who ev71 vaccine recommendations 2nd pc

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WHO/BS/2020.2388 3

ENGLISH ONLY 4

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Recommendations to assure the quality, safety and efficacy of enterovirus 6

71 vaccines (inactivated) 7

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Proposed new Recommendations 10

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NOTE: 14

15

This draft document has been prepared for the purpose of inviting comments and suggestions 16

on the proposals contained therein, which will then be considered by the Expert Committee 17

on Biological Standardization (ECBS). The distribution of this document is intended to 18

provide information on the proposed WHO Recommendations to assure the quality, safety 19

and efficacy of enterovirus 71 vaccines (inactivated) to a broad audience and to ensure the 20

transparency of the consultation process. 21

22

These WHO Recommendations were developed based on the outcomes and consensus of a 23

WHO working group meeting and informal consultation convened in 2019 and 2020 24

respectively, involving participants from national regulatory authorities, national control 25

laboratories, vaccine manufacturers and research institutes. 26

27

The text in its present form does not necessarily represent the agreed formulation of the 28

ECBS. Written comments proposing modifications to this text MUST be received by 29

2 October 2020 in the separately available comment form and should be addressed to the 30

Department of Health Products Policy and Standards, World Health Organization, 20 Avenue 31

Appia, 1211 Geneva 27, Switzerland. 32

33

Comments may also be submitted electronically to the Responsible Officer: Dr Dianliang Lei 34

at: [email protected]. 35

36

The outcome of the deliberations of the ECBS will be published in the WHO Technical 37

Report Series. The final agreed formulation of the document will be edited to be in 38

conformity with the second edition of the WHO style guide (KMS/WHP/13.1). 39

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WHO/BS/2020.2388

1 © World Health Organization 2020 2

3 All rights reserved. 4 5 The designations employed and the presentation of the material in this draft document do not imply the 6 expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status 7 of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or 8 boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full 9 agreement. 10 11 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed 12 or recommended by the World Health Organization in preference to others of a similar nature that are not 13 mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital 14 letters. 15 16 All reasonable precautions have been taken by the World Health Organization to verify the information 17 contained in this draft document. However, the printed material is being distributed without warranty of any 18 kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the 19 reader. In no event shall the World Health Organization be liable for damages arising from its use. 20 21 This draft document does not necessarily represent the decisions or the stated policy of the World Health 22 Organization. The named authors [or editors as appropriate] alone are responsible for the views expressed 23 herein. 24 25

WHO/BS/2020.2388

Recommendations to assure the quality, safety and efficacy of enterovirus 1


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5

Introduction 7 6

7

Purpose and scope 8 8

9

Terminology 8 10

11

General considerations 10 12

13

International reference materials 12 14

15

Part A. Manufacturing recommendations 13 16

17

A.1 Definitions 13 18

A.2 General manufacturing recommendations 14 19

A.3 Control of source materials 14 20

A.4 Control of vaccine production 18 21

A.5 Filling and containers 28 22

A.6 Control tests on the final lot 29 23

A.7 Records 31 24

A.8 Retained samples 31 25

A.9 Labelling 31 26

A.10 Distribution and transport 32 27

A.11 Stability testing, storage and expiry date 32 28

29

Part B. Nonclinical evaluation of enterovirus 71 vaccines (inactivated) 33 30

31

B.1 Product characterization and process development 33 32

B.2 Nonclinical immunogenicity and protection studies 34 33

B.3 Nonclinical safety studies 35 34

35

Part C. Clinical evaluation of enterovirus 71 vaccines (inactivated) 35 36

37

C.1 Introduction 35 38

C.2 Assays 36 39

C.3 Immunogenicity 37 40

C.4 Efficacy 38 41

C.5 Safety 42 42

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WHO/BS/2020.2388

Part D. Recommendations for NRAs 42 1

2

D.1 General recommendations 42 3

D.2 Official release and certification 43 4

5

Authors and acknowledgements 43 6

7

References 44 8

9

Appendix 1 Model summary protocol for the manufacturing and control of 10

enterovirus 71 vaccines (inactivated) 54 11

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Appendix 2 Model NRA Lot Release Certificate for enterovirus 71 vaccines 13

(inactivated) 64 14

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WHO/BS/2020.2388

Recommendations published by the World Health Organization (WHO) are intended to be 1

scientific and advisory in nature. Each of the following sections constitutes recommendations 2

for national regulatory authorities (NRAs) and for manufacturers of biological products. If an 3

NRA so desires, these WHO Recommendations may be adopted as definitive national 4

requirements, or modifications may be justified and made by the NRA. It is recommended 5

that modifications to these WHO Recommendations are made only on condition that such 6

modifications ensure that the product is at least as safe and efficacious as that prepared in 7

accordance with the recommendations set out below. The parts of each section printed in 8

small type are comments or examples intended to provide additional guidance to 9

manufacturers and NRAs. 10

11

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WHO/BS/2020.2388

Abbreviations 1

2

3

BPL beta-propiolactone 4

ELISA enzyme-linked immunosorbent assay 5

EV71 enterovirus 71 6

HFMD hand, foot and mouth disease 7

ICP immune correlate of protection 8

Ig immunoglobulin 9

IPV inactivated poliomyelitis vaccine 10

MCB master cell bank 11

NAT nucleic acid amplification technique 12

NCL national control laboratory 13

NIBSC National Institute for Biological Standards and Control 14

NIFDC National Institutes for Food and Drug Control 15

NRA national regulatory authority 16

PCR polymerase chain reaction 17

PSGL-1 P-selectin glycoprotein ligand-1 18

RD rhabdomyosarcoma 19

SCARB2 scavenger receptor class B member 2 20

SCARB2 scavenger receptor class B member 2 (gene) 21

WCB working cell bank 22

23

WHO/BS/2020.2388

Introduction 1

2

Enterovirus 71 (EV71) was first isolated from the faeces of a female suffering from 3

encephalitis in 1969 in California (1). However, a retrospective study conducted in the 4

Netherlands indicated that the virus could have emerged as early as 1963 (2); a finding 5

consistent with reports of possible worldwide EV71 epidemics in the late twentieth century 6

(3). EV71 has since been associated with a range of diseases, including hand, foot and mouth 7

disease (HFMD), throughout the world and with epidemics in Asia, Europe and the 8

Americas. The virus causes a range of effects from asymptomatic infection and mild HFMD 9

to neurological disease with severe central nervous system complications and 10

cardiopulmonary failure. In severe cases mortality rates can be high, especially in children 11

aged 5 years and younger. EV71 is considered to be the most severe neurotoxic enterovirus 12

and severe EV71 disease has become a major public health problem in China. In 2011, the 13

WHO Regional Office for the Western Pacific issued A guide to clinical management and 14

public health response for hand, foot and mouth disease (HFMD) (4) to support the 15

treatment, prevention and control of HFMD. 16

17

Several vaccines against EV71 are currently under development and three inactivated EV71 18

vaccines have been licensed in China (5–10). The WHO Expert Committee on Biological 19

Standardization discussed the EV71 situation at its 67th meeting in 2016 and considered it to 20

be of major regional significance (11). The Committee noted that the joint efforts of the 21

National Institutes for Food and Drug Control (NIFDC) and the National Institute for 22

Biological Standards and Control (NIBSC) had resulted in the development of the First WHO 23

International Standard for anti-EV71 serum (human), and recommended that consideration 24

also be given to the development of a WHO written standard for EV71 vaccines. In addition, 25

the First WHO International Standard for EV71 inactivated vaccine was established by the 26

Committee in October 2019 following a collaborative study led by NIBSC and NIFDC (12, 27

13). National standards for antigen content and neutralizing antibody responses for evaluating 28

EV71 vaccines are also available in China (14) where they have supported the development 29

and clinical assessment of such vaccines. In 2018, the Committee also endorsed a proposal to 30

develop WHO international standards for enterovirus RNA for nucleic acid amplification 31

technique (NAT)-based assays for EV71 (15, 16). 32

33

Following requests from regulators and other stakeholders for WHO to develop 34

recommendations to assure the quality, safety and efficacy of EV71 vaccines, a series of 35

meetings was convened by WHO to review the current status of their development and 36

licensure (17). These meetings were attended by experts from around the world involved in 37

the research, manufacture, regulatory assessment and approval, and control testing and 38

release of EV71 vaccines. Participants were drawn from academia, national regulatory 39

authorities (NRAs), national control laboratories (NCLs) and industry. The recommendations 40

provided in the current document for the production, quality control and evaluation of 41

inactivated EV71 vaccines have been based upon the experiences gained during the 42

development and production of the first three licensed EV71 vaccines in China, the candidate 43

WHO/BS/2020.2388

EV71 vaccines now under development (5–8, 18–22) and other inactivated viral vaccines, 1

such as inactivated poliomyelitis vaccines (IPVs) and hepatitis A vaccines (23, 24). 2

