TRS 1039 - 9789240046870 Eng

W H O Te c h n i c a l R e p o r t S e r i e s
1039
WHO Expert Committee

on Biological
Standardization
Seventy-fourth report
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W H O Te c h n i c a l R e p o r t S e r i e s
1 0 3 9
WHO Expert Committee

on Biological
Standardization
Seventy-fourth 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 Expert Committee on Biological Standardization: seventy-fourth report
(WHO Technical Report Series, No. 1039)
ISBN 978-92-4-004687-0 (electronic version)
ISBN 978-92-4-004688-7 (print version)
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Layout by Interligar
Contents
Abbreviations xiii
1. Introduction 1
2. General 4
2.1 Strategic directions in biological standardization 4
2.1.1 WHO overview 4
2.1.2 Vaccines, biotherapeutics, and cell and gene therapy products: recent and
planned activities in biological standardization 5
2.1.3 WHO EUL of COVID-19 vaccines: an update 8
2.1.4 Update from the WHO Product Development for Vaccines Advisory Committee 10
2.1.5 Report of the October meeting of the Strategic Advisory Group of Experts on
Immunization 12
2.1.6 Blood products and in vitro diagnostics: recent and planned activities
in biological standardization 13
2.1.7 Update on the use of convalescent plasma for COVID-19 15
2.1.8 Snakebite envenoming 17
2.2 Feedback from custodian laboratories 19
2.2.1 Developments and scientific issues identified by custodians of
WHO international reference standards 19
3. International Recommendations, Guidelines and other matters
related to the manufacture, quality control and evaluation of
biological products 24
3.1 General 24
3.1.1 Assessment of stability in international collaborative studies 24
3.2 Blood products and related substances 25
3.2.1 Educational modules on clinical use of blood – first tranche 25
3.2.2 WHO policy brief: the urgent need to implement patient blood management 27
3.2.3 WHO guidance on screening donated blood for transfusion-transmissible
infectious agents 28
3.2.4 WHO tools for the stepwise implementation of a haemovigilance system 30
3.3 Cell and gene therapy products 31
3.3.1 Standardization of cell and gene therapy products 31
3.3.2 WHO white paper on regulatory convergence for CGTPs 32
3.4 Vaccines and related substances 34
3.4.1 Amendment to the WHO Recommendations to assure the quality, safety and
efficacy of live attenuated yellow fever vaccines 34
3.4.2 Evaluation of the quality, safety and efficacy of messenger RNA vaccines for
the prevention of infectious diseases: regulatory considerations 35
4. International reference materials – biotherapeutics other than blood
products 38
4.1 WHO international reference standards for biotherapeutics other than blood products 38
4.1.1 Third WHO International Standard for follicle-stimulating hormone (human,
recombinant) for bioassay 38
4.2 Proposed new projects and updates – biotherapeutics other than blood products 39
4.2.1 Proposed Second WHO International Standard for interleukin-6 (human,
recombinant) 39
5. International reference materials – blood products and related
substances 41
5.1 WHO international reference standards for blood products and related substances 41
5.1.1 Third WHO International Standard for von Willebrand factor (concentrate) 41
5.1.2 Fourth WHO International Standard for ferritin (human, recombinant) 43
5.2 Proposed new projects and updates – blood products and related substances 44
5.2.1 Proposed Sixth WHO International Standard for blood coagulation factor IX
(concentrate) 44
6. International reference materials – in vitro diagnostics 47
6.1 WHO international reference standards for in vitro diagnostics 47
6.1.1 First WHO International Standard for Mycobacterium tuberculosis (H37Rv)
DNA for NAT-based assays 47
6.1.2 First WHO International Standard for varicella zoster virus DNA for NAT-based
assays 48
6.1.3 First WHO International Standard for anti-Lassa virus immunoglobulin G; and
First WHO International Reference Panel for anti-Lassa virus immunoglobulin G 49
6.1.4 First WHO International Standard for anti-thyroid peroxidase antibodies 51
6.2 Proposed new projects and updates – in vitro diagnostics 52
6.2.1 Proposed Second WHO International Standard for alpha-fetoprotein
(human cord serum) 52
6.2.2 Proposed First WHO International Standard for anti-thyroglobulin antibodies 53
6.2.3 Proposed Fifth WHO International Standard for hepatitis B virus DNA for
NAT-based assays 54
6.2.4 Update on replacement challenges for the First WHO International Standard
for anti-rubella immunoglobulin 56
7. International reference materials – standards for use in
high-throughput sequencing technologies 58
7.1 Proposed new projects and updates – standards for use in high-throughput
sequencing technologies 58
7.1.1 Proposed test protocol and WHO international reference reagents for whole-
genome sequencing in the routine lot release of OPV and control of sIPV 58
8. International reference materials – standards for use in public health
emergencies 60
8.1 Proposed new projects and updates – standards for use in public health
emergencies 60
8.1.1 Proposed First WHO International Reference Panel for antibodies to
SARS-CoV-2 variants of concern 60
8.1.2 Proposed Second WHO International Standard for anti-SARS-CoV-2
immunoglobulin 61
8.1.3 Update on the development of the First WHO International Standard for
SARS-CoV-2 antigen 63
iv
9. International reference materials – vaccines and related substances 65
9.1 WHO international reference standards for vaccines and related substances 65
9.1.1 Second WHO International Standard for diphtheria antitoxin (equine) 65
9.2 Proposed new projects and updates – vaccines and related substances 66
9.2.1 Proposed Fifth WHO International Standard for pertussis vaccine (whole cell) 66
9.2.2 Proposed WHO International Reference Reagent for diphtheria CRM197 antigen 67
Annex 1
WHO Recommendations, Guidelines and other documents related to the manufacture,
quality control and evaluation of biological products 69
Annex 2
Recommendations to assure the quality, safety and efficacy of live attenuated yellow
fever vaccines
Amendment to Annex 5 of WHO Technical Report Series, No. 978 75
Annex 3
Evaluation of the quality, safety and efficacy of messenger RNA vaccines for the
prevention of infectious diseases: regulatory considerations 85
Annex 4
New and replacement WHO international reference standards for biological products 155
v
vi
WHO Expert Committee on Biological Standardization
Seventy-fourth meeting held via video conferencing on 18 to 22 October 2021
Committee members 1
Dr K.M. Boukef, University of Monastir, Monastir, Tunisia
Dr C. Burns, National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom
Dr N. Choudhury, Health City Hospital, Guwahati, India
Professor K. Cichutek, Paul-Ehrlich-Institut, Langen, Germany (Vice-chair; and Chair for the
vaccines and biotherapeutics track)
Dr M. Darko,2 Food and Drugs Authority, Accra, Ghana
Dr A.E. del Pozo, Hospital de Pediatria Garrahan, Buenos Aires, Argentina
Dr I. Feavers, Consultant, Nacton, the United Kingdom (Rapporteur for the plenary sessions
and for the vaccines and biotherapeutics track)
Professor S. Hindawi, King Abdulaziz University, Jeddah, Saudi Arabia (Chair for the blood
products and in vitro diagnostics track)
Mrs T. Jivapaisarnpong, Advisor, King Mongkut’s University of Technology Thonburi,
Bangkok, Thailand (Chair)
Dr G. Kang, Christian Medical College, Vellore, India
Ms C. Morris, National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom
Mr V.R. Reddy,2 South African National Blood Service, Weltevreden Park, South Africa
Dr Y. Sohn, Seoul National University, Seoul, Republic of Korea
Dr D. Teo, Visiting Consultant, Blood Services Group, Health Sciences Authority, Singapore,
Singapore (Rapporteur for the blood products and in vitro diagnostics track)
Dr J. Wang, National Institutes for Food and Drug Control, Beijing, China
Dr Y. Wang, National Institutes for Food and Drug Control, Beijing, China
Dr C. Witten, Center for Biologics Evaluation and Research, Food and Drug Administration,
Silver Spring, MD, United States of America (the USA)
1
The decisions of the Committee were taken in closed session with only members of the Committee
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.
2
Unable to attend.
vii
WHO Expert Committee on Biological Standardization Seventy-fourth report
Temporary advisors
Dr P. Aprea, National Administration of Drugs, Food and Medical Technology, Buenos Aires,
Argentina
Dr A.D. Barrett, University of Texas Medical Branch, Galveston, TX, the USA
Dr C.A. Bravery, Consultant, London, the United Kingdom
Dr M. Buda, European Directorate for the Quality of Medicines & Healthcare, Strasbourg,
France
Dr E. Griffiths, Consultant, Kingston upon Thames, the United Kingdom
Dr A. Hilger, Paul-Ehrlich-Institut, Langen, Germany
Dr A. Kitchen, Consultant, Billericay, the United Kingdom
Dr H. Klein, National Institutes of Health, Bethesda, MD, the USA
Dr M.A. Liu, Consultant, Lafayette, CA, the USA
Dr L. Mallet, European Directorate for the Quality of Medicines & Healthcare, Strasbourg,
France
Dr P. Marks, Center for Biologics Evaluation and Research, Food and Drug Administration,
Silver Spring, MD, the USA
Dr J. Martin, National Institute for Biological Standards and Control, Potters Bar, the UK
Dr H. Meyer, Paul-Ehrlich-Institut, Langen, Germany
Dr P. Minor, St Albans, the United Kingdom
Professor D.H. Muljono, Eijkman Institute for Molecular Biology, Jakarta, Indonesia
Dr M. Page, National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom
Dr K. Peden, Center for Biologics Evaluation and Research, Food and Drug Administration,
Silver Spring, MD, the USA
WHO Technical Report Series, No. 1039, 2022
Dr P. Salmikangas, Consultant, Klaukkala, Finland

Dr S. Sankarankutty, Health Sciences Authority, Singapore, Singapore
Dr R. Sheets, Consultant, Silver Spring, MD, the USA
Dr C. So-Osman, Sanquin, Netherlands
Dr P. Strengers, Consultant, Amsterdam, Netherlands
Dr N. Verdun, Center for Biologics Evaluation and Research, Food and Drug Administration,
Rockville, MD, the USA
Dr A.L. Waddell, Stanley, the United Kingdom (Editor of the report of the Committee)
Dr K-W Wan, Medicines and Healthcare products Regulatory Agency, London, the United
Kingdom
viii
Dr S. Wendel, Hospital Sirio-Libanês, São Paulo, Brazil

Dr B. Whitaker, Food and Drug Administration, Silver Spring, MD, the USA
Dr M. Wierer, European Directorate for the Quality of Medicines & Healthcare, Strasbourg,
France
Dr T. Wu, Biologic and Radiopharmaceutical Drugs Directorate, Health Canada, Ottawa,
Canada
Dr M. Xu, National Institutes for Food and Drug Control, Beijing, China
State actors
Dr N. Almond, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr M. Bailey, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr K. Chumakov, Center for Biologics Evaluation and Research, Food and Drug
Administration, Bethesda, MD, the USA
Dr A. Douglas-Bardsley, National Institute for Biological Standards and Control, Potters
Bar, the United Kingdom
Dr B. Fox, National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom
Dr J, Fryer, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr S. Govind, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr L. Hassall, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr M.M. Ho, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr K. Ishii, National Institute of Infectious Diseases, Tokyo, Japan
Dr A. Ishii-Watabe, National Institute of Health Sciences, Kawasaki, Japan
Dr S. Kempster, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr K.M. Lee, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr G. Mattiuzzo, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr M. Moore, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
ix
Dr M. Nübling, Paul-Ehrlich-Institut, Langen, Germany

Dr M. Ochiai, National Institute of Infectious Diseases, Tokyo, Japan
Dr K. Partridge, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr G. Raychaudhuri, Center for Biologics Evaluation and Research, Food and Drug
Administration, Silver Spring, MD, the USA
Dr P. Rigsby, National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom
Dr N. Rose, National Institute for Biological Standards and Control, Potters Bar, the United
Kingdom
Dr M. Rosu-Myles, Biologic and Radiopharmaceutical Drugs Directorate, Health Canada,
Ottawa, Canada
Dr C. Schärer, Swiss Agency for Therapeutic Products, Bern, Switzerland
Dr K.H. Sohn, Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea
Dr J. Southern, Representative of the South African Health Products Regulatory Authority,
Simon’s Town, South Africa
Dr P. Stickings, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr Y. Takahashi, National Institute of Infectious Diseases, Tokyo, Japan
Dr C. Thelwell, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr G. Unger, Paul-Ehrlich-Institut, Langen, Germany
Dr A. Vasheghani, Food and Drug Organization, Tehran, the Islamic Republic of Iran
Dr M. Wadhwa, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Dr S. Williams, National Institute for Biological Standards and Control, Potters Bar, the
United Kingdom
Observers from non-state actors in official relations

International Alliance for Biological Standardization
Professor J-H. Trouvin, Paris, France
International Federation of Pharmaceutical Manufacturers & Associations
Dr M. Gencoglu
International Society of Blood Transfusion
Dr M. Busch, the USA
x
Professor E. Wood, Melbourne, Australia

International Society on Thrombosis and Haemostasis
Dr J. Meijers, Amsterdam, Netherlands
United States Pharmacopeial Convention
Dr F. Atouf, Rockville, MD, the USA
Dr K. Carrick, Rockville, MD, the USA
Representation from intergovernmental and other entities

Africa Society for Blood Transfusion
Dr M. Farouk, Cairo, Egypt
Chinese Pharmacopeia
Dr X. Zhao, Beijing, China
Coalition for Epidemic Preparedness Innovations
Dr V. Bernasconi, Oslo, Norway
Dr W. Dowling, Oslo, Norway
Dr P. Kristiansen, Oslo, Norway
Developing Countries Vaccine Manufacturers Network
Dr P. Carneiro, Instituto Butantan, São Paulo, Brazil
International Generic and Biosimilar Medicines Association
Dr M. Baldrighi, Medicines for Europe
Dr M. Schiestl, Sandoz
Dr K. Watson, Celltrion
International Foundation for Patient Blood Management
Dr A. Hofmann, University Hospital Zurich, Zurich, Switzerland
Plasma Protein Therapeutics Association
Dr D. Misztela, Brussels, Belgium
World Health Organization (WHO)

Access to Medicines and Health Products (MHP)
Dr M. Simão, Assistant Director-General
Health Products Policy and Standards (MHP/HPS)
Dr C. Ondari, Director
Technical Standards and Specifications (MHP/HPS/TSS)
Dr I. Knezevic (Secretary to the Committee; Lead for the vaccines and biotherapeutics track)
Dr Y. Maryuningsih (Lead for the blood products and in vitro diagnostics track)
Mr S. Chatzixiros
Ms S. Jenner
xi
Dr R. Isbrucker
Dr H-N. Kang
Dr D. Lei
Dr S.H. Yoo
Dr J. Yu
Dr T. Zhou
Medical Devices and Diagnostics (MHP/HPS/ATM/MDD)
Dr F.G. Moussy
In Vitro Diagnostics Assessment (MHP/RPQ/PQT/IVD)
Dr U. Stroher
Vaccines and Immunization Devices Assessment (MHP/RPQ/PQT/VAX)
Dr C. Rodriguez-Hernandez
Dr D. Williams, Consultant on venom and antivenom
Agenda, Policy and Strategy (UHL/IVB/APS)
Dr J. Hombach
Product and Delivery Research (UHL/IVB/PDR)
Dr E. Sparrow
Representation from WHO regional offices 3

WHO Regional Office for Africa
Dr B.D. Akanmori
WHO Regional Office for the Americas
Dr M.L. Pombo
Dr M. Beltran
WHO Regional Office for the Eastern Mediterranean
Dr H. Langar
Dr Y. Abdella
WHO Regional Office for the Western Pacific
Dr J. Shin
3
Unable to attend: WHO Regional Office for South-East Asia and WHO Regional Office for Europe.
xii
Abbreviations
ACT WHO Access to COVID-19 Tools (Accelerator)
AFP alpha-fetoprotein
AG-BRA Advisory Group for Blood Regulation, Availability and Safety
ATMP advanced therapy medicinal product
BAU binding antigen unit
BCG bacillus Calmette–Guérin
CBER Center for Biologics Evaluation and Research
CCP COVID-19 convalescent plasma
CEPI Coalition for Epidemic Preparedness Innovations
CGTP cell and gene therapy product
COVID-19 coronavirus disease 2019
CRM197 cross-reacting material 197
Ct cycle threshold
cVDPV2 circulating vaccine-derived poliovirus serotype 2
DAT diphtheria antitoxin
DNA deoxyribonucleic acid
EDQM European Directorate for the Quality of Medicines &
HealthCare
ELISA enzyme-linked immunosorbent assay
EUL emergency use listing
FDA US Food and Drug Administration
FSH follicle-stimulating hormone
FIX blood coagulation factor IX
GBT Plus Blood WHO Global Benchmarking Tool Plus Blood
GP glycoprotein
HBV hepatitis B virus
HIV human immunodeficiency virus
ICDRA International Conference of Drug Regulatory Authorities
xiii
IFU instructions for use

IL-6 interleukin-6
IPV inactivated poliomyelitis vaccine
ISBT International Society of Blood Transfusion
IU International Unit(s)
IVD in vitro diagnostic
LASV Lassa virus
LMIC low- and middle-income countries
mAb monoclonal antibody
MAPREC mutant analysis by polymerase chain reaction and restriction
enzyme cleavage
mRNA messenger RNA
NAT nucleic acid amplification technique
NIBSC National Institute for Biological Standards and Control
NP nucleoprotein
NRA national regulatory authority
OPV oral poliomyelitis vaccine
PBM patient blood management
PCR polymerase chain reaction
PDVAC WHO Product Development for Vaccines Advisory Committee
PEI Paul-Ehrlich-Institut
Ph. Eur. European Pharmacopoeia
Ph. Eur. BRP European Pharmacopoeia Biological Reference Preparation
RDT rapid diagnostic test
RNA ribonucleic acid
RSV respiratory syncytial virus
SAGE Strategic Advisory Group of Experts (on Immunization)
SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
sIPV Sabin IPV
xiv
Abbreviations
TB tuberculosis
TgAb thyroglobulin antibody
TPO thyroid peroxidase
TPP target product profile
VOC variant of concern
VWF von Willebrand factor
VZV varicella zoster virus
xv
1. Introduction
The seventy-fourth meeting of the WHO Expert Committee on Biological
Standardization was held from 18 to 22 October 2021 via video conference due to
the travel restrictions imposed during the coronavirus disease 2019 (COVID 19)
pandemic. The meeting was opened on behalf of the Director-General of WHO
and the Assistant Director-General, Access to Medicines and Health Products, by
Dr Clive Ondari, Director, Health Products Policy and Standards. Dr Ondari began
by welcoming Committee members, meeting participants and observers, noting
that this was one of the most long-standing WHO expert committees. Dr Ondari
also noted the particularly large number of new members of the Committee for
the current meeting and expressed his gratitude for the commitment and support
of all members in these challenging times. Dr Ondari singled out Dr Harvey
Klein for particular thanks following his recent retirement from the Committee,
and expressed appreciation for his considerable contribution to the WHO Blood
Regulators Network and to the blood-related activities of the Committee over the
past 15 years.
Dr Ondari noted that the work of WHO had inevitably been impacted
by the additional demands arising from the COVID-19 pandemic. Nevertheless,
there remained a pressing need for WHO to maintain its wide range of global
public health activities. Reflecting on the broad agenda of the current meeting, Dr
Ondari looked forward to receiving the expert advice of the Committee in these
areas, and thanked the secretariat for their efforts in preparing for the meeting.
Dr Ondari acknowledged the hard work of all of the WHO expert committees in
addressing both the COVID-19 and non-COVID-19 activities of WHO.
The WHO response to COVID-19 was continuing at all levels of
the organization, and was geared towards accelerating the development and
availability of diagnostics, therapeutics and vaccines. Dr Ondari noted the crucial
ongoing contribution of the Committee to all three of these pillars of the WHO
Access to COVID-19 Tools (ACT) Accelerator, and expressed the view that the
speed of the Committee in facilitating the development of WHO written and
measurement standards for COVID-19 had in part being made possible by its
previous experience of other emerging pathogens. In this regard, the Executive
Board of the World Health Assembly had placed on record its appreciation for
the rapid development of the WHO regulatory considerations document on the
evaluation of messenger RNA (mRNA) vaccines. Dr Ondari also highlighted
the pivotal role of the National Institute for Biological Standards and Control
(NIBSC) in providing measurement standards to support COVID-19 vaccine
clinical trials with unprecedented speed. In addition, upcoming WHO guidance
documents on the evaluation and use of monoclonal antibodies (mAbs) for the
treatment of infectious diseases, including COVID-19, will make an important
contribution to the therapeutics pillar of the ACT Accelerator. The advice of
1
the Committee would also continue to be needed on the use of COVID-19

convalescent plasma (CCP) given that this approach was currently being used
widely in many low- and middle-income countries (LMIC) to treat the disease
despite inadequate clinical trial data. Dr Ondari informed the Committee that
a new advisory group – the Advisory Group for Blood Regulation, Availability
and Safety (AG-BRAS) – had been established to drive global improvements in
the safety and accessibility of blood products. This new advisory group would be
at the disposal of the Committee and Dr Ondari expressed his gratitude to Dr
Anneliese Hilger for her leadership as its chair.
Dr Ondari briefly reviewed the agenda of the meeting noting that several
cross-cutting activities with other WHO committees and groups would be
discussed, including policy matters arising from the Strategic Advisory Group of
Experts on Immunization (SAGE), the emergency use listing (EUL) of COVID-19
vaccines and priorities in vaccine development. Regarding biotherapeutics,
an update was to be provided on the upcoming revised WHO Guidelines on
evaluation of biosimilars and on the importance of this revision in accelerating
universal access to biosimilar medicines by 2030. Dr Ondari concluded by saying
that he also looked forward to receiving the Committee’s input on planned WHO
activities in the developing area of cell and gene therapies.
Dr Ivana Knezevic, Secretary to the Committee, thanked Dr Ondari for
his opening remarks. Dr Knezevic reminded meeting participants that the setting
of norms and standards and promoting their implementation is a core function
of WHO and a vitally important element in providing WHO leadership in global
health matters. For example, since 2020, a wide range of activities related to the
COVID-19 pandemic had been initiated by WHO, including the development of
WHO written and measurement standards for consideration and endorsement
by the Committee. As noted above by Dr Ondari, the important contribution
made by the Committee to the prompt adoption and rapid availability of WHO
guidance on the evaluation of mRNA vaccines had been recognized by the
Executive Board of the World Health Assembly in January 2021.

Noting that this was the fourth meeting of the Committee to be held via
video conference, Dr Knezevic reflected on the large attendances at the three
previous meetings held in 2020. Going forward, there would now be two meetings
per year – in April and October. Although this decision had partly been driven by
the demand for standards related to COVID-19 response efforts, it was felt that
biannual meetings would more generally facilitate the timely establishment of WHO
written and measurement standards, and the endorsement of project proposals. In
order to provide the necessary breadth of expertise and ensure all WHO regions
were represented, the Expert Advisory Panel on Biological Standardization from
which Committee members were selected would continue to be expanded.
Dr Knezevic then outlined the procedures and working arrangements
for the present meeting. An open information-sharing session involving all
2
Introduction
participants including non-state actors would be held on Monday 18 October

2021. Committee members, regulatory authority representatives and subject
matter experts from governmental organizations would then participate in
the main meeting from Monday 18 October to Thursday 21 October 2021. On
Tuesday and Wednesday, the agenda would be divided into two parallel tracks
with vaccines and biotherapeutics discussed in one track and blood products
and in vitro diagnostics in the other. All final decisions and recommendations
on the adoption of WHO written standards and the establishment of WHO
measurement standards would be made in a closed session held on Friday 22
October attended only by Committee members and WHO staff. Dr Knezevic
concluded by thanking WHO staff, WHO drafting and working group members,
colleagues from WHO collaborating centres and custodian laboratories, and the
many individual experts present for their invaluable support.
Following the conclusion of the open information-sharing session,
the meeting officials were elected. In the absence of dissent, Mrs Teeranart
Jivapaisarnpong was elected as Chair and Professor Klaus Cichutek as Vice-chair
for the plenary sessions, with Dr Ian Feavers as Rapporteur. Professor Cichutek
was elected as Chair for the vaccines and biotherapeutics track with Dr Feavers
elected as Rapporteur. Professor Salwa Hindawi was elected as Chair for the blood
products and in vitro diagnostics track with Dr Diana Teo elected as Rapporteur.
Dr Knezevic presented the declarations of interests completed by all members
of the Committee and by WHO temporary advisers and participants. After
evaluation, WHO had concluded that none of the interests declared constituted
a significant conflict of interest and that the individuals concerned would be
allowed to participate fully in the meeting.
The Committee then adopted the proposed agenda and timetable
(WHO/BS/2021.2012).
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2. General
2.1 Strategic directions in biological standardization
2.1.1 WHO overview
Dr Knezevic began her overview by noting the link between the work of
the Committee and that of other WHO expert committees and groups,
and by outlining the WHO and other resources relevant to WHO biological
standardization activities. Within WHO, the Norms and Standards for Biologicals
(NSB) and the Blood and other Products of Human Origin (BTT) teams were
part of the Technical Standards and Specifications Unit which itself was part
of the Health Policy and Standards Department. Other resources included
WHO collaborating centres, stakeholders, external experts and the Expert
Advisory Panel on Biological Standardization. Among recent improvements,
BTT had been strengthened and the Expert Advisory Panel expanded to ensure
the availability of expertise in new areas of work and to improve geographical
representation. Dr Knezevic then outlined the four strategic priorities of the
WHO five-year plan to help build effective and efficient regulatory systems,
namely: (a) strengthening national and regional regulatory systems; (b)
improving regulatory preparedness for public health emergencies; (c) reinforcing
and expanding WHO prequalification and product risk assessment; and (d)
increasing the impact of WHO regulatory support activities.
Dr Knezevic then highlighted several recent cross-cutting WHO
COVID-19 activities in the context of the three pillars of the ACT Accelerator.
In the case of the vaccines pillar (COVAX), such activities had included the
development of WHO written and measurement standards, and collaboration
with a range of working groups set up by WHO and partner organizations.
WHO standards were now cited in the criteria for EUL of COVID-19 vaccines
and therefore play an important role in WHO prequalification of such vaccines.
The holding of prequalification meetings with manufacturers had helped to

ensure the correct use of the relevant measurement standards and interpretation
of written standards. For the diagnostics pillar, activities had included the
development of WHO antigen standards for rapid diagnostic tests (RDTs) as well
as an antibody standard and reference panel to support diagnostic assays and
seroepidemiological studies. With regard to the therapeutics pillar, two guidance
documents on mAbs were now being developed. The first of these focused on
manufacturing aspects and was currently the subject of public consultation on
the WHO website while the second, on the nonclinical and clinical evaluation
of mAbs for use in treating infectious diseases, would shortly be posted on the
website for comments. In addition, following the establishment of the First WHO
International Standard for anti-SARS-CoV-2 immunoglobulin in 2020 and a
recent Cochrane rapid review on the use of CCP, consideration was now being
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given to the revision of the current WHO interim guidance on maintaining a safe
and adequate blood supply during the pandemic, and on the safe collection of CCP.
Reflecting on the importance of WHO collaborating centres during
the pandemic, Dr Knezevic specifically highlighted the rapid development and
evaluation of WHO measurement standards undertaken by NIBSC with the
support of other collaborating centres and laboratories worldwide, as well as
the key contributions made by WHO collaborating centres to the production of
WHO written standards. Dr Knezevic noted that NIBSC in the United Kingdom,
the National Institutes for Food and Drug Control in China and the Paul-Ehrlich-
Institut in Germany would complete their re-designations as WHO collaborating
centres in 2021.
Dr Knezevic continued by briefly reviewing the current status of
WHO EUL of COVID-19 vaccines (see section 2.1.3 below), reiterating that
this clearly emphasized the importance of biological standards in the WHO
prequalification process. In the context of the therapeutics pillar of the ACT
Accelerator, the WHO Therapeutics and COVID-19 living guideline provides
conditional recommendations on the use of the neutralizing mAbs casirivimab
and imdevimab, as well as recommendations for or against the use of a range
of other proposed therapeutic products. WHO had also facilitated a number
of interactions among COVID-19 stakeholders, including product developers,
regulatory groups and networks, and national regulators. In several cases, such
activities had provided direct opportunities to promote the appropriate use of
WHO biological standards.
Dr Knezevic then briefly summarized the current status of regulatory
systems in different jurisdictions, noting that 28% of countries had now met
the World Health Assembly resolution WHA67.20 goal of having a stable, well-
functioning and integrated regulatory system as assessed by the WHO Global
Benchmarking Tool. Dr Knezevic concluded by reflecting on the evolving
scientific and regulatory challenges now being faced, and on the importance of
international cooperation in ensuring the safety, quality and efficacy of locally
used medical products, making best use of available resources and expertise,
avoiding duplication, and concentrating regulatory efforts and resources where
they were most needed.
2.1.2 Vaccines, biotherapeutics, and cell and gene therapy products:

recent and planned activities in biological standardization
Dr Knezevic reported on recent and planned WHO standardization activities for
vaccines, biotherapeutics, and cell and gene therapy products (CGTPs). Currently,
a total of 103 WHO written standards set out the key principles for evaluating a
broad range of biological products, and thus provide a basis both for the setting
of national requirements and for WHO prequalification. These documents are
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monitored and where necessary revised in order to reflect significant advances in

scientific knowledge and experience. WHO then supports the implementation of
these written standards by regulators and manufacturers through workshops and
relevant training. Dr Knezevic reminded the Committee that during its meetings
in 2020, four new or revised WHO written standards for vaccines had been
recommended for adoption by WHO.
Dr Knezevic informed the Committee that guidance had now been
provided on the WHO Biologicals website on the application of existing WHO
guiding principles to the evaluation of COVID-19 vaccines. WHO had also
conducted a review of the scientific evidence supporting the development of
mRNA vaccines and had published a review of the scientific and regulatory
aspects of their development and evaluation. These and other efforts were
intended to facilitate international convergence of manufacturing and regulatory
practices, thereby improving access to this important new class of vaccine. Dr
Knezevic presented an overview of COVID-19 mRNA vaccines currently in
clinical development and noted that their success was driving the development
of mRNA vaccines for other infectious diseases. At the current meeting, the
Committee would be asked to consider the new WHO guidance document on
evaluating the quality, safety and efficacy of mRNA vaccines for the prevention
of infectious diseases (see section 3.4.2 below) along with an amendment to the
current WHO Recommendations to assure the quality, safety and efficacy of live
attenuated yellow fever vaccines (see section 3.4.1 below).
Written standards scheduled for consideration by the Committee in
2022 included revised WHO Guidelines on evaluation of biosimilars, the two-
part regulatory considerations document on mAbs for use in treating infectious
diseases outlined above in section 2.1.1 and a manual on the development
of secondary antibody standards. The Committee was updated on recent
progress in the revision of the WHO Guidelines on evaluation of biosimilars
which was now scheduled for submission to the Committee in April 2022. In
addition, in order to ensure consistency across the range of related documents,
WHO guidance on biosimilar mAbs and the WHO Questions and Answers
document on biosimilars would also be revised in 2022 for submission to the
Committee in 2022–2023. Other new or revised WHO written standards likely
to be submitted to the Committee from 2022 onwards included: (a) revised
WHO Recommendations for oral poliomyelitis vaccines (OPVs) and other
revised WHO guidance on poliomyelitis vaccine production and evaluation; (b)
revised WHO Guidelines on dengue vaccines and on rotavirus vaccines; and
(c) revised WHO Recommendations for the preparation, characterization and
establishment of international and other biological reference standards. Requests
had also been received from external stakeholders for the revision of the current
WHO written standards on post-approval changes to vaccines and on pandemic
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influenza preparedness, and of the WHO global model regulatory framework for
medical devices including in vitro diagnostics (IVDs). Work had also started on
developing WHO guidance on the standardization of enteric vaccines and would
continue during 2022.
Dr Knezevic then turned to the WHO measurement standards
developed since 2013 – including the three standards established on the advice
of the Committee in 2020 to support the development of COVID-19 vaccines,
therapeutics and diagnostics. It was anticipated that use of the WHO COVID-19
antibody standards would facilitate the harmonized interpretation of clinical
study data by allowing antibody titres to be expressed in International Units (IU)
thereby supporting efforts to identify an immune correlate of protection defined
in IU/mL. The announcing of the establishment of the First WHO International
Standard for anti-SARS-CoV-2 immunoglobulin in the scientific press and its
subsequent rapid worldwide distribution highlighted its importance as a key tool
for vaccine developers, national reference laboratories and academic groups.
Among the other main outcomes of recent Committee meetings had been the
endorsement of a proposal to develop a WHO standard for SARS-CoV-2 antigen
to support the development, assessment and comparability of antigen-based
RDTs and an update on progress would be provided at the current meeting (see
section 8.1.3 below).
Going forward, WHO would continue to provide technical support
to users of its COVID-19 standards, for example through dissemination of its
Considerations for evaluation of COVID-19 vaccines document which provides
advice to manufacturers on both the process and criteria that will be used by
WHO to evaluate COVID-19 vaccines submitted either for prequalification or
EUL. Technical assistance will also be provided to users of WHO COVID-19
standards through COVAX, the vaccines pillar of the ACT Accelerator, and
through WHO participation in the recently established Technical Advisory
Group on COVID-19 Vaccine Composition.
Dr Knezevic concluded by outlining recent WHO efforts in the
standardization of CGTPs. In February 2020, WHO had initiated the
development of a white paper on the fundamental principles and issues related to
the regulatory oversight of CGTPs and an update on progress would be provided
at the current meeting (see sections 3.3.1 and 3.3.2 below). A survey had also
been conducted of CGTP regulation in jurisdictions with relevant marketing
authorization experience to better understand the current global regulatory
landscape and inform the development of future WHO guidance on these
advanced products. The survey results will be analyzed and published in 2022.
In addition to these preparatory steps, the Committee was reminded that in 2019
it had recommended the establishment of two WHO measurement standards
relating to the integration of lentiviral vectors, and had subsequently endorsed
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further measurement standards projects in this area, including on replication-

competent lentiviruses, pluripotent stem cell identity for flow cytometry and
mesenchymal stromal cells.
2.1.3 WHO EUL of COVID-19 vaccines: an update

The Committee was updated on the WHO EUL of COVID-19 vaccines by Dr
Carmen Rodriguez. After briefly summarizing the main features of both WHO
prequalification and EUL, Dr Rodriguez highlighted the reliance of both processes
on the support of WHO-listed authorities, especially through the provision of
regulatory oversight of abbreviated processes.
Following the onset of the COVID-19 pandemic, WHO had reviewed the
activities that needed to be aligned to expedite the assessment and availability of
vaccines in countries. Vaccine development criteria and submission requirements
had been identified and an assessment process established and documented.
In-country approval and post-approval monitoring of safety, quality and
effectiveness would then follow based on national regulatory reliance on WHO
prequalification and EUL. Such a “roadmap” had allowed for product-specific
regulatory alignment in these crucial areas, with further alignment and support
activities now ongoing.
Dr Rodriguez briefly reviewed the current WHO EUL COVID-19
vaccines and the broad range of post-listing monitoring and reporting
commitments that manufacturers were required to undertake. Among these
ongoing commitments, manufacturers were expected to provide updated vaccine
efficacy and effectiveness data, along with monthly safety reports and benefit–
risk evaluations every 6 months. Other post-listing commitments undertaken
by manufacturers included serious adverse events reporting, quality complaint
disclosure and reporting of any post-listing changes that may impact vaccine
quality, safety or efficacy, or any constraints in production or quality control that
might affect the emergency use condition granted to the product.

