ms D.T.M. Hang, Ministry of Health,
Viet Nam; Dr H. Langar, WHO Regional Office for the Eastern Mediterranean,
Egypt; Dr P. Lauer, Sanofi Pasteur, France; Dr C.K. Lee, Korea Food and Drug
Administration, Republic of Korea; Dr H. Leng, Medicines Regulatory Authority,
South Africa; Dr M.G. Lopez Santos, Comisión Federal para la Protección contra
Riesgos Sanitarios, Mexico; V.G. Maqueda, Biologist, Buenos Aires, Argentina;
Dr A. Mihaylova, Bulgarian Drug Agency, Bulgaria; Dr J. Miteva, Bulgarian
Drug Agency, Bulgaria; Dr S. Pagliusi, DCVMN International, Switzerland;
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Dr V.A. Pessanha, Oswaldo Cruz Foundation, Brazil; Mrs K. Porkaew, Ministry

of Public Health, Thailand; Dr S. Ramanan, Amgen, the USA; Dr P. Rampignon,
GSK Vaccines, Belgium; Dr D. Rebeski, The Biovac Institute, South Africa;
Dr M. Refaat, Central Administration for Pharmaceutical Affairs, Egypt;
Dr S.O. Rumiano, Consultant, Buenos Aires, Argentina; Dr A.L. Salvati, Agenzia
Italiana del Farmaco, Italy; Dr J. Shin, WHO Regional Office for the Western
Pacific, Philippines; Dr W. Stevens, Health Canada, Canada; Dr I. Susanti, PT
Bio Farma, Indonesia; Ms C.S. Takata, Butantan Institute, Brazil; Dr S. Uddin,
Directorate General of Drug Administration, Bangladesh; Dr Y. Wang, National
Institutes for Food and Drug Control, China; Mr B. Wibisono, National Agency
of Drug and Food Control, Indonesia; and Dr A.K. Broojerdi, Dr D. Lei, Dr I.
Streipa-Nauman and Dr D.J. Wood, World Health Organization, Switzerland.
The document WHO/BS/2015.2253, incorporating comments received
from regulators and industry following public consultation on the WHO
Biologicals website, was prepared by V.G. Maqueda, Biologist, Buenos Aires,
Argentina; Dr B. Yáñez Chamizo, Centro para el Control Estatal de Medicamentos,
Equipos y Dispositivos Médicos, Cuba; Dr S. Fakhrzadeh, Ministry of Health and
Medical Education, the Islamic Republic of Iran; Dr S.O. Rumiano, Consultant,
Buenos Aires, Argentina; Dr Y. Wang, National Institutes for Food and Drug
Control, China; Dr A. Chawla, Consultant, Greater Noida, India; Dr M. Refaat,
Central Administration for Pharmaceutical Affairs, Egypt; Dr A. Khadem, Pasteur
Institute of Iran, the Islamic Republic of Iran; and Dr M. Chafai, Dr D. Lei, Dr D.
Mubangizi and Dr I.R. Thrussell, World Health Organization, Switzerland.
Further changes were subsequently made to document WHO/BS/
2015.2253 by the WHO Expert Committee on Biological Standardization.
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Annex 3
Regulatory assessment of approved rDNA-derived
biotherapeutics
Addendum to Annex 4 of WHO Technical Report Series, No. 987
1. Introduction 134
2. Regulatory expectations for rDNA-derived biotherapeutics,
including similar biotherapeutic products 134
3. Review of products on the market 135
4. Points to consider in a stepwise regulatory assessment 137
5. Regulatory actions 139
6. Authors and acknowledgements 140
7. References 144
131
Guidance documents published by WHO are intended to be

scientific and advisory in nature. Each of the following sections
constitutes guidance for national regulatory authorities (NRAs) and
for manufacturers of biological products.
132
Annex 3
Abbreviations
ALIFAR Asociación Latinoamericana de Industrias Farmacéuticas
EGA European Generic Medicines Association
ICDRA International Conference of Drug Regulatory Authorities
& Associations
IGPA International Generic Pharmaceutical Alliance
RBP reference biotherapeutic product
SBP similar biotherapeutic product
133
1. Introduction
This WHO guidance document considers the regulatory assessment needed to
address situations where, for various reasons, biotherapeutic protein products
prepared by recombinant deoxyribonucleic acid (DNA) technology (rDNA-
derived biotherapeutics) were licensed with data packages that do not follow
current international regulatory standards for these biologicals. This includes,
for example, biotherapeutic products licensed via a generic pathway or with
limited analytical, nonclinical and/or clinical data (1, 2). At its 2010 meeting
in Singapore (3) the International Conference of Drug Regulatory Authorities
(ICDRA) discussed such situations and requested WHO assistance in developing
approaches for evaluating these already-licensed products in accordance with
current WHO guidelines. In May 2014 the Sixty-seventh World Health Assembly
adopted two relevant resolutions: one on promoting access to biotherapeutic
products and ensuring their quality, safety and efficacy (4) and the other on
regulatory systems strengthening (5) in which WHO was requested to provide
guidance, especially on dealing with increasingly complex biological products.
Although primarily addressing rDNA-derived biotherapeutic protein
products, some aspects of this document may also be relevant to other
biotherapeutics.
2. Regulatory expectations for rDNA-

derived biotherapeutics, including
similar biotherapeutic products
The regulatory expectations for rDNA-derived biotherapeutics can be found in
the relevant WHO Guidelines adopted by the 2013 WHO Expert Committee
on Biological Standardization (6). Following extensive consultation at the global
level since 2004, WHO Guidelines on the evaluation of similar biotherapeutic

products (SBPs) were adopted by the Committee in 2009 (7). These latter
WHO Guidelines emphasize the need for a head-to-head demonstration of the
“similarity” of such products to reference biotherapeutic products (RBPs) of
assured quality, safety and efficacy that have been licensed on the basis of a full
licensing dossier. A head-to-head comparability exercise between a candidate
SBP and an RBP is essential to justify a reduced nonclinical and clinical package
for licensing (7). Studies should be designed to demonstrate comparability and
to detect any potential difference in quality, nonclinical and clinical attributes
between the SBP and RBP rather than simply to confirm the safety and efficacy
of the two products. It should be ensured that any differences that are detected
have no clinically meaningful impact on product performance.
134
Annex 3
If a head-to-head comparison of the SBP with the RBP as outlined in

the WHO Guidelines for SBPs (7) is not performed throughout the development
process then the final product should not be referred to as an SBP (8, 9). SBPs
are not “generic medicines” and the approval process used for small-molecule
generics is not applicable.
3. Review of products on the market

Problems have been identified in some countries where, for various reasons,
biotherapeutic products were licensed using data which no longer meet current
WHO regulatory expectations – such as biotherapeutic products licensed as
generics or as small-molecule drugs. In many cases pharmacovigilance systems
in such countries are weak or even nonexistent, with the result that little is known
about the safety and efficacy of individual products. In addition, the terminology
used for such products is confusing and their traceability poor (10, 11). In some
countries, the coexistence on the market of these products and SBPs, as well as
rDNA-derived biotherapeutics licensed with full data packages, is a matter of
concern. This was the situation for both erythropoietin (12) and heparin (13).
Some updating of national regulations has occurred to take account of the
recognized difficulties and changes made in international regulatory expectations
(14–17). Special considerations apply to the production and control of biological
medicines, including biotherapeutics, which do not apply to chemical drugs. This
is because of the biological nature of the starting materials, the manufacturing
processes and the test methods needed to characterize batches of the product
– as well as the highly complex molecular structure of products themselves.
Nonclinical and clinical evaluations are key components of the regulatory
assessment of all biotherapeutics. Products already approved under the pre-
existing regulations will need to be reassessed to ensure that they meet the
new requirements.
National regulatory authorities (NRAs) should undertake a stepwise
regulatory review of all biotherapeutic products already authorized for marketing,
as follows:
1. First, NRAs should identify products that have been licensed with
data which do not meet current WHO regulatory expectations.
2. Second, an assessment of identified products and gaps, based on
the product-specific considerations listed below in section 4, should
be carried out in order to decide upon the appropriate action
needed to remedy the situation, and to determine the timelines for
implementing this action. This will inevitably involve a risk–benefit
assessment of the situation.
135
3. Third, manufacturers should submit a plan of action for dealing

with the problem to the NRA within a defined – but short –
period of time. The plan of action should consist of an analysis of
available and missing data in accordance with WHO Guidelines
(6, 7), as well as a description of measures (which may include
interim assessments) and proposed timelines needed to address the
identified gaps.
4. Fourth, NRAs should evaluate the plan of action proposed by the
manufacturer and reach agreement with the manufacturer on
the next steps for generating missing data and for their (possibly
stepwise) submission to the NRA.
5. Fifth, NRAs should assess the submitted data (for example, quality/
manufacturing, nonclinical and clinical data as needed) using
a stepwise approach – possibly in several separate packages at
different times – and decide upon the appropriate regulatory action
to take based upon the assessment outcome.
The timeline for completing the overall review exercise will depend
upon the time needed to generate and provide the missing information, taking
into consideration the product-specific points outlined below in section 4.
For example, in 2009 one NRA clarified the “appropriate regulatory pathway”
for dealing with changes in the regulatory oversight of low molecular weight
heparins 1 to reflect the fact that in future they would be regulated in that country
as biologicals and not as small-molecule pharmaceuticals (16). In addition,
it was announced that any biosimilar heparin submissions should follow the
regulatory framework for biosimilars and not the generic pathway. A transition
period of 12 months was set to allow manufacturers to update their files to reflect
the data required for biologicals. Manufacturers were also required to report on
how much of their licensed product was sold in the country per year following
the official start date of the revised regulatory approach.
Similar transitional provisions have been made by other NRAs when
updating the regulations for biotherapeutics, including biosimilars (15, 17).
Low molecular weight heparin is not an rDNA-derived biotherapeutic product but is highlighted here
1
as an example of a reviewed product on the market. It is not considered to be a biological product in

some countries.
136
Annex 3
4. Points to consider in a stepwise regulatory assessment

A particular licensed product should be allowed to remain on the market during
the review process unless specific causes or events lead the NRA to make its
own judgment to suspend market availability of the product during the review
process. Consideration should be given to the following points when deciding
upon the appropriate regulatory actions:
■■ NRAs should consider: (a) the number of products on the market
which have been licensed without adequate quality, nonclinical and/
or clinical data; and (b) the availability of alternative therapeutics
on that market licensed locally with an adequate data package and/
or licensed by an experienced NRA that meet the standards of the
relevant WHO guidelines (see next bullet point).
■■ It is important to find out if the product in question is manufactured
and licensed in a country with a jurisdiction which has, and follows,
well-established regulatory frameworks, including as appropriate all
the principles set out in the relevant WHO Guidelines for rDNA-
derived biotherapeutics (6) and in WHO Guidelines for SBPs (7).
Account should also be taken of whether the jurisdiction concerned
has considerable experience in the evaluation of biotherapeutic
products (including SBPs) and post-marketing surveillance
activities. If a product is manufactured and/or licensed in a country
with considerable experience in these areas then this provides some
degree of confidence regarding product quality, safety and efficacy.
In addition, it is important to ascertain whether the actual product
authorized in the country with limited regulatory experience is
comparable – with respect to manufacturing process and controls,
recent good manufacturing practices inspection and labelling –
to the product licensed, supplied and used in the manufacturing
country with the more experienced jurisdiction. It is also important
to determine whether registration of the product in question has
been rejected, cancelled or suspended by other experienced NRAs.
■■ It is also important to know the extent to which the registration
dossier of the biotherapeutic product meets the recommendations
set out in the above WHO Guidelines (6, 7). Attention should be
paid to any key differences between national requirements and the
WHO Guidelines – such as the lack of a head-to-head comparability
exercise for an SBP. The NRA should provide manufacturers with a
critical dataset for the re-registration of such products. Changes in
regulatory requirements may be needed, as well as amendments to
137
the legal framework of the country concerned, to enable such new

requirements to be implemented.
■■ The necessity and extent of use of a biotherapeutic product along
with the availability of alternative products (if any) should be
ascertained. This would include, for example, assessing whether
the product was essential for treating certain patients and what the
clinical outcomes would be if the product was taken off the market.
This assessment should cover: (a) the disease that is being treated;
(b) whether the condition is life threatening; (c) the consequences of
treating or not treating, or of stopping treatment in patients already
using the product; (d) the risk of switching between therapeutic
alternatives; (e) the likelihood (and potential consequences, if any)
of supply problems on clinical outcomes should the product be
taken off the market; and (f) the type of patient population (for
example, paediatric, adult or older persons).
■■ The seriousness of a potential lack of efficacy should be considered,
as should possible safety issues (including higher efficacy) that
may result from the continued use of the product under review.
This should include an assessment of the severity of the potential
impact on a patient of an immunogenic effect arising from the
use of the product and an assessment of any adverse effects. Such
effects might include cross-reactivity with native proteins caused
by biotherapeutic products – such as pure red cell aplasia caused by
erythropoietin (1).
■■ Consideration should also be given to the ability of the
pharmacovigilance system in the country to detect and monitor any
potential adverse reactions and/or efficacy problems (such as reduced
clinical effectiveness) associated with the biotherapeutic product.
■■ Criteria for the evaluation of functional pharmacovigilance

systems have been developed by WHO (18). Given the poor
pharmacovigilance systems in many countries, as well as
terminology difficulties, it may not be possible to obtain sufficient
data to demonstrate that a particular product was the cause of an
adverse reaction or that patients may be at risk from the use of
products that are clinically untested or were tested in inadequately
designed studies. Traceability is a key element in monitoring the
safety and efficacy of biologicals as it enables pharmacovigilance
measures to be put in place.
■■ The expertise and capacity of regulators responsible for licensing
biotherapeutic products are critically important factors in the
138
Annex 3
appropriate evaluation of these products. Collaboration between

NRAs, including work-sharing agreements and joint reviews with
other NRAs, should also be explored (for example, see 2, 19, 20).
■■ Consideration should be given to ensuring transparency with
respect to informing health-care professionals, pharmacists and
patients of the review process and its timelines. This could be done
through website posting (16), via a symbol and some text in the
product information or any other means the NRA is allowed to use,
highlighting the need to align the licensing process with current
international expectations. This could also provide an opportunity
to request users to report any safety and/or efficacy issues.
5. Regulatory actions
On the basis of the outcomes of the regulatory assessment, the NRA should
decide upon the appropriate actions to be taken. The decisions and actions of
NRAs may differ depending upon the assessments made according the points
listed above in section 4, which will be jurisdiction specific. In a stepwise
approach, product supply would not be compromised and authorization might
be regularized after the defined time period during which the product would
have undergone further regulatory evaluation, and on condition that it was
shown to have an acceptable risk–benefit profile.
Capacity-building will be needed where resources and expertise are
considered inadequate. Where the number and level of experience of staff
available to undertake an overall review are limited, consideration could be
given to mentoring or to work-sharing arrangement amongst NRAs. In the case
of mentoring, support could be provided through WHO from an experienced
authority that uses well-established processes that accord with relevant WHO
guidelines. In addition, the sharing of information between NRAs regarding
the basis for regulatory decisions on biotherapeutic products (including SBPs)
and the availability of publically available evaluation reports are considered
important sources of support for regulatory authorities that are less experienced
in dealing with these highly complex products, and may accelerate product
assessment. Communicating the details of what information was reviewed
and how it was incorporated into decision-making is also important for
prescribers, patients and other stakeholders, and can help promote confidence
in biotherapeutic products. The summary basis of decision documents produced
by some regulatory agencies, such as Health Canada, the European Medicines
Agency and the United States Food and Drug Administration, are examples of
informative documents.
139
The stepwise regulatory assessment approach outlined in this document

is flexible and designed to increase the accessibility of biotherapeutic products
of assured quality, safety and efficacy, as called for in the relevant 2014 World
Health Assembly resolutions (4, 5).
6. Authors and acknowledgements

The first draft of this guidance document was prepared by Dr E. Griffiths,
Consultant, Kingston-upon-Thames, the United Kingdom; and Dr H-N. Kang
and Dr I. Knezevic, World Health Organization, Switzerland.
Comments were then received from the following reviewers: Mrs A.
Abas, Ministry of Health Malaysia, Malaysia; Dr W.S. Alhaqaish, Jordan Food
and Drug Administration, Jordan; Dr N. Annibali, Asociación Latinoamericana
de Industrias Farmacéuticas (ALIFAR), Argentina; Mrs J. Bernat (provided
consolidated International Federation of Pharmaceutical Manufacturers &
Associations (IFPMA) comments), Switzerland; Dr B. Boonyapiwat, Ministry of
Public Health, Thailand; Dr G. Castillero, Ministry of Health, Panama; Dr J.M.
Cousiño, Federacion Latinoamericana de la Industria Farmacéutica, Chile; Dr F.
Muñoz Espinoza, Instituto de Salud Pública de Chile, Chile; Dr M.T. Ibarz,
Instituto Nacional de Higiene “Rafael Rangel”, Venezuela; Dr A. Klein, Health
Canada, Canada; Dr C. Njue, Health Canada, Canada; Ms D Pérez, Instituto
Nacional de Higiene “Rafael Rangel”, Venezuela; Dr G.R. Soni, Ministry of Health
and Family Welfare, India; Mr H. Vásquez, Dirección General de Medicamentos,
Insumos y Drogas, Peru.
The second draft was prepared by Dr E. Griffiths, Consultant, Kingston-
upon-Thames, the United Kingdom; and Dr H-N. Kang, World Health
Organization, Switzerland taking into consideration comments received from the
above reviewers as well as the discussions at the Second WHO Implementation
Workshop on Quality Assessment of Similar Biotherapeutic Products held
in Xiamen, China, 28–30 May 2012 and attended by: Mrs A. Abas, Ministry
of Health Malaysia, Malaysia; Dr W.S. Alhaqaish, Jordan Food and Drug
Administration, Jordan; Ms J. Archer (International Generic Pharmaceutical
Alliance (IGPA) representative), Hospira, Australia; Dr B. Boonyapiwat, Ministry
of Public Health, Thailand; Dr L. Gomes Castanheira, Agência Nacional de
Vigilância Sanitária, Brazil; Dr R. Chakrabarti (United States Pharmacopeial
Convention representative), United States Pharmacopeial Convention–India,
India; Dr W. Chang, State Food and Drug Administration, China; Mr D. Cheng,
Beijing Four Rings Bio-Pharmaceutical Co., Ltd, China; Dr Y. Choi, Korea Food
and Drug Administration, Republic of Korea; Ms J. Dahlan, National Agency of
Drug and Food Control, Indonesia; Mr G. Eich (IFPMA representative), Amgen
Inc. Corporate Services/Global Regulatory Affairs & Safety, the USA; Dr K. Gao,
National Institutes for Food and Drug Control, China; Mr T. Go, Health Sciences
140
Annex 3
Authority, Singapore; Dr E. Griffiths, Consultant, Kingston-upon-Thames, the

United Kingdom; Dr L. Gu, Shenyang Sunshine Pharmaceutical Co., Ltd, China;
Dr Z. Guo (Chinese Pharmacopoeia representative), Chinese Pharmacopoeia
Commission, China; Dr N. Hassannia, Food and Drug Organization, the Islamic
Republic of Iran; Dr K. Ho, Agence Nationale de Sécurité du Médicament et
des Produits de Santé, France; Dr S. Hufton, National Institute for Biological
Standards and Control, the United Kingdom; Mrs W. Jariyapan, Ministry of
Public Health, Thailand; Mr R. Jian, Health Sciences Authority, Singapore; Dr J.
Joung, Korea Food and Drug Administration, Republic of Korea; Dr Y. Kishioka
(Japanese Pharmacopoeia representative), Pharmaceutical and Medical Devices
Agency, Japan; Mr J. Leong, Health Sciences Authority, Singapore; Dr J. Li,
Shanghai CP-Guojian Pharmaceutical Co., Ltd, China; Dr C. Liang, National
Institutes for Food and Drug Control, China; Dr J. Luo, State Food and Drug
Administration, China; Mrs V. Madrigal, Recepta Biopharma, Brazil; Dr C. Njue,
Health Canada, Canada; Mrs Y. Hechavarria Nunez, Centro para el Control
Estatal de la Calidad de los Medicamentos, Cuba; Dr P.H. Pan (Developing
Countries Vaccine Manufacturers Network (DCVMN) representative), Innovax
Biotech Co., Ltd, China; Dr R. Perez, Biotech Pharmaceutical Co., Ltd, China;
Dr S. Pluschkell (IFPMA representative), Pfizer Inc., the USA; Professor C.
Rao, National Institutes for Food and Drug Control, China; Dr M. Schiestl
(IGPA representative), Sandoz GmbH, Austria; Dr T. Schreitmueller (IFPMA
representative), F. Hoffmann-La Roche, Ltd, Switzerland; Dr S. Shani, Ministry
of Health and Social Welfare, India; Dr Q. Shen, National Institutes for Food
and Drug Control, China; Dr X. Shen, China Bio-Tech Group, China; Dr G.R.
Soni, Ministry of Health and Family Welfare, India; Dr L. Sun, Xiamen Amoytop
Biotech Co., Ltd, China; Dr R. Thorpe, National Institute for Biological Standards
and Control, the United Kingdom; Mrs C. Ulm (European Generic Medicines
Association (EGA) representative), Mylan GmbH, Switzerland; Dr A. Vallin,
Centre of Molecular Immunology, Cuba; Dr J. Wang, Health Canada, Canada;
Dr J. Wang, National Institutes for Food and Drug Control, China; Dr M. Xu,
National Institutes for Food and Drug Control, China; Dr S. Zhang, State Food
and Drug Administration, China; and Dr M.H. Friede, Dr H-N. Kang and Dr I.
Knezevic, World Health Organization, Switzerland.
The resulting draft document was posted on the WHO Biologicals
website for the first round of public consultation from 11 February to 12 March
2014. The draft document was also discussed at the First WHO Implementation
Workshop on Evaluation of Biotherapeutic Products held in Seoul, Republic of
Korea, 13–14 May 2014.
The third draft was prepared by Dr E. Griffiths, Consultant, Kingston-
upon-Thames, the United Kingdom; and Dr H-N. Kang, World Health
Organization, Switzerland, taking into account comments received from
following reviewers: Mrs A. Abas, Ministry of Health Malaysia, Malaysia;
141
ALIFAR, Argentina; Mrs J. Bernat (provided consolidated IFPMA comments),

Switzerland; Dr B. Boonyapiwat, Ministry of Public Health, Thailand; Dr Y. Choi,
Ministry of Food and Drug Safety, Republic of Korea; Dr F. Muñoz Espinoza,
Instituto de Salud Pública de Chile, Chile; Dr H-K. Heim, Bundesinstitut
für Arzneimittel und Medizinprodukte, Germany; Dr J. Joung, Ministry of
Food and Drug Safety, Republic of Korea; Dr Y. Kishioka, Pharmaceutical
and Medical Devices Agency, Japan; Dr A. Klein, Health Canada, Canada;
Ms S. Kox (provided consolidated EGA comments), Belgium; Dr P. Kurki,
Finnish Medicines Agency, Finland; Mrs V. Madrigal, Recepta Biopharma,
Brazil; Dr C. Njue, Health Canada, Canada; Mrs Y. Hechavarria Nunez,
Centro para el Control Estatal de la Calidad de los Medicamentos, Cuba; Dr J.
Shin, WHO Regional Office for the Western Pacific, Philippines; Dr W. Tan,
Genzume Singapore, Singapore; Dr R. Thorpe, Consultant, Welwyn, the United
Kingdom; Dr J. Wang, Health Canada, Canada; Dr M. Weise, Bundesinstitut für
Arzneimittel und Medizinprodukte, Germany; Dr S. Xie, China Food and Drug
Administration, China.
The draft document was then posted on the WHO Biologicals website
for the second round of public consultation from 16 December 2014 to 30
January 2015.
The document WHO/BS/2015.2251 was prepared by Dr E. Griffiths,
Consultant, Kingston-upon-Thames, the United Kingdom; and Dr H-N.
Kang, World Health Organization, Switzerland taking into account comments
received from the following reviewers: Dr R. Aaron, Tanzania Food and Drugs
Authority, United Republic of Tanzania; Mrs A. Abas, Ministry of Health
Malaysia, Malaysia; Dr A. Abdelaziz, Jordan Food And Drug Administration,
Jordan; Dr R. Abete (provided consolidated ALIFAR comments), Argentina;
Dr M. Aboulwafa, National Organization for Research and Control of
Biological Products, Egypt; Mrs J. Bernat (provided consolidated IFPMA
comments), Switzerland; Dr Y. Choi, Ministry of Food and Drug Safety,
Republic of Korea; Dr J. Gangakhedkar, Central Drugs Standard Control

Organization, India; Dr K. Gao, National Institutes for Food and Drug Control,
China; Ms C. Gongora Torres (provided consolidated comments), Ministry
of Health and Social Protection, Colombia; Dr T. Guo, National Institutes
for Food and Drug Control, China; Dr H. Hamedifar, CinnaGen Co., the
Islamic Republic of Iran; Dr H-K. Heim, Bundesinstitut für Arzneimittel und
Medizinprodukte, Germany; Dr J. Joung, Ministry of Food and Drug Safety,
Republic of Korea; Dr B. Kim, Ministry of Food and Drug Safety, Republic
of Korea; Dr Y. Kishioka, Pharmaceutical and Medical Devices Agency, Japan;
Ms S. Kox (provided consolidated EGA comments), Belgium; Dr P. Kurki, Finnish
Medicines Agency, Finland; Dr C. Liang, National Institutes for Food and
Drug Control, China; Dr N. Lyoko, Zambia Medicines Regulatory Authority,
Zambia; Mr R. Mezzasalma (provided consolidated European Association for
142
Annex 3
BioIndustries comments), Belgium; Dr R. Mody, United States Pharmacopeial

Convention–India, India; Mr F. Montes de Oca, (provided consolidated
Argentine Association of Industrial Pharmacy and Biochemistry comments),
Argentina; Dr V. Murthy, Apotex Inc., Canada; Dr C. Njue, Health Canada,
Canada; Dr D. Pathankar, United States Pharmacopeial Convention–India,
India; Dr Z. Sauna, United States Food and Drug Administration, the USA;
Dr T. Schreitmueller (provided consolidated Federación Latinoamericana de la
Industria Farmacéutica comments), Mexico; Dr M. Seigelchifer, United States
Pharmacopeial Convention–India, India; Ms J. Shim (provided consolidated
Korea Biomedicine Industry Association comments), Republic of Korea; Dr J.
Southern (Developing Country Vaccine Regulators’ Network representative),
South Africa; Dr S. Suh, Ministry of Food and Drug Safety, Republic of Korea;
Dr M. Wadhwa, National Institute for Biological Standards and Control, the
United Kingdom; Dr J. Wang, Health Canada, Canada.
Also taken into consideration were comments and advice provided
during a WHO informal consultation on regulatory risk assessment for
biotherapeutic products held in Geneva, Switzerland, 29–30 April 2015
and attended by: Mrs A. Abas, Ministry of Health Malaysia, Malaysia; Dr A.
Abdelaziz, Jordan Food and Drug Administration, Jordan; Dr K. Bangarurajan,
Central Drug Standards Control Organization, India; Mrs J. Bernat (provided
consolidated IFPMA comments), Switzerland; Dr B. Boonyapiwat, Ministry of
Public Health, Thailand; Ms J. Dahlan, National Agency of Drug and Food
Control, Indonesia; Mrs D. Darko, Food and Drug Authority, Ghana; Mr D.
Goryachev, Ministry of Health, Russia; Dr E. Griffiths, Consultant, Kingston-
upon-Thames, the United Kingdom; Dr K. Heidenreich (IFPMA representative),
Novartis International AG, Switzerland; Dr H-K. Heim, Federal Institute for
Drugs and Medical Devices, Germany; Dr C. Ilonze, National Agency for Food
and Drug Administration and Control, Nigeria; Mr R. Ivanov, Biocad, Russia;
Mrs W. Jariyapan, Ministry of Public Health, Thailand; Dr J. Joung, Ministry
of Food and Drug Safety, Republic of Korea; Dr D. Khokal, Health Sciences
Authority, Singapore; Dr P. Kurki (European Medicines Agency Biosimilar
Medicinal Products Working Party representative), Finnish Medicines Agency,
Finland; Ms S. Kox (provided consolidated EGA comments), Belgium; Ms I.
Lyadova, Ministry of Health, Russia; Ms S. Marlina, National Agency of Drug
and Food Control, Indonesia; Dr J. Meriakol, Ministry of Health, Kenya; Mr K.
Nam, Ministry of Food and Drug Safety, Republic of Korea; Dr C. Njue, Health
Canada, Canada; Mrs Y. Nunez, Centro para el Control Estatal de la Calidad de
los Medicamentos, Cuba; Ms H. Pedersen, WHO Regional Office for Europe,
Denmark; Dr M. Pombo, Pan American Health Organization, the USA; Dr A.
Qu (DCVMN representative), Shanghai Institute of Biological Products Co., Ltd,
China; Dr S. Ramanan (IFPMA representative), Amgen Inc., the USA; Dr M.
Schiestl (EGA representative), Sandoz GmbH, Austria; Dr E. Spitzer (ALIFAR
143
representative), Argentina; Ms I. Susanti (DCVMN representative), PT Bio

Farma, Indonesia; Dr R. Thorpe, Consultant, Welwyn, the United Kingdom;
Dr C. Vaca Gonzalez, Ministry of Health and Social Protection, Colombia;
Ms B. Valente, Agência Nacional de Vigilância Sanitária, Brazil; Ms N. Vergel,
Instituto Nacional de Vigilancia de Medicamentos y Alimentos, Colombia;
Dr M. Wadhwa, National Institute for Biological Standards and Control, the
United Kingdom; Dr J. Wang, Health Canada, Canada; Dr J. Wang, National
Institutes for Food and Drug Control, China; Dr L. Wang, National Institutes
for Food and Drug Control, China; Dr K. Watson (IFPMA representative),
AbbVie, Ltd, the United Kingdom; Dr C. Webster (EGA representative), Baxter
Healthcare Corporation, the United Kingdom; Dr S. Xie, China Food and Drug
Administration, China; Dr T. Yamaguchi, Pharmaceuticals and Medical Devices
Agency, Japan; Dr A. Zhang (DCVMN representative), China National Biotec
Group Co., Ltd, China; and Dr K. Gao, Dr H-N. Kang and Dr I. Knezevic, World
Health Organization, Switzerland.
The document WHO/BS/2015.2251 was then posted on the WHO
Biologicals website for the third round of public consultation from 3 July to 14
September 2015, and comments were received from the following reviewers:
Mrs J. Bernat (provided consolidated IFPMA comments), Switzerland; Dr S.
Creekmore, National Institutes of Health, the USA; Dr R. Dominguez Morales,
Centro para el Control Estatal de la Calidad de los Medicamentos, Cuba; Mr F.
Fon Mendez (provided consolidated Asociación Mexicana de Industrias de
Investigación Farmacéutica comments), Mexico; Dr E.P. Fuenter, Consultant,
Argentina; Ms K. Holcombe, Bio, the USA; Dr J. Joung, Korea Ministry of
Food and Drug Safety, Republic of Korea; Dr D. Khokal, Health Sciences
Authority, Singapore; Ms S. Kox (provided consolidated EGA comments),
Belgium; Dr Z. Kusynova (provided consolidated International Pharmaceutical
Federation comments), Netherlands; Mr M.A. Maito, (provided consolidated
ALIFAR comments), Argentina; Dr J. Wang, Health Canada, Canada; Dr K.
Zoon, National Institutes of Health, the USA.