3

Purpose and scope 4

5

These WHO Recommendations provide guidance to NRAs and manufacturers on the 6

manufacturing processes, quality control and nonclinical and clinical evaluations needed to 7

assure the quality, safety and efficacy of inactivated EV71 vaccines. 8

9

The guidance applies to EV71 vaccines prepared by the inactivation of whole EV71 virus for 10

prophylactic use, grown in mammalian cells in culture, and using formaldehyde or other 11

chemical inactivation procedures. 12

13

The document does not cover recombinant and other forms of subunit vaccines, vectored 14

vaccines, virus-like particle vaccines or bivalent EV71-CA16 vaccines, which are at an early 15

stage of development. However, some aspects outlined in this document may be relevant and 16

may be taken into consideration during the development of such vaccines. 17

18

These WHO Recommendations should be read in conjunction with current WHO guidance 19

documents on the nonclinical (25) and clinical (26) evaluation of vaccines, good 20

manufacturing practices for biological products (27), characterization of cell banks (28), 21

nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines (29) and lot release (30). 22

23

Manufacturers and regulators should also take note of the decision of the Committee in 2018 24

to discontinue the inclusion of the innocuity test (also referred to as the abnormal toxicity test 25

or general safety test) in routine lot release testing requirements for all vaccines in WHO 26

Recommendations, Guidelines and other guidance documents for biological products (31, 27

32). As this test is no longer recommended, it is not included in the routine testing 28

requirements for EV71 vaccines provided in the current document. 29

30

Terminology 31

32

The definitions given below apply to the terms as used in these WHO Recommendations. 33

These terms may have different meanings in other contexts. 34

35

Adjuvant: a vaccine adjuvant is a substance, or combination of substances, that is 36

used in conjunction with a vaccine antigen to enhance (for example, increase, accelerate, 37

prolong and/or possibly target) the specific immune response to the vaccine antigen and the 38

clinical effectiveness of the vaccine. 39

Adventitious agents: contaminating microorganisms of the cell culture, or source 40

materials used in its culture, that may include bacteria, fungi, mycoplasmas, transmissible 41

spongiform encephalopathy agents and endogenous and exogenous viruses that have been 42

unintentionally introduced into the manufacturing process. 43

WHO/BS/2020.2388

Cell bank: a collection of vials of cells of uniform composition (though not 1

necessarily clonal) derived from a single tissue or cell and used for the production of a 2

vaccine, either directly or via a cell bank system. 3

Cell seed: a quantity of well-characterized cells derived from a single tissue or cell of 4

human or animal origin and stored frozen in liquid nitrogen in aliquots of uniform 5

composition, one or more of which may be used for the production of a master cell bank. 6

EV71 antigen: the virus-specific antigen produced in infected cell cultures and 7

purified from such cultures. It can be assayed by methods such as enzyme-linked 8

immunosorbent assay (ELISA) using EV71-specific antibodies. The antigen may consist of 9

empty or full virus particles or both. The full and empty particles differ in their antigenic 10

reactivity and both may be present in the final vaccine. 11

Final bulk: a formulated vaccine preparation from which the final containers are 12

filled. The final bulk may be prepared from one or more purified antigen bulks formulated to 13

contain all excipients and homogenous with respect to composition. 14

Final lot: a collection of sealed final containers of finished vaccine that is 15

homogeneous with respect to the risk of contamination during the filling process. A final lot 16

must therefore have been filled from a formulated bulk in one continuous working session. 17

Immunogenicity: the capacity of a vaccine to induce antibody-mediated and/or cell-18

mediated immunity and/or immunological memory. 19

Master cell bank (MCB): a quantity of well-characterized cells of human or animal 20

origin derived from a cell seed at a specific population doubling level or passage level, 21

dispensed into multiple containers, cryopreserved and stored frozen under defined conditions 22

(such as the vapour or liquid phase of liquid nitrogen) in aliquots of uniform composition. 23

The MCB is prepared from a single homogeneously mixed pool of cells and is used to derive 24

all working cell banks. The testing performed on a replacement MCB (derived from the 25

same clone or from an existing master or working cell bank) is the same as for the initial 26

MCB, unless a justified exception is made. 27

Master seed lot: a quantity of virus suspension that has been processed at the same 28

time to ensure a uniform composition, and passaged for a specific number of times that does 29

not exceed the maximum approved by the NRA. It is characterized to the extent necessary to 30

support development of the working seed lot. 31

Purified inactivated bulk: a purified pool of virus harvests in which the virus has 32

been inactivated through the use of a validated method either before or after purification. It 33

may be prepared from one single harvest or a number of single harvests and may yield one 34

or more final bulks. 35

Seed lot: a preparation of live viruses constituting the starting material for the vaccine 36

antigen. A seed lot is of uniform composition (though not necessarily clonal), is derived from 37

a single culture process and is aliquoted into appropriate storage containers, from which all 38

future vaccine production will be derived either directly or via a seed lot system. 39

Single harvest: a quantity or suspension derived from a batch of production cells 40

inoculated with the same seed lot and processed together in a single production run. 41

Working cell bank (WCB): a quantity of cells of uniform composition derived from 42

one or more ampoules of the MCB at a finite passage level, stored frozen at −70 °C or below 43

WHO/BS/2020.2388

in aliquots, one or more of which are used for vaccine production. All containers are treated 1

identically and once removed from storage are not returned to the stock. 2

Working seed lot: a quantity of virus of uniform composition derived from the 3

master seed lot used at a passage level approved by the NRA for the manufacturing of 4

vaccine. 5

6

General considerations 7

8

HFMD was first reported in New Zealand in 1957 (33) and occurs mostly in young children, 9

with a peak incidence at about 2 years of age. The common mild disease involves lesions on 10

the mucosal surfaces of the mouth and spots on the palms of the hands and soles of the feet 11

which resolve in a few days; this is not life threatening. However, a more severe and 12

potentially fatal form of the disease was reported in 1969 (1) which is now recognized to 13

encompass meningitis/encephalitis, autonomic nervous system dysregulation, cardiovascular 14

collapse and pulmonary oedema. The overall mortality rate of HFMD is of the order of one 15

per 1000 to 10 000 cases. 16

17

The frequency of reported HFMD cases is geographically highly variable with most cases 18

occurring in East Asian countries, particularly China but including Viet Nam, Thailand, 19

Singapore, Malaysia and the Republic of Korea. Normally, only few cases of severe disease 20

are reported in Europe or the USA with reports of mild HFMD also being less common – 21

though underreporting of the latter is very likely. Typically, the total combined number of 22

cases in Europe and the USA is of the order of several hundred per year, whereas in 2008 23

there were 488 955 cases and 128 deaths in China alone (4, 34, 35). HFMD is now a 24

reportable disease in China (in contrast to Europe and the USA) and during the period 2013–25

2018 between 300 000 and 400 000 cases occurred in May–June each year, with a small 26

number of deaths (4, 34, 35). The reason for the differences in disease burden in different 27

geographical areas is not yet clear. 28

29

The causative agents of HFMD are picornaviruses, most often of human enterovirus species 30

A. The picornaviruses belong to the Picornaviridae, a family of small non-enveloped viruses 31

with a single-stranded positive-sense RNA genome of about 7500 nucleotides. The 32

enteroviruses are a recognized taxon by the International Committee on Taxonomy of Viruses 33

and are sub-classified into numerous species including the four human enteroviruses A, B, C 34

and D (36). The archetypal human picornavirus is poliovirus (belonging to species C) but the 35

most common cause of HFMD is species A enteroviruses, chiefly the Coxsackie A viruses 36

and EV71. The relative frequency of different enterovirus species found varies from year to 37

year when clinical isolates or environmental samples such as sewage are examined. 38

39

In recent years, Coxsackie viruses such as A16, A10 and A6 have caused most of the HFMD 40

cases in Asia. However, other human enteroviruses of the Picornaviridae family have also 41

being implicated and as the different serotypes are antigenically distinct, the development of 42

a vaccine based on a single serotype to protect against all mild HFMD cases is unlikely using 43

existing technologies. There is therefore interest in the development of combination vaccines 44

WHO/BS/2020.2388

containing several serotypes. However, while mild HFMD is caused by many strains of 1

enterovirus, the great majority of severe disease in recent years has been caused by EV71 2

which accounts for 70% of severe HFMD cases and 90% of HFMD-related deaths in China 3

(35). As a result, EV71 has been the main focus of vaccine development. 4

5

EV71 isolates can be clustered according to their genomic sequence into at least eight 6

genogroups (A–H) (36, 37) but belong to one serotype. Genogroups B and C have been of 7

greatest interest because of their frequency of isolation and implication in disease in Eastern 8

Asia, and can each be sub-classified into five subgenogroups (C1–C5 and B1–B5). C4 is by 9

far the major genogroup circulating in China, while B4, B5 and C5 are found in other Asian 10

countries. In contrast, strains of genogroups C1 and C2 are predominantly found in Europe 11

where severe disease is uncommon. It is possible that this has some effect on the disease 12

burden, with C4 being particularly virulent. However, an outbreak of HFMD with severe 13

disease caused by a C1 genogroup strain occurred in Spain in 2016 (38, 39). 14

15

A valid animal model would be useful in vaccine development to measure protective efficacy 16

and potency, as well as to resolve issues related to virulence. As yet, the available models are 17

imperfect. Neonatal mice are susceptible to EV71 by intracerebral inoculation and neonatal 18