To date, regulatory approval of COVID-19 vaccines has been based
on data submitted from clinical trials based on efficacy end-points. Data from
these trials and from studies of immunity to natural infection were still being
analysed to identify immune correlates of protection, which to date have not
been determined and which may vary between vaccine platforms. With the
roll-out of effective vaccines, clinical end-point efficacy studies were however
becoming increasingly difficult and were no longer feasible in many regions. As
an alternative approach, comparative immunogenicity (immunobridging) study
designs were being considered on the assumption that neutralizing antibodies
were the vaccine-induced immune marker most likely to imply protection against
SARS-CoV-2 infection – an assumption that explains why some regulators were
willing to accept post-approval changes to current vaccines. The evaluation of new
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COVID-19 vaccines would however present a particular challenge and although

innovative approaches have been proposed there was currently no consensus
among regulators on the best approach to take. With regard to additional and
booster doses, some regulators had selectively approved the use of an additional
vaccine dose to complete the primary immunization schedule for certain
immunocompromised groups. Currently, there was no regulatory consensus
on the use of booster doses for fully vaccinated individuals in the general
population, though a small number of regulators have amended their emergency
authorizations to approve a booster dose of specific vaccines for subsets of the
general population. Dr Rodriguez concluded by listing several issues on which
the Committee may be able to offer specific advice, including the need for further
WHO guidance in a number of key areas.
Responding to questions from the Committee, Dr Rodriguez confirmed
that WHO EUL was valid for 1 year before reassessment based on the nature
and status of the public health emergency. A number of COVID-19 vaccine
manufacturers were currently seeking marketing authorization and would go
through the prequalification process. Clarification was also given that although
prequalification required the continued oversight of a mature regulatory
authority, the authority of record on an EUL was the regulator in the country of
origin of the product.
Acknowledging the challenge of approving innovative vaccines when
Phase III trials based on clinical end-points were not feasible, the Committee
noted that regulators were not prepared to commit to immunobridging studies
and to date no product had been approved based on data from such studies.
WHO was monitoring this issue and if regulatory consensus emerged on the use
of immunobridging data then WHO guidance would be needed. Reflecting on the
considerable complexity of immunobridging criteria and the ongoing discussions
among international regulators, the Committee felt that immunobridging studies
would be less likely to be needed once an immune correlate of protection had
been established, and that regulatory guidance should only be developed once the
situation became clear. Noting the importance of the First WHO International
Standard for anti-SARS-Cov-2 immunoglobulin in efforts to establish an immune
correlate of protection, the Committee applauded the considerable efforts of
WHO in supporting users of its measurement standards and promoting the use
of IU for serological assays. It was suggested that a similar approach might be
taken to promoting the use of IU in the diagnostics area.
The Committee reviewed the request made for additional guidance and
noted that WHO advocated the application of its existing guiding principles
to COVID-19 vaccines as far as possible. WHO should therefore prioritize the
development of guidance that was either unavailable or which could not be
inferred from guidance on other vaccines or infectious diseases. It was noted,
9
for example, that guiding principles for the evaluation of viral vectored vaccines
were already provided in the existing WHO Guidelines on the quality, safety and
efficacy of Ebola vaccines.
2.1.4 Update from the WHO Product Development

for Vaccines Advisory Committee
The Committee was updated on the activities of the WHO Product Development
for Vaccines Advisory Committee (PDVAC), which has continued to convene
virtually throughout the pandemic. With regard to tuberculosis vaccines, the
majority of candidate vaccines in the current pipeline were intended to boost
waning immunity to bacillus Calmette–Guérin (BCG) in adolescents and adults.
PDVAC had advised WHO to develop a roadmap of the steps required for the
prospective introduction of vaccines already in or approaching Phase III trials.
As both next-generation BCG and non-BCG tuberculosis vaccines were under
development, PDVAC had also suggested that WHO consider reviewing, and
where necessary revising, its existing written standards relevant to tuberculosis,
while also noting the current lack of measurement standards for non-BCG
tuberculosis vaccines.
The Committee was then reminded that WHO guidelines on the quality,
safety and efficacy of group B streptococcus vaccines were being developed that,
among other aims, would set out the standards required for WHO prequalification.
A workshop had been held and a series of meetings with regulators and policy-
makers planned for 2022 to discuss potential licensure strategies based on the
identification of an immune correlate of protection. Work was also continuing on
the harmonization of assays for measuring the immunological response to group
B streptococcus infection and vaccination, including the development of a WHO
measurement standard based on pooled serum from individuals immunized
with a hexavalent candidate vaccine that was expected to proceed to Phase III
trials in 2023.
PDVAC has also continued to monitor the development of RSV
immunization products, with several candidate vaccines now in Phase III
trials, including maternal vaccines and mAbs intended to provide passive
immunization to young infants. WHO Preferred Product Characteristics for RSV
vaccines and mAbs were published in 2017 and 2021 respectively. Regulatory
submissions for the most advanced candidate mAbs are expected towards the
end of 2022, and an RSV-specific supplement to the upcoming WHO regulatory
considerations document on the nonclinical and clinical evaluation of mAbs for
infectious diseases (see section 2.1.1 above) is planned. To accelerate access to
these products in LMIC, WHO is raising awareness of the burden of RSV and of
prospective products for disease prevention, and is developing tools to facilitate
in-country decision-making.
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PDVAC has also continued to monitor the development of mAbs

against other infectious diseases. For example, several mAbs against human
immunodeficiency virus (HIV) were being developed as longer-acting
alternatives to daily oral pre-exposure prophylaxis. In collaboration with global
HIV, hepatitis and sexually transmitted infections programmes, WHO Preferred
Product Characteristics for broadly neutralizing mAbs for HIV prevention had
been drafted and subjected to public consultation, with publication scheduled
for early 2022. In addition, two rabies mAb products were now on the market
with others in clinical development. In 2018, the WHO position paper on rabies
was updated to include a recommendation to use rabies mAbs as an alternative
to blood-derived rabies immunoglobulin. In 2021, rabies mAbs were added to
the WHO Model List of Essential Medicines and a rabies-specific supplement to
the WHO regulatory considerations document on the nonclinical and clinical
evaluation of mAbs for infectious diseases is planned.
The update concluded with a review of the enteric vaccine development
pipeline, noting in particular the prospects for Salmonella combination vaccines
in the future. With regard to the ongoing development of a WHO Shigella
reference serum to support assay standardization, the utility of controlled human
infection models in vaccine development was raised. Although WHO regulatory
considerations for human challenge trials have been published, there may be a
need for specific guidance on trials involving enteric pathogens.
The Committee welcomed the recent developments outlined which
had generated a lot of interest, particularly with regard to the development of
novel vaccines and other products. Nevertheless, despite the availability of
new platforms, many challenges remain, including the need to elicit the most
appropriate immune responses, the lack of a correlate of protection for tuberculosis
vaccines and the need to identify protective epitopes for HIV. Commenting
specifically on the development of mAbs against HIV, the Committee was
assured that several longer-acting alternatives to daily prophylaxis were in the
pipeline. The Committee then asked about the prospect of further mRNA vaccine
developments following the success of this approach for COVID-19 vaccines but
was informed that PDVAC had not yet specifically discussed this issue.
With regard to written standards, the Committee noted the proposal to
incorporate disease-specific supplements into the upcoming WHO guidance
on nonclinical and clinical evaluation of mAbs for infectious diseases, while
also emphasizing the current need to focus on COVID-19. The Committee also
acknowledged that novel tuberculosis and recombinant BCG vaccine developments
would probably require the revision or amendment of existing WHO guidance
and noted the absence of WHO measurement standards for prospective non-
BCG vaccines. The Committee urged WHO to continue to regularly review the
need for both written and measurement standards in this area.
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2.1.5 Report of the October meeting of the Strategic

Advisory Group of Experts on Immunization
Dr Joachim Hombach updated the Committee on matters discussed at the
October meeting of the Strategic Advisory Group of Experts on Immunization
(SAGE). With regard to polio vaccination it had been noted that during the first 9
months of 2021 there had only been two paralytic cases of wild poliovirus type 1
and 326 cases of circulating vaccine-derived poliovirus, predominantly serotype
2 (cVDPV2). Given the desire of countries to use only inactivated poliomyelitis
vaccine (IPV), SAGE recommended a primary three-dose IPV series for low-risk
areas with an optional two-dose series for the lowest risk areas. It was further
agreed that the same early schedules currently recommended for the whole cell
pertussis pentavalent vaccine would also be applicable to the hexavalent vaccine
containing IPV. SAGE also endorsed the wider use under WHO EUL of novel
OPVs against type 2 polioviruses based on genetically stabilized viruses to allow
countries to promptly respond to outbreaks caused by cVDPV2.
Dr Hombach then drew the attention of the Committee to the interim
SAGE recommendation applicable to all WHO EUL COVID-19 vaccines that
moderately or severely immunocompromised people should receive an additional
dose of vaccine as part of an extended primary series. Interim recommendations
had also been issued on the use of an extended primary immunization series for
inactivated COVID-19 vaccines produced by certain manufacturers to address
their lower efficacy in older people. Dr Hombach also informed the Committee
that an inactivated COVID-19 vaccine (Covaxin) formulated with a novel
adjuvant had been authorized for use in adults by the regulatory authority of India
and subsequently in 14 other jurisdictions. Preliminary results indicated that the
vaccine was safe and highly effective against hospitalization and death while the
delta variant was predominant in the population. However, data on the use of the
vaccine during pregnancy were limited, with developmental and reproductive
toxicology data still anticipated and there was no WHO recommendation to use
this vaccine in pregnant women. No policy recommendations would be issued
until the vaccine had been granted WHO EUL.
In a joint session, SAGE and the Malaria Policy Advisory Group had
recommended a four-dose schedule of the RTS,S/AS01 malaria vaccine for
children from the age of 5 months in regions with moderate to high malaria
transmission, with the possibility of a five-dose schedule in areas with highly
seasonal malaria. These recommendations were based on the results of a pilot
implementation programme conducted in Ghana, Kenya and Malawi involving
more than 800 000 children. Malaria vaccine delivery through routine childhood
immunization programmes has the ability to reach vulnerable children not
protected by other interventions and in the pilot programme led to statistically
significant reductions in both severe hospitalized malaria cases and hospitalization
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with malaria infection. There was also no evidence that safety signals noted
during Phase III trials had been caused by the vaccine.
SAGE also reinforced the importance of seasonal influenza vaccination,
particularly among the most vulnerable groups, with vaccination in both prior
and current seasons producing higher levels of protection than no or partial
vaccination. In addition, co-administration of inactivated seasonal influenza
vaccine with any WHO EUL COVID-19 vaccine would be acceptable and
expected to maximize the uptake of both vaccines. SAGE also stressed the need
to improve access to hepatitis E vaccine. Although a subunit vaccine has been
available since 2015, it is not widely used and manufacturers were urged to apply
for WHO prequalification. To help address the current lack of data to support
its use, WHO had been urged to work with Gavi, the Vaccine Alliance, on the
inclusion of hepatitis E vaccination for outbreak response, and on hepatitis E
diagnosis and surveillance in Gavi-eligible countries.
During discussion, it was clarified that the data on the impact of prior
vaccination with seasonal influenza vaccine on subsequent levels of protection
had been derived from a systematic review and were based on vaccinations in
consecutive years. Reflecting on the low incidence of poliovirus infections, the
Committee also asked whether bivalent vaccination campaigns were continuing
and was informed that the majority of countries still used bivalent OPV in their
routine schedule, with IPV only recommended for very low risk countries.
The Committee then enquired about the issues to be discussed at
upcoming SAGE sessions and was informed that a decision-making framework
for the implementation of booster doses of COVID-19 vaccines would shortly be
proposed. This was a complex issue as decisions are ultimately made in-country
and are vaccine specific. As it would be premature to issue vaccine-by-vaccine
recommendations at present, the decision-making framework was being put
forward. Discussion then moved on to the possibility of SAGE recommendations
being issued on heterologous boosting and childhood immunization in the
context of COVID-19 vaccine supply issues. The Committee was informed that
SAGE was undertaking a systematic review of the available data and it now looked
likely that it would recommend a more flexible approach to the use of different
booster vaccines in due course. Although children were at low risk of severe
disease, SAGE was considering a recommendation to immunize adolescents
based on the burden of disease and on the potential to reduce transmission as
vaccine supplies increased.
2.1.6 Blood products and in vitro diagnostics: recent and

planned activities in biological standardization
Dr Yuyun Maryuningsih began by informing the Committee of the establishment
of the Advisory Group for Blood Regulation, Availability and Safety (AG-BRAS)
13
in July 2021. This advisory group had been established to provide technical
advice and recommendations to WHO in the fields of blood regulation and
transfusion medicine. As highlighted by the adoption of several World Health
Assembly resolutions, there is a global need to ensure the safety and availability of
blood products. Furthermore, the development of the WHO Action framework
to advance universal access to safe, effective and quality-assured blood products
2020–2023 now provides a global strategic direction. In January 2020, the merging
of the WHO blood products and blood safety and transplantation teams resulted
in the combined and strengthened WHO Blood and other Products of Human
Origin (BTT) team, and an associated need for wide-ranging expert guidance from
experts in the field. The new advisory group would reflect the need for a suitable
diversity of expertise and experience, while also ensuring representation from all
six WHO regions. Currently comprising 25 members serving in their individual
capacities, the functions of AG-BRAS were: (a) to advise on the development
of WHO norms, standards and guidelines for ensuring the safety, quality and
availability of blood products; (b) to advise on scaling up the implementation
of WHO policies and strategies, and strengthening national systems for blood
supply and regulation; and (c) to provide scientific assessments of current and
emerging threats to the safety and availability of blood and blood products. To
date, two virtual meetings of the advisory group had been conducted, with a third
scheduled in late 2021 to develop its workplan.
Dr Maryuningsih then summarized the outcomes of a recent workshop
on blood and blood products held as part of a virtual meeting of the International
Conference of Drug Regulatory Authorities (ICDRA). Meeting recommendations
to countries included a call for ministries of health to provide effective
leadership and governance in the development of national blood regulation
systems. Countries were also urged to use the WHO Global Benchmarking
Tool Plus Blood (GBT Plus Blood) to identify gaps and needs in national blood
regulation, with training on the use of this tool having been conducted in nine
countries. ICDRA also recommended that national governance mechanisms are
established and implemented to support collaboration among regulators, blood
establishments and hospitals in developing and improving well-functioning
blood systems. ICDRA recommendations for WHO included: (a) ensuring the
provision of support to countries in developing and strengthening their national
blood regulatory systems as a WHO Action framework priority; (b) providing
periodical reports on the progress achieved in implementing the framework;
and (c) supporting the implementation of WHO guidance and blood policies at
country level, particularly in LMIC.
Dr Maryuningsih then provided an update on the current status of several
WHO documents developed in 2021 that were intended to facilitate achievement
of the high-level strategic objectives set out in the WHO Action framework. A
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policy brief on the urgent need to implement patient blood management (see
section 3.2.2 below) and educational modules on clinical use of blood (see section
3.2.1 below) had both been completed with publication expected by the end of
2021. In addition, planning clearance had been obtained for a WHO guidance
document on screening donated blood for transfusion-transmissible infectious
agents, and for tools for the stepwise implementation of a haemovigilance
system, with both resources now under development. WHO had also organized
or contributed to a series of webinars to promote the use of recently published
WHO guidance in this area, and had held a training workshop on the use of the
WHO GBT Plus Blood tool. Dr Maryuningsih noted that consideration would
also need to be given to updating the WHO interim guidance on maintaining
a safe and adequate blood supply and collecting convalescent plasma in the
context of the COVID-19 pandemic. Dr Maryuningsih concluded by reporting
on the eighth meeting of the WHO network of collaborating centres for blood
products and in vitro diagnostics held in September 2021 at which the relevant
WHO measurement standards to be considered by the Committee at the current
meeting had been discussed.
The Committee sought clarification of the envisaged role of AG-BRAS,
particularly with regard to the existing WHO collaborating centre process for
proposing and developing WHO measurement standards for blood products for
review by the Committee. Clarification was provided that AG-BRAS would focus
on WHO written standards rather than measurement standards, and that WHO
would continue to rely on its network of collaborating centres for the latter.
Further clarification was given that in addition to the need for technical advice and
recommendations on both blood regulation and transfusion medicine following
the formation of the BTT team, WHO norms and regulations governing WHO-
managed networks and engagement with expert bodies also required balanced
geographical and other representation in its advisory mechanisms. It was noted
that several members of the long-established WHO Blood Regulators Network
were also now members of AG-BRAS, and it was expected that the establishment
of AG-BRAS would help rationalize all of the various advisory mechanisms in
this field that WHO had relied on in the past.
The Committee acknowledged the many past contributions made by
the WHO Blood Regulators Network in strengthening harmonization and
standardization efforts in blood product safety and quality, expressed its support
for the new advisory group going forward and looked forward to its contributions
in furthering progress in this area.
2.1.7 Update on the use of convalescent plasma for COVID-19

Dr Cynthia So-Osman updated the Committee on the use of CCP for the treatment
of COVID-19. Both CCP and hyperimmune serum containing polyclonal
15
immunoglobulin G (hyper IgG) had been used early in the COVID-19 pandemic
based on the reported efficacy of convalescent plasma used for diseases such
as severe acute respiratory syndrome in 2003, A(H1N1) influenza in 2009, and
Middle East respiratory syndrome in 2012. However, definitive evidence of the
effectiveness of this approach for COVID-19 was not available. In addition, drugs
shown to be effective in initiating recovery from COVID-19 (such as tocilizumab
and dexamethasone) were not specifically directed towards clearing the virus.
Dr So-Osman outlined the three product types currently available – namely
CCP, hyper IgG and mAbs. CCP can be prepared very quickly once convalescent
donors are available, while hyper IgG and mAbs both take significantly longer
to be produced. In addition, the relatively high costs of producing hyper IgG
and mAbs would likely constrain their availability. The Committee was informed
that Cochrane living systematic reviews were currently in progress for all three
products with the aim of searching the literature for definitive evidence of their
effectiveness.
The first Cochrane living systematic review on the use of CCP or hyper
IgG to treat COVID-19 had been published in May 2020, with a subsequent
review published in July 2020. Both reviews found no difference in primary
outcome (effectiveness) among patients who received or did not receive CCP.
A more recent review had been published in May 2021 based on 13 studies
involving a total of 48 509 participants (including the large RECOVERY study
with more than 11 000 patients). These studies had mostly been carried out in
hospitals, primarily among patients with moderate to severe COVID-19. It was
firmly concluded that CCP conferred no benefit in treating such patients. It was
however unclear whether CCP was of any benefit in treating those with mild or
asymptomatic disease. With 130 studies still ongoing, unpublished or recently
published, these living systematic reviews would continue.
Dr So-Osman reflected on why CCP appeared not to work in these
studies and highlighted the Dutch CONCOVID study which had shown no
difference in viral clearance between hospitalized COVID-19 patients provided
with standard care and those treated with CCP. However, many of the patients
had developed antibodies by day 7 of infection and it was hypothesized that
CCP might be effective if transfused earlier and at a high titre. The results of
studies involving the use of CCP within 7 days of disease onset, and its use in
immunocompromised seronegative patients, had now been published or were
being finalized and would be included in the next Cochrane living systematic
review update. In February 2021, the US Food and Drug Administration (FDA)
issued a Letter of Authorization which stipulated that only early and high-titre
CCP treatments could be used.
Future Cochrane initiatives would evaluate the use of CCP and hyper
IgG to prevent SARS-CoV-2 infection and the use of hyper IgG in treating
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General
COVID-19. The first review of mAb use in COVID-19 patients had been
published in September 2021 and found insufficient evidence to support the use
of this approach in the overall patient population – though it may have a role to
play in specific patient subgroups.
The Committee observed that there were some indications that the very
early use of high-titre CCP in people at high risk of severe disease may lead to a
less severe disease course. Dr So-Osman noted that of four reported studies on
the use of CCP during the pre-hospitalization stage and during early symptomatic
infection, only one had shown any benefit, with the remaining studies reporting
no difference in outcome. Dr So-Osman further clarified that the Cochrane
reviews had primarily involved randomized control trials which allowed for
specific patient subgroups (for example, immunocompromised patients) to
be evaluated and the results aggregated to improve the available data. A Phase
III randomized control study of CCP had recently been concluded that could
potentially also contribute to the subgroup data. The Committee queried whether
it was possible to identify the specific level of antibody required for treatment
efficacy and was referred to the CONCOR-1 Study in which patients had been
divided into groups receiving either high, medium or low antibody titre CCP,
and which had shown no evidence of benefit. The Committee concluded that
the use of CCP was a developing field and with many studies still ongoing, firm
conclusions on its efficacy in different patient subgroups were yet to be reached.
2.1.8 Snakebite envenoming

Dr David Williams updated the Committee on the progress and priorities of
the snakebite envenoming programme at WHO. As part of efforts to improve
access to safe and effective treatments, a technical and scientific advisory group
had been established to develop target product profiles (TPPs) to guide the
development of conventional plasma-derived antivenoms, next-generation
products and small molecule inhibitors. Four such TPPs, for animal-plasma-
derived antivenoms for Africa, would shortly go out for public consultation
and TPPs for other products would be developed throughout 2022. Key
considerations in the preparation of TPPs include the need to ensure: (a)
adequate amounts of active pharmaceutical ingredient; (b) appropriate protein
content; (c) initial doses sufficient to neutralize the average adult venom yield
for each intended species; and (d) acceptable product stability in countries
where the products will be used. Dr Williams then summarized the outcome of
laboratory evaluations of the composition and activity of six products assessed
as part of WHO benefit–risk assessment of sub-Saharan antivenoms. A high
degree of variability had been found in terms of both protein content and active
pharmaceutical ingredient content, with four of the products (based on F(ab′)2
fragments) failing to meet their own specifications in this regard, and three
17
such products containing significant amounts of low molecular weight proteins,

indicating poor process and quality control.
Dr Williams informed the Committee that following advances in the field
there was now a need to review and revise the current WHO Guidelines for the
production, control and regulation of snake antivenom immunoglobulins. One of
the outcomes of the WHO benefit–risk assessments of sub-Saharan antivenoms
had been the recognition of the need for a fundamental reassessment of how
antivenoms were designed and how venoms were selected as immunogens.
Given the substantial benefits offered by intact IgG antivenoms in terms of safety,
production efficiency, viral safety, quality, efficacy and cost, a critical review of the
use of F(ab′)2 and other fragments was now warranted. The assessments had also
indicated that the current WHO guidance on the quality control, stability and
storage of antivenoms would benefit from revision, along with improved guidance
on the preclinical and clinical evaluation of antivenoms to reflect new technologies
and methods, including potential methods for replacing in vivo testing in the
context of the ethical use of animals. The revision process would begin in 2022
with the aim of presenting the updated document to the Committee in 2023.
A key barrier to the development of a WHO prequalification procedure
for antivenoms is the lack of reference materials for all regions, with wide species
and geographical variations presenting a particular challenge. Although reference
venoms are available in eastern Asian and some Latin American countries, they
are not available in sub-Saharan Africa or South Asia, where the incidence of
snakebites is highest. WHO is exploring the development of venom reference
standards for at least six medically important African snake species representing
the full spectrum of envenoming syndromes. A network of national control
laboratories with relevant experience will be needed to work with laboratories
at the forefront of snake venom and antivenom characterization to expedite the
development of such reference standards. A WHO prequalification process could
then be implemented that incorporated the current bespoke antivenom benefit–

risk assessment procedure along with elements of existing WHO prequalification
procedures. A protocol for the development of reference materials in this area will
be presented at a future Committee meeting and discussion of this and related
WHO activities with the Committee in 2022 would be timely.
Dr Williams concluded by informing the Committee of a substantial upgrade
to the WHO snakebite information and data platform. Information and data had
now been better integrated and a range of tools now under development would
shortly be incorporated. These include a DHIS2 data-reporting module on snakebite
burden and antivenom use, a tool to improve access to health care, antivenoms and
other health commodities, and an antivenom cost-effectiveness tool.
Reflecting on the challenges and complexity of developing antivenom
reference materials, the Committee speculated as to whether the proposed
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General
regional standards would have to take into account differences in snake species,
but agreed that the approach taken should focus on the venom rather than the
snake itself. The use of a well-characterized pool of venom as the immunogen
would help to address the inherent variations.
2.2 Feedback from custodian laboratories

2.2.1 Developments and scientific issues identified by custodians
of WHO international reference standards
Center for Biologics Evaluation and Research (CBER), Silver Springs, MD USA
Dr Celia Witten reviewed the standardization activities of CBER during the
last year. CBER COVID-19-related activities had included participation in the
collaborative studies to establish WHO international standards both for nucleic
acid amplification technique (NAT)-based assays and serological assays. In the
context of COVID-19 vaccine development, CBER had participated in WHO
technical working groups on viruses, reagents and immunoassays, and animal
models, with support also provided to WHO workshops on the standardization
of clinical trials and their methodologies. Furthermore, CBER would also
be participating in the forthcoming collaborative studies to establish the First
WHO International Reference Panel for antibodies to SARS-CoV-2 variants of
concern and the Second WHO International Standard for anti-SARS-CoV-2
immunoglobulin (see sections 8.1.1 and 8.1.2 below).
In other areas, CBER had seen an increase in requests for the reference
viruses used to validate high-throughput sequencing as an alternative to in vivo
adventitious virus detection. Replacements stocks were now being developed and
two more viruses (coronavirus and parvovirus) will be included in an expanded
panel, with the results of a collaborative bridging study to be presented to the
Committee for its consideration in due course. Dr Witten also updated the
Committee on a project it had endorsed in 2018 to develop universal reagents
for the potency testing of IPV based on human and mouse mAbs specific for the
D-antigen. These mAbs and other reagents had now been assessed in preliminary
studies and sent to NIBSC for filling so that a collaborative study could start in
late 2021.
With regard to the current meeting, CBER had participated in the
collaborative studies on the First WHO International Standard for anti-Lassa virus
immunoglobulin G (see section 6.1.3 below) and the Third WHO International
Standard for von Willebrand factor (concentrate) (see section 5.1.1 below)
and had been involved in the drafting of the WHO regulatory considerations
document on mRNA vaccines (see section 3.4.2 below). In addition, CBER
was participating in the ongoing revision of the WHO Recommendations for
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OPVs and in the collaborative study to replace animal neurovirulence testing

with whole-genome high-throughput sequencing for routine OPV lot release
and monitoring of IPV production consistency – the outcomes of both of which
were expected to be presented to the Committee in 2022. CBER staff had also
developed the first draft of the white paper on CGTPs scheduled for discussion
at the current meeting (see section 3.3.2 below). Upcoming standardization
activities include participation in a collaborative study to support establishment
of a First WHO International Standard for activated blood coagulation factor X
(human), and in projects to replace the current WHO international standards for
blood coagulation factor VIII (concentrate) and factor IX (concentrate).
European Directorate for the Quality of Medicines &

HealthCare (EDQM), Strasbourg, France
Dr Laurent Mallet updated the Committee on recent standardization activities
at EDQM. Dr Mallet began by reminding the Committee that EDQM is the
custodian laboratory for international standards for antibiotics. Twenty three
international standards for old antibiotics were currently available, eight of
which were on the WHO Model List of Essential Medicines. No issues had
been identified in relation to these standards since the previous meetings of the
Committee in 2020. Since EDQM took over the responsibility for these standards
from NIBSC in 2006, 10 replacement standards had been established.
Dr Mallet then provided an overview of the goals and selected ongoing
activities of the EDQM biological standardisation programme. Among the
projects with implications for the application of the 3Rs principles (Replacement,
Reduction, Refinement) regarding the use of animals in research was an
international collaborative study to validate a quantitative sandwich enzyme-
linked immunosorbent assay (ELISA) as an in vitro alternative to the in vivo
potency test for rabies vaccines. A set of samples consisting of 11 vaccines and
five virus strains, along with a standard operating procedure and reagents, had
been distributed to 31 participants worldwide to evaluate the transferability and
robustness of the method. The results of the study were expected to be reported
to the Committee in 2022. A second project with direct implications for the 3Rs
principles was an initiative to eliminate animal-based pyrogen testing from the
European Pharmacopoeia (Ph. Eur.). Despite changes to relevant texts aimed
at encouraging users to perform in vitro tests (such as the monocyte activation
test) the rabbit pyrogen test continued to be widely used. Dr Mallet set out a
timeline for the complete removal of the rabbit pyrogen test from the Ph. Eur. by
2026 as an essential step towards the exclusive use of in vitro tests for the control
of pyrogens. The current animal-based tests would be replaced by risk-based
analysis of the potential presence of non-endotoxin pyrogens in the product or
intermediate being tested. If the presence of non-endotoxin pyrogens could be
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General
ruled out in this way, then the revised Ph. Eur. will recommend the use of the
bacterial endotoxin test alone; otherwise, the monocyte activation test will be
recommended, either on its own or in addition to the bacterial endotoxin test.
The Committee acknowledged the progress made towards the removal
of the rabbit pyrogen test from the Ph. Eur. and strongly supported moves to
establish the monocyte activation test as an alternative to animal testing for
pyrogens worldwide. Noting that the use of fresh human blood in the test
remained a challenge, it highlighted the need for research into the development of
alternatives based on the use of suitable cell lines and the application of reporter
gene technology. Regarding the development of a quantitative sandwich ELISA
as an alternative to the in vivo potency test for rabies vaccines, the Committee
noted that essential reagents for the assay were available commercially and their
supply was assured. The Committee further noted that ensuring the ability of the
replacement assay to detect subpotent vaccine batches would be critical.
National Institute for Biological Standards and Control

(NIBSC), Potters bar, the United Kingdom
Before beginning his review of recent standardization activities at NIBSC, Dr Marc
Bailey first informed the Committee of an ongoing transformation programme
at the Medicines and Healthcare products Regulatory Agency (MHRA) of which
NIBSC was part.
Dr Bailey then updated the Committee on a specific issue that had arisen
with regard to the First WHO International Reference Panel for lentiviral vector
copy number, established on the recommendation of the Committee in 2019.
Significant differences had been reported between data obtained by droplet digital
polymerase chain reaction (PCR) and quantitative PCR when using the standard,
which would potentially impact on all PCR-based methods. Independent
laboratory analysis had confirmed the presence of method bias. The proposed
solution was to change the unitage originally used for the standard (lentiviral
copies/cell) to IU/ampoule. This would overcome the problem of the standard
being method-dependent, make it consistent with similar WHO standards and
reinforce the IU concept in the advanced therapy medicinal product (ATMP)
community. A correspondingly revised supplement or new report would be
submitted to the Committee for its consideration in 2022.
Dr Bailey went on to update the Committee on NIBSC activities in
relation to COVID-19 reference materials, noting in particular the rapid
depletion of stocks of the First WHO International Standard for anti-SARS-
CoV-2 immunoglobulin and its proposed replacement scheduled for discussion
at the current meeting (see section 8.1.2 below). For now, NIBSC was distributing
secondary standards calibrated against the current standard. Dr Bailey concluded
by reviewing the list of reference materials currently scheduled for submission
21
by NIBSC for discussion by the Committee in early 2022, highlighting the

challenging deadline for such submissions and noting that more projects would
likely be submitted were the meeting to be rescheduled for slightly later in the year.
The Committee began by expressing its profound concern with regard
to the potential upcoming changes to the current financing, structure and
operating environment of NIBSC. As the foremost WHO collaborating centre
in the development of international reference materials, the activities of NIBSC
were pivotal not only to the work of the Committee but to the advancement
of regulatory science worldwide. The role played by NIBSC in developing and
distributing WHO international reference materials was vital in ensuring the
national licensing and release of a huge range of much-needed diagnostics,
vaccines, biotherapeutics and other biological products. In addition, as one of
only four designated essential regulatory laboratories for influenza, NIBSC
plays a central role in both seasonal and pandemic influenza surveillance and
response activities each year. NIBSC also provides crucial support to the Global
Polio Eradication Initiative through its central role in the development of new
poliomyelitis vaccines and as the world’s main custodian of poliovirus strains.
Without the contribution made by NIBSC the hard-won gains in improving
access to vital medicines in some of the poorest countries in the world will
be jeopardized and the prospect of achieving many of the goals and targets of
internationally agreed initiatives such as the sustainable development goals will
be materially damaged. The Committee strongly supported a proposal that WHO
send as a matter of urgency a letter to the chief executive of the MHRA and chair
of its board, setting out the importance of the work of NIBSC in achieving the
global health aspirations of the international community.
Paul-Ehrlich-Institut (PEI), Langen, Germany

Dr Heidi Meyer updated the meeting on the recent standardization activities of
PEI. These had included participation in the development or revision of a number

of WHO written standards, including the new WHO guidance document on
evaluating the quality, safety and efficacy of mRNA vaccines for the prevention
of infectious diseases (see section 3.4.2 below) and the amendment to the
current WHO Recommendations to assure the quality, safety and efficacy of live
attenuated yellow fever vaccines (see section 3.4.1 below) both of which would
be discussed at the current meeting. PEI was also contributing to the ongoing
revision of the WHO Guidelines on evaluation of biosimilars.
Dr Meyer then explained how PEI staff had supported the WHO
prequalification programme by carrying out product summary file reviews of
vaccines and performing lot-release testing of several WHO prequalified vaccines.
PEI had also provided support to the WHO prequalification programme for IVDs
by assessing various SARS-CoV-2 nucleic acid amplification tests for WHO EUL.
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General
Dr Meyer concluded by reporting on the results of a comparative evaluation of

the sensitivity of SARS-CoV-2 antigen RDTs. As of September 2021, 226 different
SARS-CoV-2 antigen RDTs had been evaluated using a panel of 50 positive samples
covering a wide range of viral load (cycle threshold (Ct) = 17–36). The results
revealed a wide range of sensitivity (0/50–43/50 detected) while also highlighting
the criticality of the pre-analytical swabbing steps. Of the 226 RDTs evaluated,
20% failed to meet a sensitivity target of detecting more than 75% of samples with
a Ct less than 25. It was also noted that the batch-to-batch consistency of some
antigen RDTs was poor and that the use of different names for the same original
products hampered traceability. The comparative evaluation of antigen RDTs
would continue.
Noting the wide range of sensitivity found across the SARS-CoV-2
antigen RDTs evaluated so far, the Committee emphasized the importance of
ensuring RDT sensitivity and specificity, and highlighted the potential need for
further WHO guidance on this issue.
23
3. International Recommendations, Guidelines and
other matters related to the manufacture, quality
control and evaluation of biological products
3.1 General
3.1.1 Assessment of stability in international collaborative studies
The Committee was presented with an overview of the key issues in assessing
the stability of WHO international reference standards during their development
and evaluation in international collaborative studies. The biological activity of
most such standards was designated in arbitrary “International Units” (IU) and
their degradation cannot therefore be measured directly as by definition they are
the primary standard for the respective biological activity. Although biological
standards were often highly stable lyophilized preparations, it was nevertheless
important to evaluate their predicted degradation, typically using data from
accelerated thermal degradation studies. In this approach, the potency of test
samples stored at various temperatures between 4–56 °C is assayed at several time
points, usually over a period of between 6 months and 2 years. The potency of
these samples relative to that of baseline samples stored at −20 °C is then used
to predict the loss in activity expected at −20 °C using the Arrhenius equation.
To date, a predicted potency loss of less than 0.1% per year has been considered
acceptable.
There are, however, a number of known shortcomings in the use of the
Arrhenius equation to predict the rate of loss of biological activity. Although
many reference materials appear to exhibit Arrhenius-like behaviour over a
limited range of temperatures, the relationship is approximate and may not be
sustained over a wider temperature range. Estimates obtained by this method can
also have poor precision, while highly stable materials may not lose potency even
when stored at elevated temperatures. In addition, assumptions made about the

course of the degradation process cannot be tested statistically.
The WHO Recommendations for the preparation, characterization and
establishment of international and other biological reference standards sets
out the stability data and related information required by WHO to support
establishment of its international reference standards. This includes information
on participating laboratories, assays used, details of the study design and statistical
considerations. In addition to the stability of the material during storage, it is also
recommended that the stability of the reconstituted standard is determined.
Using the example of the measurement standards to be discussed at the
current meeting, the Committee was provided with an overview of potential
stability study outcomes. Ideally, the predicted degradation rate will demonstrate
acceptable stability but in some cases this rate is either too high to assure stability
24
International Recommendations, Guidelines and other matters
or there was no loss of activity at elevated temperatures. In other cases there may
be no appropriate model for stability prediction or insufficient time to complete
an accelerated thermal degradation study, especially when a standard is urgently
required. For real-time stability monitoring, options include monitoring the
potency of the standard over time relative to another appropriate preparation
and/or monitoring other parameters of the standard (such as physicochemical
characterization data). As the precise situation will vary for different standards, a
clear vision is required of the stability monitoring goals, potential outcomes, and
the assay and statistical tools to be used.
Noting that the vast majority of reference materials were stable and
relatively few turned out to be unsatisfactory, the Committee discussed the
key issues raised and the stability data expected in collaborative study reports.
Although monitoring the stability of reference materials in real time was
considered ideal, the comprehensive post-establishment stability monitoring
of WHO international standards was considered too resource intensive to be
achieved in practice. Furthermore, as the acceptable annual loss of potency
varied among standard preparations this should be considered on a case-by-
case basis rather than regarding 0.1% per year as a requirement for all reference
materials. Observing that the acceptable level of stability typically relates to the
intended use of the standard and to the precision of the assays involved, it was
agreed that a decision tree to inform the design of stability studies rather than
simply following the Arrhenius model should be included when the current
WHO Recommendations were revised. This would help project leaders to know
what the Committee expected in cases where accelerated thermal degradation
studies failed to predict a degradation rate or where the predicted degradation
rate was too high to assure stability. The Committee commented on the wealth of
experience now available on how the characteristics and formulation of different
reference materials affect their stability, suggesting the possibility of more-flexible
approaches to stability studies conducted as part of collaborative studies. It was
noted in this regard that the stability of reconstituted reference materials was
increasingly being assessed in collaborative studies. The Committee concluded
by highlighting the importance of feedback from users of reference materials in
identifying any emerging stability issues
3.2 Blood products and related substances

3.2.1 Educational modules on clinical use of blood – first tranche
The WHO Educational modules on clinical use of blood have been developed as
an update to the WHO Clinical use of blood in medicine, obstetrics, paediatrics,
surgery and anaesthesia, trauma and burns which has been widely used by a
diverse community of health professionals since its publication in 2001. The
Educational modules will provide a new practical resource for a similarly broad
25
global audience to help guide decisions and improve training and knowledge in
the field of transfusion medicine. It is intended that the modules will thus support
implementation of the WHO Action framework to advance universal access to
safe, effective and quality-assured blood products 2020–2023, and complement
the WHO Aide-mémoire for national health programmes on clinical use of blood
and other resources.
The Committee was informed that the module development process
had been a collaborative effort between WHO and the International Society of
Blood Transfusion (ISBT). Materials were developed by an international panel
of authors with broad expertise in transfusion medicine, with feedback provided
by a panel of critical readers drawn from countries worldwide and by the WHO/
ISBT steering committee. This process had ensured that critical inputs could
be invited on the modules to ensure their relevance across the diverse range of
settings in which they might be used.
A brief outline of the nine modules comprising the first tranche of
materials was provided and clarification given that these educational materials
were intended to provide simple and clear information that could be readily
grasped by a general audience, including students. As the aim was to provide an
introduction to the topic rather than an encyclopaedic text, the materials focus
on important principles, key points and practical application, and were illustrated
with clear diagrams and examples of clinical scenarios.
Organized as a series of modules that could be individually updated
as needed, the materials would be made available in both electronic and hard
copy formats, and would be accessible online. Publication of the first tranche of
modules was planned for the end of 2021 and would be followed by a series of
webinars hosted by ISBT in collaboration with WHO. The modules would be
promoted and disseminated by WHO and through ISBT and other policy and
professional networks. Based on the extensive use of the 2001 WHO document
and the positive feedback received, a similarly wide uptake was anticipated for
the modules and their translation into multiple languages was anticipated. In
addition, a second tranche of modules was now in preparation and would likely
be finalized shortly. Topics in the second tranche would complement those in the
first tranche and would include the principles of appropriate use of blood and
blood products and patient blood management, major haemorrhage and massive
transfusion, trauma resuscitation, platelet transfusion, plasma transfusion,
adverse events and transfusion in under-resourced countries.
The Committee recognized the timeliness and importance of the
Educational modules as an update of the 2001 document. Given the global reach
of the earlier document, the Committee welcomed the steps taken to ensure
adequate representation of all regions in the authorship and review processes.
This included Latin America where the previous document, translated into
26
Portuguese and Spanish, had been widely used. The Committee was assured that
representation from all regions would be ensured in both the finalization of the
first tranche of modules and the development and review of the second tranche.
A number of additional topics for inclusion were then suggested by the
Committee, including a separate module on the blood donor. In response, it was
clarified that references to blood donors were generally restricted to stewardship
and responsibility for the blood as the focus of the materials was on clinical
decision-making given the target audience. However, it was recognized that
the topic of walking donors overlaps both the donor and clinical settings and
could be considered in future updates. Other topics of current interest were also
highlighted including the use of convalescent plasma and advanced therapies, but
in such fast-moving fields requiring regular updating only the broader principles
and aspects could realistically be included in what was intended to become a
reference resource of established information and practices.
3.2.2 WHO policy brief: the urgent need to implement

patient blood management
The Committee was informed of the development of a WHO policy brief intended
to support implementation of the WHO Action framework to advance universal
access to safe, effective and quality-assured blood products 2020–2023. This
policy brief specifically relates to Strategic Objective 4 of the Action framework
on implementation of patient blood management (PBM) to optimize the clinical
practice of transfusion.
Anaemia and blood loss taken together represent one of the biggest
public health and health economic burdens at the global level, with an estimated
2.9 billion individuals with anaemia and/or micronutrient deficiencies, and more
than 600 million individuals with chronic or acute blood loss and/or bleeding
disorders. There is therefore an urgent need for countries to implement PBM to help
address this largely preventable and greatly underestimated threat to population
health. By addressing the problems of anaemia, blood loss and coagulopathy
through a patient-centred, systematic and evidence-based approach, PBM has
the potential to significantly improve global population health and the clinical
outcomes of hundreds of millions of surgical, medical and obstetric patients,
while significantly reducing health resource utilization and health care costs.
The Committee was provided with an overview of the three pillars of
PBM and clarification was given of the importance of distinguishing PBM from
the concept of optimal blood use. Optimal blood use programmes are based on
ensuring clinically indicated transfusions at the minimal effective dose and have
a narrower focus compared to the broader clinical approach of PBM. The goal
of PBM was not to reduce or restrict the use of blood transfusions or any other
therapy but rather to place emphasis on the patient’s own blood as a valuable
27
resource even before transfusion is considered. A thorough understanding of

such differences will help to shift the focus from the blood product to the patient.
There is now a significant body of scientific evidence of the clinical
benefits of therapeutic strategies based on PBM, including improved clinical
outcomes. A strong economic argument can also be made in terms of improved
cost-effectiveness, alleviated cost constraints and almost immediate returns on
investment. Additionally, there is an ethical obligation not to ignore and withhold
any medical model that is beneficial not only for society at large but also for
highly vulnerable populations, individual patients and blood donors. The WHO
policy brief therefore calls on countries to adopt a national PBM policy, introduce
the necessary governance, and reallocate resources to improve population
health status and individual patient outcomes, thus reducing overall health
care expenditures. As a next step in this initiative, the development of WHO
implementation guidelines for PBM was planned for 2022–2023 involving a large
multidisciplinary and multi-profession team drawn from all relevant sectors.
The Committee acknowledged the importance of PBM and commended
the next step to develop WHO guidelines on its implementation. Strong concern
was expressed that, if accurate, the estimated total number of people worldwide
with anaemia and/or micronutrient deficiency would represent approximately
one third of the global population. If so, there was a clear and urgent need to
strengthen PBM and its implementation in countries.
3.2.3 WHO guidance on screening donated blood for

transfusion-transmissible infectious agents
The Committee was informed of progress made in the updating of the 2010
WHO document Screening donated blood for transfusion-transmissible
infections: recommendations. The update process had been initiated and guided
by the outcomes of a 2018 WHO review meeting involving independent experts
in the field. The updated document, scheduled for publication by the end of
2021, is intended to assist countries that require support in developing a national
screening programme to maximize the microbial safety of blood donations.
Guidance will be provided on the key infectious agents for which all donations
should be screened, the most appropriate methodology and screening targets,
and the systems required to support effective and reliable screening. The updated
guidance is also intended to assist national blood systems in advocating for the
political support and resources needed.
The Committee was provided with an overview of the key elements and
general guidance provided in the updated document. Key elements include critical
and screening-specific elements of infectious disease screening programmes,
while the general guidance covers important broader aspects of blood services
and their activities which help to support effective screening programmes. The
28
Committee was informed that the overall aims of the update process had been
to ensure that the core guidance remained appropriate while also bringing up
to date the supporting evidence, background information and messages. In
addition, a number of core messages had been strengthened, and the terminology
made clearer and more precise. Developments in understanding and technology
had also been incorporated, along with guidance on the setting of acceptable
risk levels. Specific guidance was now provided on molecular screening and on
a number of “additional” infectious agents for which screening may be required.
During discussion, the Committee enquired whether mention had been
made of the work of the Committee and WHO collaborating centres in developing
assay calibrants and reference materials, and whether these were of relevance.
Confirmation was given that these were indeed relevant to the updated guidance
now provided on quality assurance and quality control. Although the work of the
Committee was not specifically mentioned in the text, reference had been made
to WHO programmes on ensuring quality assurance and quality control, and to
the provision of reference materials. A suggestion to add information, including
website links, on the relevant WHO reference materials now available to support
evaluation of assay performance and assay validation was accepted and would be
included in the final version of the document.
In light of the concerns raised about RDTs and their promotion and use
in many countries, the Committee enquired whether mention of these had been
made in the updated document. Clarification was given that although RDTs were
not directly advocated in the document, they can have a role to play in some
situations. The issue was not the RDTs per se but ensuring their proper evaluation
and validation for their intended use. The Committee emphasized the vital
importance of this issue and the generally poor understanding of the variability
in quality of such tests, citing the SARS-CoV-2 and HIV RDTs as examples. It
was acknowledged that the issues around RDT evaluation and validation were
considerable, and that a process of global evaluation and rapid dissemination of
the outcomes would significantly benefit blood services worldwide.
The Committee noted that the updated document focused primarily on
the screening of donations, and asked if pathogen inactivation might not be a
better approach, considering the threat of future emerging viruses. Clarification
was given that although pathogen inactivation had a role and was mentioned as
part of the overall structure in countries that use the approach, it would not be
of value to many countries until it could be applied effectively across all products
and the associated issues of reduced yield and high cost addressed. The review
group had felt that this evolving situation could not properly be addressed in
the updated document. In addition, advocating for its use might be unhelpful
and could detract from the key messages to be conveyed to the envisaged target
audience of the document.
29
3.2.4 WHO tools for the stepwise implementation of a haemovigilance system