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17. Ministerial Resolution, No. 181-2015. Proyecto de reglamento que regula la presentacion
y contenido de los documentos requeridos en la inscripcion y reinscripcion de productos
biologicos similares. Articulo 8–9. Lima, Ministry of Health; 2015 (ftp://ftp2.minsa.gob.pe/
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18. The safety of medicines in public health programs: Pharmacovigilance an essential tool. Geneva:
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efforts towards harmonization of regulation. Biologicals. 2011;39(5):348 (http://www.sciencedirect.
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20. Kohler M. Regulatory pathways in the European Union. MAbs. 2011;3(3):241–2 (http://www.ncbi.
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Annex 4
Recommendations to assure the quality, safety and
efficacy of recombinant human papillomavirus virus-like
particle vaccines
Replacement of Annex 1 of WHO Technical Report Series, No. 962
Introduction 151
Scope 152
General considerations 152
Terminology 157
Part A. Manufacturing recommendations 160
A.1 Definitions and international reference materials 160
A.2 General manufacturing recommendations 161
A.3 Control of source materials 162
A.4 Control of HPV VLP production 168
A.5 Control of purified monovalent antigen bulk 173
A.6 Control of adsorbed monovalent antigen bulk 176
A.7 Control of final bulk 177
A.8 Filling and containers 179
A.9 Control tests on the final lot 179
A.10 Records 182
A.11 Retained samples 182
A.12 Labelling 182
A.13 Distribution and transport 182
A.14 Stability testing, storage and expiry date 183
Part B. Nonclinical evaluation of recombinant HPV VLP vaccines 184
B.1 Product characterization and process development 184
B.2 Pharmacodynamic studies 185
B.3 Toxicology studies 186
Part C. Clinical evaluation of recombinant HPV VLP vaccines 186
C.1 Introduction 186
C.2 Immunological data 187
C.3 Virological data 195
C.4 Histological data 198
C.5 Evaluation of vaccine efficacy in different settings 198
C.6 Cross-protection 201
147
C.7 Safety 202

C.8 Post-licensing evaluation 202
Part D. Recommendations for NRAs 204
D.1 General recommendations 204
D.2 Official release and certification 204
Authors and acknowledgements 205
References 208
Appendix 1 Model protocol for the manufacturing and control of recombinant
human papillomavirus virus-like particle vaccines 216
Appendix 2 Model NRA Lot Release Certificate for recombinant human
papillomavirus virus-like particle vaccines 232
Recommendations published by WHO are intended to be scientific

and advisory in nature. Each of the following sections constitutes
recommendations for national regulatory authorities (NRAs) and
for manufacturers of biological products. If an NRA so desires, these
WHO Recommendations may be adopted as definitive national
requirements, or modifications may be justified and made by the NRA. It
is recommended that modifications to these WHO Recommendations
are made only on condition that such modifications ensure that the
product is at least as safe and efficacious as that prepared in accordance
with the recommendations set out below. The parts of each section
printed in small type are comments or examples intended to provide
additional guidance to manufacturers and NRAs.
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Annex 4
Abbreviations
CIN cervical intraepithelial neoplasia
CIN2–3 CIN grades 2 or 3
CIN2+ cervical intraepithelial neoplasia grade 2 or worse
cLIA competitive Luminex immunoassay
CMI cell-mediated immunity
EIA enzyme immunoassay
GCP good clinical practice
GMC geometric mean concentration
GMT geometric mean titre
HPV human papillomavirus
IARC WHO International Agency for Research on Cancer
ICP immune correlate of protection
& Associations
IU International Unit(s)
LAL limulus amebocyte lysate
LLOD lower limit of detection
LLOQ lower limit of quantification
MOI multiplicity of infection
MPL monophosphoryl lipid A
NAT nucleic acid amplification technique
NCL national control laboratory
PAGE polyacrylamide gel electrophoresis
PCR polymerase chain reaction
149

RBC red blood cell
RRP recurrent respiratory papillomatosis
SAGE WHO Strategic Advisory Group of Experts
SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEC–HPLC size-exclusion chromatography – high-performance liquid
chromatography
spp. species
TEM transmission electron microscopy
VLP virus-like particle
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Annex 4
Introduction
WHO Guidelines to assure the quality, safety and efficacy of recombinant human
papillomavirus (HPV) virus-like particle (VLP) vaccines were first adopted by
the WHO Expert Committee on Biological Standardization in 2006 (1) and were
based largely on experience gained from clinical trials undertaken on the first
two licensed HPV vaccines.
The factors that have prompted this revision include the substantial
amount of data accumulated during vaccine implementation, the development
of prophylactic vaccines with extended valency and the use of other production
methods. In addition, the increasing availability and routine use of HPV VLP
vaccines composed of L1 capsid protein and containing at least types 16 and
18 have important implications for trial designs and end-points for clinical
evaluation of new prophylactic HPV vaccines.
A series of meetings was convened by WHO to review the scientific
evidence needed to initiate and inform the revision process. These meetings were
attended by experts from around the world involved in the research, manufacture,
licensing/authorization, control-testing and release of HPV vaccines. Participants
were drawn from academia, national regulatory authorities (NRAs), national
control laboratories (NCLs) and industry, and included representatives of the
WHO Global HPV LabNet – an initiative that worked towards the international
standardization of HPV testing during 2006–2011. These experts reviewed new
HPV vaccines under development, and the scientific basis and evidence for
accepting alternative end-points for evaluating the clinical efficacy of candidate
HPV vaccines. The first meeting held was in February 2013 and considered issues
relating to the development and evaluation of clinical end-points for trials of new
HPV vaccines and other issues to be addressed in the proposed revision. At a
meeting held at the WHO International Agency for Research on Cancer (IARC),
Lyon, France in September 2013 a Working Group discussed whether it might be
appropriate to consider using a virological end-point – rather than a disease end-
point such as cervical intraepithelial neoplasia (CIN) grade 2 or worse (CIN2+) –
as the primary end-point for future clinical efficacy trials, and the circumstances
under which immunobridging trials might be sufficient for licensure (2, 3).
A third meeting held at WHO headquarters in November 2013 reviewed and
discussed the outcomes of the IARC scientific meeting on appropriate clinical
end-points, reviewed vaccines currently in the development pipeline, and
assessed regulatory and laboratory needs for licensing the vaccines (4).
Major issues addressed in these resulting WHO Recommendations
include updates of:
■■ terminology;
■■ general considerations and other sections to reflect the up-to-date
development of HPV vaccines;
151
■■ the section on international standards and reference preparations;

■■ Part A, in order to include bacteria as a cell substrate;
■■ Part B, in order to include a new subsection highlighting tests
needed for nonclinical evaluation, and to elaborate regulatory
requirements for both proof-of-concept pharmacology studies and
toxicological testing;
■■ Part C, in order to reflect appropriate and feasible end-points in
future trials;
■■ appendices.
Additional changes have also been made to bring the document into
line with other WHO Recommendations, Guidelines and guidance documents
published since the 2006 adoption of the WHO Guidelines on recombinant
HPV VLP vaccines (1).
Scope
These WHO Recommendations provide guidance to NRAs and manufacturers
on the manufacturing process, and on nonclinical and clinical aspects, of
recombinant HPV VLP vaccines to assure their quality, safety and efficacy.
The scope of the present document encompasses recombinant HPV
VLP vaccines for prophylactic use which contain the L1 capsid protein of one
or more HPV types.
The document does not cover vaccines targeted to L2 capsid proteins
as antigens, as appropriate serological assays have not yet been standardized
and clinical vaccine trials have not started. Non-VLP vaccines (for example,
other forms of subunit vaccines, vectored vaccines and L1 capsomers) and
investigational therapeutic HPV vaccines, which are at an early stage of
development, are also not included. However, some aspects discussed below may
be relevant and may be taken into consideration during vaccine development.

This document should be read in conjunction with other relevant WHO
guidance such as that on nonclinical (5) and clinical (6) evaluation of vaccines.
Other WHO guidance, such as that on the evaluation of animal cell cultures
as substrates for the manufacture of biological medicinal products and for the
characterization of cell banks (7) and nonclinical evaluation of vaccine adjuvants
and adjuvanted vaccines (8), should also be considered.
General considerations
HPV is not a single virus; rather, it includes a group of closely related small,
non-enveloped deoxyribonucleic acid (DNA) viruses in the Papillomaviridae
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Annex 4
family. The circular, double-stranded viral genome is approximately 8 kb in

length. The genome encodes for six early protein-coding sequences responsible
for virus replication and two late proteins (L1 and L2) which are the viral
structural capsid proteins. L1 is the major structural protein. L1 proteins
associate to form pentameric structures called capsomers (9). Mature virus
particles comprise 72 capsomers arranged in icosahedral symmetry. The
minor capsid protein, L2, is present in as many as 72 molecules per mature
virus particle (10). L2 is not required for particle formation, but plays a role in
encapsidating the genome. HPV infection, replication and particle maturation
occur in the stratified squamous epithelia of skin and mucous membranes (11).
Over 190 different types of HPV have been identified and molecularly
characterized (12). These HPVs cause a variety of epithelial diseases in humans,
ranging from benign warts to cancers (including of the cervix, vagina, vulva,
penis, anus and oropharynx). HPV types associated with the development of
cervical cancer are labelled as high risk for oncogenicity.1 Other HPV types,
such as types 6 and 11 which are associated with genital warts, are considered as
low risk for oncogenicity.
The majority of HPV infections with both high-risk and low-risk
types are asymptomatic, self-limiting and resolve spontaneously due to the
host immune response. However, in rare instances HPV infection persists.
If persistent infection with high-risk types is not detected and treated then
progression to invasive carcinoma may occur at the site of infection. The interval
between acquisition of HPV infection and malignant (invasive) progression is
generally at least 10 years. As high-risk HPV types are detected in virtually all
cervical cancers, it is scientifically accepted that the persistent viral infection is
necessary though not sufficient for the development of cancer (13, 14). The basis
for progression to invasive carcinoma is not well understood. Environmental
and physiological cofactors such as high parity, hormonal contraceptives and
smoking may increase the risk for cancer development in people with persistent
infection (15–17). Nevertheless, individuals without identified risk factors who
have persistent infection can also develop cervical cancer.
IARC currently defines 12 high-risk HPV types that are associated
with cancers in humans – namely HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58 and 59 (Group 1) – and an additional type as probably carcinogenic –
namely HPV type 68 (Group 2A) (14). HPV type 66 was formerly classified as
oncogenic but recent findings have significantly weakened the evidence for this.
Additional data on rare HPV types currently considered possibly oncogenic to
The terms oncogenic/oncogenicity and carcinogenic/carcinogenicity are both used in the scientific
1
literature on HPVs. In this document the terms oncogenic/oncogenicity are used.

153
humans (Group 2B) suggest that some, but not all, could be upgraded (18). The
distribution and prevalence of the above HPV types in patients with cancer are
generally consistent around the world. Two of the high-risk HPV types (16 and
18) account for approximately 70% of all cervical cancers globally (14). Most
anal cancers are also associated with persistent HPV infection, with HPV type 16
representing an even higher fraction (90%) of HPV-positive cancers of the anus
than is the case for cervical cancer (approximately 50%). In addition, these high-
risk HPV types are associated with a significant fraction of cancers of the vagina,
vulva, penis and oropharynx. The incidence of cervical cancer is substantially
higher than that of all other HPV-related cancers; cervical cancer is the second
most common cancer among women aged 15–44 years.
Low-risk HPV types cause genital warts, recurrent respiratory
papillomatosis (RRP) and low-grade cervical dysplasia. Genital warts affect
both males and females. Data on the worldwide burden of genital warts are
not available, but in developed countries the epidemiology is similar to other
sexually transmitted infections, peaking in young ages (15–24 years) (19). While
not malignant, these lesions are associated with physical and psychological
morbidity and may be difficult to treat. RRP is a devastating, although rare,
disease that manifests as recurrent, rapidly growing benign laryngeal tumours
that require frequent excision to prevent airway obstruction. HPV types 6 and
11 are responsible for over 90% of genital warts and RRP cases, and for 9–12%
of low-grade cervical dysplastic lesions.
Identification of a viral agent such as HPV as a major cause of diseases
implies that prophylactic vaccines or interventions against the viral agent should
prevent the disease(s) it causes. Initial studies in animal models showed that
inoculation with species-specific papillomaviruses induced an immune response
that conferred protection against homologous virus challenge. However, native
papillomaviruses are not good substrates for vaccine development as they
cannot be grown in standard cell culture. Subsequent studies showed that L1
protein produced in heterologous expression systems, such as yeast or insect

cells, self-assembles into VLPs that are morphologically similar to authentic
HPV virions but contain no viral DNA. In animal studies, VLPs were shown to
protect against experimental infection with a high dose of homologous virus (20,
21). HPV VLPs are highly immunogenic in mice and rabbits, and the resulting
antibodies have been shown to be neutralizing and type-restricted when tested
in a pseudovirion neutralization assay. In animal studies, immunization with
denatured VLPs did not produce neutralizing antibodies and did not give
protection against experimental virus challenge, indicating that neutralizing
epitopes are conformation-dependent.
Protection in animals has been demonstrated through passive transfer
of antibodies in serum (20–22). Neutralizing antibodies are believed to be the
primary mediator of this protection. Enhanced regression of established HPV
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lesions, which requires cell-mediated immunity (CMI), was not observed

after VLP vaccination. Therefore, it seems unlikely that CMI is substantially
involved as a direct effector mechanism of protection (23). The specific assays
that have been developed to evaluate the humoral immune response to HPV
include VLP‑based enzyme immunoassay (EIA), competitive immunoassay
with labelled neutralizing monoclonal antibodies and pseudovirion-based
neutralization assays (24).
The revision of the former WHO Guidelines to produce these WHO
Recommendations has been driven by the experience acquired in using the first
two licensed HPV L1 VLP vaccines in many countries, and by the development
of an extended version of one of these vaccines to include additional HPV
types. These vaccines are made up of recombinant protein L1 VLPs and contain
adjuvant to enhance their immunogenicity. The vaccines are delivered via
intramuscular injection. The products differ in the types of HPV L1 proteins
included as antigens, substrates used for production, adjuvant properties and
final formulation. The three vaccines are:
■■ A bivalent vaccine comprising HPV types 16 and 18 VLPs self-
assembled from L1 protein expressed and purified from insect cells
infected with a recombinant baculovirus; the vaccine is formulated
with a novel adjuvant (AS04) which contains aluminium hydroxide
and monophosphoryl lipid A (MPL) with each dose delivering 20 µg
of each VLP.
■■ A quadrivalent vaccine comprising HPV types 6, 11, 16 and 18
VLPs self-assembled from L1 protein expressed and purified from
Saccharomyces cerevisiae (baker’s yeast) containing L1 expression
plasmids; the vaccine is formulated with amorphous aluminium
hydroxyphosphate sulfate adjuvant with each dose delivering 20 µg of
HPV types 6 and 18 VLPs and 40 µg of HPV types 11 and 16 VLPs.
■■ An extended version of the quadrivalent vaccine that contains five
additional oncogenic HPV types (types 31, 33, 45, 52 and 58); the
vaccine is manufactured as for the quadrivalent vaccine and contains
the same adjuvant.
The bivalent vaccine expressed from recombinant baculovirus in insect
cells was the first vaccine to be developed in this host expression system. Other
novel expression systems, such as Escherichia coli or Pichia yeast, may be
introduced. Testing of novel cell substrates may have some unique requirements.
Each VLP type in a multivalent vaccine should be produced and purified
separately, and then mixed together for the final formulation. L1 protein in its
native form is not glycosylated (25). The currently available vaccines do not
utilize expression systems that glycosylate the VLP, and glycosylation does not
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appear to be important. Other expression systems without post-translation

glycosylation, such as the E. coli expression system, could also be used.
L1 VLPs may include other recombinant proteins (for example, L2) that
serve as a stabilizer rather than as an antigen (26). Conformational epitopes of
L1 VLPs are required for eliciting neutralizing antibody response in the host.
Disassembly of the L1 VLPs into capsomers and reassembly of the L1 capsomers
back into VLPs may contribute to purification of some types of HPV VLPs and
lead to more-stable VLPs.
Purified L1 VLP preparations will need to be characterized biochemically
and immunologically to determine L1 concentration, purity, post-translational
modification and assembly state. L1 VLPs incorporate nucleic acids of their
producer cells to varying degrees, and so the amount of residual host cellular
DNA incorporated needs to be determined. Removal of host cellular DNA
may be required if the amount exceeds the limits set by NRAs for the specific
production system.
Regulatory approvals for the bivalent and quadrivalent vaccines
were based on double-blind efficacy studies in females aged 16–26 years
using unvaccinated control groups – namely groups that received placebo or
another vaccine with no potential to protect against HPV. The primary end-
points for these Phase III clinical trials were based on histological detection of
precancerous epithelial changes attributable to specific HPV types as a surrogate
for risk of progression to malignant lesions (CIN grades 2 or 3 (CIN2–3) and
adenocarcinoma in situ). Secondary end-points included 6- or 12-month type-
specific persistence of HPV detection. Primary efficacy was demonstrated in
women who were not previously exposed to the vaccine types. Immunogenicity
and safety were established in younger males and females (ages 9–15 years), and
this information was used to bridge efficacy claims to this population. On the
basis of demonstrated safety and efficacy against cervical cancer precursors,
the WHO Strategic Advisory Group of Experts (SAGE) on Immunization
recommended that the primary target population should be girls within the age
range of 9 or 10 years through to 13 years – that is, before the age of initiation
of sexual activity and exposure to HPV (11).
The initial product licences were for 3-dose schedules (0, 1 or 2 months
and 6 months). Subsequently the European Medicines Agency approved:
(a) a 2-dose schedule for the bivalent vaccine for females aged 9–14 years; and
(b) a 2-dose schedule for the quadrivalent vaccine for females aged 9–13 years.
For both the bivalent and quadrivalent HPV vaccines, SAGE recommended a
2-dose schedule with a 6-month interval between doses for females younger
than 15 years. Those who are ≥ 15 years at the time of the second dose are also
adequately covered by 2 doses (11).
The extended version of the quadrivalent vaccine includes five additional
HPV types (31, 33, 45, 52 and 58). All nine HPV VLPs are prepared from L1
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protein expressed and purified from S. cerevisiae containing L1 expression

plasmids. The licensure of this 9-valent vaccine was based on demonstration of
efficacy against the additional HPV types (using a composite histological end-
point) and a comparison of immune responses to the four HPV types in the
quadrivalent vaccine.
The currently available HPV vaccines are manufactured in single-dose
or 2-dose vials or prefilled syringes without the addition of preservative. In
future, the availability of multi-dose vaccine vials should facilitate the adoption
of innovative vaccination strategies targeting pre-adolescents and adolescents
in developing countries. If these vaccines do not contain preservative, their use
should be time-restricted as is the case for reconstituted vaccines such as Bacillus
Calmette-Guerin vaccine and measles-containing vaccines (27). If a preservative
were to be added its effect on antigenicity and immunogenicity must be assessed
to ensure that the preservative does not affect immune response. Thiomersal, an
organo-mercury compound that was a widely used preservative in vaccines, was
found to destroy the major neutralizing epitope of HPV type 16 L1 VLPs when
added to the vaccine formulation (28).
Current vaccine formulations use intramuscular administration. Other
routes of administration – such as nasal, cutaneous or oral – may be investigated
to facilitate vaccination by avoiding the use of needles. It is crucial that nonclinical
studies demonstrate immunogenicity and the production of neutralizing
antibodies for any new products and any new routes of administration.
Terminology
The definitions given below apply to the terms as used in these WHO
Recommendations. They may have different meanings in other contexts.
Adjuvant: a substance or a combination of substances used in
conjunction with a vaccine antigen to enhance (for example, increase, accelerate,
prolong and/or possibly target) the specific immune response to the vaccine
antigen and the clinical effectiveness of the vaccine. This may also be called a
mineral vehicle or immunostimulant.
Adsorbed monovalent antigen bulk: a batch of purified monovalent
antigen bulk adsorbed on adjuvant. Different batches of adsorbed monovalent
antigen bulks may be pooled before collection into a single vessel.
If a novel adjuvant is used that does not involve adsorption of the VLP
to the adjuvant, the term “adjuvanted monovalent antigen bulk” may
be used.
Adventitious agents: contaminating microorganisms that may include

bacteria, fungi, mycoplasmas, and endogenous and exogenous viruses, that have
been unintentionally introduced into the manufacturing process.
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Cell bank: a collection of containers containing aliquots of a suspension

of cells from a single pool of uniform composition, stored frozen under defined
conditions (typically −60 °C or below for yeast or bacteria and in liquid nitrogen
for insect or mammalian cell lines).
The individual containers (for example, ampoules or vials) should be
representative of the pool of cells from which they are taken and should
be frozen on the same day following the same procedure and using the
same equipment and reagents.
Cell seed: a quantity of vials containing well-characterized cells derived

from a single tissue or cell of human or animal origin, stored frozen in liquid
nitrogen in aliquots of uniform composition, one or more of which are used for
the production of a master cell bank.
Cell substrate: cells used to manufacture a biological product.
Comparator vaccine: a licensed vaccine with established efficacy or with
traceability to a vaccine with established efficacy that is tested in parallel with a
candidate vaccine and serves as an active control in nonclinical or clinical testing.
Composite end-points: combine two or more single end-points in one
outcome to demonstrate overall treatment effects. Subjects who have experienced
any of the events specified in the single end-points are considered to have
experienced the composite end-point (29).
Control cell culture: cells amplified from the working cell bank and
split from those intended for use in production immediately prior to intentional
viral infection (for example, with baculovirus expression vector) and grown in
parallel for the purpose of serving as test material.
Expression construct: defined as the expression vector containing the
coding sequence of the recombinant protein (30).
Expression system: the host cell with its expression construct and
the cell culture process that is capable of expressing protein encoded by the
expression construct. Expression systems can be bacterial-based, baculovirus-

insect-cell-based, mammalian-based or yeast-based.
Final bulk: the formulated vaccine present in a container from which
the final containers are filled. The final bulk may be prepared from one or more
adsorbed monovalent antigen bulks and may contain VLP antigens from one or
multiple HPV types. If prepared from one or more monovalent antigen bulks,
mixing should result in a uniform preparation to ensure that the final containers
are homogenous.
Final lot: a collection of sealed final containers of finished vaccine that
is homogeneous with respect to the risk of contamination during the filling
process. All of the final containers must therefore have been filled from a single
vessel of final bulk in one working session.
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HPV L1 protein: the major structural capsid protein of human

papillomavirus, of which 360 molecules are found in the native virion associated
in 72 pentameric capsomers.
Immune correlate of protection (ICP): an immune response parameter
of a type (for example, serum antibodies) and amount above which an individual
is most probably protected. For many vaccines, functional antibodies above a
threshold geometric mean titre (GMT) or geometric mean concentration (GMC)
serve as an ICP.
Immunobridging studies: studies intended to support the extrapolation
of efficacy from one formulation, population or dose regimen to another based
on comparison of relevant immune responses.
Inoculum intermediate: a quantity of recombinant baculovirus of
uniform composition, derived from the recombinant baculovirus working
seed lot. The inoculum intermediate has a defined shelf-life. It is intended to be
used to initiate the production of recombinant L1 proteins from insect cells.
L1 virus-like particle (VLP): a non-infectious, non-replicating, non-
enveloped, icosahedral capsid particle that resembles native virions but does
not contain viral DNA. The regular arrays of L1 pentameric capsomers retain
conformational epitopes.
Master cell bank (MCB): a quantity of well-characterized cells of animal
or other origin, derived from a cell seed at a specific population doubling level
(PDL) or passage level, dispensed into multiple containers, cryopreserved and
stored frozen under defined conditions (typically −60 °C or below for yeast or
bacteria, and in liquid nitrogen for insect or mammalian cell lines). The MCB is
prepared from a single homogeneously mixed pool of cells. In some cases, such
as genetically engineered cells, this may be prepared from a selected cell clone
established under defined conditions. The MCB is used to derive all working
cell banks for the anticipated lifetime of the vaccine product.
Parental cells: cells that are manipulated to give rise to a cell substrate.
Manipulation may be simply the expansion of a primary cell culture to
provide early passage cells, or a more complex activity such as developing
a transfected clone. Both processes would provide a cell seed. The parental
cells may refer to any stage prior to the preparation of the cell seed.
Production cell culture: a collection of cell cultures used for the

production of HPV L1 antigen that have been prepared together from one or
more containers of the working cell bank.
Purified monovalent antigen bulk: a batch of purified antigen of a
single HPV type. Different batches of purified monovalent antigen bulks may be
pooled into a single vessel.
Recombinant baculovirus master seed lot: a quantity of recombinant
baculovirus of uniform composition derived from an original baculovirus
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expression construct, processed at one time and passaged for a documented

number of times.
Recombinant baculovirus working seed lot: a quantity of recombinant
baculovirus of uniform composition derived from the recombinant baculovirus
master seed lot by a limited number of passages. The recombinant baculovirus
working seed lot may be used to prepare inoculum intermediates or alternatively
to initiate the production of recombinant L1 proteins.
Single antigen harvest: a concentrated cell suspension or supernatant
containing the intended HPV antigens of one virus type harvested from cell
cultures prepared from a single production run.
Single harvest pool: a homogeneous pool of multiple single harvests of
the intended HPV antigens of one virus type, collected into a single vessel prior
to purification.
Working cell bank (WCB): a quantity of well-characterized cells of
animal or other origin, derived from the MCB at a specific PDL or passage level,
dispensed into multiple containers and stored under defined culture conditions
(typically −60 °C or below for yeast or bacteria, and in liquid nitrogen for insect
or mammalian cell lines). The WCB is prepared from a single homogeneously
mixed pool of cells. One or more of the WCB containers are used for each
production culture. Multiple WCBs may be made and used during the lifetime
of the vaccine product.
Part A. Manufacturing recommendations

A.1 Definitions and international reference materials
A.1.1 International name and proper name
The international name should be “recombinant human papillomavirus virus-
like particle vaccine” followed in parentheses by the type specificity and the
name of the recombinant protein (for example, types 16 and 18 L1 proteins). The
proper name should be equivalent to the international name in the language of
the country of origin.
The use of the international name should be limited to vaccines that
meet the specifications elaborated below.
A.1.2 Descriptive definition

The recombinant HPV VLP vaccine is a sterile liquid vaccine preparation that
contains purified VLPs composed of the recombinant major capsid proteins (L1)
of one or more HPV types. The VLPs may be formulated with a suitable adjuvant.
Such vaccines are for prophylactic use.
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A.1.3 International reference materials

International Standards for HPV types 16 and 18 antibodies are available. These
materials may be used in immunoassays utilizing VLP binding assays and
pseudovirion neutralization tests of adequate sensitivity (31, 32).
International Standards for HPV types 16 and 18 DNA are also available.
These standards are suitable for the calibration of in-house or working standards
for the amplification and detection of HPV types 16 and 18 DNA (33).
The reference materials listed above are available from the National
Institute for Biological Standards and Control, Potters Bar, the United
Kingdom.
The WHO Catalogue of International Reference Preparations should be consulted

for the latest list of appropriate WHO International Standards and reference
materials (34). The Human papillomavirus laboratory manual (24) provides
guidance on the preparation and use of secondary standards calibrated against
International Standards for HPV DNA and antibodies.
International Standards and Reference Reagents for the control of HPV
VLP vaccine antigen content and potency are not available. Therefore, product-
specific reference preparations may be used.
A.2 General manufacturing recommendations