(but not adult) rhesus monkeys develop symptoms of HFMD on infection. Adult or infant 19

mice are not susceptible to infection. Infant rhesus monkeys have been demonstrated to 20

develop HFMD symptoms upon inoculation with the virus and could therefore be used as a 21

model of protection (40, 41). The neurovirulence of EV71 was demonstrated in cynomolgus 22

monkeys and this model would be useful for challenge-protection studies for candidate EV71 23

vaccines (42, 43). Picornaviruses are believed to use specific receptors to infect human cells. 24

Human P-selectin glycoprotein ligand-1 (PSGL-1) is expressed in leukocytes and involved in 25

their binding to endothelial cells in the early stages of inflammation, and has been identified 26

as a receptor for EV71. However, the disease produced by clinical EV71 strains in transgenic 27

mice carrying PSGL-1 was not enhanced compared to non-transgenic strains. Human 28

scavenger receptor class B member 2 (SCARB2) has also been identified as a receptor for 29

EV71. Transgenic mice carrying the SCARB2 gene are more susceptible to infection and 30

disease than non-transgenic controls but the effect is not dramatic – two-week old transgenic 31

mice develop mild symptoms and then recover (44–47). 32

33

Three vaccines against EV71 have been licensed in China, all using C4 genogroup strains. 34

Candidate vaccines containing B4 and B5 genogroups are in development elsewhere but have 35

not yet reached the licensing stage. In addition, the development of vaccines against 36

Coxsackie A16, A6 and A10 is being considered with a view to developing 37

combination/multivalent vaccines. The efficacy of the three licensed EV71 vaccines after two 38

doses ranges from 90.0% to 97.4% after one year of surveillance (5–7) to 95.1% after two 39

year follow-up (8). 40

41

Licensed EV71 vaccines are produced from virus grown on mammalian cells and inactivated 42

by validated methods – similar to the approach used for IPVs and hepatitis A vaccines. Tissue 43

culture grown virus harvests comprise two types of particle forms; one containing the RNA 44

WHO/BS/2020.2388

genome (full) and one that does not (empty). In the case of both polioviruses and EV71 1

viruses, the two particle forms have different antigenic and immunogenic properties; 2

poliomyelitis vaccines are purified so that they contain little if any empty virus particles but 3

EV71 vaccines contain both types; potentially complicating potency assays. The atomic 4

structures of both full and empty particles of EV71 viruses and polioviruses have been 5

resolved by X-ray crystallography and cryogenic electron microscopy (48, 49). 6

7

Specific issues in the development of inactivated EV71 vaccines include: 8

9

▪ The degree to which a vaccine based on one genogroup will protect against the others 10

is not established. Although there is good cross-neutralization between genogroups, 11

including by sera induced by vaccination (50, 51), it has not been established that this 12

translates into good cross-protection in humans. One recent collaborative study 13

indicated that assays of antigen content work acceptably on all genogroups tested. 14

However clinical cross-protection has not been demonstrated. Thus, the C4 15

genogroup vaccines may or may not protect against other genogroups. 16

▪ There is a lack of a convenient and convincing animal model, with the model most 17

accurately reflecting human disease at present being infant rhesus monkeys. This 18

makes the study of protective efficacy and immunogenic potency difficult other than 19

by clinical trial. Neonatal mice are susceptible to disease and transgenic mice 20

carrying the SCARB2 gene which encodes EV71 receptors have been developed and 21

can prove useful without fully imitating human pathogenesis. 22

▪ Virological issues include the existence of full and empty particle forms in the 23

licensed products. These two particle forms differ in their antigenic and 24

immunogenic properties, thus complicating potency assays. It is not clear whether 25

current national and international vaccine reference standards and antigen potency 26

assays are suitable for specifically detecting one or other of the particle forms (52). 27

28

International reference materials 29

30

Subsequent sections of this document refer to WHO reference materials that may be used in 31

laboratory or clinical evaluations. The following key standards are currently used in the 32

control of EV71 vaccines. 33

34

▪ A WHO international standard for anti-EV71 serum is available for the 35

standardization of diagnostic tests for use in seroprevalence studies and for assessing 36

immunity. This standard was established by the WHO Expert Committee on 37

Biological Standardization in 2015 as the First WHO International Standard for anti-38

EV71 serum (human) (NIBSC code 14/140) with an assigned value of 39

1000 IU/ampoule (53, 54). The preparation is held and distributed by NIBSC and 40

NIFDC. 41

▪ A WHO international reference reagent for EV71 neutralization assays (low-titre) is 42

also available for use in the standardization of virus neutralization assays. This 43

WHO/BS/2020.2388

reference reagent was established by the Committee in 2015 as the First WHO 1

International Reference Reagent for EV71 neutralization assays (NIBSC code 2

13/238) with an assigned value of 300 IU/ampoule (53, 54). The preparation is held 3

and distributed by NIBSC and NIFDC. 4

▪ In 2019, the First WHO International Standard for EV71 inactivated vaccine (NIBSC 5

code 18/116) was established by the Committee with an assigned unitage of 6

3625 IU/ampoule (12, 13). In addition, WHO international reference reagents for 7

EV71 genogroups C4 and B4 inactivated vaccine (NIBSC codes 18/120 and 18/156 8

respectively) were established with assigned unitages of 300 and 250 IU/ampoule 9

respectively (12, 13). These preparations are held and distributed by NIBSC. The 10

First WHO International Standard for EV71 inactivated vaccine is intended for use in 11

in vitro assays to measure the antigen content of vaccine products through the 12

calibration of secondary reference preparations. However, it is known that full and 13

empty virus particles (known to be present in EV71 vaccine preparations from all 14

manufacturers) differ in their antigenicity and immunogenicity. The proportion of 15

empty/full virus particles in the WHO international standard is not known. It is also 16

not known whether this matters for the overall assessment of vaccines or if the 17

current international standard is suitable for accurately measuring antigen content 18

across manufacturers. International standards and reference reagents for the control 19

of in vivo potency assays are under investigation (13). 20

▪ Product-specific national standards for EV71 neutralizing antibody and EV71 antigen 21

were established by NIFDC following collaborative studies conducted by NIFDC and 22

the three main vaccine manufacturers in China (14). This has helped to ensure the 23

accuracy, comparability and repeatability of anti-EV71 neutralizing antibody and 24

EV71 antigen detection assays, and hence improve EV71 vaccine standardization. In 25

addition, NIFDC has also developed a new national reference for in vivo vaccine 26

potency. These preparations are held and distributed by NIFDC. 27

28

Part A. Manufacturing recommendations 29

30

A.1 Definitions 31

32

A.1.1 International name and proper name 33

34

The international name of the vaccine should be “enterovirus 71 vaccine (inactivated)”. The 35

proper name should be the equivalent of the international name in the language of the country 36

of origin. 37

38

The use of the international name should be limited to vaccines that meet the specifications 39

given below. 40

41

A.1.2 Descriptive definition 42

43

WHO/BS/2020.2388

An EV71 vaccine (inactivated) consists of a sterile suspension of EV71 grown in cell 1

cultures, concentrated, purified and inactivated. The antigen may be formulated for delivery 2

with a suitable adjuvant. The preparation should meet all of the specifications given below. 3

4

A.2 General manufacturing recommendations 5

6

The general guidance provided in WHO good manufacturing practices for pharmaceutical 7

products: main principles (55) and WHO good manufacturing practices for biological 8

products (27) should be applied to the design, establishment, operation, control and 9

maintenance of manufacturing facilities for EV71 vaccine. 10

11

Staff involved in the production and quality control of inactivated EV71 vaccine should be 12

shown to be immune to EV71. 13

14

A.3 Control of source materials 15

16

A.3.1 Virus strains and seed lot system 17

18

A.3.1.1 Virus strains 19

20

Strains of EV71 used in the production of EV71 vaccine should be identified by historical 21

records, which should include information on their origin and subsequent manipulation or 22

passage (for example, the genogroup and subgenogroup of EV71). Strain identity should be 23

determined by infectivity tests and immunological methods, and by full or partial genomic 24

sequencing. 25

26

Only virus strains that are approved by the NRA and that yield a vaccine complying with the 27

recommendations set out in these WHO Recommendations should be used. 28

29

A.3.1.2 Virus seed lot system 30

31

Vaccine production should be based on the virus seed lot system. Unless otherwise justified 32

and authorized, the virus in the final vaccine should not have undergone more passages from 33

the virus master seed lot than were used to prepare a vaccine shown to be satisfactory with 34

respect to safety and efficacy. 35

36

Virus master and working seed lots should be stored in dedicated temperature-monitored 37

freezers at a temperature that ensures stability during storage (for example, at or below 38

−60 °C). 39

40

A.3.1.3 Tests on virus master and working seed lots 41

42

WHO/BS/2020.2388

Each virus master and working seed lot used for the production of vaccine lots should be 1

subjected to the tests listed in this section. 2

3

Each virus master and working seed lot should have been derived from materials that comply 4

with the recommendations made in sections A.3.2 and A.3.3 below and should be approved 5

by the NRA. 6

7

A.3.1.3.1 Tests for adventitious agents 8

9

The virus master and working seed lots used for the production of vaccine lots should be free 10

from adventitious agents. 11

12

A sample of at least 20 mL of each virus master and working seed lot should be tested for the 13

presence of adventitious agents. The sample should be neutralized by a high-titred antiserum 14

against EV71. 15

16

If polyclonal antisera are used, the immunizing antigen used for the preparation of the antiserum should 17 not be the same as the production seed. 18 19 The immunizing antigen should be shown to be free from adventitious agents and should be grown in 20 cell cultures free from adventitious microbial agents that might elicit antibodies that could inhibit the 21 growth of any adventitious agents present. 22