The Committee was informed of the development of a WHO document intended
to support implementation of the WHO Action framework to advance universal
access to safe, effective and quality-assured blood products 2020–2023. The
document relates to one of the outcomes of Strategic Objective 4 of the Action
framework, which includes the putting in place of haemovigilance systems at
both national and organizational levels. Specifically, the document identifies
and enumerates the haemovigilance tools and other resources currently used in
countries worldwide. These resources will be made available to all countries through
a freely accessible online repository to support the stepwise implementation of a
national haemovigilance system. It is envisaged that countries at different stages
of developing a haemovigilance system, including countries with already well-
established systems, will benefit from free access to such resources.
The document builds upon two earlier WHO documents – the WHO
Aide-mémoire for ministries of health: national haemovigilance system and
the WHO guide to establishing a national haemovigilance system. Document
development had started in late 2020 with the establishment of a working
group comprising more than 20 experts from the International Haemovigilance
Network, ISBT, national haemovigilance systems and WHO. Working group
members collected and contributed relevant tools and resources from across
the global haemovigilance community. Both document development and the
collection of related resources were based on a highly inclusive approach involving
broad stakeholder engagement and feedback. In addition, as haemovigilance
can be initiated and enhanced even by committed individuals within a variety
of organizations, the target audience for the document was very broad and
included national level and other policy-makers, blood establishment staff, blood
donor organizations, public health institutions, patient groups, scientific and
professional bodies, and developmental partners and international organizations.
In 2020, a survey had been conducted to identify the priority needs in this
area, with responses received from 87 countries across all six WHO regions. The
provision of educational and training tools and modules in the context of WHO
technical support was identified as the top priority. As this involves a number
of technical and other challenges, including the need to avoid tools becoming
outdated or inaccessible, it had been decided that the ISBT Haemovigilance
website would act as the primary repository, with a smaller set of resources placed
in the WHO Notify Library. The Committee was presented with an overview of
the document structure and contents, which included a directory of the tools and
resources available on the ISBT website. A number of the templates and example
materials were available in English and French, with plans in place to include other
languages. A brief demonstration was then given of how users could navigate the
directory when looking for specific resources. In 2021, the document had been
30
widely shared, with positive feedback and suggestions received. Any identified
gaps in the tools would now be addressed and a final draft document produced
and submitted for WHO clearance by the end of 2021.
The Committee commended the contribution the document would make
to the establishment and strengthening of haemovigilance systems, especially
in developing countries. One important area where guidance was needed was
the analysis of haemovigilance data and follow-up actions to be taken. It was a
common misperception in some countries that the simple provision of data to a
medical information system was equivalent to haemovigilance. The Committee
was informed that this issue had been observed among some survey respondents
at all levels of the haemovigilance system. As a result, this issue had been addressed
in the document which emphasizes that comprehensive haemovigilance was not
just about collecting data but also included data analysis and feedback to hospitals
and blood centres so that improvements could be made. With regard to the need
for ongoing maintenance of the online tools and resources, the Committee was
informed that discussions were taking place to identify the best approach.
The Committee enquired as to the reasons for such a broad target
audience, noting for example that patient groups are very different from blood
establishments and ministries of health. The Committee wondered if it might be
more effective to develop specific tools and resources for each group. In response,
it was highlighted that many countries lack strong national health authorities
and that even though the document was primarily intended for national systems
staff, stakeholders at all system levels had been included. In addition, it was
important to recognize that small initial steps could be taken in some countries
by individuals, some of whom may be involved in efforts to improve patient and
donor safety. The Committee noted that in some cases the Ministry of Health will
even delegate responsibility for the implementing of a haemovigilance system,
and expressed its agreement with the overall approach that had been taken.
3.3 Cell and gene therapy products

3.3.1 Standardization of cell and gene therapy products
The Committee was provided with an overview of developments in cell and gene
therapy products (CGTPs) worldwide. Following outstanding efficacy results for
many of the first-authorized ATMPs the number of such products was increasing
globally with manufacturers increasingly approaching multiple regulatory
authorities. This was now a rapidly evolving area, with 53 clinical trials of gene-
editing approaches primarily based on CRISPR/Cas9 technology either active or
completed – though the evaluation of long-term safety issues remained ongoing. In
addition, the first CD19 CAR T-cell products were now in commercial use, leading
the way for evaluation of other such products, including CAR T-cells for solid
tumours. Current issues in relation to this product type include ensuring specificity
31
for the target antigen, challenges in manufacturing and potential toxicity. Similarly,
the number of approved ex vivo retrovirus and lentivirus gene therapy products
was also increasing – with critical challenges here including the risk of integrational
mutagenesis or oncogenesis, and the need to determine optimal treatment timing. In
addition, adeno-associated viruses were now being widely used for the treatment of
monogenic diseases, with ongoing issues including the need to ensure persistence of
the desired effect while avoiding immune responses directed against the virus vector.
By 2020, more than 1200 ATMP clinical trials were ongoing, with rapid
progress being made through the clinical trial phases. As a result, there would
likely be numerous such products submitted for regulatory approval in the near
future. The Committee was reminded of the inherent and unique risks associated
with ATMPs due to their biological complexity and the current lack of long-term
safety data for the majority of approaches. Early risk identification and mitigation
approaches would therefore be key requirements going forward. In addition,
numerous manufacturing, quality control, testing and legislative issues continue
to impact on the clinical development of ATMPs. Among these, the generation of
clinical evidence to support licensure is particularly challenging with regard to the
choice of comparators, dose and end-points. In the case of severe conditions, many
therapies are potentially curative or ameliorating but no relevant standard of care
exists for comparison, while studies often involve “last-line” patients for whom
best supportive care is not attractive. In the case of orphan treatments, the patient
populations eligible for clinical trials were frequently small and heterogeneous.
After being provided with an overview of existing regulatory frameworks
and guidance documents for CGTPs in different jurisdictions, the Committee
discussed a number of specific regulatory aspects in this area. Among these, the
challenge of developing suitable animal-based and in vitro approaches to ensure
the safety of ATMPs prior to their use in humans was highlighted. In addition,
with regard to the evaluation of point-of-care products, the Committee noted
that it was difficult to control products in hospitals and this issue had yet to be
adequately resolved by the major regulators. It was suggested that this and other
issues raised in this presentation, including the need for early risk identification
and mitigation, should be addressed in the WHO white paper now under
development (see section 3.3.2 below).
3.3.2 WHO white paper on regulatory convergence for CGTPs

The Committee was updated on the development of a WHO white paper on
regulatory convergence for CGTPs. During a WHO working group meeting on
CGTPs held in February 2020, consensus had been reached that CGTPs could
be divided into two categories. The first category consists of products that are
well established and relatively uncomplicated (such as skin or bone grafts) and
where safety and efficacy are assumed. The primary issue with such products
32
was preventing the transmission of infectious diseases. Although significant

issues remain to be addressed in cases where multiple components are mixed
or where stem cells are used, there is now a high degree of commonality among
different regulatory authorities in the ways in which this category of minimally
manipulated cells is regulated. The second category involves a far higher level
of complexity that raises a number of complex regulatory considerations with
regard to the manufacturing and quality control of the associated ATMPs. Such
considerations include whether manufacturing is centralized or decentralized,
ensuring appropriate good manufacturing practice for ATMPs, and aligning
different standards and practices across different regulatory jurisdictions.
Furthermore, establishing the critical quality attributes of such products will
be crucial and will require the establishment of well-defined TPPs. Other
manufacturing issues warranting regulatory consideration include how ATMPs
are transported and the delivery systems used to administer them. In addition
to manufacturing issues, the nonclinical development of ATMPs and the clinical
evidence needed to support their approval will be crucially important elements of
any prospective regulatory framework. For some ATMPs with potentially long-
lasting impact, post-marketing pharmacovigilance will be especially important.
Finally, a range of educational, ethical, scientific, political and economic factors
would also need to be considered in the context of ATMP regulation.
The Committee was informed that the principal purpose of the white
paper was to develop a common language and risk-based classification for these
highly complex products, thus facilitating regulatory convergence and dialogue
between countries on the measures needed to improve their global accessibility.
The white paper was broadly divided into the two categories of product outlined
above. Clarification was given that RNA products such as prophylactic vaccines
that result in transient protein expression were considered to be outside the scope
of the white paper. A first round of public consultation in late 2021 will provide
an opportunity for all stakeholders to comment on the proposed document.
The Committee expressed its strong support for the development of
the WHO white paper, noting that this once again highlighted the proliferation
of these increasingly complex and challenging products, several of which had
already come before the Committee for its consideration. Noting as an example
the interchangeable use of “tissue products” and “tissue engineering products”
in different regulatory jurisdictions, the Committee welcomed the proposed
harmonization of terminology. However, without additional specialized expertise,
providing WHO with fully informed guidance in this field will present a significant
challenge. WHO was therefore urged to consider expanding the expertise both
of the Committee and of its Biologicals team to provide the specialist knowledge
required to address the formidable regulatory and other challenges associated
with this complex class of biological product.
33
3.4 Vaccines and related substances

3.4.1 Amendment to the WHO Recommendations to assure the quality,
safety and efficacy of live attenuated yellow fever vaccines
Yellow fever is a viral haemorrhagic fever endemic to countries in Africa, and
Central and South America. It is transmitted from human to human via mosquitoes
and can result in high case-fatality rates among non-vaccinated young children
and the elderly. Highly effective live attenuated yellow fever vaccines derived from
strain 17D have been in use since the late 1930s and a wealth of data is available
on their safety. However, rare serious adverse reactions associated with their use
include neurological and viscerotropic disease. It is therefore crucially important
to assess the levels of neurotropism and viscerotropism exhibited by new virus
master or working seeds compared to those exhibited by vaccines shown to be
clinically safe. Appendix 2 of the current WHO Recommendations to assure
the quality, safety and efficacy of live attenuated yellow fever vaccines sets out
how virus master and working seed lots should be tested for viscerotropism,
immunogenicity and neurotropism in non-human primates, both in terms of
clinical evidence and histological lesions, based on comparison against a reference
virus approved by the NRA.
In 2018, one yellow fever vaccine manufacturer reported that three working
seed lots had been tested in monkeys using a single reference and discrepancies
observed between the clinical scoring results of two lots. Further investigation
had concluded that histological scoring was more reliable than clinical scoring.
Differences in the clinical scores for the same reference virus (PV26) obtained in
2011 and 2018 provided further evidence of the inconsistency of clinical scoring
even when using the same material. Following these observed discrepancies,
WHO was requested to amend the current guidance in such a way as to be
consistent with the approach used to test the neurovirulence of OPV seed lots in
which clinical signs are recorded but do not contribute to the pass/fail criteria.
At its meeting in August 2020, the Committee had supported a proposal

to set up a working group comprising regulators, manufacturers and academics
to review the WHO guidance on the clinical evaluation of non-human primates
provided in Appendix 2. While acknowledging that the neurotropism test was
subjective and analysis could be challenging, the working group agreed that
clinical evaluation nonetheless provided important information and should be
retained as part of the test. The resulting amendments to Appendix 2 were: (a)
improved guidance on non-human primate testing with respect to observation
and assessment of clinical signs; (b) provision of recommendations regarding
failure outcomes for the clinical signs; and (c) a recommendation to monitor
reference virus performance.
The Committee sought clarification regarding the extent of agreement
between the histological and clinical signs, and wondered if vaccine assessment
34
might simply rely on histological evaluation. The Committee was informed that
this possibility had been discussed by the working group but manufacturers still
felt that clinical signs provided useful information and there was insufficient data
to support the discarding of clinical scoring at present. Having encouraged the
development of high-throughput sequencing technologies to assess the safety of
OPV at its recent meetings, the Committee was also interested in the potential
application of this approach to yellow fever vaccine testing. Clarification was given
that although the implementation of this technology was considered highly likely
in future, there was currently insufficient data on the critical mutations or lot-to-
lot consistency of viral sequences to introduce this as a release test at present.
The Committee was then provided with an overview of a number of
issues addressed by the drafting group following feedback received during public
consultation. These included: (a) the need to be clear on whether testing was for
“neurotropism” or “neurovirulence”; (b) the feasibility of conducting randomized,
double-blind controlled tests in non-human primates; and (c) the potential benefit of
telemetry to make testing more objective. Acknowledging that “neurotropism” was
the term used throughout the current main text of the Recommendations, and that
only the appendix was to be amended, the Committee accepted the proposal to add
a definition of neurotropism to the beginning of the amendment. The Committee
did not feel that randomized, double-blind controlled tests were feasible with non-
human primates and this requirement should therefore not be added. Accepting a
manufacturer’s argument that the benefits of telemetry remain to be demonstrated,
the Committee agreed that the text referring to telemetry should be deleted but a
sentence should be inserted in small print encouraging manufacturers to explore
the possible use of telemetry to render assessment more objective.
The Committee noted that new reference preparations of yellow fever
vaccine virus strains were also now needed for use in non-human primate testing.
However, the development of such standards would be challenging and should
first be discussed with manufacturers and other stakeholders. The Committee
further noted that other yellow fever vaccine platforms were being developed and
that although no attempt had been made at the current time to review the 2010
WHO Recommendations in their entirety, an open stakeholder meeting might
usefully be organized to determine the need for such a revision.
The Committee then recommended that the document WHO/
BS/2021.2401 be adopted and annexed to its report (Annex 2).
3.4.2 Evaluation of the quality, safety and efficacy of messenger RNA vaccines
for the prevention of infectious diseases: regulatory considerations
Recent advances in the manufacturing and stabilization of mRNA have established
the approach as an important vaccine technology and the speed with which
candidate mRNA vaccines can be developed makes them eminently suitable for
35
use during public health emergencies. The unprecedented pace of development

and clinical evaluation of candidate COVID-19 mRNA vaccines has highlighted
the urgent need for WHO guidance on evaluating the quality, safety and efficacy
of such vaccines. During discussions of the recently revised WHO Guidelines
on the quality, safety and efficacy of plasmid DNA vaccines, the Committee had
expressed its support for the development of a separate WHO guidance document
on regulatory considerations for mRNA vaccines that could be updated as more
scientific and clinical data became available. It was proposed that the document
would cover the manufacture, quality control, and nonclinical and clinical
evaluation of mRNA vaccines intended for the prevention of infectious diseases
in humans.
The development of the resulting document had presented several
challenges. Although the methods of mRNA vaccine production are generally
understood, the precise details of their manufacture and control remain
confidential, and the technologies employed can differ significantly. Moreover,
compared to other vaccines there is only limited experience of their nonclinical
and clinical evaluation. Another challenge had been the need to develop the
document within a highly compressed timeline in order to support vaccine
development and regulatory approval during the ongoing pandemic. Although its
drafting had been expedited to promptly offer regulatory guidance on COVID-19
mRNA vaccine development and evaluation, the document would be broadly
applicable to mRNA vaccines against other infectious diseases. The scope of the
document was limited to mRNA and self-amplifying mRNA packaged in lipid
nanoparticles for the in vivo expression of antigen to elicit an active immune
response. Replicating agents, viral vectors, other RNA replicons, mRNA vaccines
intended for therapeutic purposes and mRNA products expressing mAbs were
outside the scope of the current document.
The Committee was provided with a detailed overview of the structure
and content of the proposed document, and of the document development

process. Given the considerable level of interest in developing COVID-19 mRNA
vaccines, two rounds of public consultation had generated numerous comments.
Specific issues addressed included the need for a more comprehensive definition
of the relatively new concept of “platform technology”. While acknowledging
that the definition used was more stringent than was generally understood in the
scientific community, the Committee agreed that from a regulatory perspective
the wording used was appropriate. Other terminology issues addressed included
reaching agreement on the use of “drug substance” and “drug product” instead
of “antigen” and “final vaccine” to reflect the fact that although the mRNA
encodes the antigen it is not itself the antigen. The Committee agreed with the
consensus that had been reached by the drafting group on these and several
other aspects of mRNA vaccine manufacturing. Noting that manufacturers may
36
regard some aspects of mRNA production as being similar to a pharmaceutical,

the Committee recommended that the document clearly indicate that good
manufacturing practices for biologicals would be the expected standard. The
Committee further noted that in vivo potency tests for mRNA vaccines were
unreliable and should not be encouraged. Potency measurements should reflect
the clinical performance of a vaccine, with potency assays used for any strain
changes validated. The Committee went on to review the overall document and
made several general suggestions. Among these, it was suggested that the more
usual regulatory term “benefit–risk” (rather than “risk–benefit”) should be used
in this and future WHO written standards.
After receiving reassurances that several relevant aspects not explicitly
covered in the proposed document were already sufficiently addressed in existing
and more general WHO guidelines on nonclinical and clinical evaluation, the
Committee recommended that the document WHO/BS/2021.2402 be adopted
and annexed to its report (Annex 3).
37
4. International reference materials –
biotherapeutics other than blood products
4.1 WHO international reference standards for
4.1.1 Third WHO International Standard for follicle-stimulating
hormone (human, recombinant) for bioassay
Follicle-stimulating hormone (FSH) is a glycoprotein produced in the anterior
pituitary gland. It is involved in the regulation of follicular growth, pubertal
maturation and reproductive processes, working in synergy with luteinizing
hormone to regulate ovulation. FSH is used in fertility treatments such as in
vitro fertilization. Early FSH products, which were extracted from urine, were of
variable purity and have largely been replaced by recombinant products. Today,
a number of such human recombinant products are on the market including a
number of biosimilars. The potency of therapeutic FSH is typically determined
using an in vivo bioassay with the dose expressed in IU traceable to the WHO
international standard. Stocks of the Second WHO International Standard
for follicle-stimulating hormone (human, recombinant) for bioassay (NIBSC
code 08/282) were now critically low, prompting the production of a candidate
replacement material to ensure the continuity of the IU for FSH products.
A quantity of manufacturer-donated purified human recombinant FSH
was formulated and filled in 0.5mL aliquots into glass ampoules for evaluation of
its suitability as a replacement standard material. The candidate material (NIBSC
code 20/218) was evaluated in an international collaborative study involving
six laboratories in six different countries. Using a method provided in the study
protocol, each laboratory carried out independent in vivo bioassays to estimate
the content of the candidate material 20/218 relative to the current WHO
international standard 08/282. Results from 16 valid assays indicated an overall

geometric mean value of 137 IU/ampoule FSH for the candidate material. Low
intra- and inter-laboratory variability (geometric coefficient of variation = 1–8%)
showed that estimates were in good agreement. No significant loss of activity was
observed in samples stored at elevated temperatures for 6 months. Although this
prevented estimation of the rate of loss of activity per annum, the lack of observed
degradation, together with the proven stability of the previous FSH standards
with a similar formulation, suggested that the material would likely exhibit good
long-term stability when stored at −20°C. Further bioassays would be performed
in 12 months in order to allow prediction of the annual rate of loss of activity.
The Committee asked for more information on the precise assays
performed by each of the collaborative study participants and was assured that
there was only one bioassay available at present. Therefore, all laboratories had
38
International reference materials – biotherapeutics other than blood products
performed the Steelman-Pohley assay as described in Ph. Eur., with the exception
of one laboratory which made a minor adjustment to the sample diluent. The
Committee considered the report of the study (WHO/BS/2021.2410) and
recommended that the candidate material 20/218 be established as the Third
WHO International Standard for follicle-stimulating hormone (human,
recombinant) for bioassay, with an assigned unitage of 137 IU/ampoule. The
Committee requested that the report of the study be revised to reflect the minor
assay adjustment made by one of the laboratories and this was duly done (WHO/
BS/2021.2410 rev.1).
4.2 Proposed new projects and updates –

4.2.1 Proposed Second WHO International Standard
for interleukin-6 (human, recombinant)
Interleukin-6 (IL-6) is a pleiotropic cytokine associated with regulation of
inflammation, haematopoiesis, cancer progression and immune responses. As
IL-6 stimulates the inflammatory and autoimmune processes in many diseases,
therapeutic anti-IL-6 products continue to be developed for a diverse range of
treatments. The current First WHO International Standard for interleukin-6
(human, recombinant) was established in 1992 and is used in a broad range of
applications by manufacturers of both biotherapeutics and immunoassays, as well
as by researchers. It is a critical reagent in cell-based assays used for the potency
testing of therapeutic mAbs and for the potency testing of IL-6 preparations used
as cell culture supplements for CGTPs. It is also used to calibrate immunoassays
used to measure serum IL-6 levels, including during the nonclinical and clinical
evaluation of immunotherapies. The Committee was informed that the increased
use of the current international standard in recent years, which had been
exacerbated by the COVID-19 pandemic, had led to the rapid depletion of stocks
which, at the current rate of use, will be exhausted by the end of 2022. There was
therefore an urgent need for a replacement international standard.
It was proposed that recombinant human IL-6, sourced from a commercial
supplier, would be lyophilized in ampoules and evaluated in a collaborative study
that would include both bioassays and immunoassays. Although the current
international standard was expressed in Chinese hamster ovary cells, it is proposed
that IL-6 expressed in E. coli be used for the replacement standard. The Committee
was informed that material had now been sourced and a trial fill completed.
Upon completion of the definitive fill, an international collaborative study
involving 17 laboratories representing industry, contract research organizations
and control laboratories would assess the suitability of the candidate material
as a replacement international standard for bioassays and immunoassays, and
39
would assign a unitage relative to the current WHO international standard. It

was anticipated that the collaborative study outcomes would be submitted for
consideration by the Committee in 2022.
The Committee recognized the urgent need for this international standard
and after due consideration endorsed the proposal (WHO/BS/2021.2411) to
develop a Second WHO International Standard for interleukin-6 (human,
recombinant).
40
5. International reference materials – blood
products and related substances
blood products and related substances
5.1.1 Third WHO International Standard for von
Willebrand factor (concentrate)
Von Willebrand disease is the most frequent inherited bleeding disorder and
is caused by a deficiency and/or abnormality of von Willebrand factor (VWF).
VWF is essential for platelet subendothelial adhesion and platelet-to-platelet
interactions, and is a specific carrier of blood coagulation factor VIII in plasma.
Where transfusion therapy is necessary, disease treatment relies upon the use of
purified VWF concentrates. The WHO international standard for VWF is used for
the potency estimation and labelling of these therapeutic products. The current
Second WHO International Standard for von Willebrand factor (concentrate)
(NIBSC code 09/182) was in high demand and it was estimated that stocks would
be exhausted by mid-2022.
Candidate replacement materials had therefore been prepared using
clinical grade VWF purified from two plasma-derived VWF concentrates
(NIBSC codes 18/248 and 08/296) and one recombinant VWF product (NIBSC
code 20/156). An international collaborative study involving 48 laboratories
in 22 countries had been conducted to assign potency values for VWF:antigen
(VWF:Ag), VWF:ristocetin cofactor (VWF:RCo) and VWF:collagen binding
(VWF:CB) to the three candidate materials. Values were calculated relative to
the current WHO international standard and to the Sixth WHO International
Standard for factor VIII/VWF (plasma) to ensure continuity of the IU and confirm
the equivalence of the IU values applied to each type of standard (concentrate
and plasma). Study participants also investigated the possibility of assigning
values to the replacement standard for VWF:glycoprotein IbR (VWF:GPIbR)
and VWF:glycoprotein IbM (VWF:GPIbM) to reflect the development of newer
immunological assays based on VWF binding to immobilized recombinant GPIb
receptors. Due to the rapid uptake of these newer assays and the resulting urgent
need for international standardization, the Sixth WHO International Standard
for factor VIII/VWF (plasma) had also been established in 2018 as the WHO
International Reference Reagent for GPIbR and GPIbM methods.
The study results showed no significant variability in overall mean potency
estimates for VWF:Ag and VWF:RCo for all three candidate materials when
measured relative to either the current WHO international standard for concentrate
or the current WHO international standard for plasma. Greater variability was
observed for VWF:CB and was associated with the different methods and different
41
collagen types used. Only candidate material 18/248 showed no significant method
differences for estimates relative to the current international standard, highlighting
the importance of the “like versus like” principle of biological standardization.
Estimates calculated relative to the WHO international standard for concentrate
were observed to be less variable than estimates calculated relative to the WHO
international standard for plasma – supporting the use of a separate international
standard for each material. Candidate material 18/248 was also associated with
the lowest inter-laboratory variability for all three analytes. Mean estimates for
GPIbR and GPIbM were also calculated for the candidate materials relative to
the current WHO international standards. Candidate material 18/248 was
again associated with low inter-laboratory variability and provided the greatest
equivalence between the various estimates made.
An accelerated thermal degradation study carried out to evaluate the
stability of the candidate material 198/248 showed little loss of potency after 3
months. Real-time stability studies of the current WHO international standard
for VWF, concentrate has demonstrated no measurable loss of activity even after
12 years of storage at 20 °C. No stability issues were therefore anticipated and
ongoing monitoring would be conducted. Stability testing of the reconstituted
material indicated that it remained stable when stored on ice for 4 hours.
The Committee queried the short duration of the stability studies and the
resulting limited data and was informed that this had been due to an oversight
in placing the candidate materials into storage for studies after filling. However,
early indications from stability testing at 56 °C storage for 3 months were that
this would not be an issue – a conclusion supported by the extreme stability of
the current international standard during its lifetime. Nonetheless, the stability of
the materials would be checked again in 6 and 12 months.
The Committee questioned the inclusion of a recombinant product in the
study and were informed that licensed recombinant products were not available
when the current international standard had been established. A recombinant

material had therefore been included in the study to provide an understanding of
the relationship between the concentrate standard and the recombinant material.
Discussion then turned to the recent transition to the newer GPIbR and GPIbM
assays and the need for specific guidance on this. The Committee was informed that
this was unlikely to pose a problem for manufacturers establishing a new product
as potency could be defined using VWF:RCo, VWF:GPIbR or VWF:GPIbM
methods without the need to compare the different analyte values. However, the
potency values obtained using these different methods could not be reconciled
as they were independent of each other. As a result, manufacturers of existing
products planning to switch to a newer method would have to understand and
demonstrate the relationship between the clinically proven RCo potency values
originally used for such products and those of the new analyte to be assayed.
42
International reference materials – blood products and related substances
The Committee also discussed the clinical and industry implications of

having different patients and products assessed using different types of activity,
each dependant on different assays or references. It was observed that some
clinical laboratories were using GPIbR and GPIbM assays to monitor patients and
that in cases where a manufacturer moved toward the use of these new methods
and standards then these should also be used for patient monitoring – however,
it was unclear at present if this would happen in practice.
The Committee considered the report of the study (WHO/BS/2021.2408)
and recommended that the candidate material 18/248 be established as the Third
WHO International Standard for von Willebrand factor (concentrate) with the
following assigned values: VWF:Ag = 12.0 IU/ampoule; VWF:RCo = 8.7 IU/
ampoule; VWF:CB = 9.8 IU/ampoule; VWF:GPIbR = 8.6 IU/ampoule; and
VWF:GPIbM = 7.3 IU/ampoule.
5.1.2 Fourth WHO International Standard for ferritin (human, recombinant)

Ferritin is the main storage protein for iron in many tissues, with the highest
concentrations found in liver, spleen and bone marrow. The protein consists of a
24-subunit heterogeneous shell (composed of varying ratios of both heavy and
light chain subunits) surrounding an iron core that can contain up to 4500 iron
atoms. With its predominantly light subunit form, circulating ferritin is not itself
iron-bearing but the level of serum ferritin directly reflects the level of stored
iron, with levels being low during iron deficiency and high during iron overload.
Serum ferritin levels are normally quantitated using an antibody test that detects
the ferritin protein. The Third WHO International Standard for ferritin (human,
recombinant) (NIBSC code 94/572) has been distributed to more than 200
laboratories in 38 countries since 1997 and is used to calibrate immunoassays
and standardize the results of different assay methodologies. The Committee was
informed that stocks of this standard were now low and a replacement standard
was required.
WHO international standards for ferritin were originally prepared using
ferritin derived from human liver and spleen but due to difficulties in acquiring
suitable tissue the current WHO international standard had been prepared using
recombinant light chains. Two candidate materials for the replacement standard
were therefore prepared using lyophilized preparations of recombinant ferritin
light chains (human) expressed either in E. coli BL21(DE3) (NIBSC code 19/118)
or ExpiCHO-STM (NIBSC code 19/162). The two candidate materials were shown
to be immunologically similar to the current international standard.
An international collaborative study involving 12 laboratories in nine
countries using 11 different immunoassay platforms was conducted to assign
potency values to the two candidate materials relative to the current WHO
international standard. Three lyophilized clinical serum samples with high,
43
normal and low levels of ferritin were also obtained from the Welsh External
Quality Assurance Scheme for use in commutability studies. The sample potencies
obtained were reported by the laboratories according to the assay platform
calibrant, with these potencies then being expressed relative to the current WHO
international standard by NIBSC. It was observed that some laboratories still
claimed traceability of their results to the first and second WHO international
standards despite these not been available for almost 25 years.
Results indicated that inter-laboratory variability was reduced when the
current WHO international standard was used as a reference material. Variability
was also reduced when the results from two laboratories were excluded due to
invalid assays resulting from non-linearity and non-parallelism. All laboratories
correctly identified the commutability samples, though wider variation was
observed for results obtained for the sample with the low ferritin level as this
was below the limit of quantitation for most of the laboratory systems. Inter-
laboratory variability was lower when using either candidate reference material
compared to in-house standards.
Combined study results indicated overall mean potencies of 10.5 µg/
ampoule for candidate material 19/118 and 8.0 µg/ampoule for candidate
material 19/162 relative to the current international standard. Better overall
agreement was observed between all laboratories and between assay methods for
the potency of candidate material 19/118 compared to candidate material 19/162.
Accelerated degradation studies indicated no significant loss of activity in either
candidate material after a period of 2 years and it was anticipated that they would
remain stable under long-term storage at −20 °C.
and recommended that candidate material 19/118 be established as the Fourth
WHO International Standard for ferritin (human, recombinant) with an assigned
content of 10.5 µg/ampoule and expanded uncertainty limits of 10.2–10.8 µg/
ampoule (95% confidence; k = 2.23).
5.2 Proposed new projects and updates – blood

products and related substances
5.2.1 Proposed Sixth WHO International Standard for
blood coagulation factor IX (concentrate)
Blood coagulation factor IX (FIX) therapeutic products are used as a replacement
therapy for the bleeding disorder haemophilia B, which is caused by an FIX
deficiency. The current WHO international standard is used for the potency
labelling of all FIX (plasma-derived, recombinant and extended half-life) and
prothrombin complex concentrates used in the treatment of congenital and
acquired FIX deficiency. There are currently more than 20 FIX therapeutic
44
International reference materials – blood products and related substances
products licensed worldwide, with more under development and approximately

600 ampoules of the international standard are distributed every year, mostly
to FIX product manufacturers, IVD manufacturers, regulatory agencies and
national control laboratories.
Although the successive WHO international standards have functioned
well in the potency labelling of both plasma-derived and recombinant FIX
products, substantial discrepancies occur when assaying extended half-life
FIX products using different reagents and kits. As all licensed extended half-
life FIX products were originally potency assigned relative to the Fourth WHO
International Standard for blood coagulation factor IX (concentrate), with the
labelled IU supported by clinical trial data, the current fifth WHO international
standard had been prepared using the same plasma-derived factor concentrate to
ensure continuity of the IU – an approach that has worked well for all licensed
FIX products. In addition, the Ph. Eur. Human coagulation factor IX concentrate
Biological Reference Preparation (BRP) batch 3 also originated from the same
source as the current WHO international standard, which has supported
traceability of the IU. The Committee was informed that stocks of both reference
materials were now running low and replacements were needed.
It is proposed that the same plasma-derived FIX concentrate will once again
be used to prepare the candidate material to ensure continuity and consistency
of the IU for all FIX products. As EDQM has expressed a wish to maintain the
current harmonized approach, the same batch of material would also be used to
replace the Ph. Eur. BRP. The Committee was informed that approximately 20
000 ampoules of the current WHO international standard and BRP had been
produced in 2015 and that in order to increase the current replacement period of
6–7 years a larger batch would be required. In addition, the plasma-derived FIX
concentrate used as the bulk source material for both the fourth and fifth WHO
international standards was being phased out by the manufacturer and may not
be available for replacement standards in future. Sufficient candidate material
had therefore been donated to the NIBSC for three fills of 25 000 ampoules each,
which would ensure sufficient material for successive standards over the next
18 years. The proposed collaborative studies would be run simultaneously in
two phases, with submission of the results for consideration by the Committee
anticipated in 2022.
During discussion, clarification was given that the proposed two-phase
approach was due to known assay and other discrepancies, and that the aim
would be to ensure that the same pattern of discrepancies would be maintained
in all successive WHO international standards. Discussion then turned to the
continued availability of the source material, with concerns expressed regarding
the rate of uptake of the international standard and the time between replacement
standards. A suggestion was made to assign the full batch of candidate material to
45
the development of the successive WHO international standards while allocating

another batch of material for EDQM use. It was explained that this had been
considered but it was thought preferable to use the same batch of candidate material
for both reference materials to maintain harmonization and reduce discrepancies.
EDQM expressed its appreciation for the approach proposed by NIBSC, reiterated
its desire to maintain full harmonization and highlighted that this would also
avoid the risk of separate batches expiring at different times. EDQM undertook
to work closely with NIBSC to ensure the best use of the materials. Discussion
then took place on the optimal filling strategy and clarification given that the
current intention was to perform all three fills in 2021 to ensure the stability of the
material. Candidate materials derived from all three fills would then be evaluated
in the collaborative study to minimize the work that would be needed to establish
the future seventh and eighth WHO international standards.
After due consideration, the Committee endorsed the proposal (WHO/
BS/2021.2411) to develop a Sixth WHO International Standard for blood
coagulation factor IX (concentrate).
46
6. International reference materials – in vitro diagnostics
6.1 WHO international reference standards for in vitro diagnostics
6.1.1 First WHO International Standard for Mycobacterium
tuberculosis (H37Rv) DNA for NAT-based assays
Tuberculosis (TB) remains a major cause of death worldwide and is a particular
public health concern in LMIC with inadequate diagnostic facilities. TB is a
respiratory disease caused by the bacterium Mycobacterium tuberculosis and
timely and accurate diagnosis is crucial for effective treatment and the prevention
of transmission. The field testing of TB diagnostics is an important element in
achieving the goal set by the international health community to end global TB
epidemics by 2030. Sputum-smear microscopy and culture techniques are effective
in diagnosing highly infectious TB but less so for the early diagnosis of infection in
people with less-pronounced symptoms, with culture methods in particular having
long turnaround times. More-sensitive RDTs based on nucleic acid amplification
techniques (NATs) are now available with other new tests in development.
The Committee was reminded that experts at a 2018 WHO workshop
had discussed advances in TB diagnostics and had identified the need for a WHO
international standard for M. tuberculosis DNA to serve as the primary calibrator
for NAT-based assays. The use of the international standard would improve
the harmonization of such assays and allow different laboratories to compare
analytical data using different assay formats. At its meeting in October 2018, the
Committee had endorsed a proposal to develop a WHO international standard
based on the commonly used laboratory strain H37Rv.
A single batch of 2992 vials of lyophilized, heat-inactivated H37Rv with
a concentration of approximately 106 genome copies per mL had therefore been
prepared. Preliminary studies confirmed the consistency of filling and effectiveness
of the heat inactivation process. This candidate material (NIBSC code 20/152)
was then evaluated for its suitability to serve as a WHO international standard for
both quantitative PCR assays and RDTs in an international collaborative study
involving eight laboratories in seven countries. It was concluded that the use of
candidate material 20/152 in quantitative PCR assays reduced inter-laboratory
variability. In addition, serial dilution of the material allowed for estimation of the
end-point titres/limit of detection of various RDT kits. There was no observable
loss of genome copies when the vials were stored at elevated temperatures up to
56 °C for 12 months, while the reconstituted material was stable for up to 4 weeks
at −20 °C and up to 1 week at 4 °C.
The Committee commended the study of this necessary standard and
reflected on the difficulty of recruiting diagnostic laboratories that were currently
heavily committed to the COVID-19 response – an issue for recent collaborative
studies in other areas. The Committee did not support a suggestion that “DNA” be
47
omitted from the name of the standard as this would be inconsistent with similar
diagnostic standards. Instead, any potential clarification required of the precise
nature of the reference material should be addressed in the instructions for use
(IFU). However, it did agree that the strain designation should be included in the
name of the standard. Clarification was then given that one of the main intended
uses of the standard would be to evaluate and monitor the fitness for purpose of
RDTs and the Committee noted the importance of such standards in the WHO
prequalification of IVDs.
and recommended that the candidate material 20/152 be established as the First
WHO International Standard for Mycobacterium tuberculosis (H37Rv) DNA for
NAT-based assays with an assigned unitage of 6.3 log10 IU/vial.
6.1.2 First WHO International Standard for varicella

zoster virus DNA for NAT-based assays
Varicella zoster virus (VZV) is a member of the Herpesviridae family of DNA
viruses and is a highly contagious and widely distributed human pathogen causing
a significant public health burden. VZV is the aetiological agent of chickenpox,
which primarily occurs in childhood. However, the virus persists in the body
asymptomatically after primary infection, establishing latency in the trigeminal
and dorsal root ganglia from where it can reactivate from dormancy to cause
herpes zoster in adults. Immunocompromised patients, including transplant
patients, are at increased risk of developing herpes zoster with disseminated VZV
infection potentially life-threatening in such patients. In addition, congenital
varicella syndrome can result in a variety of problems among affected infants,
including severe damage to the nervous system. Five VZV clades distinguishable
by single nucleotide polymorphisms and exhibiting a clear geographical
distribution have now been identified.
VZV infection is primarily diagnosed using PCR-based methods that

allow for the rapid and sensitive detection of the virus in a range of clinical
samples, including serum, plasma, whole blood, vesicle fluid, vesicle swab and
cerebrospinal fluid. Quantitative viral load assays and antiviral resistance testing
for transplantation patients, as well as vaccine/wild-type differentiation tests, are
also performed in clinical settings. In 2015, the Committee had noted the need
for standardization in this area and had endorsed a proposal to develop a WHO
international standard for use with NAT-based assays.
A candidate freeze-dried VZV preparation was propagated in cell culture
from a primary paediatric clinical isolate and evaluated in an international
collaborative study involving 12 laboratories in nine countries. Using their routine
VZV NAT-based assays, each laboratory evaluated the candidate material (NIBSC
code 19/164) alongside two proprietary VZV comparator materials (Ellen and
48
International reference materials – in vitro diagnostics
v-Oka strains), as well as four clinical samples and two spiked samples to permit
a preliminary assessment of commutability. Agreement on the mean potency
estimates reported across laboratories for both qualitative and quantitative assays
significantly improved when results were expressed relative to the candidate
material. In addition, accelerated thermal degradation study data obtained at 12
months and 18 months post-production indicated that the candidate material was
stable and suitable for long-term storage. Nucleotide sequencing data revealed
a pattern of single nucleotide polymorphisms consistent with Clade 3, with no
major genome rearrangements.
Noting the reported higher variability of results relative to the v-Oka
comparator strain, the Committee enquired whether the other samples in
the collaborative study panel had been sequenced. It was informed that work
was ongoing and a wider commutability study would allow the effect of clade
differences to be assessed. However, the higher variability observed might also
have been due to the small number of study participants. The Committee noted
in particular the small number of laboratories that had contributed qualitative
assay data and, despite assurance that the results indicated that the candidate
material had harmonized results, it was suggested that further studies be carried
out post-establishment.
WHO International Standard for varicella zoster virus DNA for NAT-based
assays with an assigned unitage of 7.0 log10 IU/vial.
6.1.3 First WHO International Standard for anti-Lassa virus

immunoglobulin G; and First WHO International Reference
Panel for anti-Lassa virus immunoglobulin G
Lassa fever is a zoonotic disease that occurs as seasonal outbreaks in several West
African countries. The causative Lassa virus (LASV) is transmitted to humans
from infected rats or person-to-person through contact with contaminated bodily
fluids. Although approximately 80% of infected people are asymptomatic, around
20% of infections result in severe disease, including viral haemorrhagic fever.
Overall mortality among hospitalized cases is around 20% but higher rates have
been observed in some outbreaks. LASV has been identified by the WHO Blueprint
for Research and Development: Responding to Public Health Emergencies
of International Concern (R&D Blueprint) as a top ten priority pathogen with
outbreak potential. While the antiviral medication ribavirin has been used as a
treatment, a number of vaccines and other treatments are under development
and reliable assays are now needed for their evaluation. The establishment of
WHO international reference materials for LASV antibodies would allow for
the standardization of serological assays thus facilitating the development of
49
vaccines and therapeutics, and support epidemiological studies of the disease. In

collaboration with the Coalition for Epidemic Preparedness Innovations (CEPI),
NIBSC had now carried out a study to develop a WHO international standard and
a WHO international reference panel for anti-LASV immunoglobulin G.
Pooled convalescent plasmas had been evaluated in an international
collaborative study involving 17 laboratories in seven countries. A total of 12
samples had been evaluated, including two anti-LASV mAb mixtures, to determine
their suitability to serve as WHO international reference materials. A wide range
of neutralization assays (using live LASV or pseudotyped virus systems) and
binding assays (directed against the glycoprotein (GP), the nucleoprotein (NP) or
a combination of the two) were used. In addition to the candidate international
standard material (NIBSC code 20/202), a reference panel was also assembled
consisting of: (a) two high-titre antibody preparations from Nigeria (NIBSC code
20/228) and Sierra Leone (NIBSC code 20/244); (b) a mid-titre pool from Nigeria
(NIBSC code 20/226) and convalescent plasma from one individual with mid-
titre neutralizing, but high-titre binding antibodies (NIBSC code 20/204); and
(c) three low-titre pools from Nigeria (NIBSC code 20/226) and Sierra Leone
(NIBSC codes 20/246 and 20/248). The candidate material 20/202 was assessed as
part of a blinded sample panel that also included the reference panel, a working
standard for LASV antibody and the two mAb mixtures.
Expressing the LASV antibody titres of the study samples relative to the
candidate material 20/202 reduced inter-laboratory variability and improved
comparability of the results obtained both by neutralization assays and by binding
assays (for both viral GP and NP). The rankings assigned by study participants
to the samples in the reference panel were similar but not identical, with the low-
titre preparations challenging the sensitivity of some assays. Thus, the reference
panel proved to be a useful tool in the assessment of serological assay sensitivity
for LASV antibodies. The mAb mixture also harmonized the results of both the
neutralizing and binding assays, though slightly less effectively than the candidate
material 20/202. The stability of the lyophilized plasma pool 20/202 was assessed
in an accelerated thermal degradation study. Relative to a baseline sample stored
at −20 °C, there was minimal loss of potency for up to 1 month at 37 °C and up
to 6 months at ambient temperature (20 °C). Using the Arrhenius equation, the
predicted loss of potency for candidate material 20/202 was estimated at 0.17%
per year when stored at −20 °C.
The Committee queried whether the serum panel samples obtained from
Nigeria and Sierra Leone were sufficiently representative of the broad range of
LASV lineages. It was agreed that other samples from different sources might
usefully be added to the panel in future to improve its representativeness. Reflecting
on the challenge of producing reference materials for emerging pathogens, it was
suggested that further studies into the generation of suitable reference materials
50
using mAbs might be worthwhile. A key issue to be addressed would the current
focus placed on mAb development in the context of mechanisms of protection
against infectious disease, which would not necessarily reflect the requirements
of diagnostic assays.
WHO International Standard for anti-Lassa virus immunoglobulin G with a
unitage of 25 IU/ampoule for neutralizing antibody, 250 IU/ampoule for anti-GP
binding IgG and 250 IU/ampoule for anti-NP binding IgG. The Committee also
recommended that the proposed panel consisting of candidate materials 20/204,
20/222, 20/226, 20/228, 20/244, 20/246 and 20/248 be established as the First
WHO International Reference Panel for anti-Lassa virus immunoglobulin G
without an assigned unitage.
6.1.4 First WHO International Standard for anti-thyroid peroxidase antibodies