The general manufacturing requirements contained in WHO good manufacturing
practices for pharmaceutical products: main principles (35) and WHO good
manufacturing practices for biological products (36) should apply to the design,
establishment, operation, control and maintenance of manufacturing facilities
for recombinant HPV VLP vaccines, with the addition of the following:
■■ Production steps involving manipulations of recombinant HPV L1
VLP types should be conducted at a biosafety level consistent with
the production of recombinant microorganisms.
■■ Quality control procedures should be in place to ensure segregation of
different HPV L1 VLP types during bulk manufacturing steps, as well
as segregation from other products manufactured in the same facility.
Sufficient cleaning-validation and product-changeover data should
be available to evaluate procedures. The antigen-manufacturing
process should be validated to demonstrate production consistency.
Typically, three consecutive lots per HPV type are required.
A.2.1 Characterization of the antigen

Characterization of HPV antigen is performed on lots produced during vaccine
development, including the process-validation batches.
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The protein composition should be established by techniques such as

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or mass
spectrometry. The SDS-PAGE bands should be identified by sensitive staining
techniques and where possible by specific antibodies, or mass spectrometry could
be used to confirm the presence of the expected products of the L1 protein. The
identity of the protein should be established by peptide mapping and/or analysis
of the terminal amino acid sequences.
Since it is known that conformational epitopes are essential for efficacy,
it is necessary that the morphological characteristics of the VLPs and degree
of aggregation should be determined. VLP characterization may be done by
additional appropriate methods.
A.3 Control of source materials

A.3.1 Cell cultures for antigen production
The use of any cell line should be based on a cell bank system (7, 37). Only cells
that have been approved and registered with the NRA should be used to produce
HPV L1 VLP vaccines. The NRA should be responsible for approving the cell
bank or seed lot. An appropriate history of the cell bank should be provided.
With regard to cell cultures, the maximum number of passages (or population
doublings) allowable from the MCB, through the WCB, and through the
production in cells should be approved by the NRA.
A.3.1.1 Recombinant yeast and bacteria cells

The characteristics of the parental cells and the recombinant production strain
(parental cell transformed with the recombinant expression construct) should be
fully described and information should be given on the testing for adventitious
agents and on gene homogeneity for the MCB and WCB. A full description of the
biological characteristics of the host cell and expression vectors should be given.
This should include genetic markers of the host cell, the construction, genetics
and structure of the expression vector, and the origin and identification of the
gene that is being cloned. Some techniques (for example, deep sequencing) allow
for the entire construct to be examined, while others (for example, restriction
enzyme analysis) allow for assessment of segments (30, 38). The molecular and
physiological measures used to promote and control the expression of the cloned
gene in the host cell should be described in detail (38).
The nucleotide sequence of the gene insert and the adjacent segments
of the vector and restriction-enzyme mapping of the vector containing the gene
insert should be provided as required by the NRA.
Cells must be maintained in a frozen state that allows for recovery of
viable cells without alteration of genotype. The cells should be recovered from
the frozen state, if necessary in selective media, such that the genotype and
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phenotype consistent with the recombinant (modified) host and vector are
maintained and clearly identifiable. Cell banks should be identified and fully
characterized by means of appropriate tests.
Data – for example on plasmid restriction enzyme mapping, nutritional
requirements or antibiotic resistance (if applicable) – that demonstrate the
genetic stability of the expression system during passage of the recombinant
WCB up to or beyond the passage level used for production should be provided
to and approved by the NRA. Any instability of the expression system occurring
in the seed culture or after a production-scale run should be documented.
Stability studies should also be performed to confirm cell viability after retrieval
from storage, maintenance of the expression system, etc. These studies may be
performed as part of their routine use in production or may include samples
taken specifically for this purpose.
A.3.1.1.1 Tests on recombinant yeast and bacteria MCB and WCB

MCBs and WCBs should be tested for the absence of bacterial and fungal
contamination by appropriate tests, as specified in Part A, section 5.2 of the
WHO General requirements for the sterility of biological substances (39), or by
a method approved by the NRA, to demonstrate that only the bacteria or yeast
production strain is present and that the MCB and WCB are not contaminated
with other bacteria or fungi.
A.3.1.2 Insect cells

If insect cells are used, cell substrates and cell banks should conform with
the WHO Recommendations for the evaluation of animal cell cultures as
substrates for the manufacture of biological medicinal products and for the
characterization of cell banks (7), as appropriate to insect cells, and should be
approved by the NRA.
The MCB is made in sufficient quantities and stored in a secure
environment, and is used as the source material for making the manufacturer’s
WCB. In normal practice an MCB is expanded by serial subculture up to a passage
number (or population doubling, as appropriate) selected by the manufacturer
and approved by the NRA, at which point the cells are combined to give a single
pool which is distributed into ampoules and preserved cryogenically to form the
WCB. WCBs of insect cells may be used for recombinant baculovirus seed lot
production and antigen expression.
A.3.1.2.1 Tests on insect cell MCB and WCB

Tests on the MCB and WCB should be performed in accordance with the WHO
Recommendations for the evaluation of animal cell cultures as substrates for the
manufacture of biological medicinal products and for the characterization of
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cell banks (7). It is important to show that the cell banks are free from bacteria,
fungi, mycoplasmas, mycobacterium species (spp.), and adventitious agents
relevant to the species that may be present in raw materials used in its derivation.
For insect cell lines, special emphasis is put on potential insect-borne human
pathogens (for example, arboviruses).
Insect viruses have not been well characterized compared with other
potential adventitious agents, and there is therefore less information about them
– and specifically about their infectivity, replicative life-cycles and pathogenicity,
if any. It should be borne in mind that infection of insect cells with some insect
viruses may occur without showing cytopathic effect. Tests may include specific
nucleic acid amplification technique (NAT) tests such as polymerase chain
reaction (PCR) and other nonspecific tests such as electron microscopy and
co‑cultivation. The specificity and sensitivity of assays should be determined by
the manufacturer and approved by the NRA.
Full characterization may be performed on either the MCB or on the
WCB, with more-limited testing on the other, depending on the strategy chosen
for testing (7). Scientific advice on the testing strategy should be sought from
the NRA.
A.3.1.3 Recombinant mammalian cells

If mammalian cells are used, the cell substrates and cell banks should conform
with the WHO Recommendations for the evaluation of animal cell cultures
as substrates for the manufacture of biological medicinal products and for the
characterization of cell banks (7) and the WHO Guidelines on the quality, safety
and efficacy of biotherapeutic protein products prepared by recombinant DNA
technology (38), and should be approved by the NRA.
A.3.2 Recombinant baculovirus master and working

seeds for antigen production

The recombinant baculovirus expression vector contains the coding sequence of
the HPV protein antigen. The recombinant baculovirus used in the production
of HPV vaccines should be identified by historical records, which will include
information on the origin and identity of the gene being cloned, and on the
construction, genetics and structure of the baculovirus expression vector(s).
The production of vaccine should be based on the recombinant
baculovirus master seed lot and working seed lot system. Recombinant
baculovirus seed lots should be stored in a dedicated temperature-monitored
refrigerator at a temperature that ensures stability and security.
Only recombinant baculovirus seed lots that are approved by the NRA
should be used. The recombinant baculovirus master seed lot should be made
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in sufficient quantities to last the lifetime of the vaccine product and should
be stored in a secure environment, preferably in two geographically separate
locations. The master seed lot is used as the source material for making the
manufacturer’s recombinant baculovirus working seed lots. Either the virus
master seed lots or the virus working seed lots should be fully characterized
and tested extensively for adventitious agents, while the other may be subjected
to more-limited testing. The testing strategy and seed lots should be approved
by the NRA.
The manufacturer’s recombinant baculovirus working seed lot is used
in the production of inoculum intermediates and single antigen harvests and is
prepared from the master recombinant baculovirus seed lot. It is recommended
that a large lot of recombinant baculovirus working seed should be set aside
as the basic material that the manufacturer should use for the preparation of
each batch of vaccine. The recombinant baculovirus working seed lot should be
prepared by a defined number of passages from the recombinant baculovirus
master seed lot using a method and a passage level from the original virus seed
approved by the NRA. Once the acceptable passage level of the working seed
lot is established, it may not be changed in making future lots of working
seed without approval from the NRA.
A.3.2.1 Tests on recombinant baculovirus master and working seed lots

The expression construct should be analysed using NAT in conjunction with
other tests performed on the purified recombinant protein for assuring the
quality and consistency of the expressed HPV L1 antigens. The genetic stability
and stability of expression of the expression construct should be demonstrated
from the baculovirus master seed up to at least the highest passage level used in
production, but preferably beyond this level (30, 38).
A.3.2.1.1 Identity
Each baculovirus master and working seed lot should be identified by the HPV
type of the inserted gene using an appropriate method such as PCR. The tests
should be approved by the NRA.
A.3.2.1.2 Sterility tests for bacteria, fungi and mycoplasmas

Each recombinant baculovirus seed lot should also be tested for bacterial, fungal
and mycoplasmal contamination by appropriate tests, as specified in Part A
of the WHO General requirements for the sterility of biological substances,
sections 5.2 (39) and 5.3 (40) or by a method approved by the NRA. In addition,
the recombinant baculovirus seed lot should be tested for insect mollicutes
(mycoplasma) such as spiroplasma, entomoplasma and mesoplasma.
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NAT alone or in combination with cell culture, with an appropriate

detection method, may be used as an alternative to one or both of the
compendial mycoplasma-detection methods following suitable validation
and agreement from the NRA (7).
A.3.2.1.3 Tests for adventitious viruses

Each recombinant baculovirus seed lot should be tested in cell cultures for
adventitious viruses appropriate to the origin and the passage history of the seed
baculovirus. For tests on recombinant baculovirus-permissive indicator cells,
the neutralization of baculovirus is necessary. Antisera used for this purpose
should be free from antibodies that may neutralize adventitious viruses, and
should preferably be generated by the immunization of specific-pathogen-free
animals with an antigen made from a source (other than the production cell
line) which has itself been tested for freedom from adventitious agents. The
inoculated indicator cells should be examined microscopically for cytopathic
changes. At the end of the examination period, the cells should also be tested
for haemadsorbing viruses (see section A.4.2.1.1 below). It should be noted
that infection of indicator cells by insect viruses may not reveal any cytopathic
effect. Additional tests such as PCR, electron microscopy and co-cultivation
may therefore be performed. It is important to show that recombinant
baculovirus seeds are free of adventitious agents relevant to the species used
in their derivation with a special emphasis on potential insect-borne human
pathogens (for example, arboviruses). The specificity and sensitivity of assays
should be determined by the manufacturer and approved by the NRA.
In general, recombinant baculovirus seeds should be assessed for
absence of adventitious agents that may have been introduced during their
production, including those that may be present in the source materials used at
each of the production stages of the master and working virus seed lots. Each
virus master or working seed lot should also be tested in animals such as guinea-
pigs and/or mice. For details of these tests, see the WHO Recommendations
for the evaluation of animal cell cultures as substrates for the manufacture of
biological medicinal products and for the characterization of cell banks (7).
New molecular methods with broad detection capabilities are being
developed for the detection of adventitious agents. These methods
include: (a) degenerate NAT for whole virus families, with analysis of the
amplicons by hybridization, sequencing or mass spectrometry; (b) NAT
with random primers followed by analysis of the amplicons on large
oligonucleotide microarrays of conserved viral sequencing or digital
subtraction of expressed sequences; and (c) high-throughput or deep
sequencing. These methods may be used to supplement existing methods
or as alternative methods to in vivo and/or in vitro tests after appropriate
validation and approval by the NRA (7).
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A.3.2.1.4 Test for mycobacterium spp.

Each recombinant baculovirus seed lot should be tested for mycobacterium spp.
The test method and specifications should be approved by the NRA.
A.3.2.1.5 Tests on control cells used for production of seeds

Tests on control cell cultures should be undertaken as described below in
section A.4.2.1.
A.3.2.1.6 Recombinant baculovirus concentration

Each recombinant baculovirus seed lot should be assayed for infectivity in a
sensitive assay in an insect cell culture system. The detailed procedures for
carrying out the tests and for interpreting the results should be those approved
by the NRA.
A.3.3 Cell culture medium

If serum is used for the propagation of cells, it should be tested to demonstrate
freedom from bacteria, fungi and mycoplasmas – as specified in Part A of the
WHO General requirements for the sterility of biological substances, sections
5.2 (39) and 5.3 (40) – as well as freedom from adventitious viruses.
Detailed guidance on detecting bovine viruses in serum for establishing
MCBs and WCBs are given in Appendix 1 of the WHO Recommendations
for the evaluation of animal cell cultures as substrates for the manufacture of
biological medicinal products and for the characterization of cell banks (7)
and should be applied as appropriate. The guidance provided on detecting
bovine viruses in serum for establishing the cell banks may also be applicable
to production cell cultures. As an additional monitor of quality, sera may be
examined for endotoxin. Gamma irradiation may be used to inactivate potential
contaminant viruses, while recognizing that some viruses are relatively resistant
to gamma irradiation.
Whatever the process used, the validation study has to determine
the consistency and effectiveness of the process while maintaining serum
performance. The use of non-inactivated serum should be justified and is not
advised without strong justification. The non-inactivated serum must meet the
same criteria as the inactivated serum when tested for sterility and absence of
mycoplasmal and viral contaminants.
The source(s) of animal components used in culture medium should be
approved by the NRA. These components should comply with the current WHO
guidelines on transmissible spongiform encephalopathies in relation to biological
and pharmaceutical products (41).
Bovine or porcine trypsin used for preparing cell cultures should be
tested and found free of bacteria, fungi, mycoplasmas and adventitious viruses,
167
as appropriate. The methods used to ensure this should be approved by the
NRA. The source(s) of trypsin of bovine origin, if used, should be approved by
the NRA and should comply with the current WHO guidelines on transmissible
spongiform encephalopathies in relation to biological and pharmaceutical
products (41).
In some countries, irradiation is used to inactivate potential contaminant
viruses. If irradiation is used it is important to ensure that a reproducible
dose is delivered to all batches and to the component units of each batch.
The irradiation dose must be low enough to allow the biological properties
of the reagents to be retained but high enough to reduce virological risk.
Therefore, irradiation cannot be considered a sterilizing process (7). The
irradiation method should be validated and approved by the NRA.
Recombinant trypsin is available and should be considered; however, it

should not be assumed to be free from risk of contamination and should
be subject to the usual considerations for any reagent of biological
origin (7).
Human serum should not be used. If human serum albumin is used at any
stage of product manufacture, the NRA should be consulted regarding the
requirements, as these may differ from country to country. As a minimum,
the use of human serum albumin should meet the WHO Requirements for
the collection, processing and quality control of blood, blood components and
plasma derivatives (42). In addition, human albumin and materials of animal
origin should comply with the current WHO guidelines on transmissible
spongiform encephalopathies in relation to biological and pharmaceutical
products (41).
Penicillin and other beta-lactams should not be used at any stage of the
manufacture because they are highly sensitizing substances.
Other antibiotics may be used in the manufacture provided that the
quantity present in the final lot is acceptable to the NRA.
Non-toxic pH indicators may be added (for example, phenol red at a

concentration of 0.002%). Only substances that have been approved by
the NRA may be added.
A.4 Control of HPV VLP production

A.4.1 Control of HPV VLP production up to single antigen
harvest in yeast and bacterial expression systems
A.4.1.1 Microbial purity
Microbial purity in each fermentation vessel should be monitored at the end of
the production run by methods approved by the NRA.
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Any agent added to the fermentor or bioreactor with the intention to

feed cells or to induce or increase cell density should be approved by the NRA.
No antibiotics should be added at any stage of manufacturing unless approved
by the NRA.
A.4.2 Control of HPV VLP production up to single antigen harvest

in recombinant baculovirus systems in insect cells
Cell cultures are expanded to an appropriate scale and are then inoculated with
recombinant baculovirus at a defined multiplicity of infection (MOI). After
adsorption, the cell cultures are fed with maintenance medium and incubated
within a defined temperature range and for a defined period of time.
The range of MOI, temperature, pH and incubation period will depend
on the insect cell substrate and the specific characteristics of the recombinant
baculovirus strain. A defined range for the MOI should be established by the
manufacturer and approved by the NRA.
A single antigen harvest is obtained within a defined time period post-
inoculation. Several single antigen harvests may be pooled. If multiple single
antigen harvests are pooled, each single antigen harvest should be sampled
for testing, stabilized and stored under suitable conditions until pooling. No
antibiotics should be added at the time of harvesting or at any later stage of
manufacture. Samples of single harvest pools should be taken for testing and
should be stored at a temperature of −60 °C or below.
A.4.2.1 Tests on control cell cultures

When the insect cell suspension is grown to the scale needed for production,
but prior to infection with the recombinant baculovirus, an amount of processed
cell suspension equivalent to at least 5% or 500 ml of cell suspension (whichever
is greater) should be used to prepare control cultures of uninfected cells. If
bioreactor technology is used, the size and treatment of the cell sample to be
examined should be approved by the NRA.
The control cell cultures should be examined microscopically for the
morphological changes of the cells attributable to the presence of adventitious
agents for at least 14 days after the day of inoculation of the production cultures,
or at the time of final virus harvest if this is later. The control cell cultures should
be incubated under conditions that are essentially similar to those used for the
production cultures, with the agreement of the NRA. For insect cells, the above
incubation time may not apply because of the specificities of cells cultivated
in suspension but it should be not less than the time of collection of the single
antigen harvest. At the end of the examination period, fluids collected from the
control cell culture from each single antigen harvest should be tested for the
presence of adventitious agents as described below. Samples that are not tested
immediately should be stored at −60 °C or below.
169
If any test shows evidence of the presence of adventitious agents in

control cell cultures, the single antigen harvests prepared from these cultures
should not be used for HPV VLP production.
For the test to be valid at least 80% of the control cells should still survive
by the end of the test period and any losses should be due to nonspecific or
accidental reasons.
A.4.2.1.1 Tests for haemadsorbing viruses

At the end of the observation period at least 25% of the control cells should be
tested for the presence of haemadsorbing viruses using guinea-pig red blood
cells. If the red blood cells have been stored the duration of storage should not
have exceeded 7 days, and the temperature of storage should have been in the
range of 2–8 °C.
In some countries, the NRA requires that additional tests for
haemadsorbing viruses are to be performed using red blood cells of
other species, including from humans (blood group O), monkeys and/or
chickens (or other avian species).
All haemadsorption tests should be read after incubation for 30 minutes at

0–4 °C, and again after incubation for a further 30 minutes at 20–25 °C.
If a test with monkey red blood cells is performed, readings should also
be taken after a final incubation for 30 minutes at 34–37 °C.
For cells cultivated in suspension, the test for presence of haemadsorbing

viruses is not technically feasible. A test for presence of haemagglutinating
agents using guinea-pig red blood cells is therefore required with spent control
cell culture fluid.
For the tests to be valid not more than 20% of the culture vessels should
have been discarded by the end of the test period and any losses should be due
to nonspecific or accidental reasons.
A.4.2.1.2 Tests for other adventitious agents

At the end of the observation period a sample of the pooled fluid and/or cell
lysate from each group of control cell cultures should be tested for adventitious
agents. For this purpose, 10 ml of each pool should be tested in the same cell
line as used for the production of virus, but not the same batch of cells as those
used for the production of vaccine. In addition, 10 ml samples of each pool
should also be tested in human cells and in a simian kidney cell line.
Each sample should be inoculated into culture vessels of these cell
cultures in such a way that the dilution of the pooled fluid in the nutrient
medium does not exceed 1 in 4. The area of the cells should be at least 3 cm2
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per ml of pooled fluid. At least one culture vessel of each kind of cell culture
should remain un-inoculated as a control.
The inoculated cultures should be incubated at the appropriate growth
temperature and should be observed for cytopathic effects for a period of at
least 14 days.
For the tests to be valid not more than 20% of the culture cells should
have been discarded by the end of the test period and any losses should be due
to nonspecific or accidental reasons.
Some NRAs require that these cells should be tested for the presence of
haemadsorbing viruses at the end of the observation period.
A.4.2.1.3 Test for identity of insect cells

At the production level, the cells should be identified by means of tests
approved by the NRA. Suitable methods include, but are not limited to,
biochemical tests (for example, isoenzyme analyses), cytogenetic tests (for
example, for chromosomal markers) and tests for genetic markers (for example,
DNA fingerprinting or PCR).
A.4.3 Control of HPV VLP production up to single

antigen harvest in mammalian cells
If applicable, tests on control cells for identity and adventitious agents should be
performed in accordance with the WHO Recommendations for the evaluation
of animal cell cultures as substrates for the manufacture of biological medicinal
products and for the characterization of cell banks (7).
A.4.4 Control of single antigen harvests

A.4.4.1 Storage and intermediate hold times
Prior to and during the purification process, the cell suspension or supernatant
fluid should be maintained under conditions shown by the manufacturer
to allow them to retain the desired biological activity. Hold times should be
A.4.4.2 Tests on single antigen harvest or single harvest pool

If appropriate, tests may be conducted on a single antigen harvest or on a pool
of single antigen harvests. The protocol should be approved by the NRA.
A.4.4.2.1 Sampling
Samples required for the testing of single antigen harvests or single harvest
pools should be taken immediately on harvesting and before further processing.
If tests for sterility and adventitious agents, as described below in sections
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A.4.4.2.2 and A.4.4.2.4, are not performed immediately then the samples taken
for these tests should be kept at a temperature of −60 °C or below and subjected
to no more than one freeze–thaw cycle.
A.4.4.2.2 Sterility tests for bacteria, fungi and mycoplasmas

Each single antigen harvest or single harvest pool should be shown to be
free from bacterial and fungal contamination by appropriate tests as specified
in Part A, section 5.2 of the WHO General requirements for the sterility of
biological substances (39).
Harvests from bacterial expression systems could be positive for bacterial
contamination. Therefore, an alternative method such as the microbial
limits test might be appropriate for addressing culture purity. Such testing
should be approved by the NRA.
In addition to sterility tests for bacteria and fungi, each single antigen harvest
or single harvest pool should also be shown to be free from mycoplasmal
contamination by appropriate tests as specified in Part A, section 5.3 of the
WHO General requirements for the sterility of biological substances (40) if
insect or mammalian cells are used in production, or by a method approved by
the NRA.
NAT alone or in combination with cell culture, with an appropriate
detection method, may be used as an alternative to one or both of the
compendial mycoplasma-detection methods following suitable validation
and agreement from the NRA (7).
A.4.4.2.3 Test for identity of HPV types

Each single antigen harvest or single harvest pool should be identified as the
appropriate HPV type by immunological assay or by a molecular biology based
assay (for example, hybridization or PCR). The tests should be approved by
the NRA. Alternatively, the identity can be confirmed as part of testing of the
purified antigen.
A.4.4.2.4 Tests for adventitious agents if insect or mammalian cells are used in production
Each single antigen harvest or single harvest pool should be tested for adventitious
viruses in cell cultures selected for their appropriateness to the origin and
passage history of the insect cell substrate and recombinant baculovirus or the
mammalian cell substrate. These cell cultures should include, as a minimum, a
monkey kidney cell line and a human cell line. Antisera used for the purpose
of neutralizing the recombinant baculovirus should be free from antibodies
that may neutralize adventitious viruses, and should preferably be generated
by the immunization of specific-pathogen-free animals with an antigen made
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from a source (other than the production cell line) which has itself been tested
for freedom from adventitious agents. The inoculated indicator cells should be
examined microscopically for cytopathic changes. At the end of the examination
period, the cells should also be tested for haemadsorbing viruses (see section
A.4.2.1.1 above).
Additional testing for specific adventitious viruses may be performed,
for example by using PCR amplification techniques.
A.5 Control of purified monovalent antigen bulk

The purification process can be applied to a single antigen harvest, part of
a single antigen harvest or a pool of single antigen harvests, and should be
approved by the NRA. The maximum number of harvests that may be pooled
should also be approved by the NRA. Adequate purification may require
several purification steps based on different biophysical and/or biochemical
principles and may involve disassembly and reassembly of VLPs. The entire
process (sequence of process steps) used for the purification of the VLPs should
be appropriately validated, as described above in section A.2, and should be
approved by the NRA. Any reagents added during the purification processes
(such as DNase) should be documented.
The purified monovalent antigen bulk can be stored under conditions
shown by the manufacturer to allow it to retain the desired biological activity.
Intermediate hold times should be approved by the NRA.
A.5.1 Tests on the purified monovalent antigen bulk

Purified monovalent antigen bulks should be subjected to the tests listed below.
Some tests may be omitted if performed on the adsorbed monovalent antigen
bulk. All quality control release tests and specifications for purified monovalent
antigen bulk, unless otherwise justified, should be validated by the manufacturer
and approved by the NRA.
A.5.1.1 Identity
Each purified monovalent antigen bulk should be identified as the appropriate
HPV antigen type by a method suitable for distinguishing between HPV types
(for example, an immunological assay). The test for antigen content may also
serve as the identity test.
A.5.1.2 Purity
The degree of purity of each purified monovalent antigen bulk, and levels of
residual host cell protein, should be assessed by suitable methods. One suitable
method for analysing the proportion of potential contaminating proteins in
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the total protein of the preparation is separation of the proteins by SDS-PAGE

under reducing denaturing conditions. The protein bands within the gel should
be identified by sensitive staining techniques. The protein in each band should
be quantified by densitometric analysis at an appropriate level of sensitivity, in
order to measure the degree of purity.
Host cell protein detection by expression-specific and sensitive EIA
methods may be developed.
A.5.1.3 Protein content

Each purified monovalent antigen bulk should be tested for the total protein
content using a suitable method.
Alternatively, the total protein content may be calculated from
measurement of an earlier process intermediate.
A.5.1.4 Antigen content

The antigen content should be measured on the purified monovalent antigen
bulk or the adsorbed monovalent antigen bulk (see section A.6.3.7 below) by an
appropriate method which is type specific.
The ratio of antigen content to protein content may be calculated and
monitored for each purified monovalent antigen bulk.
VLP vaccine antigen content are not available. Therefore, product-specific
reference preparations may be used.
A.5.1.5 Sterility tests for bacteria and fungi

Each purified monovalent antigen bulk should be tested for bacterial and fungal
sterility, as specified in Part A, section 5.2 of the WHO General requirements
for the sterility of biological substances (39), or by a method approved by

the NRA. Alternatively, this test can be performed on the related adsorbed
monovalent antigen bulks if the purified bulk is not stored prior to adsorption.
A.5.1.6 Percentage of intact L1 monomer

The integrity of L1 protein is a critical quality parameter and should be carefully
monitored. The percentage of intact L1 protein of each purified monovalent
antigen bulk should be assessed by suitable methods. The purity assay (see
section A.5.1.2 above) may also serve to assess the integrity of the L1 monomer.
The percentage of intact L1 monomer is the ratio of the intact L1 monomer to
the total protein expressed as a percentage – that is, intact L1 monomer/(total
L1 + total non-L1) x 100.
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A.5.1.7 VLP size and structure

The size and structure of the VLPs are to be established and monitored. This test
may be omitted for routine lot release once consistency of production has been
established, with the agreement of the NRA.
Suitable methods for assessing VLP size and structure include
dynamic light scattering, size-exclusion chromatography – high-
performance liquid chromatography (SEC–HPLC) and transmission
electron microscopy (TEM).
A.5.1.8 Tests for reagents used during purification or other phases of manufacture
A test should be carried out to detect the presence of any potentially hazardous
reagents used during manufacture, using a method(s) approved by the NRA. This
test may be omitted for routine lot release upon demonstration that the process
consistently eliminates the reagent from the purified monovalent antigen bulks.
A.5.1.9 Tests for residual DNA derived from the expression system
The amount of residual host cell DNA derived from the expression system should
be determined in each purified monovalent antigen bulk by suitably sensitive
methods. The level of host cell DNA should not exceed the maximum level
agreed with the NRA, taking into consideration issues such as those discussed
in the WHO Recommendations for the evaluation of animal cell cultures as
substrates for the manufacture of biological medicinal products and for the
characterization of cell banks (7).
These tests may be omitted for routine lot release upon demonstration
that the process consistently inactivates the biological activity of the residual
DNA or reduces the amount and size of the contaminating residual DNA present
in the purified monovalent antigen bulks, subject to the agreement of the NRA.
A.5.1.10 Bovine serum albumin content

If bovine serum is used in mammalian or insect cell cultures for production then
residual bovine serum albumin content should be measured and a maximum
permitted concentration should be set, and approved by the NRA.
A.5.1.11 Test for viral clearance

When an insect or mammalian cell substrate is used for the production of HPV
antigens, the production process should be validated in terms of its capacity
to remove and/or inactivate adventitious viruses – as described in the Q5A
guidelines (43) of the International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use. This testing
175
is performed during vaccine manufacturing development or as part of process

validation and is not intended as an assessment for lot release.
If a replicating viral vector such as a baculovirus is used then the
production process should be validated for its capacity to eliminate (by removal
and/or inactivation) residual recombinant virus.
A.6 Control of adsorbed monovalent antigen bulk

A.6.1 Addition of adjuvant
The purified monovalent antigens may be adsorbed onto an adjuvant such as an
aluminium salt, in which case the adjuvant and the concentration used should
be approved by the NRA. If an alternative or additional adjuvant such as MPL is
used, this should also be approved by the NRA.
If a novel adjuvant is used that does not involve adsorption of the VLP
to the adjuvant, the term “adjuvanted monovalent antigen bulk” may
be used.
A.6.2 Storage
Until the adsorbed monovalent antigen bulk is formulated into the final bulk,
the suspension should be stored under conditions shown by the manufacturer
to allow it to retain the desired biological activity. Hold times should be
A.6.3 Tests on adsorbed monovalent antigen bulk

All tests and specifications for adsorbed monovalent antigen bulk, unless
otherwise justified, should be approved by the NRA.