23

The sample should be tested in susceptible cells such as Vero, human rhabdomyosarcoma 24

(RD) or human diploid cells. The tissue cultures should be incubated at 37 °C and observed 25

for 2 weeks. Within this observation period, at least one subculture of supernatant fluid 26

should be made in the same tissue culture system. The sample should be inoculated in such a 27

way that the dilution of the supernatant fluid in the nutrient medium does not exceed 1 in 4. 28

The area of the cell sheet should be at least 3 cm2 per mL of supernatant fluid. At least one 29

culture vessel of the cell cultures should remain uninoculated and should serve as a control. 30

The cells inoculated with the supernatant fluid and the uninoculated control cultures should 31

be incubated at 37 °C and observed at appropriate intervals for an additional 2 weeks. 32

33

The virus master and working seed lots pass the test if there is no evidence of the presence of 34

adventitious agents. For the test to be valid, not more than 20% of the culture vessels should 35

have been discarded for any reason by the end of the observation period. 36

37

New molecular methods with broad detection capabilities are being developed for the detection of 38 adventitious agents. These methods include: (a) degenerate NAT for whole virus families, with analysis 39 of the amplicons by hybridization, sequencing or mass spectrometry; (b) NAT with random primers 40 followed by analysis of the amplicons on large oligonucleotide micro-arrays of conserved viral 41 sequencing, or digital subtraction of expressed sequences; and (c) high-throughput sequencing. These 42 methods might be used in the future to supplement existing methods or as alternative methods to both 43 in vivo and in vitro tests after appropriate validation and with the approval of the NRA (28). 44 45

WHO/BS/2020.2388

The theoretical risk of the presence of potential human, simian, bovine or porcine adventitious agents in 1 the seed lots, which may be derived from the use of bovine serum or porcine trypsin, should be 2 assessed. If necessary, viruses such as bovine polyomavirus, porcine parvovirus or porcine circovirus 3 may be screened for using specific assays, such as molecular NAT-based assays (28). 4

5

A.3.1.3.2 Identity test 6

7

The strain identity of the seed lot should be determined by infectivity tests. The test for 8

antigen content described in section A.4.4.2.2 below can be used to identify the seed lot. 9

10

A.3.1.3.3 Virus titration 11

12

The virus concentration of the seed lot should be determined by titration of infectious virus 13

using validated tissue culture methods. This titration should be carried out in not more than 14

10-fold dilution steps using 10 cultures per dilution, or by any other arrangement yielding 15

equal precision. 16

17

The use of human RD, human diploid or Vero cells in microtitre plates is suitable for this purpose (28). 18

19

A.3.2 Cell lines 20

21

The general production precautions, as formulated in WHO good manufacturing practices for 22

biological products (27), should apply to the manufacture of EV71 vaccine, with the 23

additional requirement that, during production, only one type of cell should be introduced or 24

handled in the production area at any one time. Vaccines may be produced in a human 25

diploid cell line or in a continuous cell line. 26

27

A.3.2.1 Master cell bank (MCB) and working cell bank (WCB) 28

29

The use of a cell line for the manufacture of EV71 vaccine should be based on the cell bank 30

system. The cell seed and cell banks should conform to WHO Recommendations for the 31

evaluation of animal cell cultures as substrates for the manufacture of biological medicinal 32

products and for the characterization of cell banks (28). The MCB should be approved by the 33

NRA. The maximum number of passages (or population doublings) by which the WCB is 34

derived from the MCB and the maximum number of passages of the production cultures 35

should be established by the manufacturer and approved by the NRA. 36

37

The WHO Vero reference cell bank 10-87 is considered suitable for use as a cell seed for generating an 38 MCB (56) and is available to manufacturers on application to the Group Lead, Norms and Standards 39 for Biologicals, Technical Specifications and Standards, Department of Health Product Policy and 40 Standards, Access to Medicines and Health Products Division, World Health Organization, Geneva, 41 Switzerland. 42

43

A.3.2.2 Identity test 44

45

WHO/BS/2020.2388

Identity tests on the MCB and WCB should be performed in accordance with WHO 1

Recommendations for the evaluation of animal cell cultures as substrates for the manufacture 2

of biological medicinal products and for the characterization of cell banks (28) and should be 3

approved by the NRA. 4

5

The cell banks should be identified by means of tests such as biochemical tests (for example, 6

isoenzyme analysis), immunological tests, cytogenetic marker tests and DNA fingerprinting 7

or sequencing. The tests used should be approved by the NRA. 8

9

A.3.3 Cell culture medium 10

11

Where serum is used for the propagation of cells it should be tested to demonstrate freedom 12

from bacterial, fungal and mycoplasmal contamination – as specified in Part A, sections 5.2 13

(57) and 5.3 (58) of the WHO General requirements for the sterility of biological substances 14

– as well as freedom from infectious viruses. Suitable tests for detecting viruses in bovine 15

serum are given in Appendix 1 of the WHO Recommendations for the evaluation of animal 16

cell cultures as substrates for the manufacture of biological medicinal products and for the 17

characterization of cell banks (28). 18

19

Validated molecular tests for bovine viruses may replace the cell culture tests of bovine sera 20

if approved by the NRA. As an additional monitor of quality, sera may be examined for 21

freedom from bacteriophage and endotoxin. Gamma irradiation may be used to inactivate 22

potential contaminant viruses, while recognizing that some viruses are relatively resistant to 23

gamma irradiation. 24

25

The source(s) of animal components used in the culture medium should be approved by the 26

NRA. The components should comply with the current WHO guidelines on transmissible 27

spongiform encephalopathies in relation to biological and pharmaceutical products (59). The 28

serum protein concentration should be reduced by rinsing the cell cultures with serum-free 29

medium and/or by purification of the virus harvests. 30

31

In some countries, control tests are carried out to detect the residual animal serum content in the final 32 vaccine. 33

34

Human serum should not be used. If human serum albumin is used at any stage of product 35

manufacture, the NRA should be consulted regarding the requirements, as these may differ 36

from country to country. At a minimum, it should meet the WHO Requirements for the 37

collection, processing and quality control of blood, blood components and plasma derivatives 38

(60). In addition, human albumin and materials of animal origin should comply with the 39

current WHO guidelines on transmissible spongiform encephalopathies in relation to 40

biological and pharmaceutical products (59). 41

42

Manufacturers are encouraged to explore the possibilities of using serum-free media for the production 43 of EV71 vaccine. 44

WHO/BS/2020.2388

1

Penicillin and other beta-lactams should not be used at any stage of manufacture because they 2

are highly sensitizing substances in humans. Other antibiotics may be used during early 3

stages of production. In this case, the use of antibiotics should be well justified, and they 4

should be cleared from the manufacturing process at the stage specified in the marketing 5

authorization. Acceptable residual antibiotic levels should be approved by the NRA. 6

7

Bovine or porcine trypsin used for preparing cell cultures should be tested and found to be 8

free of cultivatable bacteria, fungi, mycoplasmas and infectious viruses, as appropriate (28). 9

The methods used to ensure this should be approved by the NRA. 10

11

In some countries, irradiation is used to inactivate potential contaminant viruses. If irradiation is used, 12 it is important to ensure that a reproducible dose is delivered to all lots and to the component units of 13 each lot. The irradiation dose must be low enough for the biological properties of the reagents to be 14 retained but also high enough to reduce virological risk. Therefore, irradiation cannot be considered a 15 sterilizing process (28). 16 17 Recombinant trypsin is available and should be considered; however, it should not be assumed to be 18 free from risk of contamination and should be subject to the usual considerations for any reagent of 19 biological origin (28). 20

21

The source(s) of trypsin of bovine origin, if used, should be approved by the NRA and should 22

comply with the current WHO guidelines on transmissible spongiform encephalopathies in 23

relation to biological and pharmaceutical products (59). 24

25

A.4 Control of vaccine production 26

27

A.4.1 Control cell cultures 28

29

A fraction of the production cell culture equivalent to at least 5% of the total or 500 mL of 30

cell suspension, or 100 million cells, at the concentration and cell passage level employed for 31

seeding vaccine production cultures, should be used to prepare control cultures. 32

33

If bioreactor technology is used, the NRA should determine the size and treatment of the cell 34

sample to be examined. 35

36

A.4.1.1 Tests of control cell cultures 37

38

The treatment of the cells set aside as control material should be similar to that of the 39

production cell cultures but they should remain uninoculated for use as control cultures for 40

the detection of any adventitious agents. 41

42

These control cell cultures should be incubated under conditions as similar as possible to the 43

inoculated cultures for at least 2 weeks, and should be tested for the presence of adventitious 44

WHO/BS/2020.2388

agents as described below. For the test to be valid, not more than 20% of the control cell 1

cultures should have been discarded for any reason by the end of the test period. 2