Autoimmune thyroid disease is the most common autoimmune disease and is
caused by anti-thyroid microsome autoimmunity. The target of such autoimmunity
is thyroid peroxidase (TPO), with antibodies to TPO present in thyroid diseases
such as Hashimoto’s thyroiditis and hyperthyroidism. Immunoassays that measure
anti-TPO antibodies are important for the diagnosis of thyroid autoimmunity
and disease. An NIBSC reference reagent (NIBSC code 66/387) developed in the
1960s had been widely used to calibrate these immunoassays but stocks were
now heavily depleted and a replacement reference material urgently needed. At
its meeting in 2018, the Committee had endorsed a proposal to develop a First
WHO International Standard for anti-thyroid peroxidase antibodies.
A pool of human serum exhibiting high anti-TPO antibody titres had
been obtained from three donors, filled into ampoules and freeze dried. The
resulting candidate material (NIBSC code 19/260) was then evaluated in an
international collaborative study involving seven laboratories in six countries
to assess its suitability to serve as a WHO international standard for anti-TPO
antibody immunoassays. Data from a total of 13 different immunoassays was
analysed. In addition, human serum and plasma samples containing a range
of anti-TPO concentrations had also been included in the study to assess the
commutability of the candidate material in native samples.
Study results indicated that the reference reagent, 66/387 and the candidate
material 19/260 behaved in a similar manner in the various immunoassays used
and were in good agreement with one another, indicating that the establishment
of candidate material 19/260 as a replacement standard would permit the
continued calibration of such assays. Relative to reference reagent 66/387, the
overall geometric mean potency for candidate material 19/260 was estimated
to be 571 IU/ml (95% confidence interval = 493–662 IU/ml), with a median
51
value of 533 IU/ml and a robust mean value of 555 IU/ml. Using a difference-
in-bias approach, the commutability of both materials was demonstrated for all
laboratory methods used in the study. The stability of candidate material 19/260
was assessed in a thermal degradation study over a period of 7 months. Based
on the Arrhenius equation, the annual loss of activity when stored at −20 °C was
predicted to be approximately 0.013%.
It was noted that as autoantibodies were inevitably donor specific, any
replacement standard obtained from different donors would differ from the
previous reference reagent. Accepting that this was an insurmountable issue, the
Committee was content that candidate material 19/260 behaved similarly to the
current reference reagent 66/387 and would harmonize the results of immunoassays.
The Committee considered the report of the study (WHO/BS/2021.2404) and
recommended that the candidate material 19/260 be established as the First WHO
International Standard for anti-thyroid peroxidase antibodies with an assigned
unitage of 555 IU/ampoule. The Committee further recommended that the IFU
should make clear that the replacement material represented a different population
of autoantibodies than the previous reference reagent 66/387.
6.2 Proposed new projects and updates – in vitro diagnostics

6.2.1 Proposed Second WHO International Standard for
alpha-fetoprotein (human cord serum)
Alpha-fetoprotein (AFP) is a 70 kDa glycoprotein in the serum albumin family
that is generally present at low levels in adults and at slightly increased levels
in pregnant women. Significantly elevated AFP levels can be seen in disease
processes such as chronic active hepatitis and hepatocellular carcinoma, and in the
amniotic fluid in the presence of congenital abnormalities such as anencephaly,
omphalocele and spina bifida. AFP levels in serum, plasma or amniotic fluid are
measured via immunoassay as part of disease diagnosis and monitoring, or for
the detection of fetal congenital abnormalities during pregnancy. The majority of

such immunoassays are calibrated against the current First WHO International
Standard for alpha-fetoprotein (human cord serum) (NIBSC code 72/225). The
Committee was informed that stocks of this international standard, which had
been established in 1975, would be exhausted in 2022 and a replacement reference
material was urgently required. The replacement reference material was expected
to be used by manufacturer, clinical and academic laboratories to calibrate their
in-house immunoassays. Based on current level of usage, the predicted demand
was 60–70 ampoules per annum.
The current WHO international standard had been prepared using
pooled human cord serum obtained from a large number of donors, and had an
assigned unitage of 100 000 IU/ampoule (0.121 mg/ampoule). The Committee
was informed that this level was very high in comparison with the dynamic
52
assay range of current immunoassays. Replacement of the current international

standard would provide an opportunity to reduce the AFP unitage to 10 000 IU/
ampoule (0.0121 mg/ampoule), subject to consultation with end users. Sourcing
of a suitable replacement material would be challenging as the material is supplied
by only a few manufacturers and is expensive. An international collaborative
study involving manufacturers and clinical laboratories performing AFP assays
would be conducted to evaluate the candidate material and to calibrate it against
the current international standard. It was anticipated that the collaborative study
outcomes would be submitted for consideration by the Committee in 2023.
The Committee expressed concern that the replacement international
standard would only be submitted for proposed establishment in 2023 despite the
expected depletion of the current international standard in 2022, and enquired
as to the implications of this. Assurance was given that steps had been taken by
NIBSC to minimize the period during which an international standard would
not be available, with laboratories currently restricted to one ampoule per year.
In addition, source materials had now been secured and initial preparations
were under way. The Committee endorsed the proposal (WHO/BS/2021.2411)
to develop a Second WHO International Standard for alpha-fetoprotein (human
cord serum).
6.2.2 Proposed First WHO International Standard

for anti-thyroglobulin antibodies
Thyroglobulin plays a major role in the synthesis, storage and release of thyroid
hormone. The presence of thyroglobulin antibodies (TgAbs) is diagnostic of
thyroid autoimmune diseases such as Hashimoto’s thyroiditis, Graves’ disease
and other hyperthyroid and hypothyroid disorders. Measurement of TgAb level
is also used to diagnose and monitor differentiated thyroid cancers. The majority
of immunoassays used to detect TgAb in serum and plasma are calibrated using
the current International Reference Preparation of anti-thyroglobulin serum
(NIBSC code 65/093) established in 1978. Since its establishment, this reference
material had been widely used and stocks were now expected to be exhausted in
2023. It was therefore proposed that a replacement WHO international standard
be developed for use as a calibrant for TgAb immunoassays. Based on current
level of usage, the predicted demand was 85–95 ampoules per annum, with an
expected fill of 1000–1500 ampoules ensuring sufficient stocks for 10–15 years.
The current international reference preparation had been produced
from a large number of plasma donors with high titres of TgAb. It was proposed
that serum, source plasma or recovered plasma from a similar group of donors
be obtained and pooled to produce a candidate replacement material. The
candidate material would be characterized and value assigned against the current
international reference preparation 65/093 in an international collaborative study
53
involving manufacturer, clinical and academic laboratories. It was anticipated

that the collaborative study outcomes would be submitted for consideration by
the Committee in 2023.
During discussion, it was ascertained that the source material to be
purchased would likely be plasma collected by plasmapheresis and tested for
several markers by the supplier. The Committee noted that there would be a shift
from the current international reference preparation to a WHO international
standard and enquired about the implications of this both for the source material
and its characterization. In response, it was surmised that the initial status of
the long-established current reference material may simply have been due to
the small number of laboratories (n = 3–4) involved in its characterization. The
characterization and unitage assignment of the WHO international standard
would involve a greater number of laboratories. It was further noted that reference
preparations had commonly been established in situations where likely uptake
levels could not be firmly determined, or where there had been uncertainty about
the need for a WHO international standard. However, it was clear in this instance
that there was both wide uptake and a recognized need for an international
standard.
The Committee endorsed the proposal (WHO/BS/2021.2411) to develop
a First WHO International Standard for anti-thyroglobulin antibodies.
6.2.3 Proposed Fifth WHO International Standard for

hepatitis B virus DNA for NAT-based assays
Hepatitis B virus (HBV) remains a major global health problem with an estimated
257 million people living with chronic HBV infection in 2015. NAT-based assays
for HBV are routinely used for the diagnosis of hepatitis B and for monitoring
antiviral therapies. Such assays are also used to screen blood donations, as well
as cells, tissues and organs, to ensure blood and transplantation safety. The
WHO international standard is used in the calibration of secondary reference

materials and in the validation of NAT-based HBV assays by blood centres,
clinical laboratories, control authorities and IVD manufacturers. The Committee
was informed that stocks of the current Fourth WHO International Standard
for hepatitis B virus DNA for NAT-based assays (NIBSC code 10/266) would
be exhausted in 2 years, and that this would also be the time period required to
replace the standard.
All previous WHO international standards for HBV DNA had been
derived from the same Eurohep R1 reference material (genotype A2, HBsAg
subtype adw2) diluted in pooled human plasma. In each case, filling had taken
place off-site in 0.5 mL volumes with a limited fill of around 2000 ampoules
resulting in a replacement period of approximately 5 years for each WHO
international standard, starting in 1999. The Committee was informed that
54
sufficient residual Eurohep R1 reference material remained to prepare a batch

of 3000–3500 ampoules based on a fill volume of 0.5 mL. An international
collaborative study involving 10–20 laboratories would be conducted to evaluate
the candidate material against the current WHO international standard.
The Committee was informed that although current laboratory analysers
require larger sample volumes, this would not necessarily require the standard
volume to be increased as its current concentration allows for dilution to obtain a
larger sample volume. Producing a batch that would last for at least 10 years would
require a minimum of 5000 ampoules. In order to achieve this, new HBV source
material would be needed which would potentially result in sequence variations
compared to the current reference material. This would necessitate sequencing of
the whole genome to ensure that any replacement material matched, as closely as
possible, the existing WHO international standard. Variations in the new source
material could also lead to a potential drift in the IU value which would need to
be minimized by: (a) selecting collaborative study participants similar to those
involved in assessing the current WHO international standard; (b) including
the First WHO International Standard for hepatitis B virus DNA for NAT-based
assays (80 ampoules remaining) in the calibration studies; and (c) using digital
PCR in the evaluation. Efforts were currently under way to source new stocks
of HBV-positive plasma. Subject to the prompt sourcing and characterization of
the new stocks, it was envisaged that the collaborative study outcomes would be
submitted for consideration by the Committee in 2023.
The Committee acknowledged the need to minimize any risks associated
with changes in source material and noted that the sourcing and analysis of any
new material would take time. After considering the various options available, the
Committee recommended that replacement of the current international standard
should proceed using the residual stocks of Eurohep R1 reference material as
this would allow sufficient time for new material to be sourced for future WHO
international standards for HBV DNA.
The Committee noted that the current WHO international standard was
based on the HBV genotype A which is highly prevalent in Europe and Africa
but less common in the Far East, where genotypes B and C were more prevalent.
Such genotype differences might affect the interpretation of assay performance in
different regions – an issue that had previously been raised for other NAT-based
assays such as those for hepatitis C virus RNA. It was suggested that calibrating
panels containing different subtypes against the proposed WHO international
standard might help to resolve this issue. In any case, the possibility of including
other genotypes in future WHO international standards of this type should be
investigated.
The Committee endorsed the proposal (WHO/BS/2021.2411) to develop a
Fifth WHO International Standard for hepatitis B virus DNA for NAT-based assays.
55
6.2.4 Update on replacement challenges for the First WHO

International Standard for anti-rubella immunoglobulin
Rubella virus, the causative agent of rubella disease, was first identified in
1962. Although the disease is typically mild, infection early in pregnancy can
result in miscarriage or congenital rubella syndrome in the infant. Although
effective vaccines are available and have been widely used, sensitive and accurate
diagnostic methods remain important in detecting infection during pregnancy
and in monitoring progress towards local and global eradication of the disease.
The Committee was reminded that, since 1966, a series of three WHO anti-
rubella measurement standards have been used. The latest of these (RUBI-1-94)
consists of normal human immunoglobulin obtained from healthy donors and
has been in use since its establishment in 1996 as the First WHO International
Standard for anti-rubella immunoglobulin.
Today, a wide and increasing range of assays is used to assess anti-rubella
antibody levels, including neutralization, haemagglutination inhibition, single
radial haemolysis, ELISAs and more recently other forms of antibody-binding
assays. It has become evident that the use of the current WHO international
standard leads to different results across laboratories using different methods.
Over time there has been a shift away from the evaluation of functional antibody
activity towards the measurement of antibody in high-throughput binding
assays that are quicker and require less skill to perform. Other issues include the
previous lowering of the cut-off point for establishing immune protection from
15 IU/mL to 10 IU/mL, with one recent study finding that different test kits use
different cut-offs and grey zone ranges – highlighting the importance of using
the same test method when comparing results or performing follow-up testing.
In 2017, the Committee reviewed the findings of a WHO consultation on
the use of the First WHO International Standard for anti-rubella immunoglobulin
and made the following recommendations: (a) the standard should continue to
be made available; (b) the IFU should clearly highlight to users the potential
lack of commutability; (c) stakeholders should reconsider the appropriateness
of quantitative anti-rubella measurement and the use of the 10 IU/mL value as
a cut-off when assessing immune protection; and (d) the use of high-specificity
qualitative assays should be considered as an alternative to antibody quantitation.
The Committee was reminded that stocks of the current WHO
international standard were now limited and likely to be exhausted within
2 years. As the use of this measurement standard had changed over time, the
Committee was asked for its advice on the best approach to its replacement
and whether a single material could realistically harmonize both functional
and binding assays, as well as meet the needs of both vaccine manufacturers
and diagnostic kit manufacturers. A proposal was made to use a panel of anti-
rubella immunoglobulin samples covering a range of antibody concentrations
56
and calibrated against the current WHO international standard, and a potential
collaborative study design was outlined that would assess usage in different
assays and possibly allow for differentiation between vaccine development and
diagnostic applications.
The Committee agreed that the failure of the current international
standard to harmonize modern diagnostic tests resulted from the lack of a
clearly defined measurand or measuring system. Consistent with its previous
discussions on this issue, the Committee discouraged the quantitative use of a
standard to assign units as this fails to harmonize assays and therefore serves no
purpose. It concluded that only qualitative assays should be used for the diagnosis
of previous infection or vaccination, and that a panel without assigned unitage
would facilitate assay development. Commenting that the rubella field was very
active, the Committee encouraged the involvement of stakeholders in decisions
regarding the development and use of the proposed reference panel.
57
7. International reference materials – standards for
use in high-throughput sequencing technologies
7.1 Proposed new projects and updates – standards for
use in high-throughput sequencing technologies
7.1.1 Proposed test protocol and WHO international
reference reagents for whole-genome sequencing in
the routine lot release of OPV and control of sIPV
Polio eradication remains a top priority for WHO and will depend upon a
continuous supply of safe and effective vaccines. A number of live attenuated
OPV and Sabin IPV (sIPV) products have now been approved for use in many
countries. In addition, following polio eradication, a significant number of
additional manufacturers are expected to begin sIPV production to meet an
increased demand for such vaccines in the near future, with demand for novel
OPVs based on genetically stabilized viruses also anticipated.
Genetic stability is a crucial consideration in ensuring the safety of
vaccines. The Sabin strains currently used to manufacture OPV accumulate
reversions during replication in humans and cell cultures that potentially increase
their virulence. Traditionally, the genetic stability of OPV was assessed using
in vivo neurovirulence tests in monkeys or in transgenic mice expressing the
human poliovirus receptor. Since 2002, the genotypic stability of such vaccines
has also been assessed using the in vitro mutant analysis by polymerase chain
reaction and restriction enzyme cleavage (MAPREC) test, which measures the
proportion of revertant mutations present in the 5′-UTR of the viral genome.
However, MAPREC is technically challenging, requires radioisotopes and only
measures one mutation in the entire genome with implications for the evaluation
of other mutations affecting viral properties.
In 2019, the Committee had been updated on the first phase of an

international collaborative study, jointly coordinated by NIBSC and the FDA, on
the use of high-throughput sequencing as an alternative to MAPREC quantification
of mutations in live viral vaccines. It was found that high-throughput sequencing
was sufficiently sensitive to detect low frequency variants in the linear range of
raw MAPREC data, with good correlation between the two methods in assessing
single point mutations. Following the success of this initial study, the Committee
was presented with an overview of a potential future approach to routine OPV
lot release based on the use of whole-genome high-throughput sequencing and a
series of proposed next steps was set out.
The first of these steps would be to conduct a second-phase international
collaborative study to explore the suitability of whole-genome high-throughput
sequencing as a replacement for neurovirulence testing by assessing test
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International reference materials – standards for use in high-throughput sequencing technologies
procedures and reference reagents for the lot release of OPV or sIPV. A series
of consistency lots of monovalent OPV will be analyzed by whole-genome
high-throughput sequencing and the results compared to those obtained in
neurovirulence testing using non-human primates or transgenic mice. The
study, sponsored by PATH, will again be jointly coordinated by NIBSC and FDA.
Several OPV manufacturers had agreed to provide vaccine lots for use in the
study and as prospective reference materials, and a statistical method was being
developed for data analysis.
The Committee discussed the relative sensitivities of the MAPREC and
high-throughput sequencing methods, noting that both were sufficiently sensitive
to detect < 1% mutations in the 5′-UTR – a level of mutation considered to be safe.
Reflecting on the limited sequence variation observed between vaccine lots, and
noting that many mutations arising in vaccine strains are well characterized, the
Committee envisaged that neurovirulence testing may eventually not be required.
Enquiring about the affordability of high-throughput sequencing for vaccine
developers and control laboratories in LMIC, the Committee was assured that
today the approach was technically straightforward, inexpensive and accessible.
Part of this proposed project would be to support manufacturers adopting this
approach through workshops and training sessions. The Committee commended
the progress that had been made and, acknowledging the importance of
implementing modern molecular methods for lot release and the considerable
benefits that would result from the elimination of in vivo neurovirulence testing,
it endorsed the proposal (WHO/BS/2021.2411) to develop a test protocol and
WHO international reference reagents for whole-genome sequencing in the
routine lot release of OPV and control of sIPV.
59
8. International reference materials – standards
for use in public health emergencies
8.1 Proposed new projects and updates – standards
for use in public health emergencies
8.1.1 Proposed First WHO International Reference Panel for
antibodies to SARS-CoV-2 variants of concern
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological
agent of the ongoing COVID-19 pandemic. At its meeting in December
2020, the Committee recommended the establishment of the First WHO
International Standard for anti-SARS-CoV-2 immunoglobulin and the First
WHO International Reference Panel for anti-SARS-CoV-2 immunoglobulin.
These reference materials are intended to facilitate COVID-19 vaccines and
therapeutics development through the harmonization of serological assay data
worldwide. However, in late 2020, SARS-CoV-2 “variants of concern” (VOCs)
began to emerge with mutations that rendered them more transmissible. The
impact of such variants on the effectiveness of vaccines and therapeutics now
requires continual evaluation.
It was proposed that a WHO international reference panel consisting of
CCP or serum from patients infected with VOCs be developed to facilitate the
development of serological assays for VOCs in order to support the continuing
development of vaccines and therapeutic strategies. Following collaboration
between WHO, CEPI and NIBSC, three donations of CCP or serum from
individuals each infected with a VOC had now been offered by a number of
sources. The sourcing and receipt of all required donations was now ongoing.
All candidate materials would be treated to inactivate any contaminating viruses,
sequenced and filled to produce the proposed five-member panel, though
flexibility may be required in terms of panel composition should a new VOC

emerge. The suitability of the panel to serve as a reference material for assessing
serological assays for SARS-CoV-2 VOCs will then be determined as part of a
proposed collaborative study to evaluate a replacement reference material for
the First WHO International Standard anti-SARS-CoV-2 immunoglobulin (see
section 8.1.2 below). To date, three of the five panel sera have been obtained,
representing the original Wuhan-like strain and the Alpha and Delta VOCs.
Once complete, the panel would be promptly evaluated, with the collaborative
study outcomes expected to be submitted for consideration by the Committee in
early 2022.
The Committee enquired about the intended primary use of the proposed
reference panel and clarification was given that it would principally support the
development of live-virus neutralization assays and was not intended to be used
60
International reference materials – standards for use in public health emergencies
in diagnostic testing. There was also no plan to assign unitages to the panel as this
was a known complex issue and would probably cause considerable confusion
among end users. Instead, data from the collaborative study would be included
in the IFU to guide the interpretation of results.
Noting the importance of the reference panel, the Committee
acknowledged the challenge of adding sera corresponding to newly emerging
VOCs, especially as more people became vaccinated. However, the Committee
also recognized the need for the timely availability of the panel, which should
not be delayed due to difficulties sourcing sera for any specific VOC. It agreed
with the proposal to develop a flexible panel with a clearly defined process
for adding further sera as new VOCs emerged. Reflecting on the challenge of
obtaining serum samples for the current panel, the Committee made a number
of suggestions regarding the sourcing of Gamma variant serum. It would also
be important to make clear how the WHO reference panel differed from similar
national reference panels now in development. It was noted that the First WHO
Repository of red blood cell transfusion relevant bacterial reference strains might
provide a useful precedent for such WHO reference materials.
The Committee endorsed the proposal (WHO/BS/2021.2411) to develop
a First WHO International Reference Panel for antibodies to SARS-CoV-2
variants of concern.
8.1.2 Proposed Second WHO International Standard

for anti-SARS-CoV-2 immunoglobulin
The First WHO International Standard for anti-SARS-CoV-2 immunoglobulin
was established on the recommendation of the Committee in December 2020.
Despite imposing a limit of five ampoules per user, more than 2400 ampoules
had been distributed by August 2021 to 581 individual customers globally and
the standard stock had been exhausted. A replacement standard is therefore
urgently needed to ensure both continuity of supply and traceability of the
assigned unitage.
The development of a replacement material presents a number of
challenges. As the IU is an arbitrary unit that does not correspond to a physical
measurement, it cannot be calculated for each variant. Users are therefore advised
to report the potency of antisera specifically for each variant used and not to make
direct comparisons between VOCs. In addition, diagnostic kit manufacturers have
requested the use of higher titre sera as vaccinees tend to exhibit higher antibody
titres than those convalescing from infection. The proposed replacement standard
would therefore consist of a pool of high-titre serum or plasma obtained from
individuals convalescing from infection with a VOC. The pool will be prepared
from selected sera sourced for the development of the WHO international
reference panel for VOCs (see section 8.1.1 above). The ideal candidate material
61
would exhibit high antibody titres against all current VOCs, and be sufficient
to fill at least 5000 ampoules. It had been observed that serum from recovered
and subsequently vaccinated individuals exhibited higher antibody titres and a
broader response to the different VOCs than convalescent sera.
Another significant challenge was the need to avoid the inappropriate
use of the reference material in antibody-binding assays. The current WHO
international standard had been assigned an IU based on neutralizing antibodies
and concern had been raised by the Committee during its establishment that
assigning the same unitage for use in antibody-binding assays based on different
antigens could result in the incorrect use of the standard. To allow for comparison
of relative antibody titres to different antigens, the material would be assigned
an arbitrary binding antigen unit (BAU). Comparison of the current WHO
international standard with clinical samples across a range of antibody-binding
assay platforms had demonstrated its commutability with clinical material but
had also highlighted the potential unsuitability of IU assignment for such assays.
As many commercial assays based on different target antigens were calibrated in
BAU and several publications had reported results accordingly, the continued use
of the unit would be useful.
Data from the collaborative study would be used to assign an IU unitage
relative to the current WHO international standard based on neutralization
assays. A value would be calculated for each VOC with arbitrary values to be
assigned should new VOCs emerge. Regarding the timeline for development of
the replacement standard, it was clarified that if the Committee recommended
the use of high-titre sera from recovered and subsequently vaccinated
individuals then such material would need to be sourced, potentially shifting the
establishment of the replacement standard from early to late 2022. Mindful that
any such change in the source material might also affect its assigned unitage, the
Committee advised that the replacement material should be as similar as possible
to the current WHO international standard and should be assessed using the
same neutralization measurement system. It was noted that the development of
future standards using suitable convalescent serum would be challenging because
of changes in the predominant VOC, the rapidly changing COVID-19 vaccination
status of donors and the declining interest in collecting CCP. Given the interest in
using high-titre sera from infected and subsequently vaccinated individuals, the
Committee suggested that such material could, if sourced in time, be included in
the collaborative study and the data subsequently reviewed.
Acknowledging the ongoing high level of demand for the current WHO
international standard and recognizing its importance in the harmonization
of serological assays globally, the Committee endorsed the proposal (WHO/
BS/2021.2411) to develop a Second WHO International Standard for anti-SARS-
CoV-2 immunoglobulin.
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International reference materials – standards for use in public health emergencies
8.1.3 Update on the development of the First WHO

International Standard for SARS-CoV-2 antigen
The Committee was updated on the development of the First WHO International
Standard for SARS-CoV-2 antigen. The antigen assay landscape for SARS-CoV-2
was now highly complex, with several different antigens targeted either alone or
in combination, and various types of test sample and numerous formats used.
Worldwide, more than 200 SARS-CoV-2 antigen tests have received regulatory
approval or are in development. A common reference reagent was urgently
required to support their development, evaluation and monitoring. The current
project had therefore been endorsed by the Committee at its meetings in late 2020
and the aim remained of presenting the outcomes of a full collaborative study for
consideration by the Committee in early 2022. Several candidate materials had now
been sourced, including whole inactivated virus, N protein expressed in E. coli and
trimeric S protein expressed in HEK293 cells, and their concentrations determined
using mass spectrometry.
A pilot study had been conducted to evaluate the source materials and
determine the most suitable antigen preparation for use as the candidate material
in the full study. Pilot study participants (n = 17) had determined the end-point
titration for each of six samples using a range of different tests. The sample set
included inactivated virus preparations, recombinant N protein and recombinant
spike protein. In most cases, tests had been provided by the manufacturer,
with some study participants also evaluating other tests routinely used in their
laboratory. Although ED50 values were estimated using the Spearman-Karber
method due to time pressures, alternative probit models and other methods
are available, and a different method would likely be used in the definitive
collaborative study. Pilot study results showed that the virus sample inactivated
with 0.01% formaldehyde was detected with the greatest sensitivity and produced
the lowest standard deviation between laboratories. Following the production of
an interim NIBSC working standard, a candidate WHO international standard
was developed based on the Delta variant inactivated with 0.01% formaldehyde.
The candidate material will be evaluated in the definitive collaborative study by
approximately 30 laboratories using a range of assay technologies.
The Committee enquired if the proposed IU of the candidate material
was to be linked to its content, as determined by mass spectroscopy. Clarification
was given that no decision had been taken in this regard and the views of the
Committee would be welcome. It was felt that as most of the tests depended upon
an antibody-antigen interaction it would be more appropriate to use IU rather
than SI units for the proposed standard. However, the use of mass spectrometry
had been very useful in determining that inactivation did not change the amount
of protein in the whole-virus samples and had highlighted potential issues with
the use of recombinant proteins in this situation, where detection by lateral flow
63
tests relied on correct folding of the antigen. Noting that the ED50 data from
the pilot study could be used to rank the sensitivity of the different tests and
that numerous publications were now available on test sensitivity using clinical
materials, it was suggested that it might be worthwhile to compare the results of
the full study with the published scientific literature to allow for a more detailed
comparative analysis of test sensitivity.
64
9. International reference materials –
vaccines and related substances
9.1.1 Second WHO International Standard for diphtheria antitoxin (equine)
Diphtheria antitoxin (DAT) products produced from equine serum are essential
medicines used for diphtheria therapy and outbreak management, and for
prophylaxis against suspected cases of diphtheria in countries where the disease
is endemic. In countries with good vaccination coverage, DAT is stockpiled for
emergency use. The use of an equine DAT standard calibrated in IU is essential
for ensuring that products meet minimum potency requirements. The First
WHO International Standard for diphtheria antitoxin (equine) was prepared in
Copenhagen in 1934 and consists of a preparation of dried hyperimmune horse
serum in ampoules. To conserve stocks, a batch of liquid standard has been produced
at NIBSC approximately every 2 years, with a diphtheria antitoxin concentration of
10 IU/mL. In 2016, the Committee had been informed that the stock of dried serum
was running low and had endorsed the preparation of a lyophilized replacement
standard that would provide a single homogenous batch sufficient for 15–20 years.
The procured candidate material (NIBSC code 18/180) consisted of refined
diphtheria immunoglobulin prepared from horse serum and was calibrated using
both in vivo and in vitro (Vero cell) toxin neutralization assays, with potency
expressed relative to the current WHO international standard. Several formulations
were evaluated in a trial fill and were freeze-dried successfully with no loss of
biological activity. An international collaborative study was conducted involving
14 laboratories in nine countries, with 10 participants providing data from in vivo
assays and eight from in vitro methods. Although sample protocols were provided,
participants were encouraged to use their existing in-house assays. Potency was
determined by comparing the dose of DAT necessary to protect against the effects
of diphtheria toxin with the quantity of a reference preparation necessary to give
the same protection. Potency estimates obtained using either the in vivo or in
vitro toxin neutralization tests were comparable, with low inter-assay variability.
Although use of the Vero cell assay is increasing, in vivo assays continue to be
used in many countries and so the potency value for candidate material 18/180
was estimated by combining all data. This gave a value of 57 IU/ampoule, which
was similar to the geometric mean potency obtained using the in vivo assays (54
IU/ampoule). The results of accelerated thermal degradation studies predicted no
significant loss in activity when stored at −20 °C indicating long-term stability of
the material. Real-time stability data indicated that the candidate material would
be suitable for use for up to 1 year after reconstitution when stored at 2–8 °C.
65
The Committee commented on the slow progress towards the use of the
Vero cell assay and was informed that many producers of diphtheria antitoxin still
opted to use the in vivo assay. Reflecting on whether the small difference (3 IU
or 5%) between the potency estimated from all available data and that using only
the in vivo assays would be problematic for borderline potency determinations,
the Committee was satisfied that this difference would be negligible in the overall
context of assay variability. The Committee considered the report of the study
(WHO/BS/2021.2407) and recommended that the candidate material 18/180 be
established as the Second WHO International Standard for diphtheria antitoxin
(equine) with an assigned unitage of 57 IU/ampoule based on calibration by in
vivo and in vitro toxin neutralization tests.
9.2 Proposed new projects and updates –

9.2.1 Proposed Fifth WHO International Standard
for pertussis vaccine (whole cell)
The strict human pathogen Bordetella pertussis is the etiological agent of whooping
cough and is transmitted in respiratory droplets. Despite high rates of vaccination
in the young, whooping cough (pertussis) remains an important cause of
morbidity and mortality globally. There are two types of pertussis vaccine – killed
whole cell and acellular (protein antigen). Although acellular pertussis vaccines
are widely used in developed countries, more doses of whole cell vaccine are used
in routine immunization programmes worldwide each year and an international
standard is therefore still required. The potency of whole cell pertussis vaccines
is measured in IU based on the current Fourth WHO International Standard for
pertussis vaccine (whole cell) established in 2006. Despite restricted sales, stocks
of this standard are now becoming heavily depleted and it is estimated that a
replacement standard will be required within the next 2 years.

A commercial vaccine producer will donate sufficient material to produce
4000–5000 ampoules of the candidate replacement material, which at the current
restricted rate of use should last for approximately 20 years. An international
collaborative study will be conducted to assess the suitability of the candidate
material, and to assign a unitage calibrated to the current WHO international
standard based on data from the Kendrick test. It is anticipated that the study
will involve 10–20 laboratories, representing national control laboratories and
manufacturers worldwide. It was envisaged that the collaborative study outcomes
would be submitted for consideration by the Committee in 2023.
The Committee discussed the significant ethical issues raised by
performing stability studies using the Kendrick test, which uses a large number of
animals, and for which there is currently no in vitro alternative. The Committee
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International reference materials – vaccines and related substances
expressed its support for the ongoing development of alternative in vitro methods
and suggested that the use of orthogonal methods be explored further. In order to
reduce the use of the Kendrick test, and concluding that accelerated degradation
studies of the proposed standard would be part of the collaborative study, the
Committee agreed that the stability of reconstituted material only needed to be
determined by the custodian laboratory. The Committee endorsed the proposal
(WHO/BS/2021.2411) to develop a Fifth WHO International Standard for
pertussis vaccine (whole cell).
9.2.2 Proposed WHO International Reference Reagent

for diphtheria CRM197 antigen
Cross-reacting material 197 (CRM197) is a genetically detoxified form of diphtheria
toxin in which a single mutation results in the loss of ADP-ribosyltransferase
activity. CRM197 is widely used as a carrier protein in a number of different
polysaccharide conjugate vaccines, including meningococcal, pneumococcal
and typhoid glycoconjugates. Although CRM197 can be produced using a
Corynebacterium diphtheriae expression system, this approach produces relatively
low yields and a number of companies are now producing recombinant CRM197
using heterologous expression systems. These alternative sources of CRM197
carrier protein are likely to become important in helping to meet an increasing
global demand for conjugate vaccines against encapsulated bacterial pathogens.
When used for vaccine manufacture, CRM197 is characterized using
immunochemical and physicochemical methods. It is proposed that a WHO
international reference reagent be developed to support the stable and well-
characterized control of these analytical methods. The Committee was informed
that sufficient recombinant CRM197 produced in E. coli had been donated
as a potential material for this purpose, and will be filled and lyophilized. A
collaborative study will be conducted to assess the suitability of the proposed
candidate material but units of activity or content will not be assigned. Although
a number of different recombinant CRM197 materials are available, it is currently
not clear whether one material would be suitable for use as a control in analytical
techniques applied to other recombinant CRM197 products. However, published
comparisons of various CRM197 materials suggest only subtle differences in
some physicochemical characteristics. The carrier protein content of conjugate
vaccines is typically quoted in SI units – normally µg/mL in bulk conjugates and
µg/dose (or range of µg/dose) in the final product – with UV spectrophotometry
being the primary method.
The Committee briefly discussed the potential differences between
CRM197 produced in different expression systems. Acknowledging that the
purpose of the collaborative study would be to identify such differences, it was
clear from the published literature that the physicochemical differences were
67
subtle, with the main differences likely to be in yield, purity and possibly post-
translational modification. The Committee recognized the considerable level of
interest in manufacturing polysaccharide-conjugate vaccines in LMIC, especially
in the WHO African Region. Any reduction in the level of containment required
during vaccine production and the increased yield associated with the novel
CRM197 expression systems would potentially reduce the cost of this important
carrier protein.
Noting the importance of demonstrating that the proposed material
could be used as a control for both native CRM197 and CRM197 produced in
other expression systems, the Committee endorsed the proposal (WHO/
BS/2021.2411) to develop a WHO International Reference Reagent for
diphtheria CRM197 antigen.
68
Annex 1
WHO Recommendations, Guidelines and other documents
related to the manufacture, quality control and evaluation
of biological products
WHO Recommendations, Guidelines and other documents are intended to
provide guidance to those responsible for the development and manufacture of
biological products as well as to others who may have to decide upon appropriate
methods of assay and control to ensure that such 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 and guidance on biological products are formulated
by international groups of experts and published in the WHO Technical Report
Series4 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
Email: [email protected]
Website: http://apps.who.int/bookorders
Individual Recommendations and Guidelines and other documents may
be obtained free of charge as offprints by writing to:
Technical Standards and Specifications unit
Department of Health Product Policy and Standards
Access to Medicines and Health Products
World Health Organization
20 avenue Appia
1211 Geneva 27
Switzerland
4
Abbreviated in the following pages to “TRS”.
69
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 UK WHO/BLG/97.1
National Biological Standards Board (1997)
Biological substances: International Standards Revised 2004, TRS 932 (2006)
and Reference Reagents
Biotherapeutic products, changes to approved Adopted 2017, TRS 1011 (2018)
biotherapeutic products: procedures and data
requirements
Biotherapeutic products, similar Adopted 2009, TRS 977 (2013)
Biotherapeutic products, similar: Adopted 2018; online document
WHO Questions and Answers 5
Biotherapeutic protein products prepared by Revised 2013, TRS 987 (2014);
recombinant DNA technology Addendum 2015, TRS 999 (2016)
Blood, blood components and plasma Revised 1992, TRS 840 (1994)
derivatives: collection, processing and quality
control
Blood and blood components: management Adopted 2016, TRS 1004 (2017)
as essential medicines
Blood components and plasma: estimation of Adopted 2016, TRS 1004 (2017)
residual risk of HIV, HBV or HCV infections
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
5
Available online at: https://www.who.int/biologicals/expert_committee/QA_for_SBPs_ECBS_2018.pdf?ua=1
70
Annex 1

documents
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, plasmid Revised 2020, TRS 1028 (2021)
Ebola vaccines Adopted 2017, TRS 1011 (2018)
Enterovirus 71 vaccines (inactivated) Adopted 2020, TRS 1030 (2021)
Haemophilus influenzae type b conjugate Revised 1998, TRS 897 (2000)

vaccines
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 1994, TRS 858 (1996)
Hepatitis B vaccines (recombinant) Revised 2010, TRS 978 (2013)
Hepatitis E vaccines (recombinant) Adopted 2018, TRS 1016 (2019)
Human immunodeficiency virus rapid diagnostic Adopted 2017, TRS 1011 (2018)
tests for professional use and/or self-testing
Technical Specifications Series for WHO
Prequalification – Diagnostic Assessment
Human interferons prepared from Adopted 1988, TRS 786 (1989)

lymphoblastoid cells
Influenza vaccines (inactivated) Revised 2003, TRS 927 (2005)
Influenza vaccines (inactivated): labelling Addendum 2016, TRS 1004 (2017)

information for use in pregnant women to Annex 3, TRS 927 (2005)
Influenza vaccines (live) Revised 2009, TRS 977 (2013)
Influenza vaccines, human, pandemic: Adopted 2007, TRS 963 (2011)

regulatory preparedness
Influenza vaccines, human, pandemic: Adopted 2016, TRS 1004 (2017)

regulatory preparedness in non-vaccine-
producing countries
71

documents
Influenza vaccines, human, pandemic: safe Adopted 2018, TRS 1016 (2019)
development and production
In vitro diagnostics (WHO-prequalified), Adopted 2020, TRS 1030 (2021)
collaborative procedure between WHO and
NRAs for assessment and accelerated national
registration
In vitro diagnostic medical devices, establishing Adopted 2017, TRS 1011 (2018)
stability of,
Technical Guidance Series for WHO
Prequalification – Diagnostic Assessment
Japanese encephalitis vaccines (inactivated) for Revised 2007, TRS 963 (2011)
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 840 (1994);
combined vaccines (live) Note 1993 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)
Monoclonal antibodies as similar biotherapeutic Adopted 2016, TRS 1004 (2017)
products
Papillomavirus vaccines (human, recombinant, Revised 2015, TRS 999 (2016)
virus-like particle)
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
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Annex 1

documents
Pneumococcal conjugate vaccines Revised 2009, TRS 977 (2013)
Poliomyelitis vaccines (inactivated) Revised 2014, TRS 993 (2015);
Amendment 2019, TRS 1024
(2020)
Poliomyelitis vaccines (oral) Revised 2012, TRS 980 (2014)
Poliomyelitis vaccines: safe production and Revised 2018, TRS 1016 (2019)
quality control Amendment 2020, TRS 1028 (2021)
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
Reference materials, secondary: for NAT-based Adopted 2016, TRS 1004 (2017)
and antigen assays: calibration against WHO
International Standards
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
Respiratory syncytial virus vaccines Adopted 2019, TRS 1024 (2020)
RNA vaccines, messenger, Adopted 2021, TRS 1039 (2022)
for prevention of infectious diseases
Rotavirus vaccines (live, attenuated, oral) Adopted 2005, TRS 941 (2007)
Smallpox vaccines Revised 2003, TRS 926 (2004)
Snake antivenom immunoglobulins Revised 2016, TRS 1004 (2017)
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 replacement
73

documents
Thromboplastins and plasma used to control Revised 2011, TRS 979 (2013)
oral anticoagulant therapy
Tick-borne encephalitis vaccines (inactivated) Adopted 1997, TRS 889 (1999)
Transmissible spongiform encephalopathies in Revised 2005, WHO (2006)
relation to biological and pharmaceutical products 6
Tuberculins Revised 1985, TRS 745 (1987)
Typhoid vaccines, conjugated Revised 2020, TRS 1030 (2021)
Typhoid vaccines (live, attenuated, Ty21a, oral) Adopted 1983, TRS 700 (1984)
Typhoid vaccines, Vi polysaccharide Adopted 1992, TRS 840 (1994)
Vaccines, changes to approved vaccines: Adopted 2014, TRS 993 (2015)
procedures and data requirements
Vaccines, clinical evaluation: regulatory Revised 2016, TRS 1004 (2017)
expectations
Vaccines, regulatory considerations: use of Adopted 2016, TRS 1004 (2017)
human challenge trials
Vaccines, lot release Adopted 2010, TRS 978 (2013)
Vaccines, nonclinical evaluation Adopted 2003, TRS 927 (2005)
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 for use under Adopted 2015, TRS 999 (2016)
extended controlled temperature conditions

Varicella vaccines (live) Revised 1993, TRS 848 (1994)
Yellow fever vaccines (live, attenuated) Revised 2010, TRS 978 (2013)
Amendment 2021, TRS 1039 (2022)
Yellow fever vaccines, laboratories approved Revised 1995, TRS 872 (1998)
by WHO for the production of
Yellow fever virus, production and testing Adopted 1985, TRS 745 (1987)
of WHO primary seed lot 213-77 and reference
batch 168-736
6
Available online at: http://www.who.int/biologicals/publications/en/whotse2003.pdf
74
Annex 2
Recommendations to assure the quality, safety and
efficacy of live attenuated yellow fever vaccines
Amendment to Annex 5 of WHO Technical Report Series, No. 978
Introduction 76
Amendment 77
Authors and acknowledgements 82
References 83
75
Introduction
The WHO Recommendations to assure the quality, safety and efficacy of live
attenuated yellow fever vaccines were adopted in 2010 (1). Appendix 2 of these
Recommendations addresses the testing of new virus master and working
seed lots in non-human primates. Specifically, the appendix sets out the ways
in which such lots should be tested for viscerotropism, immunogenicity and
neurotropism, both in terms of clinical evidence and histological lesions, based
on comparison against a reference virus approved by the NRA. Following
reported discrepancies in the clinical scoring of monkeys during the assessment
of working seed lots, WHO received a request from one yellow fever vaccine
manufacturer to align the neurotropism assessment outlined in the 2010
Recommendations with that used for the neurovirulence testing of oral
poliomyelitis vaccine seed lots in which clinical signs are recorded but do not
form part of the assessment or pass/fail criteria (2).
At its seventy-first meeting in August 2020, the WHO Expert Committee
on Biological Standardization recommended that a drafting group be established
to consult with as many yellow fever vaccine manufacturers and other stakeholders
as possible on a proposed revision of Appendix 2 of the 2010 Recommendations
(3). At its seventy-third meeting in December 2020, the Committee was updated
on the progress that had been made (4). The currently specified approach had
now been associated with several technical challenges including: (a) a paucity of
data on the performance of the test; (b) the difficulties inherent in conducting
a collaborative study involving non-human primates; (c) the lack of an
international reference standard for vaccines of the 17D-204 and 17DD lineages
and consequent use of different reference materials; (d) reported discrepancies
between clinical and histopathological assessments; (e) inconsistencies between
staff in the scoring of clinical and histopathological observations; and (f) the
sourcing of animals from different locations.