Each adsorbed monovalent antigen bulk should be tested for bacterial and fungal
sterility, as specified in Part A, section 5.2 of the WHO General requirements for
the sterility of biological substances (39), or by an alternative method approved
by the NRA.
A.6.3.2 Bacterial endotoxins

Each adsorbed monovalent antigen bulk should be tested for bacterial endotoxins
using a method approved by the NRA.
If it is inappropriate to test the adsorbed monovalent antigen bulk,
the test should be performed on the purified antigen bulk prior to
adsorption, subject to the approval of the NRA.
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A.6.3.3 Identity
Each adsorbed monovalent antigen bulk should be identified as the appropriate
HPV antigen type by a method suitable for distinguishing between HPV types
(for example, an immunological assay). The test for antigen content may also
serve as the identity test.
A.6.3.4 Adjuvant concentration

Adsorbed monovalent antigen bulk may be assayed for adjuvant content until
production consistency is demonstrated.
A.6.3.5 Degree of adsorption

The degree of adsorption (completeness of adsorption) of each adsorbed
monovalent antigen bulk should be assessed, if applicable. This test may be
omitted upon demonstration of process consistency, subject to the agreement
of the NRA.
A.6.3.6 pH
The pH value of the adsorbed monovalent antigen bulk may be monitored until
production consistency is demonstrated, subject to the agreement of the NRA.
A.6.3.7 Antigen content

The antigen content of the adsorbed monovalent antigen bulk should be
measured using appropriate methods. If this test is conducted on purified
monovalent antigen bulk it may be omitted from the testing of the adsorbed
monovalent antigen bulk.
VLP vaccine antigen content are not available. Therefore, product-specific
reference preparations may be used.
A.7 Control of final bulk

The final bulk should be aseptically prepared by combination of the adsorbed
monovalent antigen bulks which pass the tests specified in section A.6.3 above.
The antigen concentration in the final formulation should be sufficient to ensure
that the dose is consistent with that shown to be safe and effective in human
clinical trials. Should an adjuvant be added to the vaccine formulation, this
adjuvant and the concentration used should be approved by the NRA.
The operations necessary for preparing the final bulk should be
conducted in such a manner as to avoid contamination of the product. In
preparing the final bulk vaccine, any substances such as diluents, stabilizers
177
or adjuvants that are added to the product should have been shown to the
satisfaction of the NRA not to impair the safety and efficacy of the vaccine at
the concentration used. The final bulk should be stored under conditions shown
by the manufacturer to allow it to retain the desired biological activity until it is
filled into containers.
A.7.1 Tests on the final bulk

All tests and specifications for final bulk, unless otherwise justified, should be

Each final bulk should be tested for bacterial and fungal sterility, as specified
biological substances (39), or by a method approved by the NRA.
A.7.1.2 Adjuvant content

Each final bulk should be assayed for adjuvant content.
Where aluminium compounds are used, the amount of aluminium
should not exceed 1.25 mg per single human dose.
An example of a suitable test for an adjuvant such as MPL is gas
chromatography.
Tests for adjuvant content on the final bulk may be omitted if conducted
on each final lot derived from the final bulk.
A.7.1.3 Degree of adsorption

The degree of adsorption to the adjuvant (completeness of adsorption) of each
antigen present in each final bulk should be assessed, if applicable (for example,
if the adjuvant is aluminium salts).

This test may be omitted upon demonstration of process consistency or if
performed on the final lot.
A.7.1.4 Preservative content

The final bulk may be tested for the presence of preservative, if added. The
method used and the permitted concentration should be approved by the NRA.
A.7.1.5 Potency
The potency of each final bulk should be assessed with an appropriate in vivo or
in vitro method. If an in vivo potency test is used to test final lots, this test may be
omitted on the final bulk. The methods for detection of antibodies to HPV VLPs
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and the analysis of data should be approved by the NRA. The vaccine potency
should be compared with that of a reference preparation; the NRA should
determine the limits of potency and approve the reference preparation used.
For ethical reasons, it is desirable to apply the 3R principles (Replacement,
Reduction, Refinement) to the use of animals, where scientifically appropriate (44).
A.7.1.6 Osmolality
The osmolality of the final bulk may be tested. The osmolality test may be
omitted if performed on the final lot.
Alternative tests (for example, freezing point) may be used as surrogate
measures for ionic strength/osmolality.
A.8 Filling and containers

The requirements concerning filling and containers given in WHO good
manufacturing practices for biological products (36) should apply to vaccine
filled in the final form.
Care should be taken to ensure that the materials of which the container
– and if applicable the transference devices and closure – are made do not
adversely affect the quality of the vaccine.
Manufacturers should provide the NRA with adequate data to prove the
stability of the product under appropriate conditions of storage and shipping.
A.9 Control tests on the final lot

The following tests should be performed on each final lot (that is, in the final
containers). Unless otherwise justified and authorized, the tests should be
performed on labelled containers from each final lot by means of validated
methods approved by the NRA. All tests and specifications, including methods
used and permitted concentrations, unless otherwise justified, should be
A.9.1 Inspection of containers

Every container in each final lot should be inspected visually or mechanically,
and those showing abnormalities should be discarded and recorded for each
relevant abnormality. A limit should be established for the percentage of
containers rejected.
A.9.2 Appearance
The appearance of the vaccine should be described with respect to its form
and colour.
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A.9.3 Identity
All antigens present in the final lot should be identified by appropriate methods.
The potency test may serve as the identity test.
A.9.4 Sterility tests for bacteria and fungi

Each final lot should be tested for bacterial and fungal sterility, as specified
biological substances (39), or by a method approved by the NRA.
A.9.5 pH and osmolality

The pH value and osmolality of the final lot should be tested. The osmolality
test may be omitted if performed on the final bulk. The osmolality test may also
be omitted for routine lot release upon demonstration of product consistency,
subject to the approval of the NRA.
An alternative test (for example, freezing point) may be used as a surrogate
measure for ionic strength/osmolality.
A.9.6 Preservatives
Each final lot should be tested for the presence of preservative, if added.
A.9.7 Test for pyrogenic substances

Each final lot should be tested for pyrogenic substances. Where appropriate, tests
for endotoxin (for example, the limulus amebocyte lysate (LAL) test) should
be performed. However, where there is interference in the test – for example,
because of the addition of an immunostimulant such as MPL – a test for pyrogens
in rabbits should be performed.
A suitably validated monocyte-activation test may also be considered as

an alternative to the rabbit pyrogen test.
The test is conducted until consistency of production is demonstrated, subject

to the agreement of the NRA.
A.9.8 Adjuvant content

Each final lot should be assayed for adjuvant content, if applicable. Where
aluminium compounds are used, the amount of aluminium should not exceed
1.25 mg per single human dose.
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A.9.9 Protein content

The protein content should be determined. Alternatively this may be calculated
from an earlier process intermediate.
A.9.10 Degree of adsorption

The degree of adsorption to the adjuvant (completeness of adsorption) of each
antigen present in each final vaccine lot should be assessed, if applicable, and the
limit should be approved by the NRA.
This test may be omitted for routine lot release upon demonstration of
product consistency, subject to the approval of the NRA.
A.9.11 Potency
An appropriate quantitative test for potency by an in vivo or in vitro method
should be performed on samples that are representative of each final vaccine
lot. The method and the analysis of data from potency tests should be approved
by the NRA. The vaccine potency should be compared with that of a reference
preparation, and the limits of potency should be agreed with the NRA. The NRA
should approve the reference preparation used. If an in vivo potency test is used,
this test may be omitted on the final bulk. The method of testing for antigen
potency in an in vitro test could be quantitative with respect to the antigen
content or relative to a reference preparation.
Because of the diversity in the reactivity of vaccines containing HPV
VLPs produced by different manufacturing techniques and differences in the
adjuvants used for the vaccine formulation, it is unlikely that International
Standards will be suitable for the standardization of assays of vaccines from all
manufacturers. Consequently, International Standards will not be developed for
the potency of each HPV type. Manufacturers should establish a product-specific
reference preparation that is traceable to a lot of vaccine, or bulks used in the
production of such a lot, which has been shown to be efficacious in clinical trials.
The performance of this reference vaccine should be monitored by trend analysis
using relevant test parameters and the reference vaccine should be replaced when
necessary. An acceptable procedure for replacing reference vaccines should be
in place (45, 46).
A.9.12 General safety (innocuity) test

The need to test the final lots of the HPV vaccine for unexpected toxicity (also
known as abnormal toxicity) should be agreed with the NRA.
Some countries no longer require this test (47).
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A.10 Records
The requirements given in WHO good manufacturing practices for biological
products (36) should apply.
A.11 Retained samples

products (36) should apply.
A.12 Labelling
products (36) should apply, with the addition of the following information.
The label on the carton, the container or the leaflet accompanying the
container should state:
■■ that the vaccine has been prepared from recombinant yeast,

bacterial cells, mammalian cells or recombinant baculovirus/
insect cells;
■■ the type of HPV (from which L1 VLPs were derived) present in
the preparation;
■■ the potency per dose;
■■ the number of doses, if the product is issued in a multiple-dose
container;
■■ the name and maximum quantity of any antibiotic present in the
vaccine;
■■ the name and concentration of any preservative added;
■■ the name and concentration of any adjuvant added;
■■ the temperature recommended during storage and transport;

■■ the expiry date;
■■ any special dosing schedules.
A.13 Distribution and transport

products (36) should apply. Further guidance is provided in the WHO Model
guidance for the storage and transport of time- and temperature-sensitive
pharmaceutical products (48).
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A.14 Stability testing, storage and expiry date

A.14.1 Stability testing
Adequate stability studies form an essential part of vaccine development. Current
guidance on the evaluation of vaccine stability is provided in the WHO Guidelines
on stability evaluation of vaccines (49). Stability testing should be performed at
different stages of production, namely on single antigen harvests or single harvest
pools, purified monovalent antigen bulk, adsorbed monovalent antigen bulk,
final bulk (whenever materials are stored before further processing) and final
lot. Stability-indicating parameters appropriate to the stage of production should
be defined or selected. A shelf-life should be assigned to all in-process materials
during vaccine production – particularly intermediates such as single antigen
harvests, purified monovalent antigen bulk and final bulk.
The stability and expiry date of the vaccine in its final container,
maintained at the recommended storage temperature up to the expiry date,
should be demonstrated to the satisfaction of the NRA using final containers from
at least three final lots made from different adsorbed monovalent antigen bulks.
Given the complexity of these multivalent vaccines, other approaches
may be used with the approval of the NRA.
Accelerated stability tests may be undertaken to give additional information on

the overall characteristics of a vaccine, and may also aid in assessing comparability
when the manufacturer plans to change aspects of manufacturing.
The formulation of vaccine antigens and adjuvant (if used) must be stable
throughout the shelf-life of the vaccine. Acceptable limits for stability should
be agreed with the NRA. Following licensure, ongoing monitoring of vaccine
stability is recommended to support shelf-life specifications and to refine the
stability profile (49). Data should be provided to the NRA in accordance with
local regulatory requirements.
The final stability-testing programme should be approved by the NRA
and should include an agreed set of stability-indicating parameters, procedures
for the ongoing collection and sharing of stability data, and criteria for rejecting
vaccine(s).
A.14.2 Storage conditions

The final lot should be kept at 2–8 °C. If other storage conditions are used, they
should be fully validated and approved by the NRA. The vaccine should have
been shown to maintain its potency for a period equal to that between the date of
release and the expiry date. During storage, liquid vaccines should not be frozen.
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If a vaccine has been shown to be stable at temperature ranges higher

than the approved 2–8 °C range, it may be stored in a controlled temperature
chain for a defined period of time, subject to approval by the NRA.
A.14.3 Expiry date

The expiry date should be based on the shelf-life supported by stability studies
and should be approved by the NRA. The expiry date should be based on the date
of blending of final bulk, date of filling or the date of the first valid potency test
on the final lot.
Where an in vivo potency test is used, the date of the potency test is the
date on which the test animals are inoculated.
Part B. Nonclinical evaluation of recombinant

HPV VLP vaccines
Details of the design, conduct, analysis and evaluation of nonclinical studies
are available in the WHO guidelines on nonclinical evaluation of vaccines (5).
Further guidance on the general principles for nonclinical evaluation of vaccine
adjuvants and adjuvanted vaccines can be found in a separate WHO Guidelines
document (8). In particular, this latter document should be consulted to
determine the need for safety, pharmacology and bio-distribution studies, as
well as the extent of characterizing of the adjuvant alone, should a novel adjuvant
be introduced into the HPV vaccine formulation.
The guidance given in this current section will also apply to new L1 VLP
vaccines containing other HPV types in addition to the types – 16/18 (± 6/11) –
contained in the related licensed vaccine, and to new L1 VLP vaccines containing
types 16/18 (± 6/11) with and without additional types.
Prior to the clinical testing of any new HPV vaccine in humans there
should be extensive product characterization, proof-of-concept immunogenicity

studies and safety testing in animals. The extent of nonclinical evaluation
will depend on the complexity of the vaccine formulation, on a case-by-case
basis. The following specific issues should be considered in the context of the
development of an HPV L1 VLP-based vaccine.
B.1 Product characterization and process development

It is crucially important that vaccine production processes are appropriately
standardized and controlled to ensure consistency in manufacturing, and in the
collection of nonclinical data that may indicate potency and safety in humans.
The extent of product characterization may vary according to the stage of
development. The vaccine lots used in nonclinical studies should be adequately
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representative of the formulation intended for use in clinical investigation and,

ideally, should be the same lots as those used in clinical trials. If this is not feasible,
the lots used in nonclinical studies should be comparable to clinical lots with
respect to physicochemical characteristics and data, stability and formulation.
B.2 Pharmacodynamic studies

There is no adequate, relevant animal model for HPV infection as papillomaviruses
are species specific. The proof-of-concept for the use of L1 VLP-based vaccines has
been demonstrated in animal protection models using “homologous” viruses such
as cottontail rabbit papillomavirus. These preclinical challenge studies in various
animal models have indeed demonstrated that L1 VLPs are potent immunogens
that induce high titres of neutralizing antibodies and protect against associated
lesions (20, 21). Furthermore, transfer of serum from animals vaccinated with L1
VLPs provided protection in non-vaccinated animals challenged with the virus
(20–22). One mechanism-of-protection study in a murine cervicovaginal model
of infection using a surrogate virus or pseudovirion has indicated that exudation
of systemic neutralizing antibodies into the wound bed and binding to the virus
at the basement membrane probably account for the protection (22).
On the basis of these data:
■■ No further challenge studies need to be performed for monovalent
or multivalent HPV L1 VLP vaccine.
■■ Neutralizing antibodies are probably the primary mediator of
protection. It seems unlikely that CMI is substantially involved as a
direct effector mechanism of protection; thus, this aspect does not
need further elucidation to support regulatory approval.
It is recommended that the pharmacodynamic properties of an L1
VLP‑based vaccine should be assessed through immunogenicity studies (for
example, in rodents, rabbits and/or possibly in non-human primates) which
should take into account:
■■ the evaluation and characterization of the serum neutralizing
antibodies induced against each of the HPV L1 VLP types included
in the proposed vaccine – if the HPV vaccine contains new types in
addition to those in a licensed vaccine or a new vaccine formulation
intended for an alternative route of administration (for example,
aerosol), the inclusion of an additional arm(s) in the study design
may generate supportive data;
■■ in the case of the inclusion of a specific adjuvant in the vaccine, the
added benefit with respect to enhancing desired immune response
(humoral and/or cellular – for example, involvement of T-helper
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cells or induction of specific memory cells) to be studied in one or

more relevant species (8);
■■ the generation of supportive data with respect to the relative ratio of
the antigen and the adjuvant included in the vaccine formulation;
■■ the evaluation of serum cross-neutralizing antibodies to a broad
range of HPV types not included but related to those in the
proposed vaccine is optional (see section C.6 below).
B.3 Toxicology studies

Toxicology studies should be undertaken with the final vaccine formulation
in accordance with WHO guidance (5). Such studies should be performed
in relevant animal models, and should reflect the intended clinical use of
the vaccine and may include the administration of doses prior to and during
pregnancy (5). Because the target population for the HPV vaccines includes
women of childbearing age, reproductive and developmental toxicity studies
are required. Some NRAs will require submission of the pivotal data at the
time of the marketing authorization application (8). Repeated-dose toxicity
studies and developmental toxicity studies should include evaluation of the
immune response in a group of vaccinated animals, in addition to appropriate
toxicological end-points, taking into account existing WHO guidelines (5, 8).
Other toxicity studies, such as an inhalation toxicity study, may be required if
the vaccine is developed for administration through the aerosol route.
If a novel cell substrate (that is, a substrate that has not previously been
used to produce a licensed human vaccine or biological product) is used for
the production of an HPV VLP vaccine then safety aspects – such as potential
immune responses elicited by residual host cell proteins – should be investigated
in a suitable animal model. Such studies should be undertaken particularly if
the final product contains an adjuvant that might enhance responses to low
levels of residual proteins.
Part C. Clinical evaluation of recombinant

HPV VLP vaccines
C.1 Introduction
Clinical studies should adhere to the principles described in the WHO
Guidelines for good clinical practice (GCP) for trials on pharmaceutical
products (50) and the WHO Guidelines on clinical evaluation of vaccines:
regulatory expectations (6).
This section refers throughout to L1 VLP vaccines that contain at least
HPV types 16 and 18 and considers the clinical data that may be generated both
prior to initial licensure and post-licensure in different settings.
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Three types of clinical data can provide an indication of the ability of a

candidate HPV vaccine to prevent anogenital carcinomas:
■■ immunological data as discussed in section C.2 below;
■■ virological data as discussed in section C.3 below;
■■ histological data as discussed in section C.4 below.
Section C.5 summarizes the type(s) of data that may be used to
demonstrate vaccine efficacy in different settings.
These four sections (C.2–C.5) take into account the fact that histological
primary efficacy end-points have diminishing feasibility as a result of the impact
of HPV vaccines on the occurrence of infections due to types 16 and 18 and
the lower prevalence of other oncogenic HPV types in high-grade intraepithelial
neoplasms and cancers. Experience gained with the first two HPV vaccines has
supported the use of type-specific viral persistence as an alternative primary
efficacy end-point where there has been no routine HPV vaccination or where
uptake has been low. Neither histological nor viral-persistence end-points are
likely to be feasible for types 16 and 18 in countries that have included HPV
vaccines in their routine programmes with high uptake rates. These end-points
are also not expected to be feasible for documenting efficacy against other HPV
types that are much less commonly, and sometimes rarely, associated with pre-
malignant lesions. Consequently, in some settings an assessment of vaccine
efficacy can be based only on immunological data.
In addition:
■■ Section C.6 considers the data that could support claims for
protection against HPV types that are not included in a candidate
vaccine (that is, cross-protection).
■■ Section C.7 considers the assessment of safety within clinical studies.
■■ Section C.8 considers the assessment of antibody persistence,
vaccine effectiveness and safety in routine use.
C.2 Immunological data

Naturally acquired neutralizing (that is, functional) antibody to HPV has been
demonstrated to afford some type-specific protection (51). HPV L1 VLP vaccines
are thought to protect against persistent viral infection by eliciting neutralizing
antibody that can be measured in serum. Studies with intramuscular HPV L1
VLP vaccines have found an overall correlation between antibody levels in serum
and in cervical mucosal fluids (52–56). However, the antibody concentrations
in serum or at the site of infection that are required for protection are unknown
– that is, no immune correlate of protection (ICP) has been established for
HPV vaccines.
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The initial assessment of immune responses to HPV L1 VLP vaccines

should be based on measurement of neutralizing antibodies in serum
using a pseudovirion-based neutralization assay. Measurement of antibody
concentrations at anogenital sites (for example, in cervical mucosal fluid) is
not required.
C.2.1 Assays
Throughout any one clinical development programme it is preferable that the
same assays for determination of anti-HPV antibody are used and that testing is
conducted at a designated central laboratory. Ideally, the same approach should
apply to post-licensing investigations of antibody persistence. This uniformity
becomes essential within any one study and when attempting to compare
immune responses between studies. The assay (or assays, if a change in assay
during clinical development was unavoidable) used to generate the immune-
response data included in the application dossier should be fully validated. The
details and results of the validation exercise(s) should be provided.
In vitro neutralizing antibody assays involve measurement of the
inhibition of HPV pseudovirus infection of cultured cells and usually employ
type-specific pseudovirions carrying a marker plasmid to allow infected cells to
be scored easily. These neutralizing assays require expression plasmid constructs
for L1 and L2 for each viral type, and assay standardization relies on use of the
same source for these constructs. The WHO Human papillomavirus laboratory
manual (24) includes a method for HPV neutralizing assays that has shown good
inter-laboratory performance.
However, neutralization assays are labour intensive, technically
complex and not currently amenable to high throughput. Therefore, following
characterization of the neutralizing antibody response to a candidate HPV
vaccine, the use of alternative assay methods that are less technically demanding
(for example, type-specific competitive Luminex immunoassay (cLIA) or EIA)
may be acceptable subject to demonstration of strong correlation between the

results of these assays and neutralizing assays (57).
Competitive immunoassays utilizing neutralizing monoclonal antibodies
that bind to conformational epitopes on L1 are type specific and sensitive, detect
all immunoglobulin classes and do not measure antibodies to denatured L1
protein (57). However, only a subset of the total anti-VLP antibodies is measured
since binding to only one neutralizing epitope is monitored. Therefore, if these
assays are used it should be kept in mind that the results may under-represent
the total level of protective antibodies in sera.
VLP-based EIAs require the use of conformationally intact VLPs
as antigens to ensure type specificity. These assays detect all antibodies of a
specific immunoglobulin class (generally immunoglobulin G) that bind to the
VLPs. While both neutralizing and non-neutralizing antibodies are detected,
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the correlation with neutralizing assays has been generally good because the
strongest host response to vaccines developed to date is to neutralizing epitopes.
Laboratories performing HPV serology testing have to prepare and conduct
quality-control approaches for their own VLPs because no commercial assays
are available. The inability of laboratories to access common key source reagents
for serology assays presents significant challenges to the standardizing of HPV
serology results.
International Standards for serum antibodies to HPV type 16 and HPV
type 18 are available to help improve the comparability of results. The use of the
parallel-line method with standards calibrated to the International Standard is
described in the WHO Human papillomavirus laboratory manual (24) and has
been shown to improve inter-laboratory comparisons. Antibody levels should be
reported in International Units (IU) for HPV types for which an International
Standard is available. It should be kept in mind that the comparison of titres
between HPV types is not appropriate. For each assay the lower limit of detection
(LLOD) and lower limit of quantification (LLOQ) should be clearly established,
along with a justification of the cut-off applied to differentiate samples that are
reported to be seropositive and seronegative.
C.2.2 Characterization of the immune response

During the initial phase of the clinical development programme it is
recommended that the following should be documented for each HPV type in
a candidate vaccine:
■■ immune responses to the vaccine, with a focus on seroconversion
rates in subjects who were seronegative prior to vaccination;
■■ pre- to post-vaccination changes in geometric mean titres (GMTs)
and reverse cumulative distributions (these should be presented
separately for subjects who were seropositive or seronegative prior
to vaccination).
The following should be evaluated in clinical studies, unless the sponsor
demonstrates that the candidate vaccine is very similar to a licensed vaccine so
that the same sera collection times and dose regimens may apply:
■■ The kinetics of the immune response (that is, changes in antibody
levels in response to sequential doses) should be determined
to support the timing of the post-vaccination serological primary
end point.
■■ There should be an adequate exploration of immune responses to
support the number of doses and dose interval selected for various
age groups.
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Inclusion of an adjuvant, whether novel or already included in licensed

vaccine(s), should be supported by data demonstrating enhancement of the
immune response to one or more HPV types, and by an assessment of the
potential clinical significance of the antibody levels achieved in the target age
range (8). Since there is no established ICP for HPV vaccines, the potential
clinical significance of the effect of an adjuvant could be assessed by comparing
antibody levels achieved by the candidate vaccine with and without the adjuvant
with those elicited by a licensed vaccine for which efficacy has been documented
(see section C.2.3 below).
If the adjuvant in a candidate vaccine is not identical to one in any
type of licensed vaccine for which there is already substantial information
available on the mechanism of immune enhancement, its effect on the humoral
and cellular immune response (for example, involvement of T-helper cells or
induction of specific memory cells) should be extensively characterized.
Currently, there is no evidence indicating the need for booster doses
after completion of a primary series with HPV L1 VLP vaccines. Nevertheless,
this remains an open question until such time as very long-term data on vaccine
effectiveness are available (for example, more than 10 years), including in
subjects who were vaccinated several years prior to sexual debut. Consequently,
consideration could be given to a pre-emptive assessment of immune responses
to booster doses administered to relatively small subsets at planned intervals
following completion of the primary series (58). These data may be provided
after initial licensure and may also give an indirect indication of priming by the
primary series (see section C.8.1 below).
It is important for antibody levels to be measured at several pre-planned
time points after the final vaccine dose in order to construct antibody decay
curves that are sufficient to document the achievement of a plateau. These
data can be collected post-licensing during long-term follow-up of antibody
persistence in selected vaccinated cohorts (see section C.8.1 below).

The potential for immune interference between HPV VLP vaccines
and other vaccines likely to be given concomitantly should be investigated pre-
and/or post-licensing. The WHO Guidelines on clinical evaluation of vaccines:
regulatory expectations (6) should be consulted regarding the design and
analysis of these studies.
C.2.3 Analyses of immune responses in comparative studies

In different studies or within the same study – and depending on circumstances
that limit the feasibility of some options, as discussed below – the immune
response to a candidate vaccine may be compared with one or more of:
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■■ another HPV vaccine, in which case there may be some HPV types
in both vaccines (shared types) and some that are in one vaccine
only (unshared types);
■■ the same candidate vaccine but administered at a different dose,
different schedule or in a different population;
■■ another formulation of the candidate vaccine (for example, with and
without an adjuvant or with variable numbers of HPV types);
■■ data obtained from a group that does not receive HPV vaccine (that
is, a group that receives either a placebo or a non-HPV vaccine).
In each case, it is recommended that:
■■ Due to their clinical importance, comparisons of immune responses
should strongly support a conclusion that vaccine efficacy against
HPV types 16 and 18 is very likely to be comparable to that observed
for the two initially developed HPV vaccines.
■■ The primary analysis population for immune responses to each HPV
type is confined to those who are seronegative for the particular
HPV type at baseline. Therefore, the sample-size calculations should
also take into account the anticipated HPV type-specific baseline
seropositivity in the population under study.
■■ Primary comparisons should be based on antibody titres in sera
obtained at 1 month after the final dose of the intended regimen(s)
unless antibody-kinetic data suggest otherwise. If a test regimen
consists of a different number of doses from the control regimen
(for example, 2 versus 3 doses) or if the last dose is given at a
different time point (for example, at 4 months versus 6 months after
the first dose) then the primary comparison should still be based
on sera obtained at 1 month (or other time point based on kinetics)
after the final dose, whenever that occurs. Secondary analyses
should compare antibody titres measured at predefined time points
from the first dose, including a comparison once antibody levels
have reached a plateau.
C.2.3.1 Comparison with a group that does not receive HPV vaccine
Given the widespread licensing of HPV vaccines and their incorporation into
routine vaccination programmes in many countries, studies of sexually active
men or women that include a group that does not receive HPV vaccine will
be unacceptable in many settings. Comparisons with a group that does not
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receive HPV vaccine may still be possible in short-term immunogenicity studies

in which subjects are considered to be at low or no risk of HPV infection (for
example, prior to sexual debut) and in which all subjects eventually receive
active vaccination within a protocol-specified time frame. Nevertheless, before
selecting this design, the potential need for and value of comparing a candidate
vaccine with a control group that does not receive HPV vaccine should be
given careful consideration and should be balanced with ethical considerations
regarding the withholding of a safe and effective vaccine.
Post-vaccination seropositivity rates and seroconversion rates in
previously seronegative subjects have been very high with the initial HPV
vaccines, and these are sensitive end-points for studies that compare vaccinated
groups with groups that do not receive HPV vaccine. Consequently, these studies
should aim to demonstrate superiority of seroconversion rates to each HPV
type in the candidate vaccine group over the control group that does not receive
HPV vaccine. The predefined criteria for concluding superiority should take
into account the type-specific seroconversion rates that have been observed for
the initial HPV vaccines for which efficacy was demonstrated on the basis of
histological and virological data (59, 60).
The same approach is applicable whenever comparing immune
responses to HPV types that are included in a candidate HPV vaccine but are
not in the control HPV vaccine (that is, unshared types) since the control group
is not vaccinated with respect to these types.
C.2.3.2 Comparisons between vaccinated groups

Due to the expectation that post-vaccination seropositivity rates and
seroconversion rates will be very high in previously seronegative subjects, these
are not sensitive end-points for discriminating between immune responses in
vaccinated groups. Therefore GMT ratios for immune responses to each HPV
type that is included in the candidate vaccine(s) and control HPV vaccine(s)
(that is, shared types) are usually used for the primary comparisons between
vaccinated groups. HPV type-specific seroconversion rates should be included
among the secondary end-points.
As a general rule, for the purposes of establishing non-inferiority
between vaccine groups based on GMT ratios for antibody to individual HPV
types, it is suggested that the lower bound of the 95% confidence interval
around the GMT ratio (test versus reference vaccine) should not fall below 0.67.
Under certain circumstances, NRAs may consider allowing a lower bound of
0.5. In future, especially if an ICP can be identified or if a sponsor is able to
offer a sound rationale, it may be appropriate to reconsider these acceptance
criteria. In addition, any marked separations between the reverse cumulative
distributions should be discussed in terms of the potential clinical implications,
even if these occur only at the lower or upper ends of the curves.
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C.2.4 Immunobridging of vaccine efficacy

Although there is no ICP for HPV vaccines, it is sometimes appropriate or
necessary to use immunobridging to evaluate efficacy against anogenital
carcinomas, preneoplastic lesions and genital warts.
Immunobridging involves a demonstration of comparable immune
responses between a candidate vaccine and a licensed vaccine that has been
selected in accordance with the recommendations made below in section
C.2.4.1. In this way:
■■ the HPV type-specific efficacy observed with one HPV vaccine
may be bridged to an alternative schedule (for example, reducing
the number of doses or delaying the final dose) or population (for
example, children prior to sexual debut in whom efficacy cannot be
assessed), or to an extended version of that same vaccine (that is,
with added HPV types);
■■ the efficacy observed with a specific licensed HPV vaccine may be
bridged to a candidate vaccine for all HPV types that are shared
between the two vaccines.
The comparison of immune responses should be made in accordance
with the recommendations outlined above in section C.2.3.2. A successful
demonstration of non-inferiority in each age and gender subgroup relevant to the
indications approved for the control vaccine could support the same indications
for the candidate vaccine – that is, including cervical, anal and vulvo-vaginal
lesions. Nevertheless, when the inference of vaccine efficacy is based only on
immunobridging data, individual NRAs may decide to restrict the indications
of candidate vaccines compared with those of licensed comparator vaccines.
There may be situations in which the predefined non-inferiority margin
is not met for one or more HPV types. Since the implications of such a finding
for clinical efficacy cannot be determined, the possible effect on vaccine efficacy
and effectiveness will have to be considered on a case-by-case basis, taking into
account any possible merits of the candidate vaccine and/or test regimen (for
example, a reduced or more convenient schedule, or use in a specific age group
that facilitates incorporation into existing routine schedules) as well as the
relative importance of the HPV type(s) in question and/or their related cross-
reactive types prevalent in an individual NRA’s jurisdiction.
If non-inferiority is not demonstrated for one or more HPV types based
on immune responses determined shortly after the last dose, a predefined analysis
that demonstrates non-inferiority at later time points might be considered as an
alternative – for example, a comparison at 18–30 months after the last vaccine
dose, when current experience suggests onset of a plateau effect on the antibody
decay curves. However, as time progresses since the last vaccine dose, the
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difference between GMTs is likely to diminish, reducing the analytical sensitivity.