3

At the end of the observation period, the control cell cultures should be examined for 4

evidence of degeneration caused by an adventitious agent. If this examination or any of the 5

tests specified in this section shows evidence of the presence of any adventitious agent in the 6

control culture, the EV71 grown in the corresponding inoculated cultures should not be used 7

for vaccine production. 8

9

If not tested immediately, samples should be stored at −60 °C or below. 10

11

A.4.1.2 Tests for haemadsorbing viruses 12

13

At the end of the observation period, at least 25% of the control cells should be tested for the 14

presence of haemadsorbing viruses using guinea-pig red blood cells. If the latter cells have 15

been stored, the duration of storage should not have exceeded 7 days and the storage 16

temperature should have been in the range of 2–8 °C. In tests for haemadsorbing viruses, 17

calcium and magnesium ions should be absent from the medium. 18

19

Some NRAs require, as a test for haemadsorbing viruses, that other types of red blood cells, including 20 cells from humans, monkeys and chickens (or other avian species), are also used instead of guinea-pig 21 cells alone. 22

23

A reading should be taken after incubation at 2–8 °C for 30 minutes, and again after a further 24

incubation for 30 minutes at 20–25 °C. 25

26

If a test with monkey red blood cells is performed, readings should also be taken after a final incubation 27 for 30 minutes at 34–37 °C. 28 29 In some countries the sensitivity of each new lot of red blood cells is demonstrated by titration against a 30 haemagglutinin antigen before use in the test for haemadsorbing viruses. 31

32

A.4.1.3 Tests for other adventitious agents in cell supernatant fluid 33

34

At the end of the observation period, a sample of the pooled supernatant fluid from each 35

group of control cultures should be tested for adventitious agents. For this purpose, 10 mL of 36

each pool should be tested in the same cells, but not the same lot of cells, as those used for 37

the production of vaccine. 38

39

A second indicator cell line should be used to test an additional 10 mL sample of each pool. 40

When a human diploid cell line is used for production, a simian kidney cell line should be 41

used as the second indicator cell line. When a simian kidney cell line is used for production, a 42

human diploid cell line should be used as the second indicator cell line (28). 43

44

WHO/BS/2020.2388

The pooled fluid should be inoculated into culture vessels of these cell cultures in such a way 1

that the dilution of the pooled fluid in the nutrient medium does not exceed 1 part in 4. The 2

area of the cell sheet should be at least 3 cm2 per mL of pooled fluid. At least one culture 3

vessel of each kind of cell culture should remain uninoculated and should serve as a control. 4

5

The inoculated cultures should be incubated at the same temperature as that of the production 6

of virus antigen and observed at appropriate intervals for a period of at least 14 days. 7

8

Some NRAs require that, at the end of this observation period, a subculture is made in the same culture 9 system and observed for at least an additional 14 days. Furthermore, some NRAs require that these 10 cells should be tested for the presence of haemadsorbing viruses. 11

12

For the tests to be valid, not more than 20% of the culture vessels should have been discarded 13

for any reason by the end of the test period. 14

15

If any cytopathic changes due to adventitious agents occur in any of the cultures, the virus 16

harvests produced from the lot of cells from which the control cells were taken should be 17

discarded. 18

19

Some selected viruses may be screened for using specific validated assays which are 20

approved by the NRA, such as molecular NAT-based assays (28). 21

22

If these tests are not performed immediately, the samples should be kept at a temperature of 23

−60 °C or below. 24

25

A.4.1.4 Identity tests 26

27

At the production level, the control cells should be identified using tests approved by the 28

NRA. 29

30

Suitable methods include, but are not limited to, biochemical tests (for example, isoenzyme 31

analyses), immunological tests, cytogenetic tests (for example, for chromosomal markers), 32

morphological identification and tests for genetic markers (for example, DNA fingerprinting 33

or sequencing). 34

35

A.4.2 Cell cultures for vaccine production 36

37

A.4.2.1 Observation of cultures for adventitious agents 38

39

On the day of inoculation with the virus working seed lot, each cell culture or a sample from 40

each culture vessel should be examined visually for degeneration caused by infective agents. 41

If this examination shows evidence of the presence of any adventitious agent in a cell culture 42

then the culture should not be used for vaccine production. 43

44

WHO/BS/2020.2388

A.4.3 Control of single harvests 1

2

After inoculation of the production cells with virus, the culture conditions of inoculated and 3

control cell cultures should be standardized and kept within limits agreed with the NRA. 4

5

Samples required for the testing of single harvests should be taken immediately on 6

harvesting. 7

8

Samples may be taken after storage and filtration with the agreement of the NRA. 9

10

A.4.3.1 Identity test 11

12

The strain identity of the single harvest should be determined by infectivity tests. The test for 13

antigen content described in section A.4.4.2.2 below can be used to identify the single 14

harvest. 15

16

A.4.3.2 Sterility tests for bacteria, fungi and mycoplasmas 17

18

A volume of at least 10 mL of each single harvest should be tested for bacterial, fungal and 19

mycoplasmal contamination using appropriate tests, as specified in Part A, sections 5.2 (57) 20

and 5.3 (58) of the WHO General requirements for the sterility of biological substances, or by 21

methods approved by the NRA. 22

23

NAT alone or in combination with cell culture, with an appropriate detection method, may be used as 24 an alternative to one or both of the compendial mycoplasma detection methods following suitable 25 validation and the agreement of the NRA (28). 26 27 In some countries this test is performed on the purified virus harvest instead of on the single harvest. 28

29

A.4.3.3 Virus titration 30

31

The virus concentration of each single harvest should be determined by titration of infectious 32

virus using validated tissue culture methods to monitor production consistency and as a 33

starting point for monitoring the inactivation curve. This titration should be carried out in not 34

more than 10-fold dilution steps using 10 cultures per dilution, or by any other arrangement 35

yielding equal precision. 36

37

The use of human RD, human diploid or Vero cells in microtitre plates is suitable for this purpose (28). 38 The same cells should be used for virus titrations throughout the production process. 39 40 Information on virus titre will help in selecting single harvests that can be expected to meet potency 41 requirements after inactivation. 42 43 The virus titration may be carried out on the pooled harvest after demonstration of consistency of 44 production at the stage of the single harvest. 45

46

WHO/BS/2020.2388

A.4.4 Control of virus pools 1

2

Several single harvests may be mixed to prepare a pool of virus before inactivation. The order 3

in which the purification, filtration and inactivation of virus pools is conducted should be 4

carefully established by the manufacturer to ensure consistent full virus inactivation and 5

absence of residual infectivity. Based on experience of the production of IPVs, the WHO 6

Recommendations to assure the quality, safety and efficacy of poliomyelitis vaccines 7

(inactivated) (23) recommends purification, filtration and inactivation steps in that order. 8

9

The requirement for filtration before and during inactivation was introduced into the IPV production 10 process following the Cutter incident during which a number of paralytic polio cases occurred in 11 children following vaccination with a defective IPV (23). The vaccine used was later found to contain 12 aggregates which led to incomplete virus inactivation likely due to formaldehyde not accessing some 13 virus particles inside the aggregates. 14

15

Any deviation from the production sequence shown to be acceptable for IPV for a vaccine 16

against a virus similar to poliovirus (such as EV71) should be fully validated and justified in 17

terms of yielding a product of equivalent safety. 18

19

Inactivation of virus may be performed before or after purification according to current 20

approved procedures for the production of licensed vaccines. The method of purification and 21

inactivation as well as the agent used for inactivation should be appropriately validated and 22

should be approved by the NRA. 23

24

If inactivation of the virus pool is conducted after purification, please see section A.4.4.1 25

below. If inactivation of the virus pool is conducted before purification please see section 26

A.4.4.2 below. 27

28

A.4.4.1 Purification of virus pools 29

30

Each pool of virus should be purified. Removal of host cell protein should be assessed during 31

process validation (28). 32

33

An acceptable method is to clarify the virus suspension by filtration, to concentrate the virus by 34 ultrafiltration and, thereafter, to collect the virus peak after passing it through a gel-filtration column. 35 Further purification is achieved by passing the virus through an ion-exchange column. Other 36 purification procedures resulting in acceptable release criteria may be used. 37

38

A.4.4.2 Tests on virus pools (purified or not) before inactivation 39

40

A.4.4.2.1 Virus titration 41

42

The virus concentration of each virus pool should be determined by titration of infectious 43

virus using validated tissue culture methods. This titration should be carried out in not more 44

WHO/BS/2020.2388

than 10-fold dilution steps using 10 cultures per dilution, or by any other arrangement 1

yielding equal precision. 2

3

The use of human RD, human diploid or Vero cells in microtitre plates is suitable for this purpose (28). 4 The same cells should be used for virus titrations throughout the production process. 5 6 Information on virus titre will help in selecting pools that can be expected to meet potency 7 requirements following inactivation. 8

9

A.4.4.2.2 Virus antigen content 10

11

The antigen content of each virus pool should be determined using a validated 12

immunochemical method and should be calculated using a reference vaccine calibrated 13

against the First WHO International Standard for EV71 inactivated vaccine (see 14