Work on revising Appendix 2 of the 2010 WHO Recommendations
commenced in early 2021. On 18–19 March 2021, a virtual WHO working group
meeting was held to discuss a proposed draft of the revised text. Overall, there was
a consensus among manufacturers and NRAs that clinical evaluation provides
important information and should be retained as part of the neurotropism
test. However, there was also agreement that the test is somewhat subjective
and that analysis can be difficult. It was recognized that there was potential for
improvement in both test execution and analysis to increase harmonization
between organizations. Based on these working group discussions, the appendix
was revised by the WHO drafting group. Following public consultation and
further revision, the amendment to the 2010 WHO Recommendations presented
below was reviewed by the Committee at its meeting in October 2021.
76
Annex 2
No attempt was made at this time to review the 2010 WHO

Recommendations to assure the quality, safety and efficacy of live attenuated
yellow fever vaccines in their entirety and only the issues outlined above have
been addressed
Amendment
Replace Appendix 2 with the following text:
Appendix 2
Tests in non-human primates of new virus master and working seeds
Neurotropism is defined as the tendency or capacity of a microorganism to cause
disease of the nervous system and the yellow fever test in monkeys is a tropism
test by this definition. However, it also involves examination of the tendency
of the virus to cause viraemia after intracranial inoculation, which could be
interpreted as a surrogate of both viscerotropism and the ability to induce an
immune response in the same way. The test can differentiate between strains of
yellow fever vaccine viruses using all three of these criteria.
Each virus master and working seed lot should be tested for viscerotropism,
immunogenicity and neurotropism in a group of 10 test monkeys compared
against a second similar group of 10 monkeys injected with a reference virus.
The same test and reference groups will be used for all of the viscerotropism,
immunogenicity and neurotropism tests. The allocation of animals to the two
groups should be blinded to the operators throughout the experiment. For the
neurotropism test, the test monkeys inoculated intracranially with the virus seed
lot should be compared against the 10 monkeys injected with the reference virus.
Existing manufacturers should use a homologous reference – for example, where
their working seed is to be replaced by another derived from the same master
seed, the existing seed can be used as the reference material, provided it has been
shown to produce a vaccine with satisfactory properties. It is recommended
that sufficient stocks of such a reference are kept for all future anticipated
replacements of the working seed. New manufacturers using a new seed should
use a homologous preparation known to produce a satisfactory product as a
reference material. The reference virus should be approved by the NRA.
A WHO reference virus, 168-73, is available from the National Institute
for Biological Standards and Control, Potters Bar, England. This virus is of the
same lineage as the WHO primary seed 213-77 (see Appendix 1, Figure 1), but
available published data show that it behaves differently to vaccines of at least one
other lineage in the monkey test, being much less neurovirulent and producing
a higher viraemia. It is likely, though unproven, that 168-73 will be a satisfactory
reference for seeds of the 213-77 lineage. While 168-73 is not suitable as a
77
comparator for vaccines of other lineages, its inclusion in the neurotropism test
as a common material would make it possible to compare different tests, and one
lineage with another, for information.
The monkeys should be Macaca mulatta (rhesus monkeys) or Macaca
fascicularis (cynomolgus monkeys) and should have been demonstrated to be
non-immune to yellow fever virus and other flaviviruses using a relevant test
(such as the haemagglutination inhibition test, ELISA or seroneutralization assay)
immediately prior to injection of the seed virus. Tests should be performed using
healthy macaques of both sexes (weighing at least 2 kg and at least 18 months old).
The monkeys should not have been previously subjected to any experimentation.
The test dose should be injected into one frontal lobe of each monkey, under
anaesthetic, and the monkeys should be observed for a minimum of 30 days.
The test dose should consist of 0.25 mL containing not less than 5000
(3.7 log10) IU and not more than 50 000 (4.7 log10) IU, as shown by titration in
cell culture. In addition, the virus titres of the test virus seed lot and the reference
virus should be as close as possible.
Historically, the test dose has consisted of 0.25 mL containing the
equivalent of not less than 5000 and not more than 50 000 mouse LD50, as
shown by titration in cell culture.
1. Viscerotropism test
The criterion of viscerotropism (indicated by the amount of circulating virus)
should be fulfilled as follows: sera obtained from each of the test monkeys on the
second, fourth and sixth days after injection of the test dose should be inoculated at
dilutions of 1:10, 1:100 and 1:1000 into at least four cell culture vessels per dilution.
In no case should 0.03 mL of serum contain more than 500 (2.7 log10) IU and in no
more than one case should 0.03 mL of serum contain more than 100 (2.0 log10) IU.
2. Immunogenicity test
The criterion of sufficient virus-neutralizing antibody in the sera (immunogenicity)
should be fulfilled as follows: at least 90% of the test monkeys should be shown
to have become immune within 30 days following injection of the test dose, as
determined by examining their sera in the yellow fever virus neutralization test
described below. In some countries it has been shown that, at low dilutions, some
sera contain nonspecific inhibitors that interfere with this test. The NRA may
therefore require sera to be treated to remove such substances.
Dilutions of 1:10, 1:40 and 1:160 of serum from each test monkey should
be mixed with an equal volume of strain 17D vaccine virus at a dilution that has
been shown to yield an optimum number of plaques when assayed according to
one of the cell culture methods given in Appendix 4. These serum–virus mixtures
should be incubated in a water bath at 37 °C for 1 hour and then chilled in an
78
Annex 2
ice-water bath before inoculation of 0.2 mL aliquots of each mixture into each
of four separate cell culture vessels. The vessels should be handled in accordance
with one of the cell culture techniques described in Appendix 4. In addition, 10
vessels should be similarly inoculated with a pre-incubated mixture of the same
virus with an equal volume of a 1:10 dilution of monkey serum known to contain
no neutralizing antibodies to yellow fever virus. At the end of the observation
period, the mean number of plaques in the vessels containing virus and non-
immune serum should be compared with the mean number of plaques in the
vessels containing virus and serum from test monkeys. For the immunogenicity
test to be satisfied, serum at the 1:10 dilution from no more than 10% of the test
monkeys should fail to reduce the mean number of plaques by 50% as compared
with the vessels containing non-immune serum.
3. Neurotropism test
The monkeys in the test group should be compared with 10 monkeys injected
with the reference virus with respect to both clinical evidence of encephalitis and
the severity of histological lesions of the nervous system (5, 6).
The onset and duration of the febrile reaction should not differ between
monkeys injected with the test virus or with the reference virus.
3.1 Clinical evaluation

The monkeys should be examined daily for 30 days by personnel familiar with
the clinical signs of encephalitis in primates. All such signs should be recorded
individually on a daily basis. Evaluation may include observation from a distance
using closed circuit television to gather information. The use of implantable
telemetry devices (for example to produce electroencephalograms or to monitor
temperature and motor activity) may also be considered.
If necessary, the monkeys may be removed from their cages and examined
for signs of motor weakness or spasticity, as described elsewhere (6).
Signs of encephalitis – such as paresis, incoordination, lethargy, tremors

or spasticity – should be assigned numerical values for severity by the following
grading method. Each day each monkey should be given a numerical score based
on this scale:
0: no general signs or signs of CNS involvement;
1: rough coat, not eating;
2: high-pitched voice, inactive, slow moving;
3: shaky movements, tremors, incoordination, limb weakness;
4: inability to stand, limb paralysis or death.
79
Any animal unexpectedly found to be moribund, cachectic or unable to

obtain food or water must be euthanized. A monkey that dies receives the score
“4” from the day of death until day 30.
The clinical score for each monkey is the average of its daily scores;
the clinical score for a group is the arithmetic mean of the individual scores.
The timing of the development of clinical signs and their disappearance, as
well as their severity, provides evidence of the phenotypic identity of the test
vaccine virus and the reference virus. For the test material to be considered
sufficiently comparable to the reference material, as required, it should produce
no statistically different clinical signs, including in terms of the timescale of their
appearance and resolution. It is acknowledged that the clinical evaluation may
be imprecise.
3.2 Histopathological evaluation

The cervical and lumbar enlargements of the spinal cord and specific structures at
five levels of the brain should be examined (6) (see Appendix 3). The cervical and
lumbar enlargements should each be divided equally into six blocks. The blocks
should be dehydrated and embedded in paraffin wax; 15 µm sections should be
cut and stained with gallocyanin. Alternatively, 5 µm sections will be suitable
for hematoxylin and eosin (H&E) staining or Nissl staining (gallocyanin, cresyl
violet), as well as for immunohistochemistry techniques. One section, consisting
of two hemisections, should be cut from each block.
Tissue blocks 3–4 mm thick should be taken from the brain by making
the following frontal cuts:
Block I: the corpus striatum at the level of the optic chiasma;
Block II: the thalamus at the level of the mamillary bodies;
Block III: the mesencephalon at the level of the superior colliculi;
Block IV: the pons and cerebellum at the level of the superior olives;
Block V: the medulla oblongata at the midlevel of the inferior olives.
These blocks should be dehydrated and embedded in paraffin wax and
15 µm sections cut and stained with gallocyanin. Alternatively, 5 µm sections
will be suitable for H&E staining or Nissl staining (gallocyanin, cresyl violet), as
well as for immunohistochemistry techniques. A single section, consisting of two
hemisections, should be cut from each block.
Sections should be examined microscopically and numerical scores
assigned to each hemisection of the cervical and lumbar enlargements, and to
each anatomical structure (see Appendix 3) within each hemisection of the brain
blocks, according to the following grading system:
80
Annex 2
1 (minimal): 1–3 small, focal inflammatory infiltrates. A few

neurons may be changed or lost;
2 (moderate): more extensive focal inflammatory infiltrates
(neuronal changes or loss affects no more than one
third of neurons);
3 (severe): neuronal changes or loss of 33–90% of neurons, with
moderate focal or diffuse inflammatory infiltration;
4 (overwhelming): more than 90% of neurons are changed or lost,
with variable, but frequently severe, inflammatory
infiltration.
Each brain block contains several anatomical structures, which contribute
in different ways to the assessment of a test sample. For example, certain structures
differentiate more reproducibly than others between acceptable and unacceptable
yellow fever seed lots and vaccines (6). These are called “discriminator areas”,
whereas structures that are more susceptible to yellow fever virus replication
are called “target areas”. Though both rhesus and cynomolgus monkeys are
acceptable, the discriminator and target areas are different for the two species.
The major difference is that in cynomolgus monkeys the cervical and lumbar
enlargements are target areas whereas in rhesus monkeys they are discriminator
areas. The footnotes to the worksheets provided in Appendix 3 indicate in more
detail the discriminator and target areas for the two species. The worksheets also
list other anatomical structures that will be present in the brain sections but that
are not included in the evaluation of a test sample because they are rarely affected
(spared areas).
Three separate scores should be calculated for each monkey: (a)
discriminator areas only; (b) target areas only; and (c) discriminator plus target
areas. These three scores should be calculated as shown in the sample worksheets
provided in Appendix 3.
Overall mean scores should also be calculated for each group of monkeys
as the arithmetic mean of individual monkey scores for discriminator areas only,
and for discriminator plus target areas. Both of these overall mean scores should
be considered when determining virus seed lot acceptability. For the histological
criterion of the neurotropism test to be satisfied, both of the overall mean scores for
the test monkeys should not be significantly greater (at the 5% significance level)
than the overall mean scores for the monkeys injected with the reference virus.
Both the clinical and histological criteria of the neurotropism test should
be satisfied in order for the virus seed lot to meet the requirements for use in
production.
It is acknowledged that clinical observations may be more subjective than
histological scoring.
81
Manufacturers are encouraged to explore the possible use of telemetry to

render the assessment more objective.
Any failure to meet the statistical criteria should result in failure of

the batch. Any exception made to this rule should be rare and would only be
acceptable after a thorough investigation of the conducting of the tests, including
a review of historical in-house data. Clinical observation should be included in
the review and the record of the ultimate decision even if the findings do not meet
the statistical criteria for a pass. However, any decision to ignore the statistical
evaluation of clinical signs should be a rare and exceptional event involving close
discussion with the NRA.
Authors and acknowledgements

The first draft of this amendment of Appendix 2 of the WHO Recommendations
to assure the quality, safety and efficacy of live attenuated yellow fever vaccines was
prepared by a WHO drafting group comprising Dr P. Minor, St Albans, the United
Kingdom; Dr J. Martin, National Institute for Biological Standards and Control,
the United Kingdom; Professor A. D.T. Barrett, University of Texas Medical Branch
Sealy Center for Vaccine Development, the United States of America (USA); Dr G.
Cirefice, European Directorate for the Quality of Medicines & HealthCare, France;
Dr E. Grabski, Paul-Ehrlich-Institut, Germany; Mrs F. Garnier, Agence nationale
de sécurité du médicament et des produits de santé, France; Mrs V. Pithon, Agence
nationale de sécurité du médicament et des produits de santé, France; and Dr
D. Lei, World Health Organization, Switzerland, taking into consideration the
discussions and consensus reached during a WHO working group meeting to
amend the WHO Recommendations to assure the quality, safety and efficacy of
live attenuated yellow fever vaccines, held virtually via Zoom video conferencing,
18–19 March 2021 and attended by: Professor A.D.T. Barrett, University of
Texas Medical Branch Sealy Center for Vaccine Development, the USA; Dr R.M.
Bretas, Agência Nacional de Vigilância Sanitária, Brazil; Dr G. Cirefice, European
Directorate for the Quality of Medicines & HealthCare , France; Dr M. Diagne,
Direction des Laboratoires, Senegal; Dr A. Dieng, Direction de la Pharmacie et
du Médicament, Senegal; Dr E. Grabski, Paul-Ehrlich-Institut, Germany; Mrs F.
Garnier, Agence nationale de sécurité du médicament et des produits de santé,
France; Dr Y. Li, National Institutes for Food and Drug Control, China; Dr J. Martin,
National Institute for Biological Standards and Control, the United Kingdom;
Dr P. Minor, St Albans, the United Kingdom; Mrs V. Pithon, Agence nationale
de sécurité du médicament et des produits de santé, France; Dr M.F. Reis e Silva
Thees, Agência Nacional de Vigilância Sanitária, Brazil; Dr J. Wang, National
Institutes for Food and Drug Control, China; Dr Y. Wang, National Institutes for
Food and Drug Control, China; Dr M. Xu, National Institutes for Food and Drug
82
Annex 2
Control, China; Dr A. Trapkova, Federal Service for Surveillance in Healthcare,

Russian Federation; Dr D. Yakunin, Federal Service for Surveillance in Healthcare,
Russian Federation; Dr F. Cano, Agence nationale de sécurité du médicament et
des produits de santé, France; and Dr G. Cooper, National Institute for Biological
Standards and Control, the United Kingdom. The following participants attended
the meeting as representatives of vaccine manufacturers: M. da Luz Fernandes
Leal; M. da Silva Freire; R.C. Guimarães; A. Homma; and R. Marchevsky (Bio-
Manguinhos/Fiocruz, Brazil); A. Malkin; and A. Sinyugina (Chumakov Federal
Scientific Center for Research & Development of Immune-and-Biological
Products of Russian Academy of Sciences, Russian Federation); Y. Chen; H. Wang;
N. Li; X. Zhu; and C. Jia (Beijing Institute of Biological Products Co., Ltd, China);
C. Logvinoff; C. Allain; and E. Coppens (Sanofi Pasteur, France); and A.M. Diatta;
and A.A. Sall (Institut Pasteur de Dakar, Senegal). The WHO Secretariat for the
working group comprised: Dr I. Knezevic; and Dr D. Lei, Technical Specifications
and Standards Unit, Access to Medicines and Health Products Division, World
Health Organization, Switzerland; and Dr M. Janssen, Prequalification unit,
Regulation and Prequalification, World Health Organization, Switzerland.
The resulting document WHO/BS/2021.2401 was then posted on the
WHO Biologicals website for a round of public consultation from 24 June to
24 September 2021. Comments were received from vaccine manufacturers,
regulators and individual experts.
Further changes were made to document WHO/BS/2021.2401 by the
Expert Committee on Biological Standardization.
References
1. Recommendations to assure the quality, safety and efficacy of live attenuated yellow fever
vaccines. In: WHO Expert Committee on Biological Standardization: sixty-first report. Geneva:
World Health Organization; 2013: Annex 5 (WHO Technical Report Series, No. 978; https://www.
who.int/publications/i/item/9789241209786, accessed 21 June 2021).
2. Recommendations to assure the quality, safety and efficacy of poliomyelitis vaccines (oral, live,
attenuated). In: WHO Expert Committee on Biological Standardization: sixty-third report. Geneva:
World Health Organization; 2014: Annex 2 (WHO Technical Report Series, No. 980; https://www.
who.int/publications/i/item/9789241209802, accessed 21 June 2021).
3. Revision of the WHO Recommendations to assure the quality, safety and efficacy of live attenuated
yellow fever vaccines. In: WHO Expert Committee on Biological Standardization: seventy-first
report. Geneva: World Health Organization; 2021 (WHO Technical Report Series, No. 1028, section
3.2.3, pp. 20–1; https://www.who.int/publications/i/item/9789240020146, accessed 21 June 2021).
4. Amendment to the WHO Recommendations to assure the quality, safety and efficacy of live
attenuated yellow fever vaccines. In: WHO Expert Committee on Biological Standardization:
report of the seventy-second and seventy-third meetings. Geneva: World Health Organization;
2021 (WHO Technical Report Series, No. 1030, section 3.3.4, pp. 36–7; https://www.who.int/
publications/i/item/9789240024373, accessed 21 June 2021).
83
5. Fox JP, Penna HA. Behavior of 17D yellow fever virus in rhesus monkeys: relation to substrain, dose,
and neural or extraneural inoculation. American Journal of Hygiene. 1943;38(2):152–72 (abstract:
https://www.cabdirect.org/cabdirect/abstract/19442900204, accessed 9 June 2021).
6. Levenbook IS, Pelleu LJ, Elisberg BL. The monkey safety test for neurovirulence of yellow fever
vaccines: the utility of quantitative clinical evaluation and histological examination. J Biol
Stand. 1987;15(4):305–13 (abstract: https://www.sciencedirect.com/science/article/abs/pii/
S0092115787800033, accessed 9 June 2021).
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Annex 3
Evaluation of the quality, safety and efficacy of messenger
RNA vaccines for the prevention of infectious diseases:
regulatory considerations
1. Introduction 88
2. Purpose and scope 89
3. Terminology 91
4. General considerations 95
5. Special considerations 98
6. Manufacture and control of mRNA vaccines 101
6.1 General manufacturing overview 104
6.2 General information and description of vaccine construct and composition 109
6.3 Control of starting and raw materials and excipients 111
6.4 Process development and in-process controls 114
6.5 Product characterization 114
6.6 Consistency of manufacture 116
6.7 Manufacture and control of bulk purified mRNA (drug substance) 116
6.8 Manufacture and control of final formulated vaccine (drug product) 121
6.9 Records 130
6.10 Retained samples 130
6.11 Labelling 130
6.12 Distribution and transport 131
7. Nonclinical evaluation of mRNA vaccines 131
7.1 Pharmacology/immunology/proof-of-concept 132
7.2 Safety/toxicity in animal models 132
7.3 Accelerating nonclinical evaluation in the context of rapid vaccine development
against a priority pathogen during a public health emergency 135
8. Clinical evaluation of mRNA vaccines 137
8.1 Safety and immunogenicity evaluation 137
8.2 Efficacy evaluation 140
8.3 Efficacy evaluation in the context of a public health emergency in which
immune-escape and other variants arise 141
Authors and acknowledgements 141
References 145
85
Guidance documents published by the World Health Organization

(WHO) are intended to be scientific and advisory in nature. Each
of the following sections constitutes regulatory considerations for
national regulatory authorities (NRAs) and for manufacturers of
biological products.
86
Annex 3
Abbreviations
AESI adverse events of special interest
COVID-19 coronavirus disease 2019
DNA deoxyribonucleic acid
DNase deoxyribonuclease
dsRNA double-stranded RNA
GMP good manufacturing practice(s)
HPLC high-performance liquid chromatography
ICH International Council for Harmonisation of Technical
Requirements for Pharmaceuticals for Human Use
IU International Unit(s)
IVT in vitro transcription
LNP lipid nanoparticle
mRNA messenger RNA
NRA national regulatory authority
ORF open reading frame
PCR polymerase chain reaction
PEG polyethylene glycol
PEGylation polyethylene-glycol-ylation
RNA ribonucleic acid
RT-PCR reverse transcription polymerase chain reaction
sa-mRNA self-amplifying mRNA
SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
tRNA transfer RNA
UTR untranslated region
WHO World Health Organization
87
1. Introduction
Although the immunostimulatory effects of RNA have been known since the
early 1960s (1), the possibility of using direct in vivo administration of in vitro
transcribed messenger RNA (mRNA) to temporarily introduce genes expressing
proteins (including antigens) was demonstrated in 1990 following the direct
injection of “naked” nucleic acids (2). Subsequent improvements in stabilizing
mRNA, increasing the feasibility of manufacturing RNA-based products and
decreasing RNA-associated inflammatory responses have led to significant
advances in the development of mRNA vaccines and therapeutics (3–6). There
are several reasons why the mRNA platform has emerged at the forefront of
vaccine technology. Among these are the rapid speed at which mRNA candidate
vaccines can be constructed and manufactured, and the need to rapidly develop
vaccines against emerging pathogens, such as zoonotic influenza virus strains,
Zika virus and most recently severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19).
A number of publications have now discussed some of the safety,
production and regulatory issues associated with this new technology (7–14). In
addition, the rapidity with which clinical trials have progressed for COVID-19
candidate vaccines, their approval or authorization by NRAs and subsequent
widespread use have created a pressing need for WHO guidance on evaluating
the quality, safety and efficacy of mRNA products used for the prevention
of infectious diseases in humans. Such evaluations must take into account:
(a) the inherent immunological, physiochemical and structural properties
of mRNA; (b) the need for special formulations such as lipid nanoparticles
(LNPs) to ensure in vivo stability and efficient delivery; and (c) the novel cell-
free enzymatic manufacturing process. Because detailed information is not yet
available on the methods used for production, controls are not yet standardized
for safe and efficacious mRNA vaccines, and certain details remain proprietary
and thus not publicly available, it is not feasible to develop specific international
guidelines or recommendations at this time. Consequently, flexibility in the
scientific approach to regulating mRNA vaccines is currently needed. The
detailed production and control procedures, as well as any significant changes
in them that may affect the quality, safety and efficacy of mRNA vaccines, should
be discussed with and approved by the NRA on an individual case-by-case basis.
Nevertheless, the key principles described in this document are applicable to the
class of preventive mRNA vaccines against infectious diseases for human use in
general and are intended to provide guidance until more detailed information
becomes available. For mRNA vaccines that target diseases for which there are
existing vaccines and corresponding WHO guidance, it may be appropriate to
consider the relevant sections of this document for issues specific to mRNA
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Annex 3
vaccines in conjunction with the corresponding Part B (nonclinical evaluation)

and Part C (clinical evaluation) of the respective WHO Recommendations and
Guidelines for guidance on issues specific to the evaluation of vaccines against
that disease (15).
Any given manufacturer’s mRNA vaccines might potentially be viewed
as a platform technology in which the coding region can readily be changed
without necessarily having to change the manufacture or control of the resulting
new product (except for antigen-specific tests for identity, potency and stability).
However, this will depend on the resulting characteristics of the final vaccine.
If significant changes are made to the final vaccine, resulting in changes to the
critical quality attributes as well as subsequent cellular interaction, then further
consideration of the manufacturing process, controls and testing of the product
will be required.
The WHO Expert Committee on Biological Standardization discussed
these and related issues at its meetings in August and December 2020, and
expressed its support for the development of a WHO guidance document on
regulatory considerations in the evaluation of mRNA vaccines, which could be
updated as more scientific and clinical data on this novel product type became
available (16, 17).
2. Purpose and scope

This document provides information and regulatory considerations regarding
key aspects of the manufacture and quality control, and nonclinical and clinical
evaluation, of preventive mRNA vaccines against infectious disease for human
use. Sufficient information should be provided as is phase-appropriate, and
is expected to increase as product development advances. Although the most
advanced vaccines of this type are COVID-19 vaccines and are used as examples
in the text, the document should not be taken as providing guidance specific only
to COVID-19 vaccines. However, in light of the current COVID-19 pandemic
and corresponding speed of mRNA vaccine development, the document is
intended to provide special considerations for this type of preventive mRNA
vaccine as rapidly as possible. It should nevertheless be noted that there remain
gaps in the scientific understanding of the types and amount of immunogenicity
that any given mRNA vaccine might need to achieve for it to be successful,
broadly relevant and durably efficacious against the disease it is intended to
prevent. Each vaccine will therefore need to be evaluated in terms of its own
benefits and risks.
Because mRNA vaccines are novel and differ from other types of
vaccines (even other nucleic acid vaccines such as plasmid DNA vaccines), a
short introduction to mRNA-vaccine-specific topics is provided where deemed
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useful. Due to the novelty of mRNA vaccines and their manufacturing process,
a comprehensive approach has been taken to ensure that all relevant aspects can
be considered by manufacturers when developing this type of product, and by
regulators when evaluating such products.
The scope of the current document is limited to mRNA and self-
amplifying mRNA (sa-mRNA) packaged in LNPs for in vivo delivery of the
coding sequences of a target antigen relevant to active immunization for the
prevention of an infectious disease. It is acknowledged that mRNA and sa-mRNA
products in formulations other than LNPs are also in development, and parts of
this document may be applicable to those products as well.
Replicating agents, viral vectors and RNA replicons packaged in viral
proteins or encoded by plasmid DNA are outside the scope of this document.
In addition, mRNA and sa-mRNA products intended for therapeutic purposes
(that is, products for the treatment, mitigation or cure of diseases, including
infectious diseases, as opposed to active immunization for their prevention) are
also outside the scope of this document. In addition, mRNA products expressing
monoclonal antibodies (whether serving as passive immunization for disease
prevention or therapy) are also outside the scope of this document. It may be
the case that some aspects discussed in section 6 and its subsections do apply
to mRNA-based therapeutic products (including those expressing monoclonal
antibodies), as the manufacturing steps of such products may be similar to those
described for vaccines. However, because the nonclinical and clinical evaluations
of such therapeutic products would need to be based on their therapeutic
indication, it is not feasible to include regulatory considerations for them within
this document.
As there may be a need to develop multivalent mRNA vaccines or to
change the existing vaccine strain for some pathogens (for example, influenza
viruses or SARS-CoV-2), specific considerations are provided in this document
where appropriate. In addition, any general WHO guidance of relevance should

also be consulted; a number of WHO documents providing such guidance are
listed below in section 4.
Because regulatory pathways for emergency use authorization vary and
not all NRAs have such pathways, approval for emergency use is also outside of
the scope of the document. However, suggestions are provided, where possible,
for rapid vaccine development in the case of priority pathogens during public
health emergencies (see sections 7.3 and 8.3 below).
This document has been developed in light of the available knowledge to
date. Given that this is a dynamic field, both in terms of vaccine manufacturing
technologies and clinical-trial design, this document should be read in conjunction
with other relevant recent guidance, including WHO disease-specific guidelines
and recommendations, if available.
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3. Terminology
The definitions given below apply to the terms as used in this document. These
terms may have different meaning in other contexts.
Adjuvant: a substance intended to enhance the relevant immune response
and subsequent clinical efficacy of a vaccine.
Biological (or biological product): a medicine produced by a biological
system, as opposed to strictly chemical reactions. These include traditional
biologicals (such as live vaccines) and biotechnologically produced medicines
(such as monoclonal antibodies or subunit vaccines such as human papillomavirus
vaccines). In other documents, these may be referred to as biologics or biological
medicines.
Candidate vaccine: an investigational vaccine that is in the research and
clinical development stages and has not been granted marketing authorization or
licensure by a regulatory agency in the country in which such authorization or
licensure will be sought.
Design of experiments: a structured, organized method for determining
the relationship between factors affecting a process and the output of that process.
Drug product: see final vaccine.
Drug substance: the purified mRNA before final formulation. It is
prepared as a single homogeneous production batch, kept in one or more
containers designated as such and used in the preparation of the final dosage
form (final vaccine or drug product).
Double-stranded RNA (dsRNA): some viruses have genomes
comprising fully double-stranded RNA along their entire length rather than in
distinct segments (such as the secondary structure of mRNA). If present, such
dsRNA is sensed by intracellular receptors and can activate innate immune
responses. Depending on the manufacturing method, dsRNA can be generated
as a by-product during the in vitro transcription (IVT) manufacturing process
for some mRNA vaccines, though some segments may be single stranded. This
type of dsRNA is an impurity that should be removed from the mRNA during
the manufacturing process, or its amount in the product at least determined and
controlled. If the manufacturing method does not produce dsRNA, then the
control of this as an impurity is unnecessary.
Engineering run: a manufacturing campaign conducted to engineer
manufacturing methods in order to improve or confirm those methods for use in
good manufacturing practice (GMP)-compliant production. The materials made
in such a campaign are not intended for use in humans.
Excipient: a constituent of a medicine other than the active drug
substance, added in the formulation for a specific purpose. While most excipients
are considered inactive, some can have a known action or effect in certain
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circumstances. The excipients must be declared in the labelling and package leaflet
of the medicine to ensure its safe use. In the context of the current document, the
lipids that form the LNPs are excipients but the LNPs if formed separately from
the mRNA are defined as intermediates in the production of the drug product.
Final formulated bulk: an intermediate in the manufacturing process
of the final vaccine, consisting of a homogeneous preparation of the final
formulation of drug substance(s) and excipients at the concentration to be filled
into final containers. Alternatively, the final formulated bulk may be stored at a
higher concentration and diluted immediately prior to filling. In the context of this
document, the term refers to mRNA formulated with LNPs and other excipients
as needed. Note that if more than one drug substance is to be combined (as in
a multivalent or combination vaccine), their mixing would occur as part of the
preparation of this final formulated bulk.
Final lot: a collection of sealed final containers that is homogeneous with
respect to the composition of the product and the avoidance of contamination
during filling. A final lot must therefore have been filled from a final formulated
bulk in one continuous working session. A final formulated bulk might be filled
into more than one final lot.
Final vaccine (or drug product): a final dosage form (for example,
a vialled frozen or liquid suspension or lyophilized cake) that contains one or
more drug substances (active ingredient) typically formulated with excipients
and packaged for use. In the context of this document, the term refers to a
preparation of mRNA formulated with LNPs and other excipients that is filled
into final containers. If filled in concentrated form or lyophilized, a diluent is
needed. Otherwise, the final containers should be filled at the concentration for
the clinical dose (though each container might contain multiple doses). Also
referred to as “finished product” in other documents.
Good manufacturing practice (GMP): a system that ensures that
products are consistently produced and controlled to the quality standards

appropriate to their intended use and as required by the marketing authorization.
Immunogenicity: the capacity of a vaccine to elicit a measurable adaptive
immune response against a target antigen(s).
In vitro transcribed mRNA: the product of a manufacturing process
whereby mRNA is generated in vitro from a linear DNA template using a DNA-
dependent RNA polymerase enzyme (for example, a T7, T3 or Sp6 phage RNA
polymerase) and nucleoside triphosphates or modified nucleoside triphosphates.
Lipid nanoparticle (LNP): a delivery formulation consisting of various
lipid components to ensure that the mRNA is stabilized and encapsulated, for
example, to avoid extracellular degradation and to facilitate its uptake into cells
and release into the cytosol. The lipid components may include, but are not
limited to, an ionizable/cationic lipid, a helper lipid (for example, phospholipids
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and/or cholesterol) and a lipid(s) modified for example by polyethylene-glycol-

ylation (PEGylation). The LNPs and/or lipid components may also have adjuvant
activity.
Marketing authorization or approval: a formal authorization for
a medicine (including vaccines) to be marketed. Once an NRA approves a
marketing authorization application for a new medicine (different NRAs may
use different terms for such applications), the medicine may be marketed and
may be available for physicians to prescribe and/or for public health use (also
referred to as product (drug or biological) licensing, product authorization or
product registration). Once authorized or approved, the new medicine must
be manufactured, controlled and labelled as described in the authorization or
approval file.
Messenger RNA (mRNA): a single-stranded RNA molecule that is
translated in the cytoplasm of a cell into the protein that it encodes. It contains
one or more open reading frames (ORFs) that encode the protein (in the case of
vaccines, the target antigen), flanking untranslated regions (UTRs), a 5′ cap (or
alternative) and a 3′ sequence such as a poly(A) tail.
Mode-of-action and mechanism-of-action: the manner in which the
adaptive immune response elicited by the vaccine protects against the pathogen at
the cellular (mode) or molecular (mechanism) level – for example, neutralization
by neutralizing antibodies, opsonization by opsonizing antibodies or cytotoxicity
by T cells.
Modified nucleosides: naturally occurring modified nucleosides (such
as pseudouridine) that can be substituted for the usual nucleoside (in this case,
uridine) when making mRNA vaccines, with a resultant potential decrease
in inflammatory activity and/or increased in vivo stability. Another type of
modification is methylation. Nucleosides used to manufacture mRNA vaccines
might also contain unnatural modifications.
mRNA integrity: the proportion of the mRNA that is the correct size
and contains the 5′ cap and poly(A) tail. In addition, the correct sequence of the
mRNA should be confirmed.
Novel excipient: an excipient (for example, a lipid) not used before in
any medicine approved or licensed for human use or, if previously used in an
approved or licensed medicine for human use, then not using the same route of
administration (and/or present at a higher concentration) as that approved or
licensed. The word “novel” is used in this same way to describe other terms used
in this document.
Platform technology: a group of technologies used as a base upon which
other applications, processes or technologies are developed. In the context of
mRNA vaccines, a given manufacturer might have one or more platforms on
which they will develop vaccines (or therapeutics) against various diseases
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(separate individual vaccines or a combination vaccine) or pathogen strains

against the same disease (separate monovalent or mixed multivalent vaccines).
The term could also be applied to a particular drug-delivery system (such as
LNPs containing the mRNA) where identical lipids, concentrations, methods
of preparation and purification and so on are used. Use of the term “platform
technology” would be considered appropriate when: (a) the manufacturing
methods are essentially unchanged (but may be optimized for each specific
candidate vaccine); (b) the test methods (except for identity, potency and stability)
and acceptance criteria are unchanged; (c) the immunomodulatory compounds
or elements are unchanged; and (d) compliance with GMP is unchanged. One
implication of the use of platform technology to develop new candidate vaccines
is that the experience and knowledge gained, data generated (manufacturing,
control, stability and nonclinical) and validation of unchanged methods can all
be used as supportive data for the more rapid assessment and development of a
new candidate vaccine. Clinical and nonclinical data from the platform in terms
of safe starting doses or tolerable doses might also be supportive of initiating
clinical trials of the new candidate vaccine at doses already known to be tolerable
with the platform. If aspects of the platform technology have been changed, along
with the mRNA sequence, then justification should be provided as to why data
generated with the original platform should be considered supportive of the
new candidate vaccine. Because the production and control methods used for
mRNA vaccines are not yet standardized between manufacturers, information
from other manufacturers would not be supportive of a platform technology.
Such information may be considered to be similar to that for a product class
and evaluated as being supportive if justification is provided and compelling.
Furthermore, flexibility in the scientific approach to regulating mRNA vaccines is
justified because of the current lack of standardization even in the face of platform
technology use. As always, an individual case-by-case approach is justified and
should be discussed and agreed with the relevant NRA(s).