In addition, basing the comparison on antibody levels at 18–30 months after the
last dose would considerably delay the initial licensure of a vaccine.
C.2.4.1 Selection of the comparator vaccine

It is crucially important that immunobridging approaches to assess potential
vaccine efficacy are based on appropriate comparator vaccines. In principle,
the comparator vaccine should have been licensed on the basis of histological
or, at least, virological primary end-points. However, in some regulatory
jurisdictions, it may not be possible to license a candidate vaccine on the basis of
immunobridging to the efficacy demonstrated by another HPV vaccine within
a specific number of years after the comparator was first licensed. Therefore
the selection of comparator vaccine(s) should be discussed with the relevant
NRAs of the countries in which the candidate vaccine is intended for use. In
most instances it will be appropriate to compare the candidate vaccine with
a licensed vaccine that contains the same HPV types or as many shared HPV
types as possible.
In future it may be that immune responses to a candidate vaccine cannot
be directly compared with those to a vaccine that was previously shown to have
efficacy against disease-related end-points because it is no longer marketed.
For example, the original version of a vaccine that was licensed on the basis
of histological and/or virological data may have been replaced by a modified
version (for example, with additional HPV types). Using the modified version
in immunobridging studies becomes a problem if it does not elicit very similar
immune responses to the HPV types in the original version. As a result, there
is a risk that modified versions could be less immunogenic and potentially less
protective than the original vaccines that were developed. This inescapable fact
highlights the vital importance of post-licensure documentation of vaccine
effectiveness (see section C.8.2 below).
C.2.4.2 Immunobridging in specific circumstances

For the first two HPV vaccines to be developed, immunobridging was used to
support their use in children aged 9–15 years. Some NRAs have subsequently
accepted immunobridging to support a reduction from 3 to 2 doses in
specific age subsets (55, 61–69). In each of these cases, licensure was based on
demonstrating non-inferiority of immune responses in children to those in
female vaccinees in the age range within which efficacy had been demonstrated
following a 3-dose schedule.
In these bridging studies, the immune responses to HPV types 16
and 18 were higher in boys than in girls aged 9–15 years (64, 70). In contrast,
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immune responses to some HPV types have been lower in men than in women
of comparable ages, and lower in women aged 26–45 years compared to women
aged 15–26 years (71, 72). Therefore, whenever an immunobridging approach
is used, it is relevant to consider possible issues of interpretation based on age
range and gender (see section C.5 below).
An immunobridging approach to support use in immunosuppressed
populations is complicated by the likelihood of observing lower immune
responses compared to those in healthy subjects (73–75). The implications of
lower immune responses for vaccine efficacy are uncertain, as an ICP has not
been established. NRAs will have to consider whether the anticipation of some
degree of benefit in immunosuppressed populations, even if potentially lower
than in immunocompetent subjects, is sufficient to support a favourable risk–
benefit conclusion.
C.3 Virological data

The reliable determination of HPV infection and viral persistence at anogenital
sites requires the use of carefully controlled and standardized methods of sample
collection, processing and virus detection.
C.3.1 Sampling
Because HPV is cell associated, samples must contain cellular material and
separate samples must be obtained from each specific anatomical site of interest.
Methods of sample collection that have been validated in large-scale
epidemiological studies are recommended. The specific method used (for
example, in terms of number of turns and depth of insertion of the device)
should be standardized and adhered to for each study. Ideally, no changes to
the method should be made during each study or during the entire clinical
development programme. If changes are unavoidable, there should be adequate
cross-validation to support the pooling of results obtained with different
methods. The collection medium will influence the volume of sample to be
extracted as well as the method of extraction. Water or collection-medium
blanks should be processed and tested along with samples to ensure that no
cross-contamination occurs during processing (24).
The standard approach for monitoring HPV in the cervix is for samples
to be collected from the ecto-cervix and endo-cervix by clinicians after
visualization of the cervix using speculum examination (24). Although a range
of collection devices may be used, they should target the cervical transformation
zone and each device should be compatible with the selected collection medium.
Alternative methods (such as sampling only from either the ecto-cervix or
the endo-cervix, or self-sampling by study participants) may be considered if
appropriate validation is provided.
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C.3.2 Virus detection

The assays used for the determination of virological end-points in studies
intended to support licensure should provide type-specific results with high
sensitivity and specificity, and should include an internal control to monitor the
adequacy of samples (76, 77). The internal control, directed to a cellular DNA
sequence, establishes a lower limit for acceptable cell number but will not evaluate
the quality of the cellular sample. For instance, it will not distinguish between
inflammatory cells (unintended sample) and epithelial cells (intended sample).
Participating in proficiency tests, when available, and obtaining
satisfactory results is one step towards ensuring the quality of the assay (77). It
should be noted that proficiency tests that use purified DNA will not monitor
extraction efficiency, impact of possible PCR inhibitors in samples or competition
between types with large differences in copy number. The WHO Human
papillomavirus laboratory manual (24) provides guidance on quality assurance
and quality control aspects of HPV DNA testing to laboratories performing
testing for virological end-points to help ensure stability and standardization of
results over time.
C.3.3 Viral persistence

As discussed above under General considerations, the vaccine efficacy studies
that were conducted with the first HPV vaccines in sexually active young women
demonstrated a close correlation between the primary histological end-point
of high-grade cervical disease and 6-month persistent infection of the cervix
with HPV (3, 78–82). On this basis, and taking into account the similarity of
disease pathogenesis and progression at cervical and anal sites, viral persistence
based on at least two consecutive samples obtained over at least 6 months is an
acceptable alternative to histological end-points at these anatomical sites (3).
Nevertheless, HPV type-specific viral persistence is a feasible primary end-
point only when rates of persistent infections in vaccinees who were HPV DNA
negative at study baseline for the types in question are sufficiently high to allow
for studies of reasonable size. This is not expected to be a feasible end-point for
studies in populations in which HPV vaccines have been introduced into routine
immunization programmes and in which there has been a very high uptake.
It is also not likely to be a feasible end-point for HPV types that are rarely
encountered, regardless of any vaccine usage.
The demonstration of viral persistence should be based on consecutive
type-specific HPV DNA positive samples obtained from the same anatomical
site over at least 6 months from the time of the first positive result. Because the
timing of incident infections after completion of the vaccination series cannot be
predicted, an event-driven analysis is often employed. Thus, the primary analysis
is conducted when a protocol-defined number of total cases of viral persistence
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(which may be based on HPV types 16 and 18 or otherwise defined by types) has
been accumulated. This total number should be estimated to provide sufficient
statistical power to detect meaningful differences between treatment groups.
In the primary analysis, the cases of viral persistence should be counted from
a predefined period (for example, at least 2 weeks) after the final dose of the
vaccination series. A secondary analysis could be based on counting all cases
from the time of the first dose. Protocols should address how to handle sequential
results when at least one of a series of samples is negative for HPV DNA but is
followed by one or more positive samples.
C.3.3.1 Comparison with a group that does not receive HPV vaccine
For the same reasons discussed above in section C.2.3.1, studies of viral
persistence that include a group that does not receive HPV vaccine will be
unacceptable in many settings. If such a design is still considered acceptable and
if incidence rates are sufficiently high to make the study feasible (that is, due to
lack of widespread implementation of HPV vaccination in the regions where
the study will be conducted) then a superiority design could be used.
Depending on what is known about incidence rates, there could be co-
primary end-points of viral persistence for each of HPV types 16 and 18 or a
single composite primary end-point based on viral persistence for both types. For
candidate vaccines containing additional HPV types, a primary analysis could be
based on pooled data for HPV types 16 and 18 with a co-primary or secondary
analysis based on viral-persistence data pooled for all other HPV types and
supportive analyses of viral persistence for each HPV type. Separate or combined
studies addressing cervical and anal sites and/or by gender could be considered.
C.3.3.2 Comparisons between HPV vaccines

As discussed above, it is much more likely that another HPV vaccine containing
types 16 and 18, with or without additional types, would have to be administered
to the control group. Assuming that the candidate vaccine has good efficacy,
experience obtained with the first two HPV vaccines indicates that few, if any,
cases of viral persistence of any HPV type will occur in any reasonable time
frame, and it may be necessary to resort to an immunobridging approach for
assessment of vaccine efficacy. Nevertheless, whenever viral persistence is
assessed among the study end-points, the following approaches are suggested:
■■ Although persistent infection due to HPV types 16 or 18 is not likely
to be a feasible primary end-point, all cases should be monitored
in case the candidate vaccine has unexpectedly low efficacy (for
example, there could be a negative impact of additional HPV types
on the protection against HPV types 16 and 18 afforded by a highly
multivalent candidate vaccine).
197
■■ For any additional HPV types shared between the candidate and
comparator vaccines, supportive analyses should compare viral-
persistence data pooled across all shared types as well as for each
individual type.
■■ For any HPV types in the candidate vaccine only, viral persistence
may be pooled across the additional types, but supportive analyses
should be conducted for individual types.
C.4 Histological data

As discussed above in sections C.2 and C.3:
■■ Viral-persistence end-points can be used instead of histological end-
points to support the efficacy of a candidate vaccine.
■■ The acceptability of a control group that does not receive HPV
vaccine is rapidly diminishing.
■■ The feasibility of using histological end-points is influenced by
the same issues that affect viral-persistence end-points – that is,
decreasing rates of events due to widespread vaccine use and very
low rates of lesions associated with some HPV types regardless of
any vaccine usage.
If histological data are to be collected in a study, it is recommended that
the end-points used in the pivotal efficacy studies that were conducted with the
two initial HPV vaccines in women and with the one in men should be used
– that is, incident high-grade anogenital lesions associated with positivity for
oncogenic HPV types (59, 60, 83).
C.5 Evaluation of vaccine efficacy in different settings

This section summarizes approaches to assessing potential vaccine efficacy
against anogenital carcinomas in different settings. The following issues should

be noted:
■■ Depending on the local circumstances (that is, whether any HPV
vaccines have been licensed, the types of HPV L1 VLP they contain
and whether they have been incorporated into routine vaccination
programmes), individual NRAs may have alternative requirements
to the recommendations made in this section.
■■ For reasons discussed above in section C.4, histological end-points
are not considered.
■■ In all settings, only serological data can be obtained from subjects
prior to sexual debut.
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■■ This section does not cover the assessment of efficacy against genital
warts. For a candidate HPV vaccine containing types 6 and 11, the
considerations regarding whether genital warts is a feasible end-
point are the same as those outlined in section C.3.3.1 for viral
persistence as an end-point. In all other settings, efficacy against
genital warts will have to be based on demonstrating similar immune
responses to these two HPV types between the candidate vaccine
and a suitable control vaccine, as outlined in section C.2.4.
C.5.1 Efficacy against HPV types 16 and 18

Efficacy may be evaluated by one of the following approaches:
■■ viral persistence under the circumstances described in section C.3.3.1
■■ immunobridging to a licensed vaccine, as described in section C.2.4.
C.5.2 Efficacy against other HPV types

An evaluation of efficacy against other HPV types associated with anogenital
carcinomas is necessary in the following situations:
■■ Addition of one or more HPV type(s) associated with anogenital
carcinomas to a licensed vaccine that contains at least HPV types
16 and 18 L1 VLPs. In this case, comparisons should be made with
the original licensed vaccine and/or another licensed vaccine that
contains as many shared types as possible.
■■ De novo development of a vaccine that contains HPV types 16
and 18 plus additional types, in which case all the additional types
may already be included in a licensed vaccine, or one or more of
the additional types may not be present in any licensed vaccine.
Comparisons could be made with one or more of the following:
(a) a group that does not receive HPV vaccine, if this option is still
appropriate; (b) a licensed vaccine that contains HPV types 16 and
18 but does not include the additional types that are in the candidate
vaccine; (c) a licensed vaccine that contains as many shared types
as possible.
C.5.2.1 Shared HPV types

Efficacy may be evaluated by one of the following approaches:
■■ viral persistence, as described above in section C.3.3.2;
■■ demonstration of non-inferiority of immune responses to HPV
types shared between the candidate and comparator vaccines, as
described above in section C.2.3.2.
199
C.5.2.2 Unshared HPV types

■■ For HPV types in the candidate vaccine but not in the comparator
vaccine, there is the possibility of demonstrating superiority for the
candidate vaccine on the basis of a composite viral-persistence end-
point (either all additional types or all types), as described above
in section C.3.3.1. In reality, the incidence of viral persistence due
to the unshared types may be so low that it precludes this approach
because of the sample size that would be needed.
■■ If the approach described above is not feasible, the assessment of
efficacy of the unshared types can be based only on immunological
data. There are no sound interpretive criteria that can be
recommended to infer vaccine efficacy. Nevertheless, one approach
may be to demonstrate that the seroconversion rate to each unshared
type is non-inferior to whichever is lowest of the seroconversion
rates to HPV types 16 and 18 in the licensed vaccine group. It is not
appropriate to base the comparisons on the GMT ratios because
these are known to be very variable between types.
C.5.3 Modification(s) of the mode of use

Comparisons of immunological data, as described above in sections C.2.3 and
C.2.4, may be used to support licensure in the following settings:
■■ changes in schedule or age range for a licensed HPV vaccine;
■■ changes in formulation and/or route of administration of a vaccine
that includes L1 VLPs manufactured exactly as for the original
version of a licensed vaccine;
■■ de novo development of a candidate vaccine with a different age
range or schedule from that of licensed HPV vaccines, or specifically
formulated for a route of administration different from that of

licensed HPV vaccines.
Additional comments regarding specific situations are provided below.
C.5.3.1 Extending the age range downwards

The use of HPV vaccines from the age of 9 years was supported by
immunobridging, as described above in section C.2.4.2. In the absence of an
ICP that might be applied to interpret long-term antibody persistence data it
has been assumed that vaccination from 9 years of age will provide protection
in the years following sexual debut on the basis of observations that initial post-
vaccination antibody titres fall to a plateau that is maintained after several years.
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Annex 4
If a vaccine is proposed for use from < 9 years of age, it is recommended

that the potential value of vaccination even earlier in life be discussed with
NRAs. If this strategy is pursued, it is recommended that immune responses in
male and female subjects in the new target age range (for example, 6 to < 9 years)
are directly compared with those in men and women aged 15–26 years – that
is, in the age group in which efficacy has been demonstrated on the basis of
histological and virological end-points. A comparison between children aged
< 9 years and those aged 9 to < 15 years should not be the basis for licensure
since efficacy has not been established in the latter age range.
C.5.3.2 Extending the age range upwards

Studies using histological or viral-persistence end-points have been conducted
in women aged 26–45 years but not in men aged > 26 years. Interpretation of the
available data by individual NRAs has varied. As a result, different approaches
have been taken to the prescribing information in terms of whether to impose
an upper age limit or to omit any upper age limit for one or both sexes.
It is not expected that efficacy can be demonstrated in individuals
> 26 years of age on the basis of prevention of HPV-associated advanced
dysplastic lesions or cervical cancer. As the routine use of HPV vaccines increases,
the feasibility of demonstrating efficacy based on viral persistence will decrease.
An immunobridging approach may not succeed since immune responses may
be naturally lower as age increases. As a result, it is not possible to recommend
an approach to support licensure of HPV vaccines for use in subjects older than
26 years. NRAs must decide on a case-by-case basis whether additional data
would be needed to include older subjects in the indications for use, taking into
account what seems feasible and the public health considerations applicable to
the region.
C.6 Cross-protection
The sponsor may choose to assess the ability of a vaccine to elicit cross-reacting
neutralizing antibody against non-vaccine HPV types that are closely related to
the types included in a vaccine. However, experience indicates that these data
cannot be used to establish the ability of a vaccine to confer cross-protection (84).
Thus far, claims for cross-protection against related HPV types not
included in a vaccine have been based on relatively short-term histological
and viral-persistence data. Since the assessment of specific epitopes that
elicit cross-protection is not part of the HPV L1 VLP vaccine specifications,
the degree of cross-protection may be very vaccine specific. Currently, or in
the future, it may not be feasible to demonstrate cross-protection based on
virological persistence. Reliance on immunobridging to a licensed vaccine is not
straightforward because:
201
■■ available data suggest that cross-neutralizing antibody may not be

predictive for cross-protection;
■■ there would have to be a case-by-case evaluation of the validity of
extrapolating cross-protection claims between products based on
the possibility that small differences between epitopes could have an
impact on cross-protection.
Therefore, if viral persistence cannot be used as an end-point, it does not
appear that future cross-protection claims can be supported.
C.7 Safety
The general considerations for the pre-licensure assessment of safety during
clinical studies are those outlined in the WHO Guidelines on clinical evaluation
of vaccines: regulatory expectations (6).
In the specific case of HPV vaccines, it is important that the safety
database adequately covers the entire target age range and both sexes, as
applicable to the authorized indications. However, it is not necessary to generate
sufficient safety data to estimate frequencies of uncommon adverse events
in each subset. The numbers vaccinated within each sex and age subgroup
should be supported by discussion of any anticipated differences that could
preclude assumptions of similar safety profiles according to the characteristics
of vaccinees (for example, if the reactogenicity profile seems to be very different
between men and women or between adults and younger subjects).
Regardless of protocol recommendations for studies conducted among
sexually active women, numerous pregnancies have been documented in
vaccinees. Every effort should be made to estimate the stage of gestation in
relation to vaccine doses and to document the outcome of the pregnancy.
Specific studies in pregnant women are not recommended at this time (85).
Assessment of safety in the post-licensure period is discussed below in
section C.8.3.
C.8 Post-licensing evaluation

C.8.1 Immunological follow-up
The duration of immunological follow-up to document antibody persistence at
the time of initial licensure may be very limited. At the time of licensure, plans
should be in place to document antibody persistence to each HPV type in the
vaccine in the longer term. These data should be obtained from subsets of
subjects who were initially vaccinated across the entire age range of use, and for
both sexes. The final duration of follow-up should be agreed with NRAs on the
basis of knowledge of antibody decay curves and of the magnitude of the initial
immune response to vaccination (see section C.2.2 above).
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Annex 4
As discussed in section C.2.2, since it is not yet known whether

additional doses of vaccine may be needed after completion of the primary
series to assure prolonged protection, an assessment of immune responses to
booster doses at planned intervals following completion of the primary series
should be conducted (58).
C.8.2 Vaccine effectiveness and related issues

It is important to obtain data on vaccine effectiveness and the risk of an HPV
type-replacement phenomenon in the post-licensing period. Some NRAs
consider that collection of these data is a responsibility of the licence holder.
Nevertheless, the generation of reliable data usually requires activation of
large-scale public health initiatives and disease monitoring on a national or
regional basis (for example, the use of cancer registries to capture the effect of
HPV vaccines on rates of HPV-related anogenital carcinomas). Thus, the post-
licensing commitments that individual NRAs may require from licence holders
are expected to relate to cooperative efforts with appropriate bodies (for example,
public–private partnerships, where these are deemed to be acceptable).
In some regions where routine vaccination against HPV is not yet
established there may be specific oncogenic HPV types that are detected
relatively frequently (with or without HPV types 16/18 or other types) in
association with pre-cancerous anogenital lesions and anogenital carcinomas.
Nevertheless, for HPV vaccines that contain the specific type(s) in question,
pre-licensure estimates of vaccine efficacy based on histological or viral-
persistence end-points may not be available. In such regions, especially when
claims for efficacy against these types are based on immunological data only,
the responsible NRAs may consider it particularly important to obtain vaccine-
effectiveness data.
If a licensed vaccine has been granted claims for cross-protection based
on relatively short-term histological and viral-persistence data (see section C.6
above), there should be further follow-up to confirm these claims in the post-
licensure period.
C.8.3 Safety monitoring

The general considerations for safety surveillance and for the development of a
pharmacovigilance plan are the same as for all other types of vaccine (6).
Where HPV vaccines have been introduced into routine vaccination
programmes they are mostly administered to children prior to sexual debut.
However, initial catch-up programmes that include older subjects have sometimes
resulted in inadvertent vaccination during early pregnancy. Safety surveillance
programmes should capture the outcomes of these pregnancies (85).
203
There may be some instances in which NRAs require specific post-

licensing safety studies to be conducted by the licence holder in order to address
particular concerns. The design and time frame for reporting these studies
should be agreed at the time of initial licensure.
Part D. Recommendations for NRAs

D.1 General recommendations
The general recommendations for NRAs and NCLs given in the WHO
Guidelines for national authorities on quality assurance for biological products
(86) and Guidelines for independent lot release of vaccines by regulatory
authorities (87) should apply. These recommendations specify that no new
biological substance should be released until consistency of batch manufacturing
and quality has been demonstrated.
The detailed production and control procedures, as well as any significant
changes in them that may affect the quality, safety and efficacy of recombinant
human papillomavirus virus-like particle vaccines, should be discussed with and
For control purposes, the relevant International Standards currently
in force should be obtained for the purpose of calibrating national, regional
and working standards (46). The NRA may obtain from the manufacturer the
product-specific or working reference to be used for lot release.
Consistency of production has been recognized as an essential
component in the quality assurance of recombinant human papillomavirus
virus-like particle vaccines. In particular, the NRA should carefully monitor
production records and quality control test results for clinical lots, as well as for
a series of consecutive lots of the vaccine.
D.2 Official release and certification

A vaccine lot should be released only if it fulfils all national requirements and/or
satisfies Part A of these WHO Recommendations (87).
A protocol for the manufacturing and control of recombinant human
papillomavirus virus-like particle vaccines, based on the model protocol provided
in Appendix 1, and signed by the responsible official of the manufacturing
establishment, should be prepared and submitted to the NRA in support of a
request for the release of a vaccine for use.
A lot release certificate signed by the appropriate NRA official
should then be provided if requested by a manufacturing establishment, and
should certify whether or not the lot of vaccine in question meets all national
requirements and/or Part A of these WHO Recommendations. The certificate
204
Annex 4
should provide sufficient information on the vaccine lot. The purpose of this
official national release certificate is to facilitate the exchange of vaccines between
countries, and should be provided to importers of the vaccines. A model NRA
Lot Release Certificate is provided in Appendix 2.
Authors and acknowledgements

The first draft of this document was prepared by a WHO drafting group
comprising Dr G. Coleman, Health Canada, Canada; Dr M. Lennon (Ferguson),
Consultant, Horning, the United Kingdom; Dr M. Powell, Medicines and
Healthcare Products Regulatory Agency, the United Kingdom; Dr J. Roberts,
United States Food and Drug Administration Center for Biologics Evaluation and
Research, the USA; Dr R. Sheets, Consultant, Silver Spring (MD), the USA; Dr Y.
Sun, Paul-Ehrlich-Institut, Germany; Dr E. Unger, Centers for Disease Control
and Prevention, the USA; Dr Y. Wang, National Institutes for Food and Drug
Control, China; Dr D. Wilkinson, National Institute for Biological Standards
and Control, the United Kingdom; and Dr T.Q. Zhou, Dr I. Knezevic and Dr U.
Fruth, World Health Organization, Switzerland, taking into consideration the
discussions of a WHO drafting group meeting held in Geneva, Switzerland,
29–30 April 2014.
A second draft was then prepared by the same authors listed above
based on email comments submitted by the following reviewers during the
period 31 July–15 September 2014: Dr A. Azhari, PT Bio Farma, Indonesia;
Dr N. Bachtiar, PT Bio Farma, Indonesia; Dr J. Dillner, Karolinska Institute,
Sweden; Dr S. Franceschi, International Agency for Research on Cancer,
France; Dr R. Herrero, International Agency for Research on Cancer, France;
Dr L. Markowitz, Centers for Disease Control and Prevention, the USA; Dr P.
Neels University of Namur, Belgium; Dr N. Nurainy, PT Bio Farma, Indonesia;
Dr A. Saah, Merck Research Laboratories, the USA; Dr J. Schiller, National
Cancer Institute, the USA; Dr V.D. Tsu, PATH, the USA; Dr S. Wang, Centers
for Disease Control and Prevention, the USA.
The third draft was prepared by Dr M. Lennon (Ferguson), Consultant,
Horning, the United Kingdom; Dr M. Powell, Medicines and Healthcare Products
Regulatory Agency, the United Kingdom; Dr R. Sheets, Consultant, Silver
Spring (MD), the USA; Dr T.Q. Zhou, World Health Organization, Switzerland;
and other members of the WHO drafting group, taking into consideration the
discussions of an informal consultation on WHO Recommendations to assure
the quality, safety and efficacy of recombinant human papillomavirus virus-
like particle vaccines held in Geneva, Switzerland, 11–12 November 2014 and
attended by: Dr J. Chen (Developing Countries Vaccine Manufacturers Network
(DCVMN) representative), INNOVAX, China; Dr G. Coleman, Health Canada,
205
Canada; Dr C. D.R.Z. Blades, Agência Nacional de Vigilância Sanitária, Brazil;

Mr. C.H. Damasceno Cabral (DCVMN representative), Instituto Butantan, Brazil;
Dr V.D. Tsu (Representative of other organizations), PATH, the USA; Dr J. Dillner,
Karolinska Institute, Stockholm, Sweden; Dr R. Forsythe (International Federation
of Pharmaceutical Manufacturers & Associations (IFPMA) representative),
GlaxoSmithKline Biologicals, Belgium; Dr R. Herrero, International Agency
for Research on Cancer, Lyon, France; Dr C. Jiang (DCVMN representative),
Biotechnology Company for High and New Technology, China; Dr W.
Kong (DCVMN representative), Biotechnology Company for High and New
Technology, China; Dr I. Kukimoto, National Institute of Infectious Diseases,
Japan; Dr M. Lennon (Ferguson), Consultant, Horning, the United Kingdom;
Dr X. Liang (DCVMN representative), Shanghai Zerun Biotechnology Co. Ltd.,
Shanghai, China; Dr L. Markowitz, Centers for Disease Control and Prevention,
the USA; Dr D. Nardelli-Haefliger, University Hospital, Lausanne, Switzerland;
Dr P. Neels, Consultant, University of Namur, Belgium; Dr E. Nkansah (African
Vaccine Regulatory Forum representative), Food and Drugs Authority, Ghana;
Dr S.Y. Oh, National Institute of Food and Drug Safety Evaluation, Republic of
Korea; Dr S. Phumiamorn, Ministry of Public Health, Thailand; Dr M. Powell,
Medicines and Healthcare Products Regulatory Agency, the United Kingdom;
Dr A. Saah (IFPMA representative), Merck Research Laboratories, the USA;
Dr L. Santos Ramalho Evangelista, Agência Nacional de Vigilância Sanitária,
Brazil; Dr J. Schiller, National Cancer Institute, the USA; Mr U. Shaligram
(DCVMN representative), Serum Institute of India Ltd, India; Dr R. Sheets,
Consultant, Silver Spring (MD), the USA; Dr L. Shi (DCVMN representative),
Shanghai Zerun Biotechnology Co., Ltd, China; Dr R. Simalango, Directorate
of Drug and Biological Product Evaluation, Indonesia; Dr F. Struyf (IFPMA
representative), GlaxoSmithKline Biologicals, Belgium; Dr Y. Sun, Paul-Ehrlich-
Institut, Germany; Dr I. Uhnoo, Public Health Agency of Sweden, Sweden;
Dr E. Unger, Centers for Disease Control and Prevention, the USA; Dr J.L.
Valdez Reyes, Comisión para la Protección contra Riesgos Sanitarios, Mexico;

Dr Y. Wang, National Institutes for Food and Drug Control, China; Dr D.
Wilkinson, National Institute for Biological Standards and Control, the United
Kingdom; Dr T. Wu (DCVMN representative), INNOVAX, Xiamen, China; and
Dr U. Fruth, Dr I. Knezevic, Dr O.C. Lapujade and Dr T.Q. Zhou, World Health
Organization, Switzerland.
The fourth draft was prepared by Dr M. Lennon (Ferguson), Consultant,
Horning, the United Kingdom; Dr M. Powell, Medicines and Healthcare
Products Regulatory Agency, the United Kingdom; Dr T.Q. Zhou, World
Health Organization, Switzerland and other members of the WHO drafting
group following comments received from Dr J. Dillner, Karolinska Institute,
Sweden; Dr R. Herrero, International Agency for Research on Cancer, France;
206
Annex 4
Dr I. Kukimoto, National Institute of Infectious Diseases, Japan; Professor D.