International reference materials above) and expressed in IU. 15

16

A.4.4.2.3 Specific activity 17

18

The ratio of virus concentration or antigen content to the total protein content (specific 19

activity) of the virus pool before inactivation should be within the limits of material shown to 20

be safe and effective in clinical trials and approved by the NRA. This would ensure a 21

consistent ratio of chemical agent to the viral protein, and thus a consistent inactivation 22

process. 23

24

A. 4.4.3 Filtration before inactivation 25

26

In order to avoid interference with the inactivation process, virus aggregation should be 27

prevented or aggregates should be removed immediately before and during the inactivation 28

process. For this reason, each virus pool should be filtered before inactivation. 29

30

Satisfactory results have been reported with several filter types but a final filtration using a 0.22 µm 31 filter should be used. 32

33

Inactivation should be initiated as soon as possible and, in any case, not later than 72 hours 34

after filtration. 35

36

It is preferable to start inactivation within 24 hours of filtration. Since the purpose of the filtration step 37 is to remove particulate matter and other interfering substances that may diminish the effectiveness of 38 the inactivation process and since aggregates tend to increase on standing after filtration, efforts should 39 be made to keep within this time limit. 40

41

A sample of the filtered virus pool should be retained and its virus titre determined as 42

described in section A.4.4.2.1 above. 43

44

The main purpose of determining the titre of filtered virus pools destined for inactivation is to provide 45 the starting titre to monitor the kinetics of inactivation. 46

WHO/BS/2020.2388

1

A.4.5 Control of purified inactivated bulk 2

3

A.4.5.1 Inactivation procedure 4

5

The virus in the filtered pools should be inactivated using a validated method approved by the 6

NRA. Prior to inactivation, the concentration of the filtered pool (based on viral titre, virus 7

antigen or protein content) should be adjusted to the acceptable range established during the 8

process validation. 9

10

Most manufacturers currently use formaldehyde as the method for inactivation but at least one 11 manufacturer is using other inactivating agents such as beta-propiolactone (BPL). 12

13

The method of inactivation should have been shown to consistently inactivate EV71 virus 14

without destroying the antigenic and immunogenic activity. Inactivation of the virus pool 15

may take place before or after purification depending on the approved production process. 16

The progress of inactivation should be monitored by suitably spaced determinations of virus 17

titres. The inactivation period should usually exceed the time taken to reduce the titre of live 18

virus to undetectable amounts by a factor of at least 2 and be agreed to by the NRA. 19

20

A second filtration should be made during the process of inactivation. This step is made after 21

the virus titre has fallen below detectable levels but before the first sample for the safety test 22

is taken. 23

24

The kinetics of viral inactivation should be established by each manufacturer and approved 25

by the NRA. During the validation studies, an inactivation curve should be established with at 26

least four time points showing the decrease in live virus concentration with time. The 27

consistency of the inactivation process should be monitored; the virus titre and antigen 28

content of each pool before, during and at the end of inactivation should be determined. 29

30

A record of consistency in the effectiveness and kinetics of inactivation should be established 31

by the production of at least five consecutive lots and, if broken, a root-cause analysis should 32

be performed and a further five consecutive filtered purified virus pools should be prepared 33

and shown to be satisfactory for establishing this record. 34

35

A.4.5.2 Purification of inactivated virus pool 36

37

If inactivation is conducted using a non-purified virus pool, the inactivated pool should be 38

purified as described in section A.4.4.1 above. 39

40

A.4.5.3 Tests on purified inactivated bulk 41

42

A.4.5.3.1 Test for effective inactivation 43

44

WHO/BS/2020.2388

After removal or neutralization (as appropriate) of the inactivating agent, the absence of 1

residual live EV71 virus should be verified by inoculating a quantity of the inactivated virus 2

bulk equivalent to 5% of the lot (or not less than 1000 doses of vaccine) into sensitive cell 3

cultures of the same type as those used for vaccine production. 4

5

The virus sample should be incubated for no less than 21 days making no fewer than two cell 6

passages during that period. The dilution of the sample in the nutrient fluid should not exceed 7

1 in 4 and the area of the cell sheet should be at least 3 cm2 per mL of sample. One or more 8

culture vessels of each lot of cultures should be set aside to serve as uninoculated control 9

culture vessels with the same medium. The sensitivity of the assay should be demonstrated. 10

11

If formaldehyde has been used as the inactivating agent, samples of vaccine for tissue culture tests are 12 generally neutralized at the time of sampling by the addition of bisulfite. Usually, the samples are 13 subsequently dialysed or another validated method used. 14 15 It is possible to conduct tissue culture tests on non-dialysed material. However, this is often found to be 16 toxic to cells, even with a dilution of 1 in 4. If in such tests, nonspecific degeneration of cells occurs, or 17 if the sensitivity of the tissue culture system is reduced, the test should be repeated on dialysed 18 material. The virus antigen content after dialysis should be determined to ascertain whether the viral 19 antigen was lost during the dialysis process. 20

21

If infectious virus is detected, the bulk should not be used for further processing. The 22

isolation of live virus from an inactivated bulk should be regarded as a break in the 23

manufacturing consistency record and a production process review and revalidation should be 24

undertaken. 25

26

It is important to demonstrate that each test retains sensitivity to detect partially inactivated 27

EV71 virus. At the end of the observation period, the cell culture used for the detection of 28

residual live virus should be challenged with a validated amount of live EV71 virus of the 29

same strain as that of the inactivated virus bulk. The details of the challenge procedure should 30

be approved by the NRA. The ability to detect infectious virus should be checked 31

concurrently for each test by including a positive control at the beginning of each test. 32

Positive control flasks should be inoculated with a low quantity of virus close to the detection 33

limit of the method. Alternatively, if no positive control is used, a challenge test should be 34

performed as described above. 35

36

The problem of detecting residual active virus in an inactivated vaccine is not the same as 37

that of measuring infective virus in untreated suspensions. Other similar viruses that have 38

been exposed to the action of formaldehyde without becoming inactivated have been shown 39

to require a much longer period to produce cytopathic changes than untreated virus. For this 40

reason, it is desirable that tissue cultures in tests for the presence of residual active virus are 41

observed for as long a time as is technically possible. A satisfactory tissue culture system for 42

this purpose depends, therefore, not only on the sensitivity of the cells used for the 43

preparation of the cultures but also on the nutrient fluid. 44

45

WHO/BS/2020.2388

Serum added to the nutrient fluid should be tested for inhibitors to EV71 at serum concentrations up to 1 50%. Only serum free from inhibitors should be used. 2 3 Maintenance of the cultures in good condition may require frequent changes of culture medium. 4 However, it should be borne in mind that early changes of fluid may result in unadsorbed virus being 5 removed and the validity of the test would thus be impaired. Therefore, the fluid should be changed no 6 earlier than 5–7 days after inoculation. 7

8

A.4.5.3.2 Sterility tests for bacteria and fungi 9

10

Each purified inactivated bulk should be tested for bacterial and fungal sterility as specified 11

in Part A, section 5.2 of the WHO General requirements for the sterility of biological 12

substances (57), or by methods approved by the NRA. 13

14

A.4.5.3.3 Antigen content 15

16

The EV71 antigen content of each purified inactivated bulk should be determined by the use 17

of a validated immunochemical method and should be calculated using a reference vaccine 18

calibrated against the First WHO International Standard for EV71 inactivated vaccine (see 19

International reference materials above). The results obtained should be expressed in IU 20

and be within the required limits established by the NRA. 21

22

A.4.5.3.4 Residual inactivating agent 23

24

The concentration of free residual formaldehyde or any other chemical used for inactivating 25

the virus should be determined by a method approved by the NRA. The maximum acceptable 26

limit should be approved by the NRA. 27

28

If BPL is used then its hydrolysation kinetics should be determined during the validation of the 29 inactivation process to ensure that no residual BPL is present in the inactivated bulk at the end of the 30 inactivation step. 31

32

A.4.5.3.5 Residual cellular DNA 33

34

If continuous cell lines are used for production, the purification procedure should have been 35

shown to consistently reduce the level of residual host cell DNA (28). The amount and size of 36

residual host cell DNA should not exceed the maximum levels agreed with the NRA, taking 37

into consideration issues such as those discussed in the WHO Recommendations for the 38

evaluation of animal cell cultures as substrates for the manufacture of biological medicinal 39

products and for the characterization of cell banks (28). Human diploid cell lines have been 40

used successfully for many years for the production of viral vaccines and the residual cellular 41

DNA deriving from these cells is not considered to pose any significant risk (28). 42

43

This test can be performed on the purified virus bulk and may be omitted from routine testing, with the 44 agreement of the NRA, if the manufacturing process is validated to achieve this specification (28). 45 46

WHO/BS/2020.2388

If assessed, the size distribution of the DNA may be considered as a characterization test, taking into 1 account the amount of DNA detectable using appropriate methods, as approved by the NRA (28). 2