Self-amplifying mRNA (sa-mRNA): an mRNA vaccine that in addition
to encoding the desired antigen(s) also encodes nonstructural proteins of certain
viruses (such as alphaviruses), either on the same molecule as the antigen or on
a separate molecule. When expressed intracellularly, these ORFs produce the
proteins of the viral replication machinery thus enabling the cell to produce
multiple copies of the mRNA encoding the antigen protein. The goal of sa-mRNA
is to increase the in vivo potency of the mRNA vaccine by increasing the amount
of protein antigen made. Other designations have been given to this form of
mRNA vaccine but in this document the term sa-mRNA will be used.
Target antigen(s): the protein(s) or portion thereof, encoded within
the mRNA of an mRNA vaccine, for which an immune response to vaccination
is expected to result in protection against one or more pathogens or strains.
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That is, protection against disease (or infection) caused by a pathogen(s) may
be conferred by the resulting immune response against the target antigen(s)
following vaccination.
Therapeutic: a treatment given after a disease or condition (or signs or
symptoms thereof) is evidenced, in contrast to the prevention of disease before
exposure (or in rare cases following exposure but before the onset of signs or
symptoms) to the infectious pathogenic organism has occurred. Although
preventive vaccines are not considered to be therapeutic in this document, it is
acknowledged that the definition of therapeutic in some regulatory jurisdictions
may differ. Therapeutics as defined here are outside the scope of the current
document.
Transfer RNA (tRNA): an RNA molecule used by ribosomes and that
acts as an adaptor involved in translating the codons of the mRNA into a protein.
4. General considerations
As with all vaccines, the intended clinical use of the mRNA vaccine should
be described, including the pathogen targeted, the target antigen(s) chosen,
disease to be prevented and the target population(s). Given the novel structure
and manufacturing of mRNA candidate vaccines (in contrast to other already
licensed vaccine types with which regulators are familiar), consideration should
be given to the following when evaluating mRNA vaccines for their quality, safety
and efficacy:
■ In particular, the relevant biological characteristics of the specific
mRNA technology used should be described – including for
example the capability of the given mRNA to trigger innate immune
responses as well as target-antigen-specific responses; the quality,
quantity and bias of the immune responses (for example, type 1
T-helper (Th1) or Th2 cell phenotype); and in vivo stability. To
justify the vaccine design, all available information on the type of
immunity (protective and immunopathogenic) considered relevant
to the specific pathogen and disease should also be described.
■ The rationale for the selection of the target antigen(s) or parts
thereof and of any proteins (for example, cytokines) that are
encoded, as well as their contribution to the proposed mode- or
mechanism-of-action (proposed protective process) of the vaccine,
should be clearly described. Likewise, the rationale for the selection
of any coding sequences added to or any modification of the target
antigen, such as those to ensure the folding of the target antigen
into a particular conformation, should be provided. The complete
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annotated sequence identifying all ORFs (including any unexpected

ORFs) and all other sequence elements (including their justification
for use) should be provided. Justifications for the use of any specific
or specially designed noncoding sequence (including poly(A) tail)
and of structural elements (such as the chosen 5′ cap structure or
alternative) should be provided. With regard to sa-mRNA, any
viral replicon genes encoded in the vaccine construct to allow
amplification of the mRNA in human cells after delivery should be
described in detail. The anticipated function and purpose of each
gene sequence encoded in the mRNA should be indicated, as well as
those of specific noncoding and structural elements, explaining their
contribution to the overall mode- or mechanism-of-action.
■ The formulation of the final vaccine product and all excipients
(including all components used for the generation of LNPs)
should be described. An appropriate rationale for the proposed
composition of the final vaccine and inclusion of excipients should
be provided. Information on the method of production of the LNPs
and the final vaccine (drug product) including information on the
critical quality attributes of the intermediates and final product,
their in-process controls and any sterilization procedure should also
be provided. Toxicological and immunogenicity data on the LNP
should also be provided.
■ For each novel excipient (see Terminology for definition) detailed
information on the rationale for its inclusion, the method of
production (including details and controls on the starting materials,
intermediates and raw materials) and data from nonclinical and/
or human clinical studies on its safety and, if required by a given
NRA, on its safety pharmacology (see section 7.2.d below) should be
provided.
■ The intended dosing, the route of administration, and a description
and justification of any novel administration device as well as any
required diluent should be provided. Relevant compatibility studies
should be performed where necessary.
■ Although any given manufacturer’s mRNA vaccine product may
be considered to be produced by a platform technology if only the
target antigen sequence is changed, the control, nonclinical testing
and clinical development of each vaccine should be considered
individually, and any special features of that candidate vaccine taken
into account. Early consultation with the NRA(s) will be key to
ensuring the efficient development of any given candidate vaccine.
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■ With regard to the development of combination or multivalent

candidate vaccines, noting the development of precedents might
be helpful. Relevant examples might include: (a) seasonal influenza
virus vaccines, which are both multivalent and undergo annual
strain changes; (b) human papillomavirus vaccines such as the
quadrivalent vaccine that was changed after initial approval to a
nonavalent (that is, nine-valent) vaccine, trivalent poliomyelitis
vaccines, multivalent rotavirus vaccines and multivalent
pneumococcal vaccines, which are used against different strains that
cause the same (or related) disease(s); or (c) diphtheria and tetanus-
toxoid-containing vaccines or measles, mumps and rubella vaccines,
which are combination vaccines used against different disease
targets. Available guidance on the development of combination
vaccines against multiple diseases may also be considered.
The current document should be read in conjunction with other relevant
WHO guidelines such as:
■ WHO guidelines on nonclinical evaluation of vaccines (18);
■ Guidelines on the nonclinical evaluation of vaccine adjuvants and
adjuvanted vaccines (19);
■ Guidelines on clinical evaluation of vaccines: regulatory
expectations (20);
■ WHO good manufacturing practices for pharmaceutical products:
main principles (21);
■ Good manufacturing practices: supplementary guidelines for the
manufacture of pharmaceutical excipients (22);
■ WHO good manufacturing practices for biological products (23);
■ WHO good manufacturing practices for sterile pharmaceutical
products (24);
■ Guidelines for good clinical practice (GCP) for trials on
pharmaceutical products (25);
■ WHO guidelines on transmissible spongiform encephalopathies in
relation to biological and pharmaceutical products (26);
■ Guidelines on stability evaluation of vaccines (27);
■ Model guidance for the storage and transport of time- and
temperature-sensitive pharmaceutical products (28);
■ Guidelines on the stability evaluation of vaccines for use under
extended controlled temperature conditions (29);
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■ Guidelines for independent lot release of vaccines by regulatory

authorities (30);
■ Guidelines on procedures and data requirements for changes to
approved vaccines (31); and
■ WHO policy statement: multi-dose vial policy (MDVP). Handling
of multi-dose vaccine vials after opening (32).
5. Special considerations
The mRNA of vaccines that are currently the most advanced in terms of clinical
development or that are currently in widespread use against COVID-19 is
produced enzymatically rather than biologically within a cell. This approach thus
differs from the production of most other biologicals with which manufacturers
and regulators are familiar (1, 33). Manufacturing either starts with linearized
DNA plasmids that have been produced in bacteria (similar to the way in which
biologicals such as plasmid DNA vaccines are produced) or with a linear DNA
molecule produced enzymatically using the polymerase chain reaction (PCR)
or other synthetic methods. Regardless of whether the manufacture of the RNA
starts with a linearized molecule generated from a plasmid DNA or from an
already linear DNA sequence, mRNA production occurs enzymatically in vitro
by means of a DNA-dependent RNA polymerase that transcribes the linear DNA
template into an mRNA molecule. The mRNA sequence generally consists of the
usual elements of cellular mRNA, such as the coding region, 5′ and 3′ untranslated
regions (UTRs) that regulate mRNA translation, a 5′ cap and a 3′ poly(A) tail.
The nucleotides used in manufacture may contain naturally occurring
nucleosides or modified or synthetic nucleosides (3, 8). Examples of alterations
that might be made to the naturally occurring nucleoside include the use of
pseudouridine or N1-methylpseudouridine in place of uridine (3, 4, 34). In
addition, altering or optimizing codon use (without changing the encoded

amino acids) may impact in vivo stability and enhance in vivo translation of the
mRNA in humans (for example, for translation by transfer RNAs (tRNAs) more
frequently found in human cells). Alternatively, codons may be selected for more
infrequent tRNAs in order to slow translation of the protein, thus permitting
desired protein folding. Some changes to the mRNA are designed both to increase
its in vivo stability and to moderate activation of the innate immune system (4).
Depending on the clinical indication, it may be desirable to decrease innate
immune responses that might lead to inflammatory reactogenicity in vivo (3, 4,
34). For some preventive vaccines, some of the innate immune response may be
considered useful for augmenting the desired adaptive immune response, and
the mRNA may be designed accordingly. The gene sequence encoding the target
antigen should contain start and stop codons and be flanked by 5′ and 3′ UTRs
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and generally have a 5′ cap and a 3′ poly(A) tail. The cap can be added to the
mRNA enzymatically or during in vitro transcription (IVT) using appropriate
cap analogues. Likewise, the 3′ poly(A) tail can be encoded in the DNA template
or added enzymatically after IVT. These design features can impact the critical
quality attributes, the manufacturing methods and the control testing of the
mRNA drug substance(s) and/or the final vaccine drug product.
Of relevance to considerations of the safety and efficacy of mRNA vaccines
are the structures adopted by the RNA in the vaccine product. Unlike DNA, which
is normally double stranded, RNA is often represented diagrammatically as being
single stranded. However, depending on its sequence, RNA can form a complex
structure consisting of short double-stranded segments with various single-
stranded loops in between. The reason this is relevant is that double-stranded
RNA (dsRNA) is a form taken by the genome of some RNA viruses and can
induce cells to trigger immune reactivity as an innate response to viral infection.
However, endogenous cellular mRNA does not induce such an effect despite
containing partial double-stranded segments. The in vivo effects, including
potential triggering of innate immunity, of an mRNA candidate vaccine should
therefore be characterized and addressed in the vaccine design, nonclinical
studies and clinical trials.
RNA-based products can take different forms. The most advanced
candidate vaccines and the widely used COVID-19 vaccines take the form
of mRNA encoding the target antigen (35, 36). Because mRNA (and RNA in
general) is subject to degradation by nucleases, the most advanced mRNA
candidate vaccines and widely used COVID-19 vaccines at the time of writing
are formulated in LNPs, which aids in vivo stability and delivery (33, 37–43).
There are different types of LNPs depending on their composition, the types of
lipids employed and the manufacturing process used (44). Some may not yet have
been employed for the delivery of mRNA (45–48). Other stabilizing and delivery
systems using polymer and polypeptide, as well as other lipid-based systems or
combination of polymer and lipid-based systems, may be developed for mRNA
delivery in the future. These drug delivery systems could also be surface modified
for tailored cellular interactions, where necessary.
It is important to note that the drug substance is the mRNA(s). The
lipids which form the LNPs are excipients of the final vaccine or drug product.
The manufacture of LNPs from the different lipids is part of the drug product
manufacturing process. It is acknowledged that some LNPs, depending on their
composition, may also have immunomodulatory effects (47–50) and some lipids
may act as adjuvants without being formulated as LNPs. Nonetheless, vaccine
adjuvants, which are immunomodulating to the vaccine, are also considered to be
excipients. Similarly, as discussed above, RNA itself can be immunomodulating.
Consequently, both components (the mRNA and the lipids in the LNPs) may
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contribute to the mode-of-action of the vaccine and the implications of this

need to be considered in the critical quality attributes and in the nonclinical and
clinical evaluations.
Some candidate vaccines may contain various mRNAs encoding different
antigens. Examples include multiple antigens from the same pathogen, the same
antigen representing different strains or serotypes of the same pathogen, or
multiple antigens from different pathogens. Such vaccine development is not
without precedent – as discussed above in section 4. As with other combination
or multivalent vaccines, each mRNA would be considered to be a separate drug
substance that will be combined into a final drug product. Also, as with other
combination or multivalent vaccines, the amount of mRNA for each target
antigen, the expression efficiency of each and the resulting immune responses,
must be balanced against the other(s) in order to avoid interference with the
expression of (and thus immune responses to) all the target antigens, and to
ensure the necessary strain- or antigen-specific immune responses to the total
vaccine. Furthermore, consideration should be given to ensuring an adequate
dose of mRNA for each target antigen using appropriate nonclinical toxicity
studies to evaluate the highest tolerable total mRNA and LNP doses, and if
applicable, justified by previous clinical experience. See sections 7.3 and 8.1
below for further discussion of this point.
Additional consideration should also be given to the manufacture, control
and stability of combination or multivalent vaccines to ensure appropriate quality
control of each drug substance and the drug product and to ensure the suitability
of the analytical procedures used to control each mRNA component (that is, each
drug substance) in the final vaccine.
Interactions between the mRNA and the LNPs may vary depending on
the length and secondary structure of the mRNA, as well as the lipid composition
of the LNPs. Therefore, the particle size, morphology, surface properties (for
example, charge) and encapsulation efficiency of the resultant LNPs containing

the mRNA could vary when a different mRNA is used. Consideration of the
critical quality attributes and physicochemical properties of both the mRNA
and the LNPs is therefore necessary to provide an understanding of the desirable
properties of the final vaccine.
Some candidate products contain the components needed to permit
the mRNA vaccine to be self-amplifying (so-called self-amplifying mRNA or
sa-mRNA) (8, 38, 51). These products include the gene sequences for replicon
proteins (to date, from alphaviruses) in addition to the target antigen, either on
the same or a different mRNA molecule. As a result, the mRNA coding for the
antigen can be replicated in vivo, leading to increased expression of the target
antigen. Current sa-mRNAs are also formulated in LNPs (38). There may be
implications for vaccine safety and efficacy due to the design of the sa-mRNA
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if the target antigen is encoded either on a separate mRNA molecule or on the

same molecule as the replicon gene sequences. For example, the particle size and
morphological characteristics of the LNPs may vary depending on the size of the
mRNA encapsulated. In addition, the total amount of mRNA needed to achieve
the same level of efficacy may vary between the two designs due to differences in
the degree of expression efficiency, as well as in the amount of dsRNA, the innate
immune response, the half-life of the mRNA and sa-MRNA, and so on – all of
which could result in a different safety profile, and hence all of which needs to be
considered in the vaccine design and evaluation.
In contrast to viral replicons (which are packaged in viral structural
proteins) sa-mRNAs are delivered in LNPs or other delivery systems. This means
that the cells that take up sa-mRNAs and those that take up viral replicons are
likely to differ as viral replicons enter their host cells via the viral receptor, while
sa-mRNAs depend on a formulation for intracellular delivery (38). RNA-based
products can also be contrasted with both viral-vectored vaccines and with live
viral vaccines (for RNA viruses) by their lack of genes encoding the structural
proteins of the virus being used as the vector or live vaccine. Thus, there are
various similar products in development, the differences between which are
largely dictated by biology or design. Other similar technologies include circular
RNA products that are in development, mRNAs that use an internal ribosome
entry site (IRES) instead of a cap and RNA encapsulated in other drug-delivery
systems using polymer and polypeptide systems (or a combination of polymer
and lipid-based systems).
As described above in sections 1 and 2, and in order to develop WHO
guidance as rapidly as possible, the scope of the current document is limited to
mRNA or sa-mRNA encapsulated in LNPs.
It should also be noted that in the case of current mRNA vaccines, various
cell types at the site of injection take up the mRNA. However, future delivery
systems may be designed that selectively target the mRNA to specific cell types
or tissues – for example, through the use of surface-modified LNPs in which a
targeting ligand/motif could be attached to the LNP surface. Any changes to the
physicochemical properties that result in different innate immunostimulatory
effects may have further implications for the safety or efficacy of the mRNA or
sa-mRNA vaccine but these are beyond the scope of the current document.
6. Manufacture and control of mRNA vaccines

All mRNA vaccines are regulated as biologicals and, as with other biologicals,
adequate control of the starting and raw materials and excipients and of the
manufacturing processes is as important as that of the final product. Regulatory
considerations therefore place considerable emphasis on the control strategy
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for the vaccine manufacturing process as well as on the comprehensive

characterization and release testing of the drug substance and the final vaccine
itself.
The general guidance contained in WHO good manufacturing practices
for pharmaceutical products: main principles (21), Good manufacturing practices:
supplementary guidelines for the manufacture of pharmaceutical excipients (22),
WHO good manufacturing practices for biological products (23) and WHO
good manufacturing practices for sterile pharmaceutical products (24) should
be applied to the design, establishment, operation, control and maintenance of
manufacturing facilities for mRNA vaccines. The primary guidance on GMP
requirements for mRNA vaccines used to prevent infectious diseases is provided in
WHO good manufacturing practices for biological products (23). This document
advises that for recombinant or biotechnology products, GMP compliance
is expected from the starting materials through to the filled, finished product.
WHO guidance should also be applied to the control of mRNA vaccine filled in
the final form, the keeping of records and retained samples (for future studies
and needs), labelling, distribution and transport, as well as storage and expiry
dating for mRNA vaccines (27–29). Quality control during the manufacturing
process relies on the implementation of quality systems, such as GMP to ensure
the production of consistent commercial vaccine lots with product characteristics
similar to those of lots shown to be safe and efficacious in clinical trials.
Throughout the process, a number of in-process monitoring and/
or control tests (with acceptable limits) should be established through a risk-
based approach (including tests to measure critical and non-critical quality
attributes, as applicable) in order to allow quality to be monitored for each batch
or lot from the beginning to the end of production. Release specifications and
characterization methods should be agreed with the NRA(s) as part of the clinical
trial authorization/approval or marketing authorization/approval. The drug
substance and drug product release specifications for marketing authorization/

approval should be set based on the testing of product resulting from the
commercial manufacturing process as well as the results obtained for the lots used
in clinical trials. Such release specifications and characterization methods should
cover critical attributes that can provide reassurance on the consistent quality
required to provide a safe and effective vaccine. This will include methods to
assess content, identity, purity, potency, quality and safety attributes, and stability.
mRNA vaccines for use in clinical trials should also be prepared under
GMP conditions appropriate for the stage of clinical development – that is, full
compliance may not be possible in initial or early development when manufacturing
and control procedures remain in development and may not yet be validated; under
emergency conditions, and based on benefit–risk assessment, it may be acceptable
to consider using starting materials that were not prepared in complete compliance
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with GMP. Appropriate attention needs to be given to ensuring the quality and
correct identity of all materials used in production and control. Particular
attention should be given to the sourcing of components of animal (including
human) derivation. Attention should also be given to ensuring freedom from, or
control of, potential adventitious agents supported by relevant evidence and risk
assessment. Many of the general requirements for the quality control of biological
products, such as tests for endotoxin, stability and sterility, should also be applied
to mRNA vaccines. The commercial specifications should be defined on the basis
of the results of tests on lots that have been shown to have acceptable performance
in clinical studies. Additional controls specific to mRNA or sa-mRNA vaccines
formulated in LNPs should be described, including controls for raw materials and
excipients and in-process controls for manufacturing intermediates.
It should be recognized that the level of detail required by a regulatory
authority increases as product development proceeds. During the initial
phases of clinical development, the information contained in a clinical trial
application should be adequate to allow for an assessment of the risks derived
from the drug product and the manufacturing process. This would include, for
example, identification of and specifications for all materials used in the process,
assessment of risks from biologically sourced materials, certification or phase-
appropriate GMP compliance of the manufacturing facility, a brief description of
the processes and tests, results of testing of vaccine lot(s) (and if applicable, for a
clinical trial application, placebo or other comparator) to be used in the proposed
clinical trial and results of preliminary stability testing. As with all vaccines, for
pivotal clinical trials the level of detail provided on the quality (manufacturing
and controls) of an mRNA vaccine would be expected to increase substantially.
While not every mRNA vaccine can be viewed as being made based on
a platform technology, a given manufacturer’s technology might to some extent
be viewed this way. In other words, if essentially no changes are made to the
manufacturing processes (other than process optimization for each candidate
vaccine), tests (except for identity or potency) or specifications, then a new
candidate mRNA vaccine might be supported by data from an earlier candidate
mRNA vaccine or licensed product. For example, this could be the case when
the only changes made are to the sequence and these changes do not change the
size or secondary structure of the resultant new mRNA or its interaction with the
LNP. Supportive data might include data gained on the manufacturing processes,
tests, specifications, stability and nonclinical and clinical safety.
Details of any changes made to product composition (for example, change
in the mRNA sequence, enhanced valency, change in excipients or addition of
preservatives) or manufacture (for example, change in process, site or scale) during
the development of clinical lots should be provided. Depending on the way in
which the final product composition was changed (for example, addition of novel
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excipients) new nonclinical studies might be warranted (see section 7 below). For
changes to the manufacturing process (such as scale-up or change to the purification
process) the comparability of the resulting drug substance and drug product with
those produced using the previous process should be evaluated. Such comparability
studies might be based on immunogenicity data from animal models, results from
physicochemical analyses, studies of process and product-related impurities, and/
or stability data. The WHO Guidelines on procedures and data requirements for
changes to approved vaccines (31) should be consulted in this regard. All changes
made to the product post-approval should follow the requirements listed in these
same Guidelines (31); other relevant guidance may also be considered such as the
ICH Harmonised Guideline on pharmaceutical product lifecycle management (52).
Defined recombinant nucleic acids used as active drug substances
in vaccines, whether of biological or synthetic origin, could be assigned an
international nonproprietary name (INN) upon request (53, 54).
6.1 General manufacturing overview

Vaccines based on mRNA represent a new type of biological product and are
manufactured differently from traditional biologicals. Most such biologicals are
propagated or produced in a cell-based (or organism-based) system whereas
the mRNA component is manufactured enzymatically via IVT. The production
process normally involves the use of a linear DNA template to direct DNA-
dependent RNA transcription with recombinant enzymes and nucleoside
triphosphates. The choice of sequence or structure not only of the ORF but also
of the UTRs, the cap and the poly(A) tail length should be justified.
Generally, once the mRNA has been transcribed the template DNA is
digested using deoxyribonuclease (DNase) prior to purification of the mRNA. If
the cap and poly(A) elements are not added during the IVT process, or if a longer
poly(A) tail is required, these can be added enzymatically following synthesis
but prior to purification (8, 34, 55, 56). In addition to removing the DNA
template, the unattached caps, unincorporated nucleotides and the enzymes
(such as RNA polymerase) used in production, all process-related and product-
related impurities (for example, dsRNA and incorrectly sized mRNA molecules)
should also be removed to the extent feasible. Attention should also be paid to
the removal of enzymes possibly involved in DNA template generation, such
as DNA polymerase and restriction enzymes (if not controlled at the level of
the DNA template). The methods of purification and their purposes should be
described and justified. Any purification processes – such as protein digestion
with proteinase(s) as an impurity-reduction step – should be validated at the
appropriate phase of development (see section 6.4 below).
In most cases, the purified mRNA would be considered to correspond
to what is termed for other vaccines “the purified bulk antigen” – even though
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the mRNA is not the actual antigen but instead mimics the transcript encoding
the antigen. This could also be thought of as the bulk biological substance or
bulk active substance and is referred to in this document as the drug substance
in order to use terminology familiar to most manufacturers and regulators to
describe the active biological element of the vaccine.
As would be expected for any vaccine, a flowchart of production should
be provided that indicates each process step, the samples taken at that process
step and the in-process control tests for which the samples are taken. The process
flowchart should also clarify the steps in the process at which manufacturing
reaches the stages of drug substance, final formulated bulk and final filled vaccine
(drug product), and at which steps in the flowchart samples are being taken for
in-process control and release testing. The tests carried out at each of these steps
should also be indicated. The duration of storage of the concentrated purified
mRNA (drug substance) or any intermediates (such as the final formulated bulk)
that are held or stored should be supported by hold-time/stability studies. As
with any vaccine, an agreed-upon number of lots of the drug product should be
placed on a stability programme.
The mRNA (drug substance) is not suitable for clinical use unless it is
protected and delivered by a given formulation (the preparation of which is part
of drug product manufacture). The formulations chosen for the most advanced
mRNA vaccines so far are based on LNPs. Although there are other approaches to
encapsulating mRNA-based products, the current document only covers systems
that use LNPs. The formulation both stabilizes the mRNA and facilitates its entry
into cells and release into the cytosol, which could be achieved by either active
or passive uptake. The LNPs may also provide an adjuvant activity (47, 49, 50).
In order to protect the mRNA from degradation by nucleases, it is incorporated
into the LNPs to make it inaccessible to such nucleases – however, the LNPs
must also release the mRNA once inside the target cell. The LNPs must also be
of a suitable size range with desirable surface properties for optimal uptake by
target cells. Hence, product development data concerning the optimization of
both the formulation and the manufacturing process should be provided. For
example, consideration should be given to the concentrations of the different
lipids, the mRNA–lipid ratio, pH of buffers/solvents, mRNA encapsulation
efficiency, and the flow rate and mixing rate of the lipids and mRNA, as well as
the thawing temperature of the different components, as these will all have an
impact on the quality of the final vaccine (drug product). In this way, the process
of encapsulation into the LNPs can be carefully controlled and the production
methods and control measures adequately described and suitably validated.
Although sa-mRNA contains the coding sequences (viral nonstructural
genes) for additional proteins that permit its in vivo amplification (but not its
packaging, which requires viral structural genes), the method of manufacture in
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which IVT is followed by purification and formulation in LNPs is essentially the

same as that described above. For sa-mRNA with the additional coding sequence
on the same molecule as the target antigen coding sequence the control measures
required for the manufacturing processes might also be similar or the same as
those for an mRNA vaccine. However, if the replicon genes are encoded on a
separate mRNA molecule, then additional manufacturing processes and quality
controls may be needed to ensure that the required mRNAs are adequately
encapsulated in the same LNPs, and these additional processes should be
described. The molar ratio of the two encapsulated mRNAs should be provided
and justified, and the method of validation described. The degree of expression
efficiency might also vary between the two approaches (for example, using two
mRNAs as opposed to a single one) and this might have implications for the
expected safety and efficacy of the vaccine design due to differences in the amount
of dsRNA, the innate immune responses elicited, the half-life of the mRNA and
so on. If the separate mRNA molecules are encapsulated separately and not mixed
prior to encapsulation, this would also need to be described and may involve
additional manufacturing processes and quality controls to ensure adequate
final mixing and an appropriate ratio of the two (or more) mRNAs. Likewise,
for multivalent mRNAs the mixing step(s) either before or after encapsulation
need to be described and controlled appropriately. For sa-mRNA in which the
mRNA encoding the replicon and the mRNA expressing the target antigen are
encoded on different molecules, it will be important that these two RNAs are co-
encapsulated in order for them to be taken up by the same cell in vivo. Therefore,
if the two RNAs are encapsulated separately and then mixed, a justification for
this approach will be required.
The key quality control points should include:
a. Starting and raw materials and excipients – including, but not
limited to: (a) a linear DNA template, which could be enzymatically
or synthetically generated (for example, by PCR) or a plasmid DNA

that has been linearized (generally by restriction endonucleases);
(b) nucleotides; (c) enzymes (for example, DNA-dependent RNA
polymerase (which is usually the T7 RNA polymerase), capping
enzyme, 2′ O-methyltransferase, poly(A) polymerase, DNase
and proteinase K); (d) buffers; (e) solvents; (f) column resins
(if column chromatography is used in purification); and/or (g)
lipids. The linear DNA template is considered to be the starting
material for the manufacture of the drug substance. The other listed
items (along with any not listed but also used in manufacture)
would be considered to be raw materials. Excipients are those
raw materials that are present as inactive ingredients in the final
drug product/vaccine. For the manufacturing of excipients,
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compliance is expected with WHO Good manufacturing practices:

supplementary guidelines for the manufacture of pharmaceutical
excipients (22).
– In particular, any animal-derived (including human-derived)
starting or raw materials or excipients, or any starting or raw
materials or excipients that were themselves produced using
animal-derived (including human-derived) raw materials
should be subject to control by appropriate sourcing, by control
testing and by risk assessment. Materials of animal origin
(including human origin) should comply with the current
WHO guidelines on transmissible spongiform encephalopathies in
relation to biological and pharmaceutical products (26).
– Attention should be given to ensuring freedom from or control
of potential adventitious agents supported by relevant evidence
and risk assessment.
b. In-process controls of the manufacturing processes and
intermediates – including the processes used to manufacture the
bulk mRNA substance (drug substance), as well as the formulation
(the LNP manufacture and encapsulation steps), final formulated
bulk and filling of the final formulated bulk (drug product); also
including either controls for or validation of the consistency of LNP
formulation (regarding, for example, their size and polydispersity),
consistency of mRNA encapsulation, and removal of partial
mRNAs and dsRNA impurities.
c. Release of the mRNA vaccine drug substance and final filled
vaccine (drug product) following manufacture.
d. Process validation – processes should be validated to demonstrate
the consistent manufacturing of the commercial final drug product
with the desired quality profile (see section 6.4 below).
Analytical methods that might be considered for assessing some of these
key quality control points are discussed in the literature – though detailed analytical
procedures and acceptance criteria for tested attributes are not yet standardized
or in the public domain. As of the time of publication of the current document,
these are considered by manufacturers to be proprietary and confidential. The
methods shown in Table 1 may be considered as examples of possible means for
characterization or control at various key quality control points (57–59).
For clinical trial use, mRNA candidate vaccines should be manufactured
under GMP conditions appropriate for the stage of clinical development. It is
expected that clinical trial material should be released on the basis of meeting
appropriate quality control standards. Full compliance with GMP would be
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expected for clinical trial material used in pivotal trials and for commercial
manufacture (18, 19).
Any manufacturing changes made during clinical development,
particularly if made following completion of pivotal safety and efficacy trials
but prior to seeking licensure, need to be described and justified. A comparative
analysis with the clinical efficacy lots should be made. For post-approval changes,
compliance with the WHO Guidelines on procedures and data requirements for
changes to approved vaccines (31) would be expected, though other relevant
guidance might also be considered such as the ICH Harmonised Guideline on
pharmaceutical product lifecycle management (52).
Table 1
Examples of possible methods for characterization or control at various key quality
control points, by potential use(s)
Examples of possible method Potential use(s)

DNA template sequencing; mRNA Identity
sequencing
Quantitative reverse transcription PCR Identity and quantification
(qRT-PCR)
Ultraviolet spectroscopy; fluorescein- Quantification and purity
based assays
Agarose or acrylamide gel electrophoresis, RNA quantification, RNA size, RNA
including capillary electrophoresis integrity, LNP surface charge and
percentage encapsulation
Chromatographic assays such as size- Quantity of mRNA, quantity of lipids,
exclusion, anion-exchange, affinity or quality of mRNA and nanoparticle
reverse-phase integrity
Mass spectrometry Quantity and nanoparticle integrity

dsRNA blot; tests for percentage capping; Purity and other quality attributes
percentage transcripts with (and size of )
poly(A) tail
Cell-free translation or cell-based Potency and expression of correct protein
expression systems
Light-scattering techniques such as Particle size distribution (purity,
dynamic or static light-scattering consistency, safety)
analysis; nanoparticle tracking analysis;
electron microscopy; size-exclusion
chromatography
Laser Doppler electrophoresis; dynamic Particle surface characterization (including
light-scattering analysis size, polydispersity and zeta potential)
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Table 1 continued
Examples of possible method Potential use(s)
Electron microscopy; atomic force Physicochemical characterization
microscopy; X-ray diffraction; differential (including surface and morphological
scanning calorimetry analysis properties)
Tests for sterility, endotoxin content Safety attributes
pH determination; gravimetric, azeotropic Quality attributes
or titrimetric method to test residual
moisture
6.2 General information and description of

vaccine construct and composition
Information should be provided that describes the mRNA drug substance and
the formulated mRNA vaccine in terms of its design, sequence and construction,
its composition (for example, lipids and other excipients), and the quantities
of each excipient used. The rationale for and function of the chosen excipients
should also be provided in the description. Where relevant, information on the
structure and molecular weight of the lipids employed and on their role in the
vaccine formulation should be included.
6.2.1 mRNA sequence and arrangement of elements
a. The annotated sequence of the DNA template should be provided.

The sequence and position or length of all elements contained
within the mRNA, including start and stop codons, flanking
UTRs, regulatory elements (for example, promoter for the RNA
polymerase) and 5′ cap and 3′ poly(A) tail, should be provided, as
well as the ORF for the target antigen. If any additional proteins
are encoded (such as those for a self-amplifying construct or a
cytokine) their sequence should be provided (see points d and e
below). The presence and function of any additional sequences
included in the construct should be described.
b. Because vaccine mRNA can be manufactured containing
nucleosides that are naturally occurring or modified or synthetic, the
sequence information should include the specific nucleosides used.
c. Additionally, if sequences are changed from the native sequence in
order to optimize codons, these changes should be described and
justified. Codons may be altered for several reasons, including to
better match the frequency of the appropriate tRNAs in human
cells, to attain a specific secondary or tertiary mRNA structure, to
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reduce innate immune responses or to increase the in vivo stability

of the mRNA.
d. As noted above, in addition to coding for the target antigen(s),
sa-mRNA also codes for a viral RNA-dependent RNA polymerase
complex. Such a construct constitutes a replicon with the result that
multiple copies of the mRNA coding for the antigen can then be
made in vivo upon delivery to and uptake by the cells of the vaccine
recipient, thus potentially increasing the potency of the vaccine.
The sequences for any such replicon should be provided and their
functions explained. If the replicon is encoded on a separate mRNA
molecule from the target antigen, then the manufacture and control
of each component should be illustrated and narratively described.
Generally these coding sequences are present on the same molecule
but if separated, then additional controls may be required and
should be described.
e. If an mRNA vaccine includes sequences that code for any other
immunomodulator (such as a cytokine) or non-coding sequences
intended to act as an immunomodulator, then such sequences and
information on their purpose should be provided.
6.2.2 Formulations and components
a. Batch formula: the batch formula for commercial production

should be provided. The amounts of each component in a single
vaccine dose should be listed. The total volume of a batch should
be defined. If more than one mRNA molecule (drug substance)
is included in the drug product (final vaccine), this should be
described, including whether the different mRNA molecules are
encapsulated in a single LNP at the same time or encapsulated

separately in LNPs that are subsequently mixed.
b. Chemical nature and formulation: the mRNA is formulated
principally for increased in vivo stability and to aid cellular uptake.
While several potential types of delivery agents exist (such as
protamine complexes, cationic liposomes and lipid-, polymer- or
lipid/polymer-based nanoparticles) the mRNA vaccines currently
in use or in the most advanced clinical trials are encapsulated
into LNPs. Characterization of these formulations, both
chemically and in terms of the physical attributes of the structural
formulation (such as nanoparticles), is required and should
address characteristics such as the consistency and stability of the
formulation and final product. Considerations of the quality of the
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lipids and critical quality attributes of the drug product should also
be included. Sufficient characterization of the mRNA-LNP and of
its uptake into target cells should be provided. This may include an
understanding of the surface chemistry, size, polydispersity, shape,
charge and protein-binding properties of the resultant mRNA-LNP
in order to ensure that adequate protection of the mRNA and the
required stability of the vaccine are achieved. Where the LNPs are
shown to have inherent immunomodulatory effects, relevant data
on the potential benefits and drawbacks should be presented. Thus
any characteristics of the formulation that might impact the safety,
immunogenicity and efficacy of the vaccine should be described
and their effects (positive or negative) should be considered during
formulation development.
c. Additional immunomodulators or adjuvants: the mRNA might
also encode specific immunomodulatory molecules such as
cytokines. Furthermore, a separate adjuvant or immunomodulatory
(stimulatory or suppressive) compound not encoded in the mRNA
might be added to the formulation or as part of the LNP. As a
general principle regarding vaccines formulated with adjuvants, a
demonstration of the contribution of such an addition to vaccine
immunogenicity should be provided (19). Quality aspects of
the separate adjuvant, if included, should also be addressed and
described.
d. Additional peptides/proteins: if additional peptides/proteins are
included to target the mRNA to antigen-presenting cells or other
specific cell types or to increase the release of the mRNA from the
endosome, the sequence and function of these additions need to be
described and evidence provided of their function to support their
proposed mechanism-of-action.
e. Additional excipients (such as preservatives): the composition,
necessity for and (in the case of preservatives) the preservative
efficacy of such additional excipients should be described and
shown not to adversely affect the properties of the LNP.
6.3 Control of starting and raw materials and excipients

As with any vaccine, appropriate attention needs to be given to the sourcing
and quality of all materials used in production (22). The raw materials should
be procured from vendors/suppliers approved by the manufacturer through the
internally defined quality systems. Suppliers of such materials should be managed
by an appropriate qualification programme.
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6.3.1 Quality of starting and raw materials and excipients

The starting and raw materials and excipients, including those used to produce
the mRNA (such as the DNA template, nucleotides (which may contain modified
nucleosides), enzymes, buffer, solvents, any columns for purification and so on)
and the lipids in the LNP should be described. Information should be provided
on their provenance, quality, control, stability and role, including the point at
which each material is used in the manufacturing process. The materials should
be suitable for use in GMP production, and reference to internationally accepted
pharmacopoeias or details on their specifications should be provided.
The processes used for the derivation of the linear DNA template and
raw materials should also be described. Although the starting material for the
production of mRNA vaccines is the linear DNA template, that template may
be derived from upstream materials such as a DNA plasmid propagated in a
recombinant cell bank (see section 6.3.1.1 below).
With respect to the LNPs, the source and quality of the lipids used in their
manufacture (especially of novel lipids present in LNPs that have not previously
been studied nonclinically or clinically) should be sufficiently detailed to permit
meaningful assessment of their safety and quality. Suitable specifications should
also still be provided for any such excipient not considered to be novel. In the
case of novel excipients (for example, cationic lipids) details of the manufacturing
process and control of the novel lipids (including the starting materials and
intermediates) should be provided, where feasible. This will include information
on and relevant justification of the proposed starting materials and any
intermediates used in the synthesis of the novel excipients. Consideration should
be given to performing a nitrosamine risk assessment on the (cationic) lipid(s),
if relevant.
Details of the manufacturing site(s) and manufacturing process, along
with the required process controls and specifications of the starting materials,
raw materials (for example, enzymes, buffers and solvents), intermediates and
final excipients (for example, lipids and salts) should be provided. Consideration
should also be given to the use and control of solvents, and to the potential
for contamination with elemental impurities (60–62). Where the recycling
of materials/solvents is proposed, this should be justified and appropriately
controlled. The level of impurities associated with the excipients should also be
suitably controlled and justified. Any purification and isolation steps should be
detailed. To assure the quality of the proposed novel excipients, their manufacturer
should also have available relevant information on the analytical methods used
for the characterization, stability monitoring and batch analyses of the materials.
Since inclusion of a PEGylated lipid plays a critical role in providing in vivo
stability and enhancing the cellular interaction of LNPs (42), adequate controls
(for example, of molecular weight, polydispersity and mole percent) should be in
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place for the PEGylated lipid. For the manufacturing of excipients, compliance is
expected with WHO Good manufacturing practices: supplementary guidelines
for the manufacture of pharmaceutical excipients (22).
6.3.1.1 Quality of linear DNA derived from plasmid as starting material

As stated above, the linear DNA template is considered to be the starting material
for the GMP production of the mRNA vaccine. If the linear DNA is prepared
from plasmid DNA, then the procedures for establishing the cell banks and
the manufacture of the plasmid DNA should be performed in accordance with
the requirements for the production of material for use in subsequent GMP
manufacture.
A cell bank system should be established, described and tested for
microbial purity (freedom from bacterial and fungal contamination) and identity.
The genetic stability of the seed bank must be demonstrated. If the poly(A) tail is
encoded in the plasmid DNA, then that region on the DNA plasmid in particular
should be tested for the rate of recombination. A purification process also needs
to be in place to reduce impurities from the DNA plasmid (such as RNA, host-cell
DNA, protein, lipids and polysaccharides). The manufacturing process needs to
be set up in such a way as to minimize the risk of microbiological contamination.
Testing of DNA plasmids (if used to generate the linear DNA) and
the linear template should include tests for genetic identity by sequencing, for
integrity (including confirmation of the desired encoded antigen sequence and
regulatory/controlling sequences) and for percentage linear DNA, as well as
tests (using appropriate reference standards) for residual genomic DNA, RNA
and protein, sterility or permissible bioburden, and endotoxin levels. In early
development, testing might be carried out only on the DNA plasmid (if used) or
on the linear DNA.
In early clinical development it may be acceptable to use well-qualified
material on the understanding that greater control will be expected to support
pivotal trials and commercial manufacture.
6.3.2 Release of starting and raw materials and excipients