Nardelli-Haefliger, University Hospital, Lausanne, Switzerland; Dr A. Saah,
Merck Research Laboratories, the USA; Dr J. Schiller, National Cancer Institute,
the USA; Drs F. Struyf and R. Forsythe, GlaxoSmithKline Biologicals, Belgium;
Dr I. Uhnoo, Public Health Agency of Sweden, Sweden; Dr E. Unger, Centers for
Disease Control and Prevention, the USA; Dr D. Wilkinson, National Institute
for Biological Standards and Control, the United Kingdom.
The document WHO/BS/2015.2252 was prepared by Dr M. Lennon
(Ferguson), Consultant, Horning, the United Kingdom and Dr T.Q. Zhou,
World Health Organization, Switzerland, and the following WHO drafting group
members: Dr G. Coleman, Health Canada, Canada; Dr M. Powell, Medicines
and Healthcare Products Regulatory Agency, the United Kingdom; Dr J. Roberts,
United States Food and Drug Administration Center for Biologics Evaluation and
Research, the USA; Dr R. Sheets, Consultant, Silver Spring (MD), the USA; Dr Y.
Sun, Paul-Ehrlich-Institut, Germany; Dr E. Unger, Centers for Disease Control
and Prevention, the USA; Dr Y. Wang, National Institutes for Food and Drug
Control, China; Dr D. Wilkinson, National Institute for Biological Standards
and Control, the United Kingdom, following public consultation on the fourth
draft document posted on the WHO Biologicals website during February and
March 2015, and taking into consideration comments provided by: Dr P. Dull,
Bill & Melinda Gates Foundation, the USA; Dr S. Gairola, Serum Institute of
India Ltd, India; Dr L. Markowitz, Centers for Disease Control and Prevention,
the USA; Dr A. Saah, Merck Research Laboratories, the USA; Dr J. Schiller,
National Cancer Institute, the USA; Dr H.J. Sharma, Serum Institute of India Ltd,
India; Dr E. Unger, Centers for Disease Control and Prevention, the USA; Dr Y.
Wang, National Institutes for Food and Drug Control, China; and the IFPMA
(comments consolidated by Dr D. Abraham, Merck & Co., Inc., the USA).
Acknowledgments are also due to the following experts who provided
written comments in response to the posting of WHO/BS/2015.2252 on the
WHO Biologicals website during July and September 2015 for further public
consultation: Dr A. Kato, National Institute of Infectious Diseases, Japan; Dr S.
Morgeaux, Agence Nationale de Sécurité du Médicament et des Produits de
Santé, France; Dr D. Pratt, United States Food and Drug Administration Center
for Biologics Evaluation and Research, the USA; Dr J. Roberts, United States
Food and Drug Administration Center for Biologics Evaluation and Research,
the USA; Dr A. Saah, Merck Research Laboratories, the USA; Dr L. Shi, Shanghai
Zerun Biotechnology Co., Ltd, China; Z. Kusynová, International Pharmaceutical
Federation, Netherlands; and the IFPMA (comments consolidated by Dr D.
Abraham, Merck & Co., Inc., the USA).
207
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the HPV PATRICIA Study Group. Cross-protective efficacy of HPV-16/18 AS04-adjuvanted
vaccine against cervical infection and precancer caused by non-vaccine oncogenic HPV types:
4-year end-of-study analysis of the randomised, double-blind PATRICIA trial. Lancet Oncol.
2012;13(1):100–10
(abstract: http://www.thelancet.com/journals/lanonc/article/PIIS1470-2045%2811%2970287-X/
fulltext, accessed 23 December 2015).
82. Radley D, Saah A, Stanley M. Persistent infection with human papillomavirus 16 or 18 is strongly
linked with high-grade cervical disease. Hum Vaccin Immunother. 2015; in press (abstract: http://
www.tandfonline.com/doi/full/10.1080/21645515.2015.1088616, accessed 24 January 2016).
83. Palefsky JM, Giuliano AR, Goldstone S, Moreira Jr ED, Aranda C, Jessen H et al. HPV vaccine
against anal HPV infection and anal intraepithelial neoplasia. N Engl J Med. 2011;365:1576–85
(http://www.nejm.org/doi/pdf/10.1056/NEJMoa1010971, accessed 23 December 2015).
84. Einstein MH, Baron M, Levin MJ, Chatterjee A, Fox B, Scholar S et al. Comparison of the
immunogenicity of the human papillomavirus (HPV)-16/18 vaccine and the HPV-6/11/16/18
vaccine for oncogenic non-vaccine types HPV-31 and HPV-45 in healthy women aged 18–45
years. Hum Vaccin. 2011;7(12):1359–73 (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338933/,
85. Vichnin M, Bonanni P, Klein NP, Garland SM, Block SL, Kjaer SK et al. An overview of quadrivalent
human papillomavirus vaccine safety: 2006 to 2015. Pediatr Infect Dis J. 2015;34(9):983–91
(abstract: http://www.ncbi.nlm.nih.gov/pubmed/26107345, accessed 23 December 2015).
86. Guidelines for national authorities on quality assurance for biological products. In: WHO
Expert Committee on Biological Standardization: forty-second report. Geneva: World Health
Organization; 1992: Annex 2 (WHO Technical Report Series, No. 822; http://www.who.int/
biologicals/publications/trs/areas/biological_products/WHO_TRS_822_A2.pdf?ua=1, accessed 23
December 2015).
87. Guidelines for independent lot release of vaccines by regulatory authorities. In: WHO Expert
2013: Annex 2 (WHO Technical Report Series, No. 978; http://www.who.int/biologicals/expert_
committee/TRS_978_61st_report.pdf?ua=1, accessed 23 December 2015).
215
Appendix 1
Model protocol for the manufacturing and control of
recombinant human papillomavirus virus-like particle
vaccines
The following protocol is intended for guidance. It indicates the information that
should be provided as a minimum by the manufacturer to the NRA. Information
and tests may be added or omitted as necessary with the approval of the NRA.
It is possible that a protocol for a specific product may differ in
detail from the model provided here. The essential point is that all relevant
details demonstrating compliance with the licence and with the relevant
WHO Recommendations for a particular product should be provided in the
protocol submitted.
The section concerning the final lot must be accompanied by a sample of
the label and a copy of the leaflet (package insert) that accompanies the vaccine
container. If the protocol is being submitted in support of a request to permit
importation, it must also be accompanied by a lot release certificate from the
NRA or from the NCL in the country in which the vaccine was produced or
released, stating that the product meets national requirements as well as the
recommendations in Part A of this document.
Summary information on the final lot

International name:
Trade name/commercial name:
Product licence (marketing authorization) number:
Country:
Name and address of manufacturer:
Name and address of licence holder, if different:
Final lot
Batch number(s)
Final lot:
Final bulk:
Type of container:
Total number of filled containers in this final lot:
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Annex 4
Number of doses per container:

Antigen concentration/volume of single human dose:
Production information
Batch number of each monovalent bulk
(purified and/or adsorbed):
Site of manufacture of each monovalent bulk:
Date of manufacture of each monovalent bulk:
Site of manufacture of adjuvant(s):
Date of manufacture of adjuvant(s):
Site of manufacture of final bulk:
Date of manufacture of final bulk:
Site of manufacture of final lot:
Date of manufacture of final lot:
Date on which last determination of potency was started or
date of start of period of validity:
Shelf-life approved (months):
Expiry date:
Storage conditions:
Release date:
A genealogy of the lot numbers of all vaccine components used in the formulation of
the final lot will be informative.
The following sections are intended for the reporting of the results of the tests
performed during the production of the vaccine, so that the complete document
will provide evidence of consistency of production. Therefore, if any test has to
be repeated this must be indicated. Any abnormal result must be recorded on a
separate sheet.
Starting materials
The information requested below is to be presented on each submission. Full details
on master and working seed lots, and cell banks are requested upon first submission
only and whenever a change has been introduced.
Control of source materials (section A.3)

Cell cultures for antigen production (section A.3.1)
Source of HPV antigen (expression system):
Master cell bank (MCB) lot number and
preparation date:
217
Population doubling level (PDL) of MCB:

Date of approval of protocols indicating compliance with
the requirements of the relevant monographs and
with the product licence:
Manufacturer’s working cell bank (WCB) lot number

and preparation date:
Population doubling level (PDL) of manufacturer’s WCB:
the requirements of the relevant monographs and with
the product licence:
Production cell lot number:
Identification of cell substrate

Method:
Specification:
Date:
Result:
Nature and concentration of antibiotics or selecting agent(s) used
in production cell culture maintenance medium:
Identification and source of starting materials used in preparing

production cells, including excipients and preservatives
(particularly any materials of human or animal origin such as
albumin or serum):
Virus seed lots if a recombinant baculovirus

expression vector is used (section A.3.2)
Sufficient detail should be provided on inoculum intermediates, including the

passage level from the master seed and the length and conditions of storage, if any.
Virus strain and reference number used to prepare the
licensed HPV vaccine:
Master seed lot number and preparation date:
Number of passages between two seeds mentioned above:
Working seed lot number and
preparation date:
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Annex 4
Passage level from master seed lot:

Each seed lot should be tested for the following:

Identity
Method:
Specification:
Date:
Result:
Bacteria and fungi

Method:
Media:
Volume inoculated:
Date of start of test:
Date of end of test:
Result:
Mycoplasmas, spiroplasma, entomoplasma and mesoplasma

Method:
Volume inoculated:
Result:
Adventitious agents
Method:
Specification:
Date:
Result:
Mycobacterium spp. (if applicable)

Method:
Media:
Volume inoculated:
Result:
219
Recombinant baculovirus concentration

Method:
Specification:
Date:
Result:
Control cell cultures if mammalian or insect cells are used for production and
recombinant viral vector cannot be neutralized, thus interfering with testing.
Provide information on control cells corresponding to each single harvest (section

A.3.2.1.5).
Ratio or proportion of control to production

cell cultures:
Volume of control cells:
Dates of observation of cultures:
Percentage rejected for nonspecific reasons:
Result:
Karyotype
Method:
Probe:
Reference cells:
Result:
Identity test by DNA fingerprinting (if applicable)

Method:
Probe:
Reference cells:
Restriction enzymes:
Result:
Haemadsorbing viruses
Type(s) of red blood cell (RBC):
Storage time and temperature of RBC:
Incubation time and temperature of RBC:
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Annex 4
Percentage cultures tested:

Result:
Tests on supernatant fluids or cell lysates for other

adventitious agents (if relevant)
Date of sampling from production cell cultures:
Type of simian cells

Quantity of sample inoculated:
Incubation temperature:
Percentage of viable culture at the end:
Result:
Type of human cells

Result:
Type(s) of other cells

Result:
Bacteria and fungi

Method:
Media:
Volume inoculated:
Result:
221
Mycoplasmas
Method:
Volume inoculated:
Result:
Single antigen harvests (or pools) (section A.4)

Batch number(s):
Date of inoculation:
Date of harvesting:
Volume(s) of cell suspension/paste or supernatant,
storage temperature, storage time and
approved storage period:
Culture purity or sterility for bacteria and fungi

Method:
Media:
Volume inoculated:
Result:
Identity of HPV type

Method:
Specification:
Date:
Result:
Consistency of yield (for example, infectivity of replicating vector

virus and/or HPV antigen concentration, if applicable)
Method:
Reference preparation:
Specification:
Date:
Result:
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Annex 4
In addition, information on the following tests should be

provided if mammalian cells or insect cells are used:
Adventitious agents
Method:
Specification:
Date:
Result:
Mycoplasmas
Method:
Volume inoculated:
Result:
Purified monovalent antigen bulk (section A.5)

Batch number(s) of purified bulk:
Date(s) of purification(s):
Volume(s), storage temperature, storage time and
Identity (if applicable)

Method:
Specification:
Date:
Result:
Composition (protein, lipid, polysaccharide, if applicable)

Method:
Specification:
Date:
Result:
Protein purity (add PAGE photographs)

Method:
Specification:
Date:
Result:
223
Protein content
Method:
Specification:
Date:
Result:
Antigen content (if applicable)

Method:
Specification:
Date:
Result:
Ratio of antigen:protein content (if applicable)

Specification:
Result:
Bacteria and fungi

Method:
Media:
Volume inoculated:
Result:
Percentage intact L1 monomer

Method:
Specification:
Date:
Result:
VLP size and structure

Report on this is needed until production consistency is demonstrated
Method:
Specification:
Date:
Result:
Tests for reagents used during purification and other

phases of manufacture (if relevant)
Method:
Specification:
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Annex 4
Date:
Result:
Residual DNA (if applicable)

Method:
Specification:
Date:
Result:
Bovine serum albumin content (if mammalian or insect cells and animal serum are
used for production).
Method:
Specification:
Date:
Result:
Viral clearance
This is performed during vaccine manufacturing development and/or process
validation and is not intended for batch release (see section A.5.1.11).
Method:
Specification:
Date:
Adsorbed monovalent antigen bulk (section A.6)

Batch number(s) of adsorbed monovalent antigen bulk:
Adsorption date:
Batch number(s) of all components used during
adjuvant adsorption:
Volume, storage temperature, storage time and
Bacteria and fungi

Method:
Media:
Volume inoculated:
Result:
225
Bacterial endotoxins
Method:
Specification:
Date:
Result:
Identity
Method:
Specification:
Date:
Result:
Adjuvant concentration (if relevant)

Method:
Specification:
Date:
Result:
Degree of adsorption (if applicable)

Method:
Specification:
Date:
Result:
pH
Method:
Specification:
Date:
Result:
Antigen content (if applicable)

Method:
Batch number of reference vaccine and
assigned potency:
Specification:
Date:
Result:
Final bulk (section A.7)

Batch number:
Date of manufacture:
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Annex 4
Batch numbers and volumes of adsorbed bulk vaccines used

for the formulation of the final bulk vaccine:
Batch number(s) and volume(s) of bulk alum diluent:
Volume, storage temperature, storage time and

Bacteria and fungi

Method:
Media:
Volume inoculated:
Result:
Adjuvants
Method:
Specification:
Date:
Result:
Degree of adsorption (if applicable)

Method:
Specification:
Date:
Result:
Preservatives (if applicable)

Method:
Specification:
Date:
Result:
Potency
If an in vitro assay of each type is used
Method:
assigned potency:
Specification:
Date:
Result:
227
If an in vivo assay is used

Species, strain, sex and weight specifications:
Dates of vaccination, bleeding:

Date of assay of each type:
assigned potency:
Vaccine doses (dilutions) and number of animals
responding at each dose for each type:
ED50 of reference and test vaccine for each type:
Potency of test vaccine versus reference vaccine for each

type with 95% confidence limits of mean:
Validity criteria for each type:
Osmolality (if applicable)

Method:
Specification:
Date:
Result:
Freezing point (if applicable)

Method:
Specification:
Date:
Result:
Final lot (section A.9)

Batch number:
Date of filling:
Type of container:
Filling volume:
Number of containers after inspection:
Appearance
Method:
Specification:
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Annex 4
Date:
Result:
Identity (each type)

Method:
Specification:
Date:
Result:
Bacteria and fungi

Method:
Media:
Volume inoculated:
Result:
pH
Method:
Specification:
Date:
Result:
Osmolality (if applicable)

Method:
Specification:
Date:
Result:
Freezing point (if applicable)

Method:
Specification:
Date:
Result:
Preservatives (if applicable)

Method:
Specification:
Date:
Result:
229
Pyrogenic substances
Method:
Specification:
Date:
Result:
Adjuvant content
Method:
Specification:
Date:
Result:
Protein content (or calculated value)

Method:
Specification:
Date:
Result:
Degree of adsorption of each type (if applicable)

Method:
Specification:
Date:
Result:
Potency
If an in vitro assay of each type is used
Method:
assigned potency:
Specification:
Date:
Result:
If an in vivo assay is used

Species, strain, sex and weight specifications:
Dates of vaccination, bleeding:

Date of assay of each type:
assigned potency:
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Annex 4
Vaccine doses (dilutions) and number of animals

responding at each dose for each type:
ED50 of reference and test vaccine for each type:
Potency of test vaccine versus reference vaccine for each

type with 95% fiducial limits of mean:
Validity criteria for each type:
Date of start of period of validity:
General safety (unless omission authorized)

Method:
Specification:
Date:
Result:
Certification by the manufacturer

Name of Head of Quality Control (typed)
Certification by the person from the control laboratory of the manufacturing
company taking overall responsibility for the production and quality control of
the vaccine.
I certify that lot no. of recombinant human papillomavirus
virus-like particle vaccine, whose number appears on the label of the final
containers, meets all national requirements and/or satisfies Part A1 of the 2015
WHO Recommendations to assure the quality, safety and efficacy of recombinant
human papillomavirus virus-like particle vaccines.2
Signature
Name (typed)
Date
Certification by the NRA

If the vaccine is to be exported, attach the NRA Lot Release Certificate (as
shown in Appendix 2), a label from a final container and an instruction leaflet
for users.
1
With the exception of provisions on distribution and shipping, which the NRA may not be in a position
to assess.
2
WHO Technical Report Series, No. 999, Annex 4.
231
Appendix 2
Model NRA Lot Release Certificate for recombinant
human papillomavirus virus-like particle vaccines
Certificate No.
The following lot(s) of recombinant human papillomavirus virus-like particle

vaccine produced by 1
in ,2
whose numbers appear on the labels of the final containers, meet all national
requirements 3 and Part A4 of the 2015 WHO Recommendations to assure the
quality, safety and efficacy of recombinant human papillomavirus virus-like
particle vaccines 5 and comply with WHO good manufacturing practices for
pharmaceutical products: main principles; 6 WHO good manufacturing practices
for biological products; 7 and Guidelines for independent lot release of vaccines
by regulatory authorities.8
The release decision is based on 9
The certificate may include the following information:

■■ name and address of manufacturer;
■■ site(s) of manufacturing;
■■ trade name and common name of product;
■■ marketing authorization number;
■■ lot number(s) (including sub-lot numbers and packaging lot
numbers if necessary);
1
Name of manufacturer.
2
Country of origin.
3
If any national requirements have not been met, specify which one(s) and indicate why the release of
the lot(s) has nevertheless been authorized by the NRA.
4
With the exception of provisions on distribution and shipping, which the NRA may not be in a position
to assess.
5
6
7
8
9
Evaluation of the summary protocol, independent laboratory testing and/or procedures specified in a
defined document etc., as appropriate.
232
Annex 4
■■ type of container used;

■■ number of doses per container;
■■ number of containers or lot size;
■■ date of start of period of validity (for example, manufacturing date)
and expiry date;
■■ storage conditions;
■■ signature and function of the person authorized to issue the
certificate;
■■ date of issue of certificate;
■■ certificate number.
The Director of the NRA (or other appropriate authority)

Name (typed)
Signature
Date
233
Annex 5
Guidelines on the stability evaluation of vaccines for use
under extended controlled temperature conditions
1. Introduction 238
2. Scope 239
3. Terminology 239
4. General considerations for the evaluation of vaccines for use
under ECTC 241
5. Stability evaluation of vaccines for use under ECTC 245
6. Monitoring ECTC 250
7. Suggested product labelling information for use under ECTC 251
8. Authors and acknowledgments 252
9. References 255
Appendix Product-specific ECTC evaluation of a model monovalent
polysaccharide conjugate vaccine 257
235
Guidelines published by WHO are intended to be scientific and

advisory in nature. Each of the following sections constitutes guidance
for national regulatory authorities (NRAs) and for manufacturers of
biological products. If an NRA so desires, these WHO Guidelines may
be adopted as definitive national requirements, or modifications
may be justified and made by the NRA.
236
Annex 5
Abbreviations
CTC controlled temperature chain
ECTC extended controlled temperature conditions
EU ELISA Unit(s)
& Associations
LB lower bound
LL lower limit
MRP minimum release potency
NLT not less than
NMR nuclear magnetic resonance
NMT not more than
PS polysaccharide
237
1. Introduction
Vaccines are complex biological products and may undergo degradation during
long-term storage under cold chain conditions (for example, 2–8 °C) and this
is typically enhanced at higher temperatures. Consequently, establishing the
stability characteristics of products is a critical element of the overall evaluation
by a national regulatory authority (NRA) to ensure that licensed vaccines
remain efficacious at the end of their shelf-life when stored under the approved
conditions. In response to the stability assessment needs identified by NRAs,
WHO developed guidelines on the stability evaluation of vaccines to assist its
Member States (1). While it is well understood that vaccine quality depends
on cold chain storage, it is also recognized that immunization programmes in
certain regions face substantial challenges in maintaining cold chains in the
field, especially during the final stage of distribution in remote areas (2, 3). To
address these distribution challenges and expand immunization programmes
into specific regions WHO developed a “controlled temperature chain” (CTC)
programme. This programme currently requires that a vaccine exhibits a stability
profile suitable for a single exposure to at least 40 °C for a minimum of 3 days
just prior to administration, while remaining compliant with the approved
vaccine specifications. Additionally, the programme requires that the CTC
provision should be included in the licensure by the relevant NRA and by WHO
prequalification (4).
During the development of these WHO Guidelines, the term “extended
controlled temperature conditions” (ECTC) was proposed to distinguish
regulatory requirements from WHO CTC programme aspects. This terminology
convention is used throughout the following guidance. An ECTC assessment
should assure the performance of a vaccine following short-term exposure
to temperatures above those of a typical cold chain and could consider any
temperature above the traditional 2–8 °C cold chain that might support
vaccine distribution. Thus ECTC is independent of the specific programmatic

requirements of the current WHO CTC programme. Vaccines licensed for
use under ECTC are required to have sufficient information on the approved
conditions (such as maximum temperature and time) on the package insert.
An example of an approved ECTC product that is also compliant with
WHO CTC programme requirements is the meningitis A conjugate vaccine
MenAfriVac. ECTC evaluation and subsequent label approval has made it
possible to distribute this vaccine to populations that would otherwise have
been difficult to immunize because of the limited availability of traditional cold
chains (5, 6). ECTC labelling allows greater flexibility in vaccination campaigns
by reducing the burden on health-care workers and, once the vaccine is removed
from the cold chain to allow for immunization in remote areas, saving the costs
of further refrigeration infrastructure and eliminating the need for wet ice.
238
Annex 5
Additionally, this on-label NRA-approved approach under the ECTC avoids

off-label vaccine administration which is inconsistent with official guidance on
best practice (7).
These WHO Guidelines arose from WHO immunization programme
requirements (2, 3) and from the resulting discussions of international vaccine
stability experts at WHO-sponsored consultations in Ottawa, Canada (8)
and Langen, Germany (9). The ECTC guidance provided here is intended to
supplement the broader WHO Guidelines on stability evaluation of vaccines
(1) and focuses on ECTC-specific issues not covered in existing guidance
with as little overlap as possible. The key elements of this document are the
applications of the mathematical modelling and statistical concepts in existing
stability guidance (1) and related publications (10, 11) to the unique short-
term requirements that apply to some cases of vaccine distribution and use
(8, 9). Early dialogue between manufacturers and regulators, as well as with
public health officials in immunization programmes, is recommended so that
those vaccines compatible with ECTC use can be evaluated for licensure by the
appropriate NRA.
These Guidelines should be read in conjunction with the existing WHO
Guidelines on stability evaluation of vaccines (1). The guidance which follows
takes the form of WHO Guidelines rather than Recommendations because
vaccines represent a heterogeneous class of agents and the stability testing
programme will need to be adapted to suit the product in question. WHO
Guidelines allow greater flexibility than Recommendations with respect to
specific issues related to particular vaccines.
2. Scope
This document provides guidance to NRAs and manufacturers on the scientific
and regulatory issues to be considered in evaluating the stability of vaccines for
use under ECTC. Evaluation criteria are provided for the approval of short-term
temperature conditions, in addition to those defined for long-term storage of
a given vaccine, in situations where the vaccine is exposed to these short-term
conditions immediately prior to administration.
This document does not provide guidance on the stability evaluation of
vaccines that are inadvertently or repeatedly exposed to temperatures for which
they were not licensed.
3. Terminology
The definitions given below apply to the terms as used in these WHO Guidelines.
They may have different meanings in other contexts.
239
Accelerated stability studies: studies designed to determine the impact

over time of exposure to temperatures higher than those recommended
for storage on the physical, chemical, biological, biopharmaceutical and
microbiological characteristics of a vaccine. When the accelerated temperature
conditions are equivalent to or higher than the ECTC under evaluation, the
accelerated stability data can be considered in support of the target ECTC.
Cold chain: a series of storage and transport links used for keeping and
distributing vaccines in good condition until use according to the approved
long-term storage condition and shelf-life. The typical temperature for the long-
term storage condition is 2–8 °C although other approved temperatures can
be specified.
Extended controlled temperature conditions (ECTC): approved short-
term temperature conditions, above those defined for long-term storage,
transportation and use, for a given product immediately prior to administration.
Any temperatures above the approved long-term storage temperatures in the
cold chain could be considered for ECTC application. The development of
CTC terminology and the proposing of the alternative ECTC terminology are
described in the Ottawa meeting report (8).
Product-release model: a model that describes the relationship between
release and expiry specifications to ensure that the product will meet defined
specifications throughout its shelf-life.
Quality attributes: physical, chemical, biological, biopharmaceutical
and microbiological attributes that can be defined, measured and continually
monitored to ensure that final product outputs remain within acceptable
quality limits.
Real-time and real-condition stability studies: studies of the physical,
chemical, biological, biopharmaceutical and microbiological characteristics
of a vaccine, during and up to the expected shelf-life and storage periods of
samples kept under expected handling and storage conditions. The results
obtained are used to recommend storage conditions and to establish the shelf-
life and/or release specifications.
Shelf-life: the period of time during which a vaccine, when stored
under approved conditions, is expected to comply with the specifications. The
shelf-life is determined by stability studies on a number of product batches and
is used to establish the expiry date of each batch of a final product.
Stability of vaccine: the ability of a vaccine to retain its physical,
chemical, biological, biopharmaceutical and microbiological properties within
specified limits to assure clinical performance throughout its shelf-life.
Stability-indicating parameters: quality parameters (direct or indirect
indicators of vaccine efficacy or safety) that are sensitive to storage conditions.
These parameters are used in stability studies to assure product quality throughout
240
Annex 5
the shelf-life. Determination of these parameters should result in quantitative

values with a detectable rate of change. Qualitative parameters such as sterility
may also be considered but cannot be included in the statistical analysis.
WHO controlled temperature chain (CTC) programme: a specific
approach to vaccine management that allows vaccines to be kept at temperatures
above the long-term storage condition for a limited period of time under
monitored and controlled conditions appropriate to the stability of the antigen.
Current WHO programme conditions for CTC include a single exposure just
prior to administration, tolerating ambient temperatures of at least 40 °C for a
limited duration of at least 3 days, with these temperature and time conditions
included in the approved label.
4. General considerations for the evaluation

of vaccines for use under ECTC
The use of vaccines under ECTC requires an appropriate vaccine stability
assessment and consideration of the feasibility of compliance with the approved
storage conditions in the field. While the stability evaluation principles
described here could potentially be applied to data to support multiple
temperature exposures for a vaccine, consideration would then need to be given
to how such exposures would be tracked for specific vaccine final containers.
When reviewing the potential difficulties in tracking multiple exposures, and
ensuring that final containers that have reached the maximum exposure limit
are discarded, it was concluded that at this time guidance for an ECTC label
should be limited to a single planned exposure of specified duration within the
labelled expiry date. Hence, once a vaccine has been stored under ECTC, it
should not be returned to normal cold chain storage (for example, at 2–8 °C) in
order to prevent the inadvertent administration of vaccine that is potentially out
of specification. As experience with ECTC stability assessment and programme
implementation expands, this conclusion could potentially be reconsidered in a
future guidance update.
An ECTC application could potentially be approved solely on the basis
of product-specific stability and other quality data when both of the following
conditions are met:
■■ the approved product specifications, supported by quality attributes
of the clinical lots, remain unchanged and the vaccine is expected to
be compliant with these specifications following normal storage for
the full shelf-life, including the ECTC exposure;
■■ the battery of tests performed to assess vaccine stability, which may
include additional characterization assays in specific cases, has
241
the capacity to detect changes in potency and/or immunogenicity