3

A.4.5.3.6 Residual chemicals 4

5

If chemical substances are used during the purification process, tests for these substances 6

should be carried out. Their concentration should not exceed the limits approved for the 7

particular product. 8

9

A.4.6 Control of final bulk 10

11

Preservatives, excipients or other substances that might be added to form the final bulk 12

should have been shown, to the satisfaction of the NRA, to have no deleterious effect on the 13

immunizing potency and safety profile of the EV71 antigens. Preservative efficacy should be 14

demonstrated during product development using a method approved by the NRA. 15

16

The operations necessary for preparing the final bulk from the purified inactivated bulk 17

should be conducted in such a manner as to avoid contamination of the product. In preparing 18

the final vaccine bulk, any substances that are added to the product (such as diluents, 19

stabilizers or adjuvants) should have been shown, to the satisfaction of the NRA, not to 20

impair the safety and efficacy of the vaccine in the concentrations used. Until the final bulk is 21

filled into containers, the final vaccine bulk suspension should be stored under conditions 22

shown by the manufacturer to retain the desired biological activity. 23

24

A.4.6.1 Sterility tests for bacteria and fungi 25

26

The final bulk should be tested for bacterial and fungal sterility as specified in Part A, section 27

5.2 of the WHO General requirements for the sterility of biological substances (57), or by 28


30

A.4.6.2 Potency tests 31

32

Each final bulk should be tested using an in vivo assay for immunogenicity and in vitro 33

antigen content assay approved by the NRA, unless this is to be performed on final product. 34

Product-specific reference preparations may be used in these tests. 35

36

The EV71 antigen content of each final bulk should be determined using a validated 37

immunochemical method and calculated using a reference vaccine calibrated against the First 38

WHO International Standard for EV71 inactivated vaccine (see International reference 39

materials above). The in vitro assay found to be the most suitable for measuring the antigen 40

content is the EV71 antigen ELISA. The results obtained should be within the required limits 41

established by the NRA. 42

43

WHO/BS/2020.2388

Once consistency of production has been established for a suitable number of consecutive 1

final bulks, the in vivo assay may be omitted for the purpose of routine lot release, with the 2

agreement of the NRA. This can occur once it has been demonstrated that the acceptance 3

criteria for the EV71 antigen determination are such that the in vitro test yields a comparable 4

result to the in vivo assay in terms of acceptance or rejection of a lot. This demonstration 5

should include testing of sub-potent lots, produced experimentally if necessary by heat 6

treatment or other means of diminishing the immunogenic activity. 7

8

If an adjuvant is used in the final bulk, a desorption or treatment step may be necessary 9

before performing the EV71 antigen ELISA. 10

11

If the final bulk is formulated with other antigens into a combination vaccine, the suitability 12

of performing the EV71 antigen ELISA on the final bulk will have to be determined. If the 13

EV71 antigen ELISA is not suitable for a particular combination, an in vivo assay should be 14

used. 15

16

The potency of the final bulk should be approved by the NRA. 17

18

A.4.6.3 Preservative content (if applicable) 19

20

If preservative is added, its concentration in the final bulk should be determined by a method 21

approved by the NRA. The preservative used and concentration permitted should be 22

approved by the NRA. The preservative should not adversely affect the quality of the 23

antigens. 24

25

A.4.6.4 Adjuvant (if applicable) 26

27

Each final vaccine bulk should be assayed for adjuvant content. This test may be omitted if it 28

is to be performed on the final lot. Where aluminium compounds are used, the aluminium 29

content should not exceed 1.25 mg per single human dose. 30

31

A.5 Filling and containers 32

33

The requirements concerning filling and containers given in WHO good manufacturing 34

practices for pharmaceutical products: main principles (55) and WHO good manufacturing 35

practices for biological products (27) should apply to vaccine filled in the final form. Single- 36

and multi-dose containers may be used. 37

38

Care should be taken to ensure that the materials of which the container and (if applicable) 39

the transference devices and closure are made do not adversely affect the quality of the 40

vaccine. 41

42

WHO/BS/2020.2388

Manufacturers should provide the NRA with adequate data to prove the stability of the 1

product under appropriate conditions of storage and transport. 2

3

A.6 Control tests on the final lot 4

5

Samples should be taken from each final lot for the tests described in the following sections. 6

The following tests should be performed on each final lot of vaccine (that is, in the final 7

containers). Unless otherwise justified and authorized, the tests should be performed on 8

labelled containers from each final lot by means of validated methods approved by the NRA. 9

All tests and specifications, including methods used and permitted concentrations, should be 10

approved by the NRA. 11

12

A.6.1 Inspection of final containers 13

14

Every container in each final lot should be inspected visually or mechanically, and those 15

showing abnormalities should be discarded and recorded for each relevant abnormality. A 16

limit should be established for the percentage of rejections permitted before triggering an 17

investigation of the cause, potentially resulting in lot failure. 18

19

A.6.1.1 Appearance 20

21

The appearance of the vaccine should be described with respect to its form and colour. 22

23

A.6.2 Identity test 24

25

An identity test should be performed on at least one labelled container from each final lot 26

using an appropriate method. The potency test described in section A.6.4 below may serve as 27

the identity test. 28

29

A.6.3 Sterility tests for bacteria and fungi 30

31

Each final lot should be tested for bacterial and fungal sterility as specified in Part A, section 32

5.2 of the WHO General requirements for the sterility of biological substances (57), or by 33


35

A.6.4 Potency test 36

37

The potency of each final lot should be determined using an in vivo assay and a validated in 38

vitro antigen content assay (see section A.4.6.2 above) if such a test has not been performed 39

on the final bulk. Potency should be calculated using a reference vaccine calibrated against 40

the First WHO International Standard for EV71 inactivated vaccine (see International 41

reference materials above) or other reference preparation. 42

43

WHO/BS/2020.2388

If the use of an adjuvant in the final bulk interferes with the assay, a desorption or treatment 1

step may be necessary. If treatment/desorption is not possible, the interference of the adjuvant 2

should be documented and an in vivo assay should be performed (see section A.4.6.2 above). 3

4

The potency of the vaccine should be approved by the NRA. 5

6

A.6.5 Preservative content 7

8

Where appropriate, the preservative content of each final lot should be determined. The 9

method used and content permitted should be approved by the NRA. This test may be omitted 10

if conducted on the final bulk. 11

12

A.6.6 Endotoxin content 13

14

The endotoxin content of each final lot should be determined using a method approved by the 15

NRA. Endotoxin levels should be consistent with levels found to be acceptable in vaccine 16

lots used in pre-licensure clinical trials and should be approved by the NRA. 17

18

A.6.7 Residual formaldehyde 19

20

The concentration of free residual formaldehyde in each final lot should be determined using 21

a method approved by the NRA. The acceptable maximum limit should be approved by the 22

NRA. This test may be omitted if performed on the final bulk. 23

24

A.6.8 pH 25

26

The pH of each final lot should be determined and should be within limits approved by the 27

NRA. 28

29

A.6.9 Adjuvant and degree of adsorption (if applicable) 30

31

If an adjuvant is used in the formulation, each final lot should be assayed for adjuvant 32

content. Where aluminium compounds are used, the aluminium content should not exceed 33

1.25 mg per single human dose. 34

35

The degree of adsorption of the antigen to the aluminium compounds (aluminium hydroxide 36

or hydrated aluminium phosphate) in each final lot should be assessed. 37

38

Both tests may be omitted for routine lot release upon demonstration of consistency of 39

production, subject to the agreement of the NRA. 40

41

A.6.10 Residual antibiotics (if applicable) 42

43

WHO/BS/2020.2388

If any antibiotics are added during vaccine production, the residual antibiotic content should 1

be determined and should be within limits approved by the NRA. This test may be omitted 2

for routine lot release once consistency of production has been established to the satisfaction 3

of the NRA. 4

5

If aluminium adsorption has an impact on the test, then testing for antibiotic content may be done at the 6 purified inactivated bulk stage. 7

8

A.6.11 Extractable volume 9

10

For vaccines filled into single-dose containers, the extractable content should be checked and 11

shown to be not less than the intended dose. 12

13

For vaccines filled into multi-dose containers, the extractable content should be checked and 14

should be shown to be sufficient for the intended number of doses. 15

16

A.7 Records 17

18

The requirements given in WHO good manufacturing practices for pharmaceutical products: 19

main principles (55) and WHO good manufacturing practices for biological products (27) 20

should apply. 21

22

A.8 Retained samples 23

24



should apply. 27

28

A.9 Labelling 29

30



should apply, and additionally the label on the container or package, or the leaflet 33

accompanying each container, should include the following information: 34

35

▪ the designation(s) of the strain(s) of EV71 contained in the vaccine 36

▪ the cell substrate used for the preparation of vaccine 37

▪ the antigen content 38

▪ the method and inactivating agent used to inactivate the virus 39

▪ the nature and amount of any stabilizer and preservative present in the vaccine 40

▪ the nature and amount of adjuvant, if applicable. 41

42

WHO/BS/2020.2388

It is desirable for the label to carry the names both of the producer and of the source of the bulk 1 material if the producer of the final vaccine did not prepare it. The nature and amount of antibiotics 2 present in the vaccine, if any, may also be included. 3