As with any vaccine, certificates of compliance (if applicable) and certificates of
analysis should be provided for all raw materials and a clear indication given of
which testing is performed by the mRNA manufacturer or whether the material is
accepted on the basis of the certificate of analysis provided by the manufacturer/
vendor/supplier of the raw material. An internal policy should be defined based
on criticality risk ranking for the in-house testing and release of raw materials
used in the manufacturing process. Starting materials should be released in
accordance with the requirements and specifications for use in subsequent GMP
manufacture.
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6.4 Process development and in-process controls

The development history of the commercial manufacturing process should be
provided. Tests and acceptance criteria for alert and action limits for critical steps
of the manufacturing process should be developed and justified to ensure, and
provide feedback on, the control of the process. In cases where a well-established
platform technology is being used, knowledge gained from the manufacture of
approved products can be considered in the justification.
Validation of the manufacturing processes should demonstrate that they
comply with their critical and key parameters and yield a product that consistently
meets the predefined quality attributes. This should include demonstration of the
reproducible and consistent clearance of process- and product-related impurities
to levels acceptable for intended use in humans.
Process validation is not generally required for a candidate vaccine used
in preliminary clinical trials, though critical steps such as aseptic processing and
sterility of the drug product should be validated or appropriately demonstrated
to be controlled during the manufacture of clinical materials.
6.5 Product characterization

A summary of the characterization of the mRNA (drug substance) and the final
vaccine (drug product) should be provided in addition to in-process and lot-
release testing. Rigorous characterization using a range of orthogonal chemical,
molecular, physical and biological methods will be essential. Characterization
refers to studies and analyses that are not performed routinely on every lot but
which allow the manufacturer to gain important knowledge of the structure,
performance and safety of their product in order to guide process and analytical
test development and improvement. This is in contrast to the in-process and lot-
release testing performed on every lot. Justification of the choice of analytical
methods for the determination of various attributes should be considered,

particularly when a different outcome would likely be obtained using alternative
techniques – for example, particle size measurement using different methods. It
is for this reason that orthogonal methods are recommended.
The sequences of the population of manufactured mRNA should be
determined and the degree of consistency of the proper sequence defined.
Consistency of manufacture is discussed further in section 6.6 below. The degree
of consistency of the capping and polyadenylation processes should also be
characterized and may need to be validated (see section 6.4 above). Demonstration
of expression of the complete encoded protein(s) without truncated or alternative
forms should be provided. In particular, if expression of truncated or alternative
forms of the target antigen is demonstrated during characterization studies
and these alternative forms would result in neo-antigens or unwanted immune
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responses, then this may require a redesign of the mRNA sequence. The degree of
consistency of encapsulation of the mRNA in the LNPs should also be addressed
during characterization. Particle-uptake studies could assist in characterizing
potential potency measures through identification of cell types that take up the
particles, mode or mechanism of uptake, and efficiency of uptake, and thus guide
selection of the type of cell-free or in vitro method that best allows for assessment
of these activities. During characterization, it should be determined whether any
of these characteristics should be controlled as critical quality attributes and/or
stability-indicating attributes.
Certain aspects of the LNPs should be very carefully characterized. These
include particle size as determined by different analytical techniques to explore
the morphological and dimensional characteristics of the LNPs containing the
mRNA. Information on the density and distribution of polyethylene glycol
(PEG) within the LNPs would also be useful to help understand the surface
properties of the mRNA-LNP. Measurement of surface charge (for example, zeta
potential) should also be considered as a method for characterizing the LNPs.
These, and other properties, will affect the in vivo stability, cellular interaction
and immunological response properties of the product; such information would
also help to confirm the consistency of the manufactured vaccine.
The immunogenicity elicited by the mRNA-encoded target antigen is a
critical characteristic of the product that should be characterized in nonclinical
studies as a means to understand the product. Additionally, if the LNPs have
inherent immunomodulatory effects these should also be characterized.
Whenever other immunomodulatory elements or genes are included in the
mRNA, their contribution to the mode-of-action (for example, immunogenicity)
of the mRNA-encoded target antigen should also be determined in nonclinical
studies in order to justify their inclusion in the characterized product design
(see section 7 below). Consideration of these aspects is important in gaining
understanding and knowledge of the product in order to optimize its design and
develop appropriate control methods.
Potential impurities that might be introduced by the starting materials,
and potential product- or process-related impurities in the purified mRNA, should
be described and investigated. Such impurities may include residual bacterial
host-cell proteins (if used to manufacture the DNA template), endotoxins,
residual bacterial host-cell RNA and chromosomal DNA (if bacteria were used to
manufacture the DNA template), enzymes (such as DNA and RNA polymerases
and restriction enzymes), unincorporated nucleotides, dsRNA, incomplete or
differently sized RNA, and other materials used in the manufacturing process.
Data should be provided on the impurities present in the purified mRNA in
order to justify the specifications set for their maximum acceptable or lowest
achievable levels. For impurities and residuals with known or potential toxic
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effects, a toxicological risk assessment is expected to be carried out. Degraded

mRNA may be assessed as part of analytical procedures such as polyacrylamide
or agarose gel electrophoresis, high-performance liquid chromatography (HPLC)
and/or capillary gel electrophoresis. The degree of consistency of the sequence
and structure of the mRNA, and its expression of a consistent protein when
transfected into cells in vitro, are important characteristics to be determined for
the drug product.
Any potential impurities (both process- and product-related) that may
arise from the lipids used in the formulation of the drug product should also be
characterized and investigated. This will permit justification of the specification
limits proposed so that these impurities are suitably controlled and are within the
clinically determined acceptable range.
6.6 Consistency of manufacture

As with other biologicals, prior to seeking marketing authorization, a number of
consecutive batches should be tested and analyzed using validated methods to
determine the consistency of manufacture. Any differences between one batch
and another outside the accepted range for the attributes tested should be noted
and investigated. The data obtained from such studies, combined with product
and process knowledge and evaluation of the criticality of variations in specific
attributes, should be used as the basis for justification of the chosen specifications.
During preliminary clinical development few lots will have been made
and demonstration of production consistency may be limited or not possible.
The ability to demonstrate consistency will increase as manufacturing experience
is gained during product development. Confirmation of the consistency of
lots is generally done during advanced development (for example when the
manufacturing process has been scaled up for commercial manufacture) but prior
to submission of application(s) for marketing authorization. However, in some
cases, scale-up for commercial manufacture may be undertaken while marketing

authorization is being sought for clinical trial-scale material. Whenever changes
to the manufacturing process are implemented, the comparability of lots,
especially to those used in pivotal studies and made by the intended commercial
process, should be demonstrated. Comparability protocols and strategies for
demonstrating comparability are discussed in the WHO Guidelines on procedures
and data requirements for changes to approved vaccines (31).
6.7 Manufacture and control of bulk purified

mRNA (drug substance)
As stated above in section 6.1, an overview of the development and manufacture
of the mRNA should include a justification for the selection of the target antigen
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gene, other gene(s) contained in the mRNA sequence, UTRs, 5′ cap, 3′ poly(A)
tail and regulatory elements used. Any gene expression or other optimization
modifications should be described. Annotated sequences of the complete DNA
template and mRNA should be provided. Both an illustrative and annotated
flowchart and a narrative description of the manufacture, in-process controls
and release tests should be provided. The detailed production and control
procedures along with any significant changes in them that may affect the
quality, safety and efficacy of the mRNA vaccine should be discussed with and
approved by the NRA.
In the case of sa-mRNA, if the replicon and target antigen are expressed
on separate mRNA molecules, this should be described and clearly illustrated in
the provided flowchart, which should also include any additional manufacturing
processes and/or quality control tests. For example, consideration should be
given to controls such as the ratio of replicon-encoding mRNA molecules to
target-antigen-encoding mRNA molecules, or to methods to ensure (or controls
to determine whether or not) both molecules are encapsulated into the same
LNP, if applicable. For sa-mRNA in which the mRNA encoding the replicon and
the mRNA expressing the target antigen are encoded on different molecules, it
will be important that these two RNAs are co-encapsulated in order for them to
be taken up by the same cell in vivo. Therefore, if the two RNAs are encapsulated
separately and then mixed, a justification for this approach will be required.
6.7.1 Control of bulk purified mRNA (drug substance)

Specifications for critical quality attributes for the identity (see section 6.7.1.1
below), purity (section 6.7.1.2), quantity and physical state (section 6.7.1.3), safety
(section 6.7.1.4) and quality (section 6.7.1.5) of the bulk purified mRNA should
be established and justified. Descriptions of the analytical methods used should
be provided, the acceptance limits defined and assay validation information
described. The results of testing of all batches produced at commercial scale
should be summarized and provided. Specifications should also be established
for stability under storage conditions.
Early in development, to support clinical trial authorization, results
from testing batches made in accordance with GMP (21–24) and, if available,
engineering runs performed to establish manufacturing procedures should be
summarized and provided. Although specifications may be limited and have
somewhat wide acceptance criteria in early development, these should be
reviewed and tightened, when appropriate, as experience in the manufacturing
process and analytical methods is gained. Not all of the tests conducted during
product characterization need to be carried out on each batch of vaccine as release
testing. Some tests are required only to obtain product and process knowledge on
a limited series of batches to establish the methods and consistency of production.
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Thus, a comprehensive analysis of the initial commercial-scale production batches

should be undertaken to establish consistency with regard to the identity, purity,
quality, safety and stability of the drug substance; thereafter, a limited series of
tests may be appropriate for quality control, as agreed with the NRA.
As experience is gained in manufacturing consistency, post-approval
changes might permit reducing the testing and the amount of supporting
information required through the use of process validation, product
characterization and/or a comparability protocol (31).
6.7.1.1 Identity
Each batch of bulk purified mRNA should be tested to confirm its identity.
Confirmation of identity could include determination of the mRNA sequence by
direct RNA sequencing, sequencing (or determining the presence or absence) of
a reverse transcription PCR (RT-PCR) product or high-throughput sequencing.
If identity is based on an RT-PCR amplicon that represents only a portion of the
complete mRNA sequence (including accessory and regulatory regions), then the
sequence chosen should be unique to that mRNA product and not be common
to any others that might be manufactured in the same facility or using the same
equipment. However, it might be more appropriate to sequence the entire mRNA
as this approach could serve to address both identity and potentially purity,
depending on the sequencing method used.
6.7.1.2 Purity and impurities

Each batch of bulk purified mRNA should be tested for purity and the result
should be within the allowable limits established. The control of impurities
should also address the materials introduced during manufacture, such as the
DNA template, unincorporated nucleotides, unincorporated caps, enzymes,
mRNA fragments and dsRNA. This may be achieved through process validation
to establish the removal of process-related impurities or through release tests

for the residual impurities. Consideration of the necessity of testing for dsRNA
should take into account the design of the manufacturing process as not all
processes produce dsRNA. The analyses should include sensitive and reliable
assays for process- and product-related impurities, and strict upper (maximum
allowable) limits should be specified for their content in the bulk purified mRNA.
Chromatographic detection methods may be considered. Residual DNA template
might be quantified by quantitative PCR. It is important that the techniques
used to demonstrate purity and to measure impurities are based on as wide a
range of physicochemical, biological and/or molecular properties as possible.
Consideration of the results of methods such as forced degradation studies may
guide decisions on which product-related impurities will need to be tested for
and/or measured during production, at release and/or in stability protocols.
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Tests for residual levels of process- or product-related impurities as part of

quality control may be reduced or discontinued once production processes have
been adequately validated for their suitable removal, and production consistency
has been demonstrated, if agreed by the NRA. Plans and specifications for the
periodic revalidation of processes should be described. Until the processes are
validated, impurities should continue to be tested for and/or measured in a number
of lots as agreed by the NRA. In the case of major changes to manufacturing,
revalidation or continued measurement would be expected for the number of
lots agreed with the NRA. Container-closure system compatibility, leachables
and extractables should also be assessed and discussed in the application for
marketing authorization.
6.7.1.3 Quantification and physical state

The integrity of the structure of the mRNA is considered to be a critical quality
attribute for release of the mRNA. Thus, control is needed of mRNA integrity, 5′
capping efficiency, 3′ poly(A) tail presence or length, percentage intact mRNA,
percentage mRNA fragments, percentage of dsRNA and so on. The need to
measure 3′ poly(A) tail presence or length depends upon the way in which this
sequence is added to the mRNA. If encoded in the DNA template, then all full-
length mRNA should include the poly(A) tail but if it is added enzymatically
after IVT, then it would be appropriate to address this attribute through testing
or process validation. Likewise, the presence of dsRNA depends on whether the
processes used are capable of producing it. Tests such as gel electrophoresis, PCR
or chromatographic detection methods might be considered for these purposes.
It should be borne in mind that quantification of the mRNA is the basis for
vaccine dosing and the presence of intact mRNA is key to the mechanism-of-
action of the vaccine. Thus, the methods used for quantifying the mRNA (for
example, ultraviolet spectrophotometry) and for quantifying the intact mRNA
(for example, gel electrophoresis) should be described.
6.7.1.4 Safety attributes

Relevant safety tests should be described. These may include tests for
endotoxins along with testing either for bacterial and fungal sterility (including
demonstration of lack of bactericidal or fungicidal activity of the test article)
or microbial bioburden (including quantity, identification of microbe species
and freedom from specified unwanted organisms). Such testing is generally not
required by an NRA for the drug substance, but if required a test for pyrogenicity
may be performed on the drug product (which may be the monocyte activation
test). Animal testing should be avoided whenever alternative satisfactory testing
is available and allowed. For scientific and ethical reasons, it is desirable to apply
the 3Rs concept of “Replace Reduce Refine” to minimize the use of animals
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in testing and consideration should be given to the use of appropriate in vitro

alternative methods for safety evaluation, as well as for other product tests. In
particular, manufacturers and regulators should take note of the decision of the
WHO Expert Committee on Biological Standardization in 2018 to discontinue
the inclusion of the general safety (innocuity) test in routine lot release testing
requirements for all vaccines in WHO Recommendations, Guidelines and other
guidance documents for biological products (63). This test should therefore not
be required or requested for either the drug substance or the drug product.
6.7.1.5 Additional quality attributes

Additional important quality attributes should be established and controlled
(such as appearance, pH and, if relevant, viscosity).
6.7.1.6 Reference materials

An in-house reference preparation (that is, working standard) should be
established for use in assay standardization and comparability assessment.
Information on the reference standards or reference materials used for testing
of the bulk purified mRNA should be provided by the time of application for
marketing authorization.
A suitable batch (that is, one that has been clinically evaluated) should be
fully characterized in terms of its chemical composition, purity, biological activity
and complete sequence, and an adequate sample retained for use as a chemical
and biological reference material. The reference material should be formulated in
an appropriate form. Storage should be under conditions at which the reference
material has been shown to be stable. A routine programme for monitoring the
stability of the material should be implemented. A plan for replacing the initial
reference material upon exhaustion should be agreed with the NRA.
In early development (for example, preliminary clinical trials) an
engineering run batch or a batch from which the lot of mRNA vaccine evaluated
in the pivotal nonclinical studies was made may serve as a reference until a suitable
clinical trial batch has been identified and characterized for use as a reference
in advanced development (for example, pivotal clinical trials) and commercial
manufacture. Whatever approach is taken should be clearly described.
6.7.1.7 Stability
A stability assessment should be conducted in accordance with the WHO
Guidelines on stability evaluation of vaccines (27). The types of studies conducted,
the protocols followed, and the study results should all be summarized in an
appropriate format such as tables or graphs along with a narrative document.
The summary should include results as well as conclusions with respect to
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appropriate storage conditions or shelf-life. Data on stability to support the shelf-

life of the bulk (or stored intermediates) and any future extension of it should be
based on long-term real-time stability studies under actual conditions. For the
transportation of intermediates or drug substance, it is expected that shipment
validation will be conducted at appropriate storage temperatures and conditions.
6.8 Manufacture and control of final

formulated vaccine (drug product)
As stated above in section 6.1, an overview of the development and manufacture
of the vaccine should include both an illustrative and annotated flowchart and a
narrative description of the manufacture, in-process controls and release tests.
The methods used to assure the proper formation of LNPs should be detailed.
Any proposed hold-time of the bulk formulation or bulk LNPs should be
appropriately specified and validated. Adequate consideration should be given
to ensuring physicochemical stability and microbial control during such hold-
times. The methods used for final formulation, fill and finish should also be
described and suitably validated.
6.8.1 Composition
The final composition of the vaccine, including the active drug substance
(mRNA) and all excipients (for example, lipids), should be described along with
the quantity of the components in each presentation – particularly if marketing
authorization is being sought for more than one dosage or dosage form. The
function of each of the components should also be described.
6.8.2 Manufacture and control of LNPs and encapsulation of mRNA

The methods used to assure the proper formation of LNPs should be described.
Appropriate product development data should be provided to support the
rationale for their proposed formulation and manufacturing process. All critical
quality attributes of the LNPs and final mRNA-LNPs should be investigated.
Where suitable, a Design of Experiments (DoE) approach could be adopted.
Their size and polydispersity, and in turn stability, are all influenced by both
the flow dynamics of the lipid and aqueous phase and the shear stress induced
during the manufacturing process. Thus, relevant studies that explore the critical
processing parameters and their operational ranges optimal for mRNA-LNP
formulation and stability of the final formulated vaccine should be performed.
This will ensure that the product is consistently manufactured to the required
quality. Any proposed hold-time of the bulk LNPs or bulk formulation should be
appropriately specified and validated. If stored, these are important intermediates
in the production of the final vaccine and should be controlled appropriately.
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Adequate consideration should be given to ensuring physicochemical stability

and microbial control during such hold-times.
The preparation of the lipids, the encapsulation of mRNA with the
lipids into LNPs, dilution and any purification steps, and subsequent filling into
suitable containers should be described and the process validated to meet the
necessary in-process specifications. Various filtration techniques (for example,
tangential flow filtration) should be considered for the removal of raw materials
used in the preparation of LNPs. Specific attention should be given to minimizing
the degradation of the mRNA during encapsulation into the LNPs and under
manufacturing conditions known to influence the stability of the LNPs and final
mRNA-LNP vaccine product (for example, the impact of thawing of the mRNA
and the freezing rate of the LNPs or mRNA-LNPs). Likewise, if lyophilized,
the conditions for freeze-drying and reconstitution should be considered and
justified. If applicable, the diluent or reconstitution solution should be described.
Suitable controls for the LNPs should also be specified and would typically
include: (a) identity, quantity and purity (including impurities) of the lipids;
(b) particle size and distribution (polydispersity); and (c) RNA encapsulation
efficiency/proportion encapsulated. In some cases, the surface properties (for
example, charge), lipid molar ratio, or cationic lipid to mRNA ratio (for example,
nitrogen to phosphate ratio) may also need to be specified to ensure consistency
and stability of the product.
It will also be important to consider the subsequent impact that any change
made to the mRNA drug substance (for example, change in sequence, length or
secondary structure) may have on the critical quality attributes of the LNPs (for
example, particle size and distribution, morphology, and surface properties) and
ultimately on the final vaccine product (for example, percentage of encapsulation
and cellular interaction/uptake). Relevant developmental data are expected
to demonstrate product consistency and to support the product optimization
process. Likewise, if platform data are intended to support development of new
candidate vaccines, the impact of the new mRNA drug substance on the critical
quality attributes of the final vaccine product should be determined.
6.8.3 Manufacture of final vaccine (drug product), filling and containers

An annotated flowchart should be provided that illustrates the manufacturing steps
from the bulk purified mRNA (drug substance) to the final vaccine (drug product).
The chart should include all steps (that is, unit operations) such as dilution of the
final formulated bulk, identification of materials and intermediates, and in-process
and quality control tests. A narrative description of each process step depicted
in the flowchart should be provided. Information should also be included on,
for example, its scale, buffers and other additives, major equipment and process
controls (including in-process tests and critical process operational parameters
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with acceptance criteria that are justified by relevant development data). Details of
the sterilization process and microbial control should also be included.
The general guidance concerning filling and containers provided in WHO
good manufacturing practices for biological products (23) should be applied to
vaccine filled in the final form. The aseptic fill process of the mRNA-LNP should
be adequately validated to ensure all critical quality attributes are maintained
and meet the required specifications. Care should be taken to ensure that the
materials of which the containers and closures (and, if applicable, the transfer
devices) are made do not adversely affect the quality of the vaccine. To this end, a
container-closure integrity test and assessment of extractables and/or leachables
for the final container-closure system are generally required for the qualification
of containers and may be needed as part of stability assessments.
If multi-dose vaccine vials are used and the vaccine does not contain
a preservative, then their use should be time restricted, as is the case for
reconstituted vaccines such as bacillus Calmette–Guérin (BCG) and measles-
containing vaccines (32). In addition, the multi-dose container should prevent
microbial contamination of the contents after opening. Relevant simulation
studies (for example, multi-puncture tests) of the container-closure system
may be required to demonstrate the suitability of the proposed system. Multi-
dose vials should be designed to meet the label claim, with acceptable overfill
to allow for correct dosing. Multi-dose vaccine vials should be evaluated for the
maximum anticipated vial septum punctures to assess the risk of compromising
vial integrity and the potential for vial contamination. The extractable volume
of multi-dose vials should be validated. If multi-dose vaccine vials are supplied
as concentrate, an additional compatibility study should be conducted using the
proposed reconstitution solutions and an appropriate post-dilution hold-time
should be established. The pre-dilution and post-dilution specifications should
be set out and justified. Manufacturers should provide the NRA with adequate
data demonstrating the stability of the product under appropriate conditions of
storage, distribution and during use.
When a final vaccine contains more than one mRNA species (for
example, in a combination or multivalent vaccine, or an sa-mRNA consisting of
separate mRNAs) there may be additional considerations in the manufacture of
that final vaccine. One such consideration will be ensuring the appropriate ratio
of the different mRNAs in the formulation to optimize the expression of each
and to minimize immune interference (in the case of combination or multivalent
vaccines). Another consideration will be whether the mRNAs will be mixed
prior to encapsulation in the LNPs or whether each mRNA will be separately
encapsulated into LNPs and then a mixture of the two or more mRNA-LNPs
prepared. In either case, the approach selected should be described and justified
with relevant data.
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6.8.4 Control of final vaccine (drug product)

Samples should be assessed from each final vaccine lot. All tests and specifications
should be approved by the NRA. Specifications for the final vaccine should
be established and justified by the manufacturer. As a principle, the final
specifications should be defined on the basis of the relevant batch data on lots that
have been shown to have acceptable performance in clinical studies. Descriptions
of analytical methods and acceptance limits for the vaccine should be provided,
including information on method validation. It is recommended that testing
should include an assessment of identity (see section 6.8.4.1 below), purity
(section 6.8.4.2), content (section 6.8.4.3), safety (section 6.8.4.4), additional
quality attributes (section 6.8.4.5) and potency (section 6.8.4.6). Stability will also
need to be established to justify the requested expiry dating.
Although specifications may be limited and have somewhat wide
acceptance criteria in early development, these should be reviewed and tightened,
when appropriate, as experience in the manufacturing process and analytical
methods is gained.
A summary of the results of the testing of all lots produced at commercial
scale should be provided. Early in development, to support clinical trial
authorization, results from testing lots made in accordance with GMP (21–
24) and, if available, engineering runs performed to establish manufacturing
procedures should be summarized and provided.
Not all of the tests conducted during product development need to be
carried out on every lot of vaccine produced at commercial scale. Some tests are
required only to obtain product and process knowledge on a limited series of lots
to establish consistency of production, as discussed in sections 6.4–6.6 above.
Several consecutive lots of vaccine, in final dosage form, should be tested and
analysed using validated methods to confirm manufacturing consistency. Any
statistically significant or scientifically meaningful differences between one lot and
another should be noted and investigated. The data obtained from such studies,
as well as clinical trial outcomes with various lots, alongside product and process
knowledge and evaluation of the criticality of variations in specific attributes,
should be used as the basis for defining the vaccine specifications and acceptance
criteria to be used for routine lot release. Thus, a comprehensive analysis of the
initial commercial production lots should be undertaken to establish consistency
with regard to the identity, purity, strength/content/quantity, safety, additional
quality parameters, potency and stability of the mRNA vaccine but thereafter a
more limited series of tests may be appropriate, if agreed with the NRA.
When a final vaccine contains more than one mRNA species (for example,
in a combination or multivalent vaccine, or an sa-mRNA consisting of separate
mRNAs) there may be additional considerations in the control of that final vaccine.
Some of these considerations will be based on the approach taken in manufacture
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– for example, whether the mRNAs were encapsulated together at the same time
as a mixture or were encapsulated separately and then the different mRNA-LNPs
mixed. This may then affect the size, charge and polydispersity of the LNPs. In
addition, validating the consistency of mixing is crucial to ensuring that each dose
contains the appropriate ratio of each of the mRNAs. Ensuring the proper ratios
in the total mRNA content of the final vaccine will be critical as the total mRNA
content is the basis for dosing. Identity testing should address the inclusion of each
mRNA, while still differentiating the vaccine from other products made in the
facility. If one drug substance or component (for example, the mRNA encoding
the replicon) is used in more than one vaccine or product made in the facility, then
such identity testing will also be crucial in preventing mix-ups.
As experience is gained in manufacturing consistency, post-approval
changes might permit reducing the testing and amount of supporting information
required through the use of process validation, product characterization and/or
a comparability protocol (31).
6.8.4.1 Identity
Each lot of vaccine should be subjected to an appropriate test to confirm the
identity of the final product and distinguish it from other products made in the
same facility or using the same equipment. If the vaccine contains more than one
mRNA species (for example, in a combination or multivalent vaccine, or an sa-
mRNA consisting of separate mRNAs), then the identity of each mRNA should
be confirmed. Confirmation of mRNA identity by sequence analysis should be
considered (see section 6.7.1.1 above).
6.8.4.2 Purity and impurities

The purity of each lot of final vaccine should be assessed and shown to be within
the specified limits. Consideration should be given to potential impurities
resulting from any component of the delivery system and to controlling aspects of
impurity such as oxidation and degradation in the final vaccine. It is unlikely that
a single test will be sufficient to detect all potential impurities. Tests for mRNA
integrity, particle size, lipid/polymer impurities and the proportion/efficiency of
mRNA encapsulated in the LNPs should be considered. Container-closure system
compatibility, leachables and extractables should also be assessed and discussed
in the application for marketing authorization (see also section 6.7.1.2 above).
6.8.4.3 Content, strength or quantity

mRNA vaccines are dosed based on quantity of the mRNA by weight. Therefore,
in addition to assessing potency (see section 6.8.4.6 below), a quantification
method for the mRNA should be described (see section 6.7.1.3 above). If the
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vaccine contains more than one mRNA species (for example, in a combination
or multivalent vaccine, or an sa-mRNA consisting of separate mRNAs), then the
quantity of each mRNA should be measured and confirmation made that the
ratio of each mRNA to the other is as intended and the total mRNA dose has not
been exceeded.
6.8.4.4 Safety attributes

Each lot of final vaccine should be tested for bacterial and fungal sterility
(including demonstration of lack of bactericidal or fungicidal activity of the
test article). If the vaccine is to be administered by a non-parenteral route,
then omission of the sterility test and inclusion of an appropriate alternative
microbial bioburden test needs to be appropriately justified. Further, a test
for endotoxin should be conducted on each lot and appropriate specifications
defined. If required by the NRA, a test for pyrogenicity may be performed
(which may be the monocyte activation test). Animal testing should be avoided
whenever alternative satisfactory testing is available and allowed. For scientific
and ethical reasons, it is desirable to minimize the use of animals in testing,
and consideration should be given to the use of appropriate in vitro alternative
methods for safety evaluation and other product control tests. In particular,
manufacturers and regulators should take note of the decision of the WHO
Expert Committee on Biological Standardization in 2018 to discontinue the
inclusion of the general safety (innocuity) test in routine lot release testing
requirements for all vaccines in WHO Recommendations, Guidelines and other
guidance documents for biological products (63). This test should therefore not
be required or requested.
6.8.4.5 Additional quality attributes

Other important quality attributes should also be established and controlled.
These can include appearance (including presence of both visible and sub-visible
particulate matter), extractable volume and pH. Depending on the product
characteristics, the control of other attributes such as osmolality or viscosity may
also be important. For the final vaccine (drug product), additional attributes
should include lipid/polymer identification and content, nanoparticle size,
mRNA–lipid ratio and polydispersity index.
With respect to nanoparticle size, multiple point control should be
adopted similar to the control of nanoparticle-based therapeutic products, and the
test used for measurement of particle size should be specified, as the results will
be dependent upon the analytical method employed. The degree of encapsulation
of the mRNA in the LNP should also be regarded as a critical quality attribute as
non-encapsulated mRNA is considered to be unstable. Confirmation should be
provided that the structure of the final product does not change due to freeze-
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thawing and dilution. Techniques such as gel or capillary electrophoresis and/or

HPLC already being performed for purity or for identity may also be useful in
assessing some quality attributes.
Other tests (such as a test for residual moisture if the vaccine is lyophilized)
may be required to confirm the physical characteristics of the product as well as
the formulation. Validation of the analytical methods used should be described to
assure the control of the identified critical quality attributes of the drug product.
6.8.4.6 Potency
The potency of each lot of the final vaccine should be determined using a suitably
quantitative and validated functional method(s). Different tests may be required
to control various aspects of potency (including functionality), which will likely
be disease specific. Immunogenicity in the vaccine recipient is a complex function
of the final vaccine properties, including delivery to target cells by its formulation
as well as expression of the mRNA-encoded protein(s) (which may include a self-
amplifying replicon component). Thus, potential in vitro potency assays may
include cell-based transfection systems or cell-free assays. Such methods would
demonstrate that the correctly sized protein of correct identity is expressed from
the mRNA. However, because potency should be analyzed on the basis of not only
the product type (in this case mRNA vaccines) but also the clinical indication of
the disease to be prevented, it is not possible to indicate a particular assay method
that should be used to measure potency. Scientific justification for the potency
test(s) selected to control the product should be provided and correlated with
clinical performance, as with all quality control tests.
When a new candidate vaccine against a new strain(s) is developed,
consideration should be given to ensuring that the potency assay(s) used is valid
for the strain change.
The potency specifications for mRNA vaccines should be set based on
the minimum dose used to demonstrate efficacy in clinical trials plus human
immunogenicity data. An upper limit should also be defined based on available
human safety data.
Animal-based assays tend to be highly variable and difficult to validate.
Consideration should therefore be given to the use of appropriate in vitro
alternative methods for potency evaluation. It is envisaged that, as with plasmid
DNA vaccines, a combination of biochemical or biophysical measures (such
as nucleic acid quantity and mRNA integrity) might be used to establish and
monitor the potency of mRNA vaccines. Manufacturers are encouraged to work
towards the goal of employing in vitro assays that are suitably quantitative and
assess function. However, it needs to be acknowledged that these measures
only account for the mRNA and not the impact of any formulation, adjuvant,
immunomodulators and so on, and the potency assessment of mRNA vaccines
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will thus need to be considered on an individual case-by-case basis. Therefore,

discussing appropriate potency measures and reaching agreement with the NRA
is advised.
6.8.4.7 Reference materials

A suitable lot of the final vaccine that has been clinically evaluated should be fully
characterized in terms of its chemical composition, purity, biological activity and
full sequence, and retained for use as an internal reference material. This material
should be used as the basis for evaluation of product quality for commercial
production lots (see also section 6.7.1.6 above).
In the future, national standards may be prepared and provided by the
NRA while international standards may become available from WHO. Should such
international standards become available it will be important to calibrate the internal
or national reference material against them. In this way, comparisons can be made
in a more reliable way whenever new reference materials need to be prepared. In
addition, the expression of results in a common unit (such as IU), when appropriate,
will also allow for the comparison of test results obtained from different laboratories,
and for different products against the same pathogen based on the same or similar
technologies (for example, different COVID-19 mRNA vaccines).
6.8.4.8 Stability testing, storage and expiry date

The relevant guidance provided in WHO good manufacturing practices for
biological products (23), WHO good manufacturing practices for sterile
pharmaceutical products (24) and WHO Guidelines on stability evaluation
of vaccines (27) appropriate for the respective mRNA vaccine should apply.
Furthermore, the WHO Guidelines on the stability evaluation of vaccines for
use under extended controlled temperature conditions (29) might also apply. The
statements concerning storage temperature and expiry date that appear on the
primary and secondary packaging should be based on experimental evidence and

should be submitted to the NRA for approval. For guidance regarding vaccine
vial monitors, the WHO Getting started with vaccine vial monitors and related
WHO guidance should be consulted (64, 65).
6.8.4.8.1 Stability
Adequate stability studies form an essential part of vaccine development.
To support commercial use, the stability of the final product in the container
proposed for use should therefore be determined and the results used to set a shelf-
life under appropriate storage conditions. Attributes that are stability-indicating
should be measured and these may include appearance (including visible and sub-
visible particulate matter), mRNA quantity, vaccine potency, mRNA integrity,
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degree of encapsulation, particle size, polydispersity and impurities associated

with the mRNA and lipids. The attributes to be measured should be described
and specifications defined and justified. Real-time stability studies should be
undertaken for this purpose – though accelerated stability studies at elevated
temperatures may provide additional and complementary supporting evidence
for the stability of the product and confirm the stability-indicating nature of the
assays used to determine stability. If long-term storage (for example, > 6 months)
at temperatures above freezing is being considered, this may require additional
analytical testing to assess potential lipid oxidation or other such changes and the
resultant impact of these changes on potency.
In addition, accelerated and stress testing data as well as platform data can
be taken into account to support the shelf-life. Stability data that support clinical
use, such as data on stability at elevated temperatures for short-term storage and
dispensing, should be generated. For multi-dose vials, in-use stability data will be
needed to provide assurance of the required microbial quality and stability of the
vaccine under in-use conditions (32).
During initial clinical development limited stability information would
be expected. For example, some regulators accept 3 months of real-time stability
of the lot of final vaccine to be used in the proposed clinical trial in the containers
that will be used for the clinical trial, or one produced in the same manner in the
same container type and size and meeting the same specifications, at the time
of application for clinical trial authorization, but this should be agreed with the
NRA. Initial clinical development may also be supported by including results
from predictive stability modelling utilizing an accelerated stability assessment
programme. Likewise, stability data on a platform technology can be supportive
for new candidate vaccines based on that platform.
If deep-freeze conditions are recommended for long-term storage, then
alternative short-term storage conditions (such as frozen and/or refrigerated)
should be explored to support vaccine distribution and dispensing. Similarly,
temperature excursion studies or transportation simulation studies may also be
expected. Container-closure system compatibility with storage stability (including
with regard to leachables and extractables) should be assessed and discussed. The
stability assessment should comply with WHO Guidelines on stability evaluation
of vaccines (27). Consideration should be given to the development of vaccine
formulations that are more thermostable to improve their global utility.
6.8.4.8.2 Storage conditions

Storage conditions should be validated. The vaccine should not be stored
for a length of time and/or at a temperature greater than that shown by the
manufacturer to be compatible with a minimal loss of potency before being
distributed by the manufacturing establishment or before being issued from
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a storage site. The maximum duration of storage should be fixed with the
approval of the NRA based on the results of stability studies and should be such
as to ensure that all quality specifications for the final product, including the
minimum potency specified on the container or package, are maintained until
the end of shelf-life. During clinical trials, this period should ideally be at least
equal to the expected duration of the vaccine administration stage in the fully
enrolled clinical trial.
6.8.4.8.3 Expiry date

The expiry date should be defined on the basis of the shelf-life of the final container
supported by real-time stability studies and should be approved by the NRA. The
expiry date should be based on the date of blending of the final formulated bulk,
the date of filling or the date of the first valid potency test on the final lot, as
appropriate, and agreed with the NRA.
6.9 Records
The relevant guidance provided in WHO good manufacturing practices for
pharmaceutical products: main principles (21) should apply, as appropriate to
the level of development of the candidate vaccine.
6.10 Retained samples

A sufficient number of samples should be retained for future studies and needs.
These needs may include but are not limited to manufacturing investigations or
development, nonclinical studies or future bridging clinical trials. A vaccine lot
used in a pivotal clinical trial may serve as a reference material and a sufficient
number of vials should be reserved and stored appropriately for that purpose.
Advanced planning is required to enable the retention of an appropriate number
of containers of the pivotal clinical trial lot.
6.11 Labelling
The guidance on labelling provided in WHO good manufacturing practices for
biological products (23) should be followed as appropriate. The label of the carton
enclosing one or more final containers, or the leaflet accompanying the container,
should include, at a minimum and as agreed with the NRA:
■ the common and trade names of the vaccine;
■ INN, if applicable;
■ the names and addresses of the manufacturer and distributer;
■ lot number;
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■ nature and content of the drug (active) substance;

■ product composition, including list of excipients;
■ a statement that specifies the nature and content of adjuvant
contained in one human dose, if any;
■ dosage form and appearance;
■ the immunization schedule and the recommended route(s) of
administration;
■ the number of doses, if the product is issued in a multi-dose
container;
■ the name and concentration of any preservative added;
■ a statement on the nature and quantity, or upper limit, of any
antibiotics present in the vaccine;
■ a statement on the trace amounts of any other residuals of clinical
relevance;
■ the temperature recommended during storage and transport;
■ container-closure information;
■ the expiry/retest date;
■ any special dosing schedules;
■ any special instructions for in-use handling – for example, necessity
for gloves to prevent exposure of product to RNases when handling
multi-dose vials, or stability on mixing of contents; and
■ contraindications, warnings and precautions, and information on
concomitant vaccine use and on known adverse events.
6.12 Distribution and transport

The guidance provided in WHO good manufacturing practices for biological
products (23) appropriate for the vaccine should apply. Further guidance is
provided in WHO Model guidance for the storage and transport of time- and
temperature-sensitive pharmaceutical products (28). Shipments should be
maintained within specified temperature ranges, as applicable, and packages
should contain cold-chain monitors, if applicable (29).
7. Nonclinical evaluation of mRNA vaccines

The nonclinical evaluation of candidate mRNA vaccines should be considered on
a product-specific basis taking into account the intended clinical use. The design,
conduct and analysis of nonclinical studies including selection of appropriate
studies relating to the “pharmacology” (in the case of vaccines, immunogenicity
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and proof-of-concept) and toxicology of the product should be based on the

following WHO guidelines:
■ WHO guidelines on nonclinical evaluation of vaccines (18); and

■ WHO Guidelines on the nonclinical evaluation of vaccine adjuvants
and adjuvanted vaccines (19).
There are several potential concerns that need to be considered during

the safety and proof-of-concept evaluations of mRNA vaccines. Because of
the novelty of this product type, numerous issues are addressed below which
may be relevant to any given mRNA vaccine. However, there may also be
future additional concerns that come to light that would need to be taken
into consideration when appropriate. Not all of these issues will necessarily
be relevant to any given mRNA vaccine, depending on its design. However, it
is incumbent upon the vaccine developer/manufacturer to provide evidence
demonstrating the proof-of-concept (for example, immunogenicity and, if an
appropriate animal model is available, challenge protection) and safety of their
candidate vaccine. The types, design and number of studies expected should be
agreed with the NRA.
7.1 Pharmacology/immunology/proof-of-concept
In addition to the types of studies discussed in the WHO guidelines above (18,
19), additional issues that the NRA might expect nonclinical studies to address
may include:
a. Durability of immune responses or immune cell phenotypes

that suggest durability, particularly those that are proposed to be
related to the candidate vaccine’s induction of protection. To assess
the durability of immune responses, characterization of immune

cell phenotypes and/or cytokine expression could be helpful in
investigating persistence and memory responses.
b. Induction of innate immune responses by RNA (such as induction
of type I interferon), which have been reported to decrease
translation of the target antigen or that could affect the need for (or
timing of) boosts or subsequent doses.
7.2 Safety/toxicity in animal models