as well as safety parameters that are predictive of vaccine clinical
performance.
Additionally, when a manufacturer has accelerated stability data
that bracket the intended ECTC exposure (for example, 40 °C), the potential
for interpolation of the data to estimate the decay rate at the target ECTC
temperature could be considered on a case-by-case basis. It should be noted
that the accelerated stability data must be from lots that represent the current
manufacturing process. Vaccines used under ECTC should be capable of
withstanding the approved planned exposure conditions regardless of the shelf-
life remaining before expiry. These evaluations must involve statistical analysis of
stability data to determine the rates of decay under both the approved long-term
storage conditions and those of an ECTC exposure. It is essential that adequate
potency is available to compensate for any decay over the full approved shelf-
life under approved long-term storage conditions, as well as under the planned
ECTC exposure (for example, 40 °C for at least 3 days). Consideration of both
potency requirements is necessary to address worst-case scenarios where the
planned exposure occurs within the shelf-life of a vaccine lot that was filled at or
near the minimum release potency (MRP).
The focus of the current document is on the assessment of potency over
the shelf-life of a vaccine given the anticipated loss of potency under ECTC.
However, it is also recognized that similar principles may be used to ensure
that potentially unsafe degradation products do not exceed approved limits.
If exposure to ECTC leads to safety concerns such as undesired degradation
products or the potential reversion of toxoids under these accelerated
temperature conditions, then such risks should be explicitly evaluated.
Product-specific potency evaluations should be based on decay rates,
MRP and an appropriate end-of-shelf-life lower limit (LL) supported by clinical
data or experience. This is described both in existing stability guidance and
subsequent papers (10, 11) and is critical for ECTC applications, to which the
same principles apply. A product-release model (Fig. 1) should be developed
on the basis of studies using the manufacturer’s assays, along with quality
data and other essential information. Labelling a vaccine for ECTC use will
require the support of the manufacturer and the approval of the appropriate
regulatory authorities.
Fig. 1 illustrates the relationship between the MRP specification of 50
ELISA Units (EU) and the shelf-life (24 months) given the rate of decay (slope)
of the potency over both the long-term storage temperature (2–8 °C) and the
maximum ECTC temperature (40 °C), and indicates that the vaccine is above
the approved LL for the potency (30 EU; supported by clinical lots) at the end
of shelf-life. The potency decay assessment should be based on appropriate
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statistical analysis of multiple lots with a given degree of confidence (for example,
95%) and should include assay variability (not shown in the figure). As noted
below in section 5, logarithmic transformation (log-transformation) of potency
data typically permits analysis of stability data by linear regression.
Fig. 1
Graphic representation of a product-release model for an ECTC application
80
70
Potency (EU = ELISA Unit)
Potency range
60 at release
MRP Potency
range of
50
2–8 °C clinical lots
Available
40
potency
40 °C
30
LL
0
0 6 12 18 24 30 36
Shelf-life (months)
Potency assays used in the assessment of stability should be fit-for-use

and are typically validated as accurate, sensitive, robust and stability-indicating.
Given the greater chance of product failure after exposure to the ECTC
temperatures being considered, the ability of an assay (and even of existing
approved assays) to detect quality-related outcomes associated with an ECTC
exposure should be re-evaluated. In some cases, supplementary potency assays or
key stability-indicating tests linked with the clinical performance of the vaccine
should be used. For example, use of the test to ascertain the percentage of free
polysaccharide (free PS) in glycoconjugate vaccines should be considered, if
not already performed. Other assays, such as those typically performed during
product stability testing, should also be considered for use in an ECTC context.
These may include assessments of quality attributes that may themselves affect
stability (for example, moisture and pH), as well as tests of container integrity
under the ECTC (for example, sterility and specific container integrity tests). It
is not possible to perform decay modelling on products with potency assays that
243
have binary outputs (for example, pass/fail). In such cases, supplemental potency
assays that are capable of showing the decay of the product’s active ingredient (or
that can provide a worst-case estimate of that decay) may be considered for use
in ECTC evaluation, recognizing the need for conservatism in interpreting the
analysis and its results. In the absence of adequate stability-indicating assays for
a vaccine, approval of an ECTC label would not be possible solely on the basis
of a quality data assessment.
While ECTC approval is not a recommendation for shipping or storage,
and unplanned excursions are not within the scope of this document, an
approved ECTC label could potentially be taken into account when making
decisions on product use in cases of temporary temperature excursions.
However, given the finite nature of available potency over the shelf-life of any
given vaccine, potency lost through unplanned excursions with specific final
containers earlier in the shelf-life would not be available to support the use of
those same containers of vaccines during a planned ECTC exposure later in the
shelf-life. This highlights the importance of maintaining the cold chain prior
to the extreme temperature conditions that the vaccine would be subjected to
during a planned ECTC exposure.
For multivalent vaccines, ECTC evaluations must consider all antigens
in the product. If one antigen is known to be less stable than other antigens
within a specific vaccine then the suitability of the product for ECTC should
be based on the least-stable antigen. Potential interference between vaccine
components, including adjuvants, stabilizers and preservatives, may also
need to be considered, as applicable. To simplify the discussion here, with the
exception of comments related to free PS, the focus of this and subsequent
sections will be on how to evaluate and manage the available potency of a
monovalent vaccine.
At release, a product must possess sufficient potency to ensure clinical
effectiveness throughout its shelf-life and to account for assay variability as well
as any product decay. If there is insufficient potency available to permit ECTC

use, or if it is desirable to extend the time of the ECTC storage condition, several
strategies could be considered for enhancing the ECTC potential of a vaccine.
For example, the shelf-life under the approved long-term storage conditions
could be reduced in order to increase the available potency that could be applied
to an ECTC exposure. An example of the use of shelf-life reduction to extend
the ECTC storage time is shown in the Appendix below. If such an approach
were used, it would be important to assign a unique product name to the ECTC
version of the vaccine to avoid confusion in the field. The case study provided
in the Appendix also illustrates that the implementation of a lower release
specification for free PS would create a larger differential between the release
and end-of-shelf-life specifications, which would enhance the ECTC potential
of the product. Enhanced ECTC potential could also be achieved by reducing
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assay variability, thus reducing the amount of potency required to account for
potential errors in initial potency assignment. Finally, with a more accurate
characterization of vaccine stability, a manufacturer could reduce the amount of
potency required to account for errors in the estimation of shelf-life.
In general, additional clinical assessment for a previously approved
product being considered in an ECTC application should be required only
where a planned ECTC exposure results in a change of product specifications.
For example, a lower end-of-shelf-life potency or a higher release specification
that is not supported by clinical experience would potentially require
further clinical evaluation. If additional clinical studies could demonstrate
that lower potencies were still effective, or that higher-than-approved target
release potencies would not result in new or more-frequent adverse events,
a manufacturer could submit a regulatory amendment and use the broader
potency ranges gained through these approaches to extend the ECTC potential
of a product. Field studies in which the clinical evaluation of a vaccine has
been performed on a product that has been exposed to temperatures higher
than the approved conditions, but without potency and quality testing using
the manufacturer’s assays, are not considered acceptable from a regulatory
perspective. Clinical studies that are intended to support ECTC applications
should be performed using a vaccine with known (or modelled) potency at the
time of the ECTC use, as determined by the manufacturer’s assays.
Finally, when a lyophilized vaccine is being considered for ECTC
applications, a stability analysis should be performed for the reconstituted
product using the rigorous statistical evaluation principles described in this
document. In situations where exposure to an ECTC storage condition could
result in changes to the visual appearance of the lyophilized product that do not
impact on its clinical performance, the manufacturer should provide relevant
supportive data to the NRA considering the ECTC label change. If approved, a
description of the potential change(s) in visual appearance should be included in
the product leaflet and/or package insert. For liquid multi-dose vials, additional
data would be required to demonstrate the antimicrobial effectiveness of the
preservative under ECTC.
5. Stability evaluation of vaccines for use under ECTC

Stability evaluation of a specific vaccine planned for use under ECTC must
generate sufficient scientifically valid data to support regulatory approval of
labelling for such use. This requires assurance that there is sufficient potency
available, even with lots near to expiry, to allow for an exposure under ECTC.
The best prediction of actual end-expiry potency of any given lot depends on
a variety of factors, including release potency of the specific lot, accuracy and
precision of the potency assay, and the results of stability studies. Consequently,
245
statistical evaluation is needed to be able to state that, with a given (usually 95%)
degree of confidence, the potency after ECTC exposure at expiry will still be
above the minimum threshold needed for product efficacy. It is only through
the use of statistical analysis that it is possible to obtain an indication of the
level of confidence in results reported at release or in potencies delivered to the
vaccine recipient. Therefore, statistical analysis is required to assure the quality
of vaccines intended for delivery in the context of ECTC. Because the major
ECTC-related concern is usually that the temperature exposure will reduce
potency to unacceptable levels, the following guidance focuses on ensuring that
the minimum required potency is maintained.
Even when products are sufficiently stable to tolerate an ECTC exposure,
poorly designed studies or inappropriate statistical analyses can reduce the
likelihood that an ECTC exposure is justified. This section describes study-
design and statistical approaches that will improve the likelihood that ECTC
exposure can be justified using sound scientific principles.
Although additional clinical studies will not be required for an ECTC
approval for most licensed products, it is essential to establish the minimum
potency specification for a specific vaccine, through initial licensing studies, for
all stability assessments. Changes in specifications (including lowering of end-
expiry potency specifications) may require supporting clinical data. Thus, the
data package for ECTC applications should include summaries of the initial
clinical studies, including the quality data for the clinical lots, to support the
end-of-shelf-life potency specification.
The data package should also include stability studies that formally
demonstrate that the minimum potency is achieved throughout the time to
expiry, including the ECTC exposure. Estimates of the rate (or slope) at which
the potency decays (hereafter, referred to as “stability estimates”) at the normal
and ECTC temperatures – and an understanding of potential errors in those
estimates – are the most important outcomes of the stability studies. The
reliability of these stability estimates, and the extent to which the release potency
of any lot can be reliably determined, depend in turn on the potency assay.
As mentioned above in section 4, stability studies to support vaccine
use in an ECTC context should use the manufacturer’s potency assay in order
to preserve a connection between the released product proposed for ECTC use
and the original clinical material used to support product efficacy. It is likely
that key parameters of the potency assay will already be known from assay
validation, including assay accuracy and precision. More reliable estimates of
in-use assay precision may sometimes be obtained by other means (for example,
by comparing actual with modelled results in the stability analyses). Other data
may also be relevant to the estimation of in-use assay precision. Because there
are several possible estimates of assay precision that could be used for ECTC-
related calculations, the choice of estimate should be scientifically justified; if
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Annex 5
a clear justification cannot be made, the more conservative estimate (from the
perspective of the ECTC label) should be used.
Statistical analysis of vaccine stability is normally based on a
mathematical model and supported by data that describe the kinetics of potency
changes at different temperatures for different periods of time. Statistical release
models must support the conclusion that the mean potency of final containers
in a given lot will, given all stability losses, meet specifications throughout the
shelf-life with a given level of confidence (usually 95%), including permitted
storage periods outside the long-term storage conditions. Typically, the rate
of change (generally loss) of vaccine potency is not a simple linear function of
time. Log-transformation of potency data usually leads to a more predictive
model that permits analysis of stability data by linear regression. Thus, in most
cases, potency data should be log-transformed before analysis. When log-
transformation is not used, scientific justification for the use of a more relevant
model should be provided. Log-transformation usually provides a preferred
model of the biological process (since for most substances the rate of decay at
any point in time depends on the quantity of substance present at that time) as
compared with direct analysis of non-log-transformed potency data.
Moreover, empirical observation supports the conclusion that potency
decay for many vaccines follows first-order kinetics, which are linear following
log-transformation – though low precision of the potency assay may make it
more difficult to determine whether stability results follow the decay model.
In addition, potency measurements are often log-normally distributed; when
this is the case, log-transformation may be required to satisfy the statistical
assumptions of the modelling, and can further improve the precision of the
stability estimate. In all cases, the decay model used should correspond to actual
product decay kinetics as observed in stability studies, and this may support the
use of non-log-transformed decay models (including linear models) if these
models can also be justified as biologically relevant. It should be noted that log-
transformation is not always the best approach for stability-indicating assays.
For example, increases in degradation products over time usually cannot be
modelled on a log scale. Visual examination of the plot of transformed and
untransformed stability data can provide an indication of whether mathematical
transformations can linearize the decay curve. Sometimes no biologically
meaningful model can be identified that fits the data, as may occur when there
are multiple phases in the decay kinetics. In this case, decay estimates during
ECTC exposures can be estimated by using only the beginning and end results
of the stability testing. If the most appropriate choice of model is unclear,
selection of the most conservative option is appropriate.
Stability studies should properly evaluate the kinetics of decay, and
should indicate that decay rates (after any transformation) are not higher at
the end of the observation period than at the beginning. These studies should
247
include a sufficient number of time points to determine the adequacy of the

decay model, while also providing robust stability estimates. Linearity can
theoretically be supported by using at least three time points – the starting
point, the end-point (corresponding to the desired ECTC exposure time) and
ideally the midpoint. However, when assay precision is not high, additional
time points will probably be needed to increase the degree of assurance that
the change in ECTC-related stability parameters truly follows a linear model.
Inclusion of additional time points can also improve estimates of the true rate
of change in potency. If linearity has already been established, more precise
estimates of decay rates under ECTC can be obtained by testing sufficient
numbers of samples at post-ECTC exposure time points as compared to pre-
ECTC exposure time points. When decay kinetics are linear, testing at time
points beyond the proposed ECTC use can also improve the precision of the
stability estimate. It is often assumed that decay rates under ECTC will be
similar near the time of release and at expiry, but (as with the rate of decay
over the normal storage period) this assumption should be verified. If the
rate of decay does vary depending on the time from release then modelling
may need to take this into account, along with consideration of the potential
uncertainties added by any assumptions that are made. Testing larger numbers
of independent samples (batches/lots) can further improve this precision and
can potentially increase the likelihood that these studies will support ECTC
use, but in all cases a minimum of three lots should be tested.
Typical stability evaluations often include an analysis for “poolability”.
The presence of one or more outliers in a stability-indicating assay may indicate
unacceptable manufacturing variability and/or could cause a combined decay
slope calculated using a small number of lots to be inaccurate. Previous guidance
advocated using the worst-case lot for the decay slope estimate when analysis
suggested that data from tested lots may not be poolable. However, using the
worst-case lot can inappropriately penalize expected variability (over which
the manufacturer has no control) and can be a disincentive to conducting more

complete testing. It is reasonable to include all tested lots in the stability analysis
so long as these lots are considered representative of the licensed (and “in-
control”) manufacturing process, and so long as a sufficient number of lots are
included to address random variability. Pooling of data from a sufficient number
of representative lots should be statistically justified and agreed with the NRA.
Stability testing normally provides information on expected rates of
decay (for the linear model, the decay slope) and standard error of the decay
slope at “n” different temperatures of exposure (modelling storage, shipping,
post-reconstitution and so on) as well as under ECTC (for time tECTC with decay
slope bECTC ). Modelling of stability test results can also provide an estimate of the
precision of the potency assay.
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A first approximation of the impact of an ECTC exposure is the 95%

confidence bound on the expected loss in potency LECTC as a result of the ECTC
exposure. This can be estimated as:
L ECTC = −t ECTC ∙ b ECTC + z 1−α ∙ (t ECTC ∙ s(b ECTC))
...where t ECTC is the time at ECTC temperatures; b ECTC is the decay slope
(a negative number) at ECTC temperatures; z 1−α is the one-sided z statistic at
the confidence level associated with the desired degree of confidence (α = 0.05
for 95% confidence bounds); and s(b ECTC) is the standard error of the decay
slope. If this amount of additional decay in potency beyond that considered
by the product-release model is considered acceptable, the product can be
accepted for ECTC use.
A more accurate and less conservative estimate can be obtained by
calculating the aggregate error associated with all assumptions in the decay
model. The product-release model shown above in Fig. 1 defines the needed
potency of the product at the time of release. From this information, it is possible
to calculate the statistical lower bound (LB) on the mean potency of a product
that is released at the minimum release potency and that is exposed to multiple
temperature conditions. This is shown as follows:1
LB 1-α = MRP + t 1 ∙ b 1 + t 2 ∙ b 2 +…+ t n ∙ b n + tECTC ∙ b ECTC - U
...where 1-α describes the statistical confidence level associated with the
lower bound (α = 0.05 for 95% confidence bounds); MRP is the manufacturer’s
minimum allowable release potency (usually log-transformed); t i is time at
temperature i (where i is a positive integer used to represent the series of
temperatures to which the vaccine may be exposed); b i is decay slope (a negative
number, or zero if positive) at temperature i; and U is the combined uncertainty
associated with the independent estimation of the numbers on the right side of
the equation. Typically:
U = z1-α ∙ sqrt ((s assay)2 + (t 1 ∙ s(b 1))2 + (t 2 ∙ s(b 2))2 +…+ (t n ∙ s(b n))2 +
(tECTC ∙ s(b ECTC))2)
...where z1-α is the one sided z statistic at the confidence level associated
with the desired degree of confidence (α = 0.05 for 95% confidence bounds);
s assay is the assay precision; and s(b i) is the precision (standard error) of the
As noted, the equations listed are for the general case that could include modelling storage, shipping,
1
post-reconstitution and so on. However, when only considering the normal storage condition and a
single ECTC exposure, an example of a simplified form of the equations would be: LB 1-α = MRP + t 1 ∙ b 1 +
t ECTC ∙ b ECTC - U.
249
decay slope at temperature i. Thus, the expected end-expiry potency is expected

to be (with 95% confidence) as low as LB0.95 – which accounts for the
manufacturer’s MRP, the estimated potency losses at the various temperatures
and the associated uncertainty.
Without an ECTC exposure, and with omission of the ECTC-associated
terms, the above equations yield the minimum potency that an already-licensed
product is expected to maintain based on the manufacturer’s release model,
throughout its time to expiry. Inclusion of the ECTC term allows a reviewer to
determine the degree to which potency is affected by the ECTC exposure and
whether or not that is acceptable on the basis of clinical experience with the
vaccine at that level of potency.
When the LL of potency, defined as the minimum potency below which
there is concern about product efficacy (considering the potency results from
the clinical lots and post-market experience) has been defined, it is preferable
to rearrange the terms of the above equation to determine the MRP required to
maintain potency through to expiry, including the ECTC exposure. In essence,
this means calculating the minimum amount of potency that must be added
to that minimum potency (LL) in order to assure product quality throughout
normal storage and handling, including ECTC exposure, as follows:
MRP = LL - t 1 ∙ b 1 - t 2 ∙ b 2 -…- t n ∙ b n - t ECTC ∙ b ECTC + U
The product may be released at a higher potency within the approved
specification which provides a convenient way to ensure that the release model
will support ECTC labelling. If ECTC exposure potential cannot be established
then several options could be considered, as outlined above in section 4 (see
also the definition of Product-release model in section 3 and the related Fig. 1
in section 4). It should be noted that the analytical principles represented in
the equations above are the same as those in existing WHO vaccine stability
guidance (1) and that they have been expanded to include ECTC exposure. It
should also be noted that the equations here are not the only ways to represent
these calculations and that other approaches that encompass similar statistical
principles could potentially be acceptable where justified.
6. Monitoring ECTC
All vaccines should be kept under the recommended long-term storage
conditions with appropriate oversight prior to ECTC exposure. Use of vaccines
under ECTC requires specific monitoring of temperature exposure (for example,
peak threshold indicator) and time, as well as formal procedures to ensure
that the approved maximum temperature and time are not exceeded. Unused
vaccines that exceed the maximum approved temperature or time should be
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disposed of by suitable procedures. ECTC temperature-monitoring systems need

to be able to distinguish vaccines that are still appropriate for use from vaccines
that have exceeded the limits imposed by the data supporting ECTC use. The
monitoring requirements for ECTC differ from those for long-term storage and
transport. The respective monitoring systems for long-term and ECTC storage
should be consistent with product stability characteristics. In order to allow an
ECTC exposure, the monitoring system should assure that approved long-term
storage conditions, especially with respect to temperature, are not exceeded.
Prior to vaccine approval for use under ECTC, the relevant stakeholders, which
may include the manufacturer, the NRA and the immunization programme,
should work together to ensure that an appropriate monitoring system is
in place.
7. Suggested product labelling

information for use under ECTC
ECTC should be described in the product leaflet and/or package insert in
order to provide information to medical practitioners. The statement on ECTC
should appear in a separate paragraph in the appropriate section of the label (for
example, Storage and Handling).
The ECTC information in the product leaflet and/or package insert
should be clear, concise and specific. If a vaccine consists of two or more
components (for example, lyophilized vaccine and diluent) then ECTC
information should be given for all of the components of the vaccine.
Information to be included in an ECTC statement should take account
of the following, if applicable:
■■ maximum allowed temperature
■■ maximum time allowed at a specific temperature
■■ in-use shelf-life after opening (or reconstitution or mixture,
if applicable)
■■ advice on unopened vials exposed to ECTC (for example,
on disposal)
■■ that only a single ECTC exposure directly prior to use or disposal,
and within the shelf-life, is permitted.
The following may serve as a model text for the product leaflet and/or
package insert text for ECTC use: The vaccine [and its diluent/solvent or other
component] may be kept for a single period of time of up to [x days or x weeks or x
months] at temperatures of up to [x °C] immediately prior to administration. At the
end of this period, the vaccine [must be disposed of]. This is not a recommendation
251
for storage but is intended to guide decision-making when exposure to higher

temperatures is planned. [After opening [or reconstitution or mixture], the vaccine
can be kept for [x hours or x days] at temperatures of up to [x °C] at which point it
must be disposed of].
8. Authors and acknowledgments

The first draft of these WHO Guidelines was prepared by Dr C. Conrad, Paul-
Ehrlich-Institut, Germany; Dr E. Griffiths, Consultant, Kingston-upon-Thames,
the United Kingdom; Mrs T. Jivapaisarnpong, Department of Medical Sciences,
Thailand; Dr J. Kim, World Health Organization, Switzerland; Dr I. Knezevic,
World Health Organization, Switzerland; Dr P. Krause, United States Food and
Drug Administration Center for Biologics Evaluation and Research, the USA;
Dr J. Shin, WHO Regional Office for the Western Pacific, Philippines; Dr D.
Smith, Health Canada, Canada; Dr J. Southern, Adviser to the Medicines Control
Council of South Africa, South Africa; Dr T. Wu, Health Canada, Canada; taking
into account comments received from: Dr B.D. Akanmori, WHO Regional Office
for Africa, Congo; Dr S.C. Da Silveira, Agência Nacional de Vigilância Sanitária,
Brazil; and Ms A-L. Kahn, Dr A. Meek, Dr C.A. Rodriguez-Hernandez and Ms S.
Zipursky, World Health Organization, Switzerland. Acknowledgement is also
given to Mr M. Walsh, Health Canada, Canada, for providing expertise on the
mathematical modelling and statistical approaches used for ECTC evaluations,
and for his contribution to the appendix on evaluation of a model monovalent
polysaccharide conjugate vaccine.
The first draft was based on the Ottawa and Langen CTC consultation
reports (8, 9) prepared by Dr M. Baca-Estrada, Health Canada, Canada; Dr C.
Conrad, Paul-Ehrlich-Institut, Germany; Dr E. Griffiths, Consultant, Kingston-
upon-Thames, the United Kingdom; Dr J. Kim, World Health Organization,
Switzerland; Dr I. Knezevic, World Health Organization, Switzerland; Dr P.
Krause, United States Food and Drug Administration Center for Biologics
Evaluation and Research, the USA; Dr M. (Ferguson) Lennon, Consultant,
Horning, the United Kingdom; Dr H. Meyer, Paul-Ehrlich-Institut, Germany;
Dr V. Oeppling, Paul-Ehrlich-Institut, Germany; Dr M. Pfleiderer, Paul-
Ehrlich-Institut, Germany; Dr J. Shin, World Health Organization, Switzerland;
Dr D. Smith, Health Canada, Canada; Dr R. Wagner, Paul-Ehrlich-Institut,
Germany; Dr T. Wu, Health Canada, Canada; Ms S. Zipursky, World Health
Organization, Switzerland.
Acknowledgments are extended to the following participants in the
consultations held in Ottawa, Canada, 4–6 December 2012 and Langen,
Germany, 4–6 June 2013: Dr M-C. Annequin, Agence nationale de sécurité
du médicament et des produits de santé, France; Dr M. Baca-Estrada, Health
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Annex 5
Canada, Canada; Dr K. Brusselmans, Scientific Institute of Public Health,

Belgium; Dr M. Chultem, Health Canada, Canada; Dr C. Cichutek, Paul-
Ehrlich-Institut, Germany; Mr W. Conklin, Merck, the USA; Dr C. Conrad,
Paul-Ehrlich-Institut, Germany; Ms D. Doucet, GlaxoSmithKline Biologicals
SA, Belgium; Dr W. Egan, Novartis, the USA; Dr L. Elmgren, Health Canada,
Canada; Dr D. Felnerova, Crucell Switzerland AG, Switzerland; Dr S. Gairola,
Serum Institute of India Ltd, India; Dr E. Griffiths, Consultant, Kingston-
upon-Thames, the United Kingdom; Dr D.A. Hokama, BioManguinhos, Brazil;
Dr W. Huang, Xiamen Innovax Biotech Co., China; Mrs T. Jivapaisarnpong,
Department of Medical Sciences, Thailand; Dr J. Korimbocus, Agence
nationale de sécurité du médicament et des produits de santé, France; Dr P.
Krause, United States Food and Drug Administration Center for Biologics
Evaluation and Research, the USA; Dr H. Langar, WHO Regional Office for
the Eastern Mediterranean, Egypt; Dr A. Laschi, Sanofi Pasteur, France; Dr C.
Lecomte, GlaxoSmithKline Vaccines, Belgium; Professor H. Leng, Consultant,
Somerset West, South Africa; Dr M. (Ferguson) Lennon, Consultant, Horning,
the United Kingdom; Ms A. Lopez, Biológicos y Reactivos de México S.A.
de C.V., Mexico; Dr A. Luethi, Crucell Switzerland AG, Switzerland; Dr W.
Matheis, Paul-Ehrlich-Institut, Germany; Dr A. Merkle, Paul-Ehrlich-Institut,
Germany; Dr H. Meyer, Paul-Ehrlich-Institut, Germany; Dr B.L.M. Moreira,
Agência Nacional de Vigilância Sanitária, Brazil; Dr K-T. Nam, Ministry
of Food and Drug Safety, Republic of Korea; Dr R. Nibbeling, Institute for
Translational Vaccinology, Netherlands; Dr V. Oeppling, Paul-Ehrlich-Institut,
Germany; Dr D.M. Pascual, Centro para el Control Estatal de la Calidad de
los Medicamentos, Cuba; Dr M. Pfleiderer, Paul-Ehrlich-Institut, Germany;
Dr M.L. Pombo, Pan American Health Organization, the USA; Dr T. Prusik,
Temptime Corporation, the USA; Dr M. Reers, Biological E Ltd, India; Mr T.
Schofield, MedImmune, the USA; Ms F.A. Setyorini, PT Bio Farma, Indonesia;
Dr S. Shani, Food and Drug Administration, India; Dr I.S. Shin, Ministry of
Food and Drug Safety, Republic of Korea; Dr S.C. Da Silveira, Agência Nacional
de Vigilância Sanitária, Brazil; Dr D. Smith, Health Canada, Canada; Dr M.
Vega, Centre for Genetic Engineering and Biotechnology, Cuba; Dr R. Wagner,
Paul-Ehrlich-Institut, Germany; Ms S. Wong, Merck, the USA; Dr T. Wu, Health
Canada, Canada; Dr M. Zeng, National Institutes for Food and Drug Control,
China; and Dr J. Kim, Dr D.G. Maire, Dr C.A. Rodriguez-Hernandez, Dr J. Shin
and Ms S. Zipursky, World Health Organization, Switzerland.
The second draft document (WHO/BS/2015.2268) was prepared by
Dr C. Conrad, Paul-Ehrlich-Institut, Germany; Dr I. Feavers, National Institute
for Biological Standards and Control, the United Kingdom; Dr K. Gao, World
Health Organization, Switzerland; Dr E. Griffiths, Consultant, Kingston-upon-
Thames, the United Kingdom; Mrs T. Jivapaisarnpong, Department of Medical
253
Sciences, Thailand; Dr J. Kim, World Health Organization, Switzerland; Dr I.