In addition to the expectations outlined in the WHO guidelines listed above (18,
19), consideration should be given to whether studies need to be designed to
address the following:
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a. Biodistribution and persistence: developing a database of

evidence about this potential concern will permit the more rapid
development of future candidate vaccines (3, 66–71). This potential
issue may also depend on whether the vaccine migrates to specific
cells or tissues. Nonclinical studies that address whether the mRNA
and the LNPs (or lipid components) distribute away from the tissue
into which the vaccine was administered, into which tissues they
distribute and how long they persist may be expected by the NRA.
Agreement on these studies should be sought from the NRA.
b. Inflammation: RNA is inflammatory via a number of pathways,
particularly via the innate immune system with its numerous
sensors for RNA. In mRNA vaccines, both the mRNA molecules
and the LNPs (which enable successful delivery and cellular
uptake) have properties that can influence and trigger the innate
immune system (72, 73). While some of this activity may be
beneficial for the immune response to the vaccine, it will be
important to monitor for both systemic and local toxicity and
inflammatory responses. Nonclinical study design needs to take
into account any immune responses, reactogenicity or toxicities
that might predict immune indicators (72, 73) of serious adverse
events or adverse events of special interest (AESI) in humans.
Additionally, other components added to aid delivery, such as PEG,
although relatively benign, can also influence the physicochemical
properties and thus the safety profile (74–77). It is therefore
important to understand the overall product profile including the
formulation and how physicochemical properties (which may
vary) can influence inflammation and the safety profile. The choice
of animal model will, as always, be critical, recognizing that anti-
RNA innate immune responses in animal models are generally
significantly milder than those observed in humans.
c. Unexpected and serious toxicities from modified nucleosides:
some antivirals and anti-cancer drugs that contained specific
unnatural nucleoside analogues with altered conformation
have caused mitochondrial toxicities, resulting in myopathy,
polyneuropathy, lactic acidosis, liver steatosis, pancreatitis,
lipodystrophy and even fatality. However, some of these clinically
observed toxicities were not observed in the nonclinical animal
models used. While the modified nucleosides used in the most
advanced mRNA vaccines (against COVID-19) are naturally
occurring, future candidate vaccines may contain modifications
that are unnatural. Thus, particularly for mRNA vaccines that
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include unnatural nucleoside modifications that have not already

been well characterized in other developed nucleic-acid-based
products, careful consideration will need to be given to how these
potential toxicities might be observed in appropriate animal
models and nonclinical studies during safety evaluation (78–80).
d. Novel lipids and novel LNPs: because the lipids used to formulate
the LNPs affect the overall charge of the particle, when using
LNPs made with novel lipids or when the LNPs are themselves
modified (for example, altered ratios or modified processes) and
these LNPs have not previously been nonclinically and clinically
tested in mRNA products encapsulated in LNPs, then evaluation
of the toxicity of the new formulation containing the novel lipids
(or any novel excipients) may be required. Furthermore, the NRA
may require that the genotoxicity and systemic toxicity of the
novel lipid component be assessed, similar to the expectations for
novel adjuvants set out in the WHO Guidelines on the nonclinical
evaluation of vaccine adjuvants and adjuvanted vaccines (19) and/or
those for new chemical entities in the ICH guideline S2 (R1) (62).
e. Novel formulations: likewise, for formulations (other than LNPs)
containing novel excipients, data on and assessment of the systemic
toxicity and genotoxicity of the formulation may be expected.
It should be noted that early theoretical concerns during plasmid DNA
vaccine development regarding the potential for integration of vaccine nucleic acids
into the host genome do not apply to mRNA vaccines for the following reasons:
■ The only known mechanism by which RNA can integrate into the
host genome requires the presence of a complex containing reverse
transcriptase and integrase.
■ Further, the design of candidate mRNA vaccines should be

considered so that they do not include specific RNA-binding sites for
primers required for the reverse transcriptase to initiate transcription.
In addition, the RNA would have to be relocated to the nucleus after
reverse transcription for the resulting product to be integrated.
■ Finally, the vaccine mRNA degrades within a relatively short time
once taken up by the body’s cells, as does the cell’s own mRNA.
During that entire time, the mRNA vaccine is expected to remain
in the cytoplasm, where it will be translated and then degraded by
normal cellular mechanisms.
Therefore, nonclinical studies do not need to be performed to specifically
address integration or genetic risks for mRNA vaccines.
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As with any vaccine that is anticipated to be used widely in pregnant

women or women of childbearing potential, the guidance provided in section
4.2.2 of the WHO Guidelines on nonclinical evaluation of vaccines (18) and
section D.2.3 of the WHO Guidelines on the nonclinical evaluation of vaccine
adjuvants and adjuvanted vaccines (19) should be consulted. The necessity for
such studies will be based on the target population for the given clinical indication
of the vaccine. Often, if required, these studies are performed during or after
pivotal clinical trials have been performed with the candidate vaccine produced
using commercial manufacturing methods and scale. As a result, data should be
available at the time of filing for marketing authorization on populations that
include women of childbearing potential, and such data should be evaluated
prior to the intentional enrolment of pregnant women in clinical studies.
If clinical data from similar candidate vaccines based on the same
platform technology are available, then agreement should be reached with the
NRA on whether or not such data are scientifically sufficient to preclude the need
for further nonclinical studies. If nonclinical safety data from similar candidate
vaccines based on the same platform technology are available, agreement should
also be reached with the NRA on whether or not such data are scientifically
sufficient to preclude the need for further nonclinical safety studies. Likewise,
nonclinical safety data (and if available, clinical data) from a monovalent drug
product formulation can support the clinical development of a multivalent drug
product formulation (for example, for different strains of the same disease antigen)
or combination vaccine (different disease antigens) in cases where the same LNP
with the same molar and mRNA–lipid ratios is used, and where the sum of all the
mRNAs in the multivalent drug product formulation will be no more than the
highest dose shown to be safe in the monovalent nonclinical safety study.
7.3 Accelerating nonclinical evaluation in the context

of rapid vaccine development against a priority
pathogen during a public health emergency
In the case of the rapid development of vaccines against a priority pathogen
during a public health emergency and when the new candidate vaccines are based
on a given manufacturer’s platform technology, consideration may be given to an
abbreviated nonclinical programme as follows:
■ Where changes are made to the sequence of the target antigen
encoded in an mRNA vaccine that has already been clinically tested
(for example, in the case of a pandemic influenza strain when a
seasonal or other potential pandemic strain antigen has been tested,
or where a variant SARS-CoV-2 spike protein arises), where the
same LNPs are used (that is, same lipid composition and mRNA–
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lipid ratio, and where the total amount of mRNAs and LNPs per
dose remain equal to or below that clinically tested) and where
an approved manufacturing process is used, then, depending on
NRA requirements, the nonclinical programme might be limited
to an immunogenicity study (or studies) or a challenge-protection
study (or studies) in a relevant animal model, if available. As much
safety information as feasible should be collected during these
immunogenicity or challenge-protection studies on the understanding
that such nonclinical proof-of-concept studies are generally
performed without full compliance to good laboratory practices
(GLP). If safety information on veterinary vaccines expressing related
antigens is available, this might also be useful and should be provided.
Any other information concerning the safety of the platform
technology used should also be provided for NRA consideration, for
example, prior toxicology and biodistribution study data.
■ Where the LNPs have been tested clinically with an unrelated
mRNA such that the target antigen is novel (that is, not related to
another antigen that has been clinically tested), then the approach
of limiting nonclinical studies to an immunogenicity or challenge-
protection study might not be sufficient. The decision regarding
what type of nonclinical safety/toxicology information should be
required might be guided by consideration of what and how much
is known about the natural disease in terms of its pathology. If
the natural disease is associated with immunopathology due to
cross-reactivity, molecular mimicry, autoimmunity, allergenicity or
immunity-associated disease enhancement, then toxicology studies
would likely be needed to ensure that the novel target antigen was
not associated with these effects. It should be noted that it may not
be possible to investigate autoimmunity in nonclinical studies (18).

Where natural disease is not associated with immunopathology or
where little is known about the natural disease, discussion with the
NRA should be undertaken on how the nonclinical programme
might be abbreviated.
■ Finally, where the LNPs and the encoded target antigen (and hence
the mRNA structure and sequence) are both novel, nonclinical
evaluation may be more complex and more extensive studies may be
required; thus, discussion with the NRA should also be undertaken
and it may not be possible to significantly abbreviate the nonclinical
programme. However, it may be possible to initiate clinical studies
while some of the required nonclinical studies are being performed
in parallel with (or slightly ahead of) clinical development.
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Decisions on abbreviating the nonclinical programme should always take into

account what is already known about related and previously tested products,
particularly if based on the same platform technology. If clinical data from a
related product(s) are available, these data are likely to be more meaningful for
evaluating the safety of the candidate vaccine in humans than data from any
given animal model or from an in vitro human model.
8. Clinical evaluation of mRNA vaccines

The clinical evaluation expectations for clinical trial authorization or marketing
authorization will be driven by the disease against which the mRNA vaccine is
being or has been developed and the vaccine mode-of-action (or mechanism-of-
action). If an immune correlate of protection has been identified this may change
the expectations compared to what might be expected in the absence of such a
correlate. Clinical studies should adhere to the principles described in the WHO
Guidelines for good clinical practice (GCP) for trials on pharmaceutical products
(25) and the WHO Guidelines on clinical evaluation of vaccines: regulatory
expectations (20). Post-marketing pharmacovigilance is also discussed in the
latter guidelines. Furthermore, these same guidelines provide considerations in
evaluating dosing regimens, clinical development plans, boosting, collection of
safety data, designs for pivotal efficacy trials (including potential end-points),
standardizing immunogenicity assays (including use of IS and reporting of data
in IU) and immunobridging to infer efficacy (20). Considerations for studies
during pregnancy are also discussed in these same guidelines.
Clinical trials should capture safety, immunogenicity and efficacy data, as
expected for any other type of vaccine, but with particular consideration given to
the potential concerns outlined below as these may be more relevant for mRNA
vaccines than for other types of vaccines that might already be licensed and with
which regulators might be more familiar.
8.1 Safety and immunogenicity evaluation

Sufficient data should be obtained from preliminary clinical studies to permit
evaluation of the following safety and immunological aspects that may be
particularly relevant to mRNA vaccines:
a. Adverse immune effects
Transient decreases in lymphocytes (Grades 1–3) a few days
after vaccination were reported in the interim human clinical
trial results of an mRNA COVID-19 vaccine, with lymphocytes
returning to baseline within 6–8 days in all participants and with
no associated clinical observations (81). Such transient drops have
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been observed for other vaccines and have resulted in no significant

deleterious effect on the immune response (82, 83). Because RNA
induces type 1 interferons, which have been associated with the
transient migration of lymphocytes into tissues, the phenomenon
of any effect on lymphocyte counts in blood may need specific
attention in preliminary clinical trials (69, 84–86). Nonetheless,
because this phenomenon may be important for the immune
response to the candidate vaccine, it may be important to observe
whether changes in leukocyte counts and subsets are associated
with any adverse clinical signs or symptoms. Thus, the monitoring
of appropriate reactogenicity parameters in the immediate post-
vaccination period is paramount. Further general guidance on
safety evaluation is provided in section 7 of the WHO Guidelines
on clinical evaluation of vaccines: regulatory expectations (20).
b. Types and scope of immune responses
In addition to the type and scope of immunogenicity described in
the WHO Guidelines on clinical evaluation of vaccines: regulatory
expectations (20), in studies in which immunogenicity is measured,
additional facets of the safety and immunogenicity of mRNA
vaccines may include:
– whether the mRNA candidate vaccine biases towards certain
types of immune responses, depending on what is known
about the natural disease and the vaccine mode-of-action. To
date, two clinical studies of COVID-19 mRNA vaccines have
noted a Th1-type bias (37, 43). This information may be useful
for predicting and understanding the impact of the immune
responses for a particular disease.
– as with any new vaccine, any instances or evidence of AESI
as defined in the WHO Guidelines on clinical evaluation of

vaccines: regulatory expectations (20) or of any other novel
adverse event should be captured in clinical trials and in
post-marketing evaluation. If so, then investigations should
be conducted into associations and potential causes, such
as whether unwanted immune responses against vaccine
components (such as RNA or lipids) are generated or, if pre-
existing in the vaccine recipient, are increased or exacerbated.
Alternatively, epitope mimicry due to the responses to the
expressed antigen(s) may need to be investigated.
Consideration should also be given to the total dose of mRNA (especially
if the vaccine is a multivalent or combination vaccine, or an sa-mRNA vaccine
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where separate mRNAs are used) and to the total dose of LNPs with regard to
the maximally tolerable dose determined during the development of an mRNA
vaccine. For platform technologies, a maximally tolerable dose for a given
population may be suggested by the dose previously determined for vaccines (or
candidate vaccines) produced using that platform.
If boosting following a primary dose or series is being considered due to
waning effectiveness, careful evaluation of any increased frequency or severity
of local or systemic reactions should be performed. As with all vaccines, it
is recommended that a careful exploration of dose, timing and number of
immunizations (primary series and boosters if needed), and kinetics and
durability of immune responses be performed in preliminary clinical trials to
guide the design of the efficacy trial(s). Discussion of these issues can be found in
section 5.5 of the WHO Guidelines on clinical evaluation of vaccines: regulatory
expectations (20). In certain situations, a determination that booster doses are
needed might not be made until post-marketing data have been collected (for
example, indicating waning immunity or protection). The above Guidelines (20)
also discuss the boosting of licensed vaccines in section 5.6.1.2.3, which addresses
the situation in which an alternative posology to the licensed product may need
to be developed for the booster. Waning protection and the necessity for booster
doses is then discussed in section 6.3.8. (20). Differences between the vaccine
and circulating strains, including the potential need to add or replace strains,
are briefly discussed in section 5.3.3 and other sections that discuss influenza
vaccines, for which strain changes are frequently made.
It should be noted that during clinical trials or widespread use of
COVID-19 mRNA vaccines, immunologically relevant adverse events of particular
note (such as anaphylaxis or anaphylactoid reactions) have been observed (87,
88). Anaphylaxis is known to occur very rarely with all vaccines and is not
unique to mRNA vaccines. It is not yet known what aspect of the formulation
is associated with immunological adverse events and it is advised, as with other
vaccines, that individuals with known allergies to specific vaccine components
should not be vaccinated with vaccines containing such components (89–92).
Myocarditis and pericarditis have also been observed during COVID-19 mRNA
vaccine pharmacovigilance and appear to be associated – though the biological
mechanism and associated vaccine component have not yet been identified (93,
94). It should further be noted that recent publications by several regulatory
authorities provide useful relevant information, including publications by the
European Medicines Agency (71, 95), the Medicines and Healthcare products
Regulatory Agency (89, 96) and the US Food and Drug Administration (92, 97).
In line with the WHO Guidelines on clinical evaluation of vaccines:
regulatory expectations (20), the establishment and implementation of active
pharmacovigilance plans are recommended. In the specific case of COVID-19
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or other vaccines deployed in the context of a public health emergency,

consideration should also be given to running a public awareness campaign on
potential adverse events. As with any new vaccine, all adverse events potentially
associated with COVID-19 vaccines are currently being further assessed as part
of pharmacovigilance activities.
Given the short period for and limited scope of safety studies as part of the
efficacy studies that have led to the current widespread use of COVID-19 mRNA
vaccines, and the still unknown long-term safety impacts of mRNAs formulated
with LNPs in large human populations, it will be important to continue monitoring
and recording rare adverse events that have an unknown relationship with the
use of such vaccines. Regulatory agencies should analyze such data for vaccines
made by different manufacturers to provide a better clinical understanding and
a more precise safety profile for mRNA vaccines in the current formulation
designs. Furthermore, manufacturers and public health agencies should consider
conducting post-introduction vaccine effectiveness studies, addressing questions
of effectiveness among specific at-risk groups, the duration of protection, and
effectiveness against both infection and transmission. As stated above, this is a
rapidly evolving area and significant new data are emerging on an ongoing basis.
When international standards expressed in IU are available for
standardizing the immune assays used in clinical evaluation of the vaccine,
they should be used to calibrate internal standards or other working reference
materials, and results should be reported in IU to improve the comparability of
results across vaccines, across studies and across different assay platforms.
8.2 Efficacy evaluation

Efficacy evaluation will depend upon the disease against which the candidate
vaccine is intended to protect, and the clinical indication determined in clinical
trials. Factors that should be considered in the evaluation of vaccine efficacy are
described in the WHO Guidelines on clinical evaluation of vaccines: regulatory

expectations (20).
It should be noted that in countries in which COVID-19 mRNA vaccines
are currently being widely used, the use of placebo controls in trials requires
special consideration. The ethical considerations regarding the conducting of
ongoing COVID-19 vaccine trials with placebo controls were discussed in open
public meetings held in December 2020 (98, 99). Trial design issues (including the
selection of appropriate comparators) are discussed in the above WHO Guidelines
(20). Further guidance is also provided in the outcome document of a WHO Expert
consultation on the use of placebos in vaccine trials (100). As with all candidate
vaccines, both the scientific merits and ethical considerations should inform the
trial design and decisions must be made in the current benefit–risk context of the
country in which regulatory authorization is being sought (101, 102). In addition,
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WHO has now published more than 70 Guidelines and Recommendations for
vaccines against specific diseases, any one or several of which may provide relevant
guidance on the evaluation of any given mRNA vaccine (15).
8.3 Efficacy evaluation in the context of a public health

emergency in which immune-escape and other variants arise
As discussed in section 5.6.2 of the WHO Guidelines on clinical evaluation
of vaccines: regulatory expectations (20) it may be feasible to consider
immunobridging between the manufacturer’s original candidate vaccine (or
subsequently marketed vaccine) and a variant candidate vaccine in order to infer
efficacy of the variant mRNA candidate vaccine based on a manufacturer’s given
platform technology in which clinical end-point efficacy has been demonstrated
for the original candidate (or marketed) vaccine. The immunobridging may have
to be supported by justification of how comparable antibody titres for the prototype
and variant vaccines would translate into similar efficacy. Consideration must be
given to the following scenarios: (a) the variant candidate vaccine will replace
the original candidate vaccine; or (b) the variant and original candidate vaccines
will be combined (that is, in a bivalent or multivalent vaccine) or administered
simultaneously or in sequence. Collection of comparative safety data during such
immunobridging studies will also be expected. Overall, the considerations for
immunobridging studies may depend upon factors such as the disease, pathogen
and induced immune response(s) – trial designs and data requirements should
thus be decided on an individual case-by-case basis.
In the specific case of COVID-19 vaccines, consideration may be given to
the guidance provided by WHO (103, 104), the European Medicines Agency (71,
95), the Medicines and Healthcare products Regulatory Agency (89, 96), the US
Food and Drug Administration (92, 97) and other regulatory authorities (105–107).
Currently, mRNA vaccines against influenza viruses are in development
and any proposed strain changes may have to take into consideration current
practices for inactivated or live attenuated influenza virus vaccines. The
WHO recommendations to assure the quality, safety, and efficacy of influenza
vaccines (human, live attenuated) for intranasal administration (108) and
WHO Recommendations for the production and control of influenza vaccine
(inactivated) (109) should be consulted.
Authors and acknowledgements

The preliminary draft of this WHO document was prepared by Dr M.A. Liu,
consultant, ProTherImmune, the USA; and Dr H-N. Kang, World Health
Organization, Switzerland. This preliminary draft was then subject to expert
working group review during the period 1–15 October 2020 and comments
141
received from: Dr. E. Grabski and Dr H. Meyer, Paul-Ehrlich-Institut, Germany;

Dr. K. Karikó, Dr A. Kuhn, Dr U. Blaschke, Dr C. Blume and Dr J. Diekmann,
BioNTech, Germany; Dr D. Kaslow, PATH Vaccine Development Global Program,
the USA; Professor S. Lu, University of Massachusetts Medical School, the USA;
Professor E.E. Ooi, Duke-NUS Medical School, Singapore; Dr K. Peden, United
States Food and Drug Administration, the USA; Professor P. Roy, London School
of Hygiene & Tropical Medicine, the United Kingdom; Dr S. Sankarankutty,
Health Sciences Authority, Singapore; Dr J. Ulmer, GSK (formerly); Dr F. Verdier,
Sanofi Pasteur, France; Dr M. Watson and Dr C. Vinals, Moderna, the USA; and
Dr T.Q. Zhou, World Health Organization, Switzerland.
The first draft was then prepared by a WHO drafting group comprising
Dr R. Sheets, consultant, the USA; Dr M.A. Liu, consultant, ProTherImmune,
the USA; Dr H. Meyer, Paul-Ehrlich-Institut, Germany; Dr K. Peden, United
States Food and Drug Administration, the USA; Dr S. Sankarankutty, Health
Sciences Authority, Singapore; and Dr T.Q. Zhou, World Health Organization,
Switzerland. The resulting draft document was then posted on the WHO
Biologicals website during December 2020 and January 2021 for a first round
of public consultation. Comments were received from: Dr S. Acharya and
Dr F. Atouf, US Pharmacopeia, the USA; Dr A. Adisa, Therapeutic Goods
Administration, Australia; Dr L. Bisset, Dr A. Cook, Dr V. Ganeva and Dr
K-W. Wan, Medicines and Healthcare products Regulatory Agency, the United
Kingdom; Dr R.M. Bretas, Agência Nacional de Vigilância Sanitária, Brazil; Dr
G. Cirefice and Dr C. Milne, European Directorate for the Quality of Medicines &
HealthCare, France; Dr I. Feavers, consultant, the United Kingdom; Dr D. Feikin,
IVB/WHO (on behalf of the Covid-19 Vaccine Effectiveness working group);
Dr G. Frank, Biotechnology Innovation Organization, the USA; Dr E. Griffiths,
consultant, the United Kingdom; Dr R.A. Hafiz, Saudi Food & Drug Authority,
Saudi Arabia; Dr W. Jaroenkunthum, Department of Medical Sciences, Thailand;
Dr K. Karikó, Dr A. Kuhn, Dr U. Blaschke, Dr C. Blume, Dr C. Lindemann

and Dr J. Diekmann, BioNTech, Germany; Dr D.C. Kaslow, PATH Vaccine
Development Global Program, the USA; Dr M. Kucuku, National Agency for
Medicines and Medical Devices, Albania; Dr U. Loizides and Dr R.G. Balocco,
International Nonproprietary Names Programme and Classification of Medical
Products, World Health Organization, Switzerland; Professor S. Lu, University
of Massachusetts Medical School, the USA; Dr I. Mahmood Al-Sabri, Ministry
of Health, Oman; Professor S.F. Malan, University of the Western Cape, South
Africa; Dr J. Maslow, GeneOne Life Science Inc., the USA; Dr S. M. Morales
Sánchez, National Food and Drug Surveillance Institute, Colombia; Professor
E.E. Ooi, Duke-NUS Medical School, Singapore; Dr C. Pohl, Schlosspark-
Klinik, Germany; Dr I. Prawahju, National Agency for Drug and Food Control,
Indonesia; Dr R. Rabe, Norwegian Medicines Agency, Norway; Professor M.
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Rizzi, University of Piemonte Orientale, Italy; Dr J.S. Robertson, consultant,

the United Kingdom; Dr N. Rose and Dr S. Schepelmann, National Institute for
Biological Standards and Control, the United Kingdom; Professor P. Roy, London
School of Medicine & Tropical Hygiene, the United Kingdom; Dr M. Savkina,
Federal State Budgetary Institution Scientific Centre for Expert Evaluation of
Medicinal Products, Russian Federation; Dr L. Tesolin, Dr K. Brusselmans and
Dr W. van Molle, Sciensano, Belgium; Dr J. Ulmer, independent consultant,
the USA; Dr L. Viviani, Humane Society International, Italy; Dr A.L. Waddell,
WHO temporary advisor, the United Kingdom; Dr M. Li and Dr W. Wei, Center
for Drug Evaluation, National Medical Products Administration, China; Dr K.
Weisser, Paul-Ehrlich-Institut, Germany; and Dr G. Zenhäusern, Swiss Agency
for Therapeutic Products, Switzerland. Dr P. Barbosa, International Federation
of Pharmaceutical Manufacturers & Associations (IFPMA) provided the
consolidated comments of subject matter experts from: GSK Vaccines, Belgium;
Pfizer Vaccines, the USA; and Sanofi Pasteur, France.
Taking into consideration the comments received, the second draft
document was prepared by Dr R. Sheets, consultant, the USA; Dr M.A. Liu,
consultant, ProTherImmune, the USA; Dr H. Meyer, Paul-Ehrlich-Institut,
Germany; Dr K. Peden, United States Food and Drug Administration, the
USA; Dr S. Sankarankutty, Health Sciences Authority, Singapore; Dr K-W. Wan,
Medicines and Healthcare products Regulatory Agency, the United Kingdom;
and Dr T.Q. Zhou, World Health Organization, Switzerland. The second draft was
then reviewed at a WHO informal consultation on regulatory considerations for
evaluation of the quality, safety and efficacy of RNA-based prophylactic vaccines
for infectious diseases, held virtually on 20–22 April 2021 and attended by: Dr
I.G. Al Gayadh and Dr R.A. Hafiz, Saudi Food & Drug Authority, Saudi Arabia;
Dr P. Aprea, Administración Nacional de Medicamentos, Alimentos y Tecnología
Medica, Argentina; Dr M. Ayiro, Pharmacy and Poisons Board, Kenya; Dr C. Bae,
Ministry of Food and Drug Safety, Republic of Korea; Dr K. Bloom, University of
the Witwatersrand and South African Medical Research Council, South Africa;
Dr K. Bok and Dr B.S. Graham, National Institutes of Health, the USA; Dr R.
Bose, Central Drugs Standard Control Organization, India; Dr J. Fernandes,
Health Canada, Canada; Dr E. Grabski and Dr H. Meyer, Paul-Ehrlich-Institut,
Germany; Dr S. Alireza Hosseini, Food and Drug Administration, the Islamic
Republic of Iran; Dr N. de Jesus Huertas Mendez, Instituto Nacional de Vigilancia
de Medicamentos y Alimentos, Colombia; Mrs T. Jivapaisarnpong, King
Mongkut’s University of Technology Thonburi, Thailand; Professor F. Krammer,
Icahn School of Medicine at Mount Sinai, the USA; Dr M.A. Liu, consultant,
ProTherImmune, the USA; Dr J. Lu, National Medical Products Administration,
China; Professor S. Lu, University of Massachusetts Medical School, the USA;
Dr A. Marti and Dr T. Schochat, Swiss Agency for Therapeutic Products,
143
Switzerland; Dr T. Matano, National Institute of Infectious Diseases, Japan; Dr

C. Milne, European Directorate for the Quality of Medicines & HealthCare,
France; Dr P. Minor, consultant, the United Kingdom; Dr W. van Molle and Dr
L. Tesolin, Sciensano, Belgium; Professor E.E. Ooi, Duke-NUS Medical School,
Singapore; Dr M. Page, Dr N. Rose and Dr S. Schepelmann, National Institute
for Biological Standards and Control, the United Kingdom; Dr K. Peden, United
States Food and Drug Administration, the USA; Dr S. Pumiamorn, Ministry of
Public Health, Thailand; Dr J.S. Robertson, independent expert (representative
of the International Nonproprietary Names Programme), the United Kingdom;
Professor P. Roy, London School of Hygiene & Tropical Medicine, the United
Kingdom; Professor K. Ruxrungtham, Chulalongkorn University, Thailand; Dr S.
Sankarankutty, Health Sciences Authority, Singapore; Dr Y. Shelar, Central Drugs
Standard Control Organization (HQ), India; Dr R. Sheets, consultant, the USA;
Dr R. Shivji, European Medicines Agency, Netherlands; Ms G.S. Silveira, Agência
Nacional de Vigilância Sanitária, Brazil; Dr V.G. Somani, Central Drugs Standard
Control Organization, India; Dr K-W. Wan, Medicines and Healthcare products
Regulatory Agency, the United Kingdom; Dr J. Wang and Dr Y. Wang, National
Institutes for Food and Drug Control, China; Dr W. Wei, Center for Drug
Evaluation, National Medical Products Administration, China; and Professor D.
Weissman, Penn Center for AIDS Research, the USA. Representatives of other
organizations: Dr S.K. Acharya, United States Pharmacopeia, the USA; Dr N.
Jackson, Coalition for Epidemic Preparedness Innovations, the United Kingdom;
Dr D. Kaslow, PATH Vaccine Development Global Program, the USA; and Dr C.
Vinals, the Biotechnology Innovation Organization, the USA. Representatives of
the Developing Countries Vaccine Manufacturers Network (DCVMN): Dr M.M.
Ahasan, Incepta Vaccine, Bangladesh; Dr D.T. Dat, Vabiotech, Viet Nam; Dr C.
Roy, Bharat Biotech, India; Dr W. Wijagkanalan, BioNet-Asia Co., Ltd, Thailand;
and Mr A. Wong, Walvax Biotechnology, China. Representatives of the IFPMA: Dr
D. Boyce, Pfizer, Ambler, PA, the USA; Dr S. Chiyukula and Dr S. Simons, Sanofi,
Cambridge, MA, the USA; Dr R. Forrat, Sanofi, Lyons, France; Dr S. Gregory
and Dr G. Maruggi, GSK, Rockville, MD, the USA; Dr S. Lockhart, Pfizer,
New York, NY, the USA; and Dr F. Takeshita, Daiichi Sankyo, Japan. Individual
manufacturers/other industries: Dr T. Class, Translate Bio, the USA; Dr S. Gould,
Charles River Laboratories, Lyons, France; Dr A. Kuhn (20 and 21 April), Dr R.
Rizzi (20 and 21 April), Dr C. Lindemann (22 April) and Dr E. Lagkadinou (22
April), BioNTech SE, Germany; Dr K. Lindert, Arcturus Therapeutics, the USA;
Dr J. Maslow, GeneOne Life Science Inc., the USA; Dr J.M. Miller and Dr D.
Parsons, Moderna, Inc., the USA ; Dr F. Neske and Dr L. Oostvogels, CureVac
AG, Germany; Mr Y. Park, GeneOne Life Science Inc., Republic of Korea; and
Dr B. Ying, Suzhou Abogen Biosciences Co., Ltd. P.R., China. WHO staff: Dr
C. Ondari, Dr I. Knezevic, Dr T.Q. Zhou, Dr M. Friede, Dr B. Giersing, Ms
144
Annex 3
E. Sparrow, Dr U. Loizides, Dr R.G. Balocco and Dr D. Wood, World Health

Organization, Switzerland; and Dr S. Escalante and Dr J. Shin, WHO Regional
Office for the Western Pacific, Philippines.
Based on the outcomes of the above informal consultation, the document
WHO/BS/2021.2402 was prepared by Dr R. Sheets, consultant, the USA; Dr M.A.
Liu, consultant, ProTherImmune, the USA; Dr H. Meyer, Paul-Ehrlich-Institut,
Germany; Dr K. Peden, United States Food and Drug Administration, the USA; Dr
S. Sankarankutty, Health Sciences Authority, Singapore; Dr K-W. Wan, Medicines
and Healthcare products Regulatory Agency, the United Kingdom; and Dr T.Q.
Zhou, World Health Organization, Switzerland. Document WHO/BS/2021.2402
was then posted on the WHO Biologicals website for a second round of public
consultation from July to September 2021 and written comments received from:
Dr S. Acharya and Dr F. Atouf, United States Pharmacopeia, the USA; Dr L. Bisset,
Medicines and Healthcare products Regulatory Agency, the United Kingdom; Dr
E. Cocuzzi, Health Canada, Canada; Dr T. Class, Translate Bio, the USA; Dr S.
Dubey, Dr J. Nussbaum, Dr L. Plitnick, Dr J. Prescott, Dr J. Wei and Dr Y. Zhang,
Merck Vaccines, the USA; Dr M. Li and Dr S. Jin, Center for Drug Evaluation,
National Medical Products Administration, China; Dr S. Gould, Charles River
Laboratories, France; Dr Z. Guo, Chinese Pharmacopoeia Commission, China;
Dr A. Gudjonsson, Dr G. Abrahamsen and Dr T.K. Andersen, Norwegian
Medicines Agency, Norway; Dr F. Neske, CureVac, Germany; A.A. Zuniga, A.M.
Santana Puentes, Instituto Nacional de Vigilancia de Medicamentos y Alimentos,
Colombia; Dr S. Muchakayala, Touchlight Genetics Ltd, the United Kingdom;
Dr S. Wendel, Hospital Sírio-Libanês Blood Bank, Brazil; Dr G. Sanyal, Dr P-A.
Gilbert and Dr D. Robinson, Bill & Melinda Gates Foundation, the USA; Dr J.
Wang, National Institutes for Food and Drug Control, China; Dr I. Zadezensky,
Moderna, the USA; and A. Kuhn, U. Blaschke, K. Karikó, D. Theisen, B. Vallazza,
C. Lindemann and E. Lagkadinou, BioNTech SE, Germany. Dr P. Barbosa,
IFPMA, provided the consolidated comments of subject matter industry experts.
Further changes were made to document WHO/BS/2021.2402 by the
WHO Expert Committee on Biological Standardization.
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108. Recommendations to assure the quality, safety and efficacy of influenza vaccines (human, live
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sixtieth report. Geneva: World Health Organization; 2013: Annex 4 (WHO Technical Report
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154
Annex 4
New and replacement WHO international reference
standards for biological products
The provision of global measurement standards is a core normative WHO activity.
WHO international reference standards are widely used by manufacturers,
regulatory authorities and academic researchers in the development and
evaluation of biological products. The timely development of new reference
standards is crucial in harnessing the benefits of scientific advances in new
biologicals and in vitro diagnosis. At the same time, management of the existing
inventory of WHO international reference standards requires an active and
carefully planned programme of work to replace established materials before
existing stocks are exhausted.
The considerations and guiding principles used to assign priorities
and develop the programme of work in this area have previously been set out
as WHO Recommendations.7 In order to facilitate and improve transparency
in the priority-setting process, a simple tool was developed as Appendix 1 of
these WHO Recommendations. This tool describes the key considerations taken
into account when assigning priorities, and allows stakeholders to review and
comment on any new proposals being considered for endorsement by the WHO
Expert Committee on Biological Standardization.
A list of current WHO international reference standards for biological
products is available at: https://www.who.int/health-topics/Biologicals#tab=tab_1.
At its meetings held via video conference on 18–22 October 2021, the
WHO Expert Committee on Biological Standardization made the changes shown
below to the previous list. Each of the WHO international reference standards
shown in this table should be used in accordance with their instructions for use
(IFU).
7
Recommendations for the preparation, characterization and establishment of international and other
biological reference standards (revised 2004). In: WHO Expert Committee on Biological Standardization:
fifty-fifth report. Geneva: World Health Organization; 2006: Annex 2 (WHO Technical Report Series, No. 932;
http://www.who.int/immunization_standards/vaccine_reference_preparations/TRS932Annex%202_
Inter%20_biol%20ef%20standards%20rev2004.pdf?ua=1, accessed 26 October 2021).
155
Additions 8
Material Unitage Status
Biotherapeutics other than blood products
Follicle-stimulating 137 IU/ampoule Third WHO International
hormone (human, Standard
recombinant) for bioassay
Blood products and related substances
von Willebrand factor VWF:Ag 12.0 IU/ampoule Third WHO
(concentrate) VWF:RCo 8.7 IU/ampoule International Standard
VWF:CB 9.8 IU/ampoule
VWF:GPIbR 8.6 IU/ampoule
VWF:GPIbM 7.3 IU/ampoule
Ferritin (human, 10.5 μg/ampoule Fourth WHO
recombinant) Expanded uncertainty International Standard
limits = 10.2–10.8 μg/
ampoule (95% confidence;
k = 2.23)
In vitro diagnostics
Mycobacterium tuberculosis 6.3 log10 IU/vial First WHO International
(H37Rv) DNA for NAT-based Standard
assays
Varicella zoster virus DNA 7.0 log10 IU/vial First WHO International
for NAT-based assays Standard
Anti-Lassa virus 25 IU/ampoule for First WHO International
immunoglobulin G neutralizing antibody Standard
250 IU/ampoule for anti-GP

binding IgG
250 IU/ampoule for anti-NP
binding IgG
Anti-Lassa virus [No unitage assigned] First WHO International
immunoglobulin G Reference Panel
Anti-thyroid peroxidase 555 IU/ampoule First WHO International
antibodies Standard
8
Unless otherwise indicated, all materials are held and distributed by the National Institute for Biological
Standards and Control, Potters Bar, Herts, EN6 3QG, the United Kingdom.
156
Annex 4
Material Unitage Status

Vaccines and related substances
Diphtheria antitoxin 57 IU/ampoule Second WHO
(equine) International Standard
157
SELECTED WHO PUBLICATIONS OF RELATED INTEREST

Report of the seventy-second and seventy-third meetings.
WHO Technical Report Series, No. 1030, 2021 (xvii + 269 pages)

Seventy-first report.
WHO Technical Report Series, 1028, 2021 (xii + 102 pages)

Seventieth report.
WHO Technical Report Series, No. 1024, 2020 (xvi + 227 pages)

Sixty-ninth report.
WHO Technical Report Series, No. 1016, 2019 (xv + 251 pages)

Sixty-eighth report.

Sixty-seventh report.
WHO Technical Report Series, No. 1004, 2017 (xviii + 591 pages)

Sixty-sixth report.
WHO Technical Report Series, No. 999, 2016 (xix + 267 pages)

Sixty-fifth report.

Sixty-fourth report.
WHO Technical Report Series, No. 987, 2014 (xviii + 266 pages)
Website: https://www.who.int/health-topics/Biologicals#tab=tab_1
To purchase WHO publications, please contact:

WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland;
email: [email protected]; order online: http://apps.who.int/bookorders
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 products used in
medicine, and the establishment of international biological
reference materials.
Following a brief introduction, the report summarizes a
number of issues brought to the attention of the Committee
at its meetings held via video conference in October 2021. Of
particular relevance to manufacturers and national regulatory
authorities are the discussions held on the development
and adoption of new and revised WHO Recommendations,
Guidelines and guidance documents. Following these
discussions, the following two documents were adopted on
the recommendation of the Committee: (a) Amendment to
the WHO Recommendations to assure the quality, safety
and efficacy of live attenuated yellow fever vaccines; and (b)
Evaluation of the quality, safety and efficacy of messenger RNA
vaccines for the prevention of infectious diseases: regulatory
considerations.
Subsequent sections of the report provide information on the
current status, proposed development and establishment of
international reference materials in the areas of: biotherapeutics
other than blood products; blood products and related
substances; in vitro diagnostics; standards for use in high-
throughput sequencing technologies; standards for use in
public health emergencies; and vaccines and related substances.
A series of annexes is then presented which includes an
updated list of all WHO Recommendations, Guidelines and
other documents related to the manufacture, quality control
and evaluation of biological products (Annex 1). The above
two WHO documents adopted on the advice of the Committee
are then presented as part of this report (Annexes 2 and 3).
Finally, all new and replacement WHO international reference
standards for biological products established during the
October 2021 meeting are summarized in Annex 4. The updated
full catalogue of WHO international reference standards is
available at: https://www.who.int/teams/health-product-and-
policy-standards/standards-and-specifications/catalogue.
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W H O Te c h n i c a l R e p o r t S e r i e s

WHO Expert Committee

To ensure the widest possible availability of authoritative information and guidance on

WHO Expert Committee

© World Health Organization 2022

Dr P. Salmikangas, Consultant, Klaukkala, Finland

Dr S. Wendel, Hospital Sirio-Libanês, São Paulo, Brazil

Dr M. Nübling, Paul-Ehrlich-Institut, Langen, Germany

Observers from non-state actors in official relations

Professor E. Wood, Melbourne, Australia

Representation from intergovernmental and other entities

World Health Organization (WHO)

Representation from WHO regional offices 3

IFU instructions for use

the Committee would also continue to be needed on the use of COVID-19

Executive Board of the World Health Assembly in January 2021.

participants including non-state actors would be held on Monday 18 October

The holding of prequalification meetings with manufacturers had helped to

2.1.2 Vaccines, biotherapeutics, and cell and gene therapy products:

monitored and where necessary revised in order to reflect significant advances in

further measurement standards projects in this area, including on replication-

2.1.3 WHO EUL of COVID-19 vaccines: an update

might affect the emergency use condition granted to the product.

COVID-19 vaccines would however present a particular challenge and although

2.1.4 Update from the WHO Product Development

PDVAC has also continued to monitor the development of mAbs

2.1.5 Report of the October meeting of the Strategic

2.1.6 Blood products and in vitro diagnostics: recent and

2.1.7 Update on the use of convalescent plasma for COVID-19

2.1.8 Snakebite envenoming

such products containing significant amounts of low molecular weight proteins,

then be implemented that incorporated the current bespoke antivenom benefit–

2.2 Feedback from custodian laboratories

OPVs and in the collaborative study to replace animal neurovirulence testing

European Directorate for the Quality of Medicines &

National Institute for Biological Standards and Control

by NIBSC for discussion by the Committee in early 2022, highlighting the

Paul-Ehrlich-Institut (PEI), Langen, Germany

PEI. These had included participation in the development or revision of a number

Dr Meyer concluded by reporting on the results of a comparative evaluation of

when stored at elevated temperatures. In addition, assumptions made about the

3.2 Blood products and related substances

3.2.2 WHO policy brief: the urgent need to implement

resource even before transfusion is considered. A thorough understanding of

3.2.3 WHO guidance on screening donated blood for

3.2.4 WHO tools for the stepwise implementation of a haemovigilance system

3.3 Cell and gene therapy products

3.3.2 WHO white paper on regulatory convergence for CGTPs

was preventing the transmission of infectious diseases. Although significant

3.4 Vaccines and related substances

At its meeting in August 2020, the Committee had supported a proposal

use during public health emergencies. The unprecedented pace of development

and content of the proposed document, and of the document development

regard some aspects of mRNA production as being similar to a pharmaceutical,

international standard 08/282. Results from 16 valid assays indicated an overall

4.2 Proposed new projects and updates –

would assign a unitage relative to the current WHO international standard. It

when the current international standard had been established. A recombinant

The Committee also discussed the clinical and industry implications of