Knezevic, World Health Organization, Switzerland; Dr P. Krause, United States
Food and Drug Administration Center for Biologics Evaluation and Research, the
USA; Dr J. Shin, WHO Regional Office for the Western Pacific, Philippines; Dr D.
Smith, Health Canada, Canada; Dr J. Southern, Adviser to the Medicines Control
Council of South Africa, South Africa; Mr M. Walsh, Health Canada, Canada;
Dr T. Wu, Health Canada, Canada, taking into account comments received from:
Dr B.D. Akanmori, WHO Regional Office for Africa, Congo; Dr A. Alsalhani,
Médecins Sans Frontières, France; Dr M-C. Annequin, Agence nationale de
sécurité du médicament et des produits de santé, France; Dr B. Bolgiano, National
Institute for Biological Standards and Control, the United Kingdom; Dr J.
Bridgewater, United States Food and Drug Administration Center for Biologics
Evaluation and Research, the USA; Dr A. Chang, Johns Hopkins University, the
USA; Dr A. Cheung, United States Food and Drug Administration Center for
Biologics Evaluation and Research, the USA; Ms D. Doucet, GlaxoSmithKline
Biologicals SA, Belgium; Dr J. Du, National Institutes for Food and Drug
Control, China; Dr W. Egan, Novartis, the USA; Dr S. Gagneten, United States
Food and Drug Administration Center for Biologics Evaluation and Research,
the USA; Dr D. Garcia, Agence nationale de sécurité du médicament et des
produits de santé, France; Dr E. Griffiths, Consultant, Kingston-upon-Thames,
the United Kingdom; Dr T. Guo, National Institutes for Food and Drug Control,
China; Mr K. Hicks (International Federation of Pharmaceutical Manufacturers &
Associations (IFPMA) representative), Sanofi Pasteur, France; Ms A. Juan-Giner,
Médecins Sans Frontières, France; Dr B-G. Kim, Ministry of Food and Drug
Safety, Republic of Korea; Dr J. Korimbocus, Agence nationale de sécurité du
médicament et des produits de santé, France; Dr T-L. Lin, United States Food and
Drug Administration Center for Biologics Evaluation and Research, the USA;
Dr M. Ramos, Public Health England, the United Kingdom; Mr T. Schofield,
MedImmune, the USA; Dr J. Shin, WHO Regional Office for the Western
Pacific, Philippines; Dr S.C. Da Silveira Andreoli, Agência Nacional de Vigilância

Sanitária, Brazil; Dr D. Smith, Health Canada, Canada; Dr T. Prusik, Temptime
Corporation, the USA; and Ms A-L. Kahn, Dr U. Kartoglu, Dr D. Petit and Ms S.
Zipursky, World Health Organization, Switzerland.
Acknowledgments are extended to the following participants in a
consultation held in Geneva, Switzerland, 24 March 2015: Mr F.S. Adeyemi,
National Agency for Food and Drug Administration and Control, Nigeria;
Dr M. Allin, Pfizer, Belgium; Ms Y. Bai, China Food and Drug Administration,
China; Dr J. Bergers, National Institute for Public Health and the Environment,
Netherlands; Ms P. Carneiro, Institute Butantan, Brazil; Dr C. Conrad, Paul-
Ehrlich-Institut, Germany; Ms D. Doucet, GlaxoSmithKline Biologicals SA,
Belgium; Ms N. Dubois, Pfizer, Belgium; Dr A. Fauconnier, Federal Agency for
Medicines and Health Products, Belgium; Dr I. Feavers, National Institute
254
Annex 5
for Biological Standards and Control, the United Kingdom; Dr S. Gairola,

Serum Institute of India, India; Mr K. Gopinathan, Biological E Ltd, India; Dr E.
Griffiths, Consultant, Kingston-upon-Thames, the United Kingdom; Dr K.
Hicks, Sanofi Pasteur, France; Mr Y. Kaushik, Bharat Biotech International,
India; Dr B-G. Kim, Ministry of Food and Drug Safety, Republic of Korea;
Dr J. Korimbocus, Agence nationale de sécurité du médicament et des produits
de santé, France; Dr P. Krause, United States Food and Drug Administration
Center for Biologics Evaluation and Research, the USA; Mr A. Kukrety, Food
and Drug Administration, India; Dr J.S. Lloyd, Immunization Supply Chain,
France; Dr A. Luethi, Crucell Switzerland AG, Switzerland; Dr B.L.M. Moreira,
Agência Nacional de Vigilância Sanitária, Brazil; Dr D.M. Pascual, Centro para
el Control Estatal de la Calidad de los Medicamentos, Cuba; Mr C. Perrin,
Médecins Sans Frontières, France; Dr T. Prusik, Temptime Corporation, the
USA; Mrs J. Rogers, Food and Drugs Authority, Ghana; Ms P.P. Said, PT Bio
Farma, Indonesia; Ms C. Schmit, GlaxoSmithKline Biologicals SA, Belgium;
Mr T. Schofield, MedImmune, the USA; Dr D. Smith, Health Canada, Canada;
Dr J. Southern, Adviser to the Medicines Control Council of South Africa,
South Africa; Ms S. Wong, Merck, the USA; Dr T. Wu, Health Canada, Canada;
and Ms A-L. Kahn, Dr U. Kartoglu, Dr J. Kim, Dr I. Knezevic, Dr A. Meek and
Dr C.A. Rodriguez-Hernandez, World Health Organization, Switzerland.
9. References
Technical Report Series, No. 962; http://who.int/biologicals/vaccines/Annex_3_WHO_TRS_962-3.
2. Meeting of the Strategic Advisory Group of Experts on Immunization (SAGE), Geneva, 10–
12 April 2012 [website]. Geneva: World Health Organization; 2012 (http://www.who.int/
immunization/sage/meetings/2012/april/presentations_background_docs/en/index.html,
3. WHO Immunization Practices Advisory Committee (IPAC), Geneva, 17–18 April 2012. Final meeting
report and recommendations. Geneva: World Health Organization; 2012 (http://www.who.int/
immunization/policy/committees/IPAC_2012_April_report.pdf?ua=1, accessed 7 February 2016).
4. Vaccine management and logistics: Controlled Temperature Chain (CTC) [website]. Geneva: World
Health Organization (http://www.who.int/immunization/programmes_systems/supply_chain/
resources/tools/en/index6.html, accessed 14 December 2015).
5. Butler D. Vaccines endure African temperatures without damage: anti-meningitis campaign
in Benin delivers more than 150,000 doses with no losses from excess heat. Nature. 19
February 2014 (http://www.nature.com/news/vaccines-endure-african-temperatures-without-
damage-1.14744#/ref-link-1, accessed 14 December 2015).
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6. Zipursky S, Djingarey MH, Lodjo J-C, Olodo L, Tiendrebeogo S, Ronveaux O. Benefits of using
vaccines out of the cold chain: delivering meningitis A vaccine in a controlled temperature chain
during the mass immunization campaign in Benin 2014. Vaccines. 2014;32:1431–5 (https://
www.researchgate.net/publication/260373472_Benefits_of_using_vaccines_out_of_the_cold_
chain_Delivering_Meningitis_A_vaccine_in_a_controlled_temperature_chain_during_the_
mass_immunization_campaign_in_Benin, accessed 14 December 2015).
7. Use of MenAfriVac™ (meningitis A vaccine) in a controlled temperature chain (CTC) during
campaigns. Geneva: World Health Organization; 2013 (WHO/IVB/13.04; http://apps.who.int/iris/
bitstream/10665/86018/1/WHO_IVB_13.04_eng.pdf?ua=1, accessed 7 February 2016).
8. WHO/Health Canada drafting group meeting on scientific and regulatory considerations on
the stability evaluation of vaccines under controlled temperature chain. Ottawa, Canada, 4–6
December 2012. Meeting report. Geneva: World Health Organization; 2012 (http://who.int/
biologicals/areas/vaccines/CTC_FINAL_OTTAWA_Web_Meeting_report_25.11.2013.pdf?ua=1,
9. WHO/Paul-Ehrlich-Institut informal consultation on scientific and regulatory considerations
on the stability evaluation of vaccines under controlled temperature chain (CTC). Paul-Ehrlich-
Institut, Langen, Germany, 4–6 June 2013. Meeting report. Geneva: World Health Organization;
2013 (http://who.int/biologicals/vaccines/CTC_Final_Mtg_Report_Langen.pdf?ua=1, accessed 7
February 2016).
10. Krause PR. Goals of stability evaluation throughout the vaccine life cycle. Biologicals.
2009;37(6):369–78 (abstract available at: http://www.sciencedirect.com/science/article/pii/
S1045105609001201, accessed 14 December 2014).
11. Schofield TL. Vaccine stability study design and analysis to support product licensure. Biologicals.
2009;37(6):387–96 (abstract available at: http://www.ncbi.nlm.nih.gov/pubmed/19717312,
256
Annex 5
Appendix
Product-specific ECTC evaluation of a model monovalent
polysaccharide conjugate vaccine
The model vaccine and the stability data presented in this appendix were
developed on the basis of Health Canada’s overall experience with conjugate
vaccines and do not represent characteristics or data from any specific product.
The analysis presented is also applicable to other stability-indicating parameters,
such as vaccine potency. The vaccine example under evaluation is a monovalent
conjugate vaccine composed of purified capsular polysaccharide (PS) covalently
attached to diphtheria toxoid protein. The final vaccine product is a non-
adjuvanted liquid formulation presented in single-dose vials. The normal storage
temperature for this model conjugate vaccine is 2–8 °C, with a time to expiry of
three years; the temperature under consideration for the ECTC application is
40 °C. The quality attributes monitored in routine stability studies intended for
licensure included total PS, free PS, molecular size distribution, free protein,
pH and sterility. Free PS is considered a key stability-indicating attribute for
polysaccharide conjugate vaccines since in general this parameter is linked to
the clinical performance of this type of vaccine. The specification for free PS for
this model conjugate vaccine was set as “not more than (NMT) 15%” at release
and “NMT 25%” at the end of the shelf-life. A review of manufacturing data
indicated that, at release, 90% of the commercial lots contained less than 10%
of free PS and 10% of lots contained free PS in the range 10–13%. In addition,
vaccine lots containing 5–25% free PS were shown to be safe and immunogenic
in clinical studies.
Stability data
Real-time and real-condition stability studies were conducted to establish the
shelf-life under normal storage conditions (2–8 °C) and to support the ECTC
application. Although a minimum of three lots is required for statistical
modelling, analysis of a larger data set (more lots) leads to more-precise
estimates. In this example, routine stability-monitoring tests were performed
for four commercial vaccine lots stored at 2–8 °C and for an additional four
commercial lots stored at 40 °C. In addition, O-acetyl content, nuclear magnetic
resonance (NMR) spectrum and immunogenicity (rabbit complement source
serum bactericidal assay and immunoglobulin G) in a mouse model were also
evaluated to characterize vaccine lots exposed to the 40 °C condition. Analysis
257
of routine monitoring data revealed that total PS, molecular size distribution,
free protein and pH were stable for all lots stored under the 2–8 °C and 40 °C
conditions. However, an increase of free PS was observed for all lots, as
summarized in Tables 1 and 2.
Table 1
Summary of free PS content at 2–8 °C
Free PS (NMT 25%)

Lot #
0 3M 6M 9M 12M 18M 24M 30M 36M
1 7.53 9.58 10.73 11.17 12.54 13.51 16.07 NT 16.05
2 7.01 9.36 10.77 10.32 10.59 11.92 14.60 14.56 15.03
3 2.38 6.01 8.13 7.46 8.94 9.37 10.08 11.09 10.88
4 5.71 6.15 7.85 7.77 9.02 10.87 14.37 12.21 13.77
NMT = not more than; M = month; NT = not tested.
Table 2
Summary of free PS content at 40 °C
Free PS (NMT 25%)

Lot #
0 1W 2W 3W 4W 6W 8W 10W 12W
5 2.01 2.38 4.81 6.18 9.39 11.39 13.55 13.67 13.88
6 1.74 5.71 4.64 5.37 8.16 9.08 9.98 11.77 14.37
7 5.43 10.48 10.49 10.59 13.94 15.35 15.66 15.57 16.88
8 5.21 8.05 9.45 9.05 12.71 15.10 15.72 15.73 17.26

NMT = not more than; W = week.
Statistical analysis
An initial analysis (1) demonstrated that the free PS data did not fit a linear
regression, with or without log-transformation (plots not provided). Because
the increase in the stability-indicating free PS is due to the hydrolysis of bound
PS, the rate of increase of free PS is the same as the rate of decrease of bound PS.
Therefore, the bound PS at each test point can be calculated from the free and
total PS on the basis of mass balance. The hydrolysis of bound PS can be analysed
as a first-order reaction at a decay rate that is proportional to the concentration
258
Annex 5
of bound PS. Consequently, the rate of increase of free PS was analysed indirectly
through the modelling of bound PS, and log-transformation of the bound PS
content at different test points yielded data that were more amenable to linear
regression analysis (1). Thus, free PS data obtained in stability studies were
converted to percentage bound PS (Tables 3 and 4) and then subjected to log-
transformation. A release model was developed to characterize the relationship
between bound PS at release and end-expiry, thus permitting evaluation of
potential ECTC use.
Table 3
Summary of bound PS content at 2–8 °C
Bound PS (NLT 75%)

Lot #
0 3M 6M 9M 12M 18M 24M 30M 36M
1 92.47 90.42 89.27 88.83 87.46 86.49 83.93 NT 83.95
2 92.99 90.64 89.23 89.68 89.41 88.08 85.40 85.44 84.97
3 97.62 93.99 91.87 92.54 91.06 90.63 89.92 88.91 89.12
4 94.29 93.85 92.15 92.23 90.98 89.13 85.63 87.79 86.23
NLT = not less than; M = month; NT = not tested.
Table 4
Summary of bound PS content at 40 °C
Bound PS (NLT 75%)

Lot #
0 1W 2W 3W 4W 6W 8W 10W 12W
5 97.99 97.62 95.19 93.82 90.61 88.61 86.45 86.33 86.12
6 98.26 94.29 95.36 94.63 91.84 90.92 90.02 88.23 85.63
7 94.57 89.52 89.51 89.41 86.06 84.65 84.34 84.43 83.12
8 94.79 91.95 90.55 90.95 87.29 84.90 84.28 84.27 82.74
NLT = not less than; W = week.
Statistical analysis was performed using R version 3.1.1 (2) to estimate

the loss of bound PS under both the 2–8 °C and 40 °C storage conditions with
95% confidence, and the key results are summarized in Tables 5 and 6. The
analysis was undertaken in the following steps:
259
1. For each stability lot, the percentage of bound PS at each test point
was log-transformed and the slope was calculated using a linear
regression model. Plots of the linear regression fit for all stability
lots are presented in Fig. A1.
2. Lots 1, 2, 3 and 4, monitored at 2–8 °C, were assessed with respect
to slope variability, which was considered acceptable for use of the
linear regression model with a pooled (mean) slope for all four lots.
The same analysis was also applied to the data set (lots 5, 6, 7 and 8)
at 40 °C, which supported the use of a pooled slope.
3. The assay precision (sassay) was estimated by the residual error
from the regression analysis using the pooled slope for the
corresponding data set.
4. The uncertainty was calculated using formulae described above in
section 5. Two examples are:
■■ the uncertainty (U) at 2–8 °C for 36 months = z 0.95 ∙ sqrt [(s assay)2
+ (t 2–8 ∙ s(b 2–8))]2
= 1.644854 ∙ sqrt [0.012728112 + (36 ∙ 0.0001845235)2] = 0.02361.
■■ U at 2–8 °C for 36 months followed by 3 days at 40 °C
= z 0.95 ∙ sqrt [(s assay)2 + (t 2–8 ∙ s(b 2–8))2 + (tECTC ∙ s(b ECTC))2]
= 1.644854 ∙ sqrt [0.012728112 + (36 ∙ 0.0001845235)2 +
(0.1 ∙ 0.008625)2]
= 0.02366.
5. The change in bound PS was estimated using the linear regression

model and the formula provided above in section 5. An example
is provided below to illustrate the calculation of the total change in
bound PS at 2–8 °C over 36 months, plus 3 days at 40 °C. This is
based on the worst-case lots which contain 85% bound PS at release.
■■ First step: the log-transformed total decay of bound PS:

= (t 2–8 ∙ b 2–8 ) + (tECTC ∙ b ECTC) − U
= 36 ∙ (−0.002430492) + 0.1 ∙ (−0.07429892) − 0.02365824 =
−0.1185858.
■■ Second step: the log-transformed bound PS at end of storage
(2–8 °C plus ECTC):
= log e (bound PS at release) + [(t 2–8 ∙ b 2–8 ) + (tECTC ∙ b ECTC) − U]
= log e85 − 0.1185858 = 4.324065.
■■ Third step: bound PS at the end of storage (2–8 °C plus ECTC) =
e 4.324065 = 75.4949.
260
Annex 5
■■ Fourth step: the decay of bound PS at 2–8 °C over 36 months

plus 3 days at 40 °C:
= bound PS at release − bound PS at the end of storage (2–8 °C
plus ECTC)
= 85 − 75.4949 = 9.5051.
Table 5
Summary of statistical analysis of bound PS data at 2–8 °C
Data set Shelf-life Pooled s(b 2–8)a s assayb Uc Decay

(months) slope (per of bound
month) PS (%)
4 lots 24 −0.003311 0.0002765 0.01146 0.02178 8.1851
(0–24M)
4 lots 36 −0.002430 0.0001845 0.01273 0.02361 8.9388
(0–36M)
a
standard error of slope.
b
assay variability, estimated as standard deviation of residuals.
c
combined uncertainty, calculated using the formula described above in section 5.
Table 6
Summary of statistical analysis of bound PS data at 2–8 °C and 40 °C
Data Pooled s(b 40)a Months Days at s assayb Uc Decay

set slope (per at 2–8 °C 40 °C of bound
month) PS (%)
4 lots −0.04482 Not Not Not Not Not Not
(0–12W) done done done done done done
4 lots −0.07430 0.007766 24 7 0.01146 0.02199 9.5207
(0–4W)
−0.07430 0.008625 36 3 0.01273 0.02366 9.5051
a
standard error of slope.
b
assay variability, estimated from the regression analysis residual error.
c
combined uncertainty, calculated using the formula described above in section 5.
W = week.
261
Fig. A1
Plot of bound PS data for four conjugate vaccine lots
Examination of the plots presented in Fig. A1 indicates that at each of

these temperature conditions the decay of bound PS for each lot appears to
be better modelled by a straight line when compared to the modelling of free
PS. This supports the use of a linear regression model of bound PS in this case.
However, it is also noted that the estimated slope of bound PS decay differs over
different study periods, especially under 40 °C. As shown in Table 6 the decay
rate is higher over 4 weeks (−0.07430/week) compared to that over 12 weeks
(−0.04482/month). Differences in rates of change for key quality attributes over
a product’s shelf-life are not uncommon; therefore it is important to highlight the
need to characterize trends when modelling the data and the need to estimate
the rate of change based on real-time data over the full study period.
Owing to limited data points at 40 °C, the rate of bound PS decay used
for ECTC application was based on the modelling of a 4-week data set, and a
conservative approach was taken to limit the total decay of bound PS to slightly
below 10%. The statistical analysis summarized in Table 6 revealed an estimated
9.5% loss of bound PS (equal to the increase of free PS) after 36 months storage
at 2–8 °C followed by 3 days at 40 °C, or 24 months storage at 2–8 °C followed
by 7 days at 40 °C.
262
Annex 5
Conclusion
The application of the “product release model” to the analysis of free PS can be
summarized as:
Release specification (15%) = End of shelf-life specification (25%)
minus estimated combined increase at 2–8 °C and 40 °C (upper
bound with 95% confidence level)
On the basis of statistical modelling and product-related information, the
following can be concluded:
■■ Different specifications for release and end of shelf-life should
be established for free PS for this model conjugate vaccine.
A specification of “NMT 25%” at the end of shelf-life is considered
acceptable on the basis of clinical lots shown to be safe and
immunogenic in clinical studies. A release specification of “NMT
15%” was considered appropriate on the basis of manufacturing
capability, which ensures a high compliance rate for commercial lots
at release.
■■ A 36-month shelf-life at 2–8 °C was determined to be appropriate
for this model conjugate vaccine. This conclusion ensures that
worst-case lots, which contain the highest level of free PS permitted
by the release specification (NMT 15%), plus the accumulation of
free PS during the storage period (approximately 8.94%), comply
with the end of the shelf-life specification (NMT 25%).
■■ A single storage period of 3 days at 40 °C, prior to immunization,
was considered acceptable because the worst-case lots, which
contain 15% free PS at release and are stored for almost 36 months
at 2–8 °C followed by an exposure of 3 days at 40 °C, were expected
to contain approximately 24.51% free PS.
■■ If a period longer than 3 days at 40 °C is needed, shortening the
shelf-life at 2–8 °C from 36 months to 24 months would allow for
a single storage period of 7 days at 40 °C. Alternatively, the release
specification of “NMT 15% free PS” might be tightened (for example
to “NMT 13% free PS”) on the basis of additional manufacturing
experience to allow for longer than 3 days of ECTC exposure while
maintaining a 3-year shelf-life at 2–8 °C.
As explained above in section 4, clinical testing of a vaccine stored under
ECTC would not be necessary as long as a battery of stability-monitoring tests
provided sufficient assurance that the critical quality attributes of the vaccine
(such as potency) met the specifications supported by clinical experience.
263
Other than routine stability-monitoring assays, the following

characterization tests were also performed to assess quality attributes related to
vaccine clinical performance after this model conjugate vaccine was stored at
40 °C for 12 weeks: (a) O-acetyl content remained stable; (b) PS structure was
confirmed using NMR; and (c) carrier protein integrity was confirmed by the
results of an in vivo immunogenicity test. It was noted that the antigen dose used
to immunize the mice was within the dose–response curve, indicating that the
in vivo test was of acceptable sensitivity. In conclusion, the available stability
data sets assessing critical quality attributes were considered sufficient to support
the application of a single storage period of 3-day ECTC (40 °C) for this model
conjugate vaccine, within the approved 3-year shelf-life at 2–8 °C.
References
1. Wickham H. ggplot2: Elegant Graphics for Data Analysis. New York: Springer-Verlag New York,
2009.
2. R Core Team. R: a language and environment for statistical computing. Vienna: R Foundation for
Statistical Computing; 2014 (available at: http://www.R-project.org/, accessed 22 July 2015).
264
Annex 6
Biological substances: WHO International Standards,
Reference Reagents and Reference Panels
A list of WHO International Standards, Reference Reagents and Reference Panels
for biological substances is available at: http://www.who.int/biologicals.
At its meeting in October 2015, the WHO Expert Committee on Biological
Standardization made the changes shown below to the previous list.
Additions1
Preparation Activity Status
Antibiotics
Streptomycin* 76 000 IU/vial Fourth WHO International
Standard
Biotherapeutics other than blood products
Tumour necrosis factor 10 000 IU/ampoule First WHO International
receptor Fc fusion protein Standard
(etanercept)
Antibodies to Nine panel members; no First WHO Reference Panel
erythropoietin (human) unitage assigned
Blood products and related substances
Anti-A and anti-B in For DRT: 128 (nominal Second WHO Reference
serum and plasma for use anti-A and anti-B titre) Reagent
in haemagglutination For IAT: 256 (nominal anti-A
assays and anti-B titre)
Blood coagulation factor 10.5 IU/ampoule Fifth WHO International
IX (concentrate) Standard
Unless otherwise indicated, all materials are held and distributed by the National Institute for Biological
1
Standards and Control, Potters Bar, Herts, EN6 3QG, the United Kingdom. Antibiotic reference preparations
identified by an * in the above list are held and distributed by the European Directorate for the Quality
of Medicines & HealthCare, Council of Europe, 7 allée Kastner, CS 30026 F-67081, Strasbourg, France.
Materials identified by an ** in the above list are held and distributed by the Paul-Ehrlich-Institut, 63225
Langen, Germany.
265

Assignment of FIX 0.9 IU/ampoule Fourth WHO International
antigen value to the Standard
current Fourth WHO
International Standard for
blood coagulation factors
II, VII, IX, X (plasma)
In vitro diagnostic device reagents
JC virus DNA for 7.0 log10 IU/ml First WHO International
NAT‑based assays Standard
BK virus DNA for 7.2 log10 IU/ml First WHO International
NAT‑based assays Standard
Hepatitis C virus RNA for 5.0 log10 IU/vial Fifth WHO International
NAT-based assays Standard
Anti-Toxoplasma gondii 160 IU/ampoule Fourth WHO International
(human) (IgG content 263 U/ Standard
ampoule)
Assignment of a 107 pmol/l First WHO International
holotranscobalamin value Standard
to the current First WHO
International Standard for
vitamin B12 and folate in
human serum
Human C-peptide 8.64 µg/ampoule First WHO International
Standard
Hepatitis E virus Eleven panel members; First WHO Reference Panel
genotypes for NAT-based no unitage assigned
assays**
Extension of the First Extended by 10 bacterial First WHO Repository

WHO Repository of strains
platelet transfusion
relevant bacterial
strains**
Ebola virus antibodies 1 U/ml First WHO reference
reagents
Ebola virus RNA for 7.5 log10 U/ml (for assays First WHO reference
NAT‑based assays targeting the np, vp35 and reagents
gp genes); and 7.7 log10 U/ml
(for assays targeting the
vp40 and l genes)
266
Annex 6

Vaccines and related substances
Anti-EV71 serum (human) 1000 IU/ampoule First WHO International
Standard
EV71 neutralization assay 300 IU/ampoule First WHO Reference
standard Reagent
Meningococcal 0.845 ± 0.043 mg/ampoule First WHO international
serogroups A and X (for serogroup A); and standards
polysaccharide 0.776 ± 0.089 mg/ampoule
(for serogroup X)
Diphtheria toxoid for use 1870 Lf/ampoule Third WHO International
in flocculation test Standard
267
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999
999

This report presents the recommendations of a WHO Expert
Committee commissioned to coordinate activities leading to the
adoption of international recommendations for the production
and control of vaccines and other biological substances, and the
establishment of international biological reference materials.
Following a brief introduction, the report summarizes a
number of general issues brought to the attention of the
Committee. The next part of the report, of particular relevance

to manufacturers and national regulatory authorities, outlines
the discussions held on the development and adoption of
new and revised WHO Recommendations, Guidelines and
on Biological
guidance documents. Following these discussions, a WHO
guidance document on Regulatory assessment of approved
rDNA-derived biotherapeutics was adopted along with WHO
Standardization
Guidelines on the stability evaluation of vaccines for use under
extended controlled temperature conditions and on WHO good
manufacturing practices for biological products. In addition,
revised WHO Recommendations to assure the quality, safety
and efficacy of recombinant human papillomavirus virus-like
particle vaccines were also adopted by the Committee.
Subsequent sections of the report provide information on the
current status and proposed development of international Sixty-sixth report
reference materials in the areas of antibiotics; biotherapeutics
other than blood products; blood products and related
substances; in vitro diagnostic device reagents; and vaccines
WHO Technical Report Series

and related substances.
A series of annexes are then presented which include an
updated list of all WHO Recommendations, Guidelines and
other documents on biological substances used in medicine
(Annex 1). The above four WHO documents adopted on
the advice of the Committee are then published as part
of this report (Annexes 2–5). Finally, all additions and
discontinuations made during the 2015 meeting to the list of
International Standards, Reference Reagents and Reference
Panels for biological substances maintained by WHO are
summarized in Annex 6. The updated full catalogue of
WHO International Reference Preparations is available at:
http://www.who.int/bloodproducts/catalogue/en/.

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What are the main topics discussed in the report?

What are the main topics discussed in the report?

What guidance documents were adopted by the committee?

What guidance documents were adopted by the committee?

999

WHO Expert Committee on Biological Standardization

WHO Expert Committee

WHO Technical Report Series

WHO Expert Committee on Biological Standardization

WHO Expert Committee

© World Health Organization 2016

Representatives of other organizations

Dr E. Charton, European Pharmacopoeia Department, Strasbourg, France

International Society of Blood Transfusion

Plasma Protein Therapeutics Association

Participated via teleconference.

the United Kingdom

Participated via teleconference.

Participated via teleconference.

Dr D. Stahl, Paul-Ehrlich-Institut, Langen, Germany

Participated via teleconference.

Participated via teleconference.

ICDRA International Conference of Drug Regulatory Authorities

IGPA International Generic Pharmaceutical Alliance

NIBSC National Institute for Biological Standards and Control

rDNA recombinant DNA

SPF specific pathogen free

to them has been relatively limited, particularly in less-resourced countries.

and moved on to the election of meeting officials. In the absence of dissent,

The Secretary to the Committee, Dr David Wood, then presented

Dr E. Griffiths (consulting); Dr P. Minor (public statements and research support); Dr J. Petricciani

(including monoclonal antibodies), convalescent whole blood and plasma,

Next steps will include the preparation of a preliminary outline of a

2.1.2 Blood products and related in vitro diagnostics: recent

2.1.3 Vaccines and biotherapeutics: recent and planned

antibody products should be developed as an addendum. An associated

for regulatory convergence were also highlighted, including through the

Dr Schaerer concluded by highlighting the range of topics that had been

2.2.2 Report of the fifth meeting of WHO collaborating centres

assays and report results in international units (IU) where an international

technique (NAT)-based testing, with such testing then being implemented in

HCV antibody testing a switch from indirect enzyme-linked immunosorbent

2.2.3 Report from the network of WHOCCs for the

currently consists of eight WHOCCs:

■■ National Institute of Food and Drug Safety Evaluation (NIFDS),

2.2.4 Report of a WHO informal consultation on international

aspects of the standardization of therapeutic monoclonal antibodies, which had

retroactive applications of units by regulators. At present, there is some degree

WHO has proceeded cautiously with a standardization programme

2.3 Feedback from custodian laboratories

National Institute for Biological Standards and Control

publish material previously presented in the reports of the Committee.

European Directorate for the Quality of Medicines &

Paul-Ehrlich-lnstitut (PEl), Langen, Germany

approaches and requirements for maintaining the emergency availability of

narcolepsy and MERS-CoV. The Ebola outbreak prompted multiple collaborative

participated in the development of physical standards and methodologies for

2.4 Cross-cutting activities of other WHO committees and groups

In relation to the development of a BQ scheme for biological products