4

A.10 Distribution and transport 5

6



should apply. Further guidance is provided in the WHO Model guidance for the storage and 9

transport of time- and temperature-sensitive pharmaceutical products (61). 10

11

A.11 Stability testing, storage and expiry date 12

13

A.11.1 Stability testing 14

15

Adequate stability studies form an essential part of vaccine development. Current guidance 16

on the evaluation of vaccine stability is provided in the WHO Guidelines on stability 17

evaluation of vaccines (62). Stability testing should be performed at different stages of 18

production when intermediate product is stored, namely on single harvests, purified 19

inactivated bulk, final bulk and final lot. Stability-indicating parameters should be defined or 20

selected appropriately according to the stage of production. During vaccine production a 21

shelf-life should be assigned to all in-process materials – particularly intermediates such as 22

single harvests, purified inactivated bulk and final bulk. 23

24

The stability of the vaccine in its final containers, maintained at the recommended storage 25

temperature up to the expiry date, should be demonstrated to the satisfaction of the NRA. As 26

a guide, containers from at least three consecutive final lots derived from different bulks may 27

be tested. 28

29

Accelerated stability tests may be undertaken to provide additional information on the overall 30

characteristics of the vaccine and may also aid in assessing comparability should the 31

manufacturer decide to change any aspect of manufacturing. 32

33

The formulation of the vaccine should be stable throughout its shelf-life. Acceptable limits 34

for stability should be agreed with the NRA. Following licensure, ongoing monitoring of 35

vaccine stability is recommended to support shelf-life specifications and to refine the stability 36

profile (62). Data should be provided to the NRA in accordance with local regulatory 37

requirements. 38

39

The final stability testing programme should be approved by the NRA and should include an 40

agreed set of stability-indicating parameters, procedures for the ongoing collection and 41

sharing of stability data, and criteria for rejecting vaccine(s). 42

43

A.11.2 Storage conditions 44

WHO/BS/2020.2388

1

EV71 vaccine (inactivated) should be stored at all times at a temperature of 2–8 °C. 2

3

If a vaccine has been shown to be stable at temperature ranges higher than the approved 2–4

8 °C range, it may be stored under extended controlled temperature conditions for a defined 5

period, subject to the agreement of the NRA (63). 6

7

A.11.3 Expiry date 8

9

The expiry date should be based on the shelf-life as supported by stability studies and 10

approved by the NRA. The start of the dating period should be based on the date of blending 11

of the final bulk, the date of filling or the date of the first valid potency test on the final lot, 12

and should be agreed with the NRA. 13

14

Where an in vivo potency test is used, the date of the potency test is the date on which the test animals 15 were inoculated with the final bulk. 16

17

Part B. Nonclinical evaluation of enterovirus 71 vaccines (inactivated) 18

19

Nonclinical evaluation of a new EV71 vaccine should follow the principles outlined in the 20

WHO guidelines on nonclinical evaluation of vaccines (25) which provide details on the 21

design, conducting, analysis and evaluation of nonclinical studies. Further guidance on the 22

general principles for the nonclinical evaluation of vaccine adjuvants and adjuvanted 23

vaccines can be found in separate WHO Guidelines (29). The following sections B.1–B.3 24

provide specific guidance on addressing important issues related to the nonclinical 25

development of a new inactivated whole EV71 virus vaccine. 26

27

B.1 Product characterization and process development 28

29

The vaccine lots used in nonclinical studies should be adequately characterized as described 30

in Part A of these WHO Recommendations, taking into consideration the stage of product 31

development. Both the antigen(s) of the vaccine and the end product need to be clearly 32

defined, and the manufacturing process carefully monitored for all crucial steps so as to 33

confirm the consistency of production. It is essential that sufficient data are generated to 34

confirm the full inactivation of vaccine virus and the absence of virulent virus in the end 35

product. Furthermore, sufficient vaccine stability data are necessary to support its suitability 36

for use in the nonclinical studies. 37

38

It is crucially important that vaccine manufacturing processes are appropriately standardized 39

and controlled to ensure consistency of production. The extent of product characterization 40

may vary according to the stage of development. To support their validity, nonclinical studies 41

should be carried out on vaccine lots that are adequately representative of the concurrent 42

clinical lots in terms of their physicochemical data, stability, qualitative and quantitative 43

impurity profiles, and formulation. 44

WHO/BS/2020.2388

1

B.2 Nonclinical immunogenicity and protection studies 2

3

B.2.1 Evaluation of immunogenicity in animal models 4

5

Unless otherwise justified, the immunogenicity of any new EV71 vaccine needs to be 6

characterized in relevant animal models (for example, mice, rats or rabbits) before proceeding 7

to human trials. These proof-of-concept nonclinical studies should reflect the clinically 8

proposed use of the vaccine, including the administration route, and should include an 9

evaluation of serum neutralizing antibody response against isolated virus strains or 10

pseudoviruses (5–7, 64, 65), and dose-range testing of the antigen. The immune response to 11

the candidate vaccine should ideally be assessed after each dose of vaccine, and whenever 12

possible against a licensed EV71 vaccine used as an active control. Data on cross-neutralizing 13

antibodies should be obtained from nonclinical immunogenicity studies using a range of 14

isolated heterologous viruses or pseudoviruses of different subgenogroups. These data may 15

guide selection of the doses, dosing regimen and administration route to be evaluated in 16

clinical trials. 17

18

When a candidate EV71 vaccine is formulated with a new adjuvant, a rationale for the 19

selection of the adjuvant should be provided and the benefit of its inclusion in the vaccine 20

formulation should be demonstrated by the immunogenicity data. 21

22

The immunogenicity studies in animals may additionally be considered, when appropriate, as 23

part of a comparability exercise to demonstrate the reproducibility of the manufacturing 24

process whenever major changes are introduced during the different stages of process 25

development or during the validation phase of a new candidate EV71 vaccine manufacturing 26

process. 27

28

B.2.2 Challenge-protection studies 29

30

Current evidence suggests that serological immune responses play an essential role in 31

mediating protection by formalin-inactivated whole EV71 virus vaccines. Animal studies 32

conducted in newborn mice, transgenic mice and nonhuman primates have demonstrated that 33

vaccination with inactivated EV71 vaccines induces protective immunity against EV71 and 34

that protection in challenged animals is primarily mediated by neutralizing antibodies (41, 47, 35

50, 66). Importantly, human efficacy trials conducted with several formalin-inactivated EV71 36

vaccines have shown a strong correlation between vaccine-induced serum neutralizing 37

antibodies and protection against EV71-associated diseases (5–7). Based on these 38

observations, it is considered that, for a similarly manufactured candidate EV71 vaccine, no 39

further challenge-protection studies in animal models need to be performed. 40

41

However, challenge-protection studies may be useful for any candidate EV71 vaccine based 42

on a novel production process or intended to have novel mechanisms of action. 43

WHO/BS/2020.2388

1

Since evidence from epidemiological studies and clinical trials for cross-protection against 2

EV71 disease has thus far been limited, any claims of cross-protection should in general be 3

supported by appropriate animal data. Specifically, challenge-protection studies should be 4

conducted in appropriate animal models to evaluate the potential for protection against 5

heterologous viruses of different genogroups, as this could indicate the breadth of protection. 6

7

B.3 Nonclinical safety studies 8

9

For a new EV71 vaccine based on inactivated whole EV71 virus, a repeat-dose toxicity study 10

in a relevant animal species is generally needed to assess potential local and systemic toxicity 11

and any other undesirable effects. Omission of standalone local tolerance and single-dose 12

toxicity studies is possible if assessment of acute toxic effects and local tolerance has been 13

incorporated into the repeat-dose toxicity study. 14

15

If the candidate vaccine contains a novel adjuvant, the principles set out in the WHO 16

Guidelines on the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines (29) 17

should be followed. For example, consideration should be given to assessing the toxicity of 18

the adjuvant alone. 19

20

For candidate EV71 vaccines manufactured using novel cell substrates, efforts should be 21

made during the nonclinical safety study to explore biomarkers indicative of potential allergic 22

reactions – for example, by measuring type 2 CD4+ T-helper cell responses. 23

24

Part C. Clinical evaluation of enterovirus 71 vaccines (inactivated) 25

26

C.1 Introduction 27

28

Clinical trials should adhere to the principles described in the WHO Guidelines for good 29

clinical practice (GCP) for trials on pharmaceutical products (67) and the WHO Guidelines 30

on clinical evaluation of vaccines: regulatory expectations (26). This section focuses only on 31

issues relevant or specific to the clinical development of inactivated EV71 vaccines. 32

33

At present, no efficacy data on cross-protection are available from completed clinical trials 34

with inactivated EV71 vaccines due to the distinct regional circulation of specific 35

subgenogroups. In addition, no internationally recognized immune correlate of protection 36

(ICP) or surrogate marker of protection has been established. Although immunogenicity 37

results from clinical trials suggest that a neutralizing antibody titre of 1:16 to 1:32 might be 38

related to protection, further analysis based upon the use of a scaled logit model has indicated 39

that significantly higher levels of neutralizing antibodies might be needed to achieve 40

protection (5–7, 51, 64). 41

42

WHO/BS/2020.2388

If cross-protection against heterologous EV71 viruses is to be claimed then appropriate 1

nonclinical and/or clinical studies should be conducted to evaluate the potential for such 2

cross-protection (see section B.2.1 above). In addition, data demonstrating the ability of 3

antibodies obtained from vaccinated individuals to neutralize various subgenogroups of EV71 4

viruses in vitro, including recently circulating isolates, are expected to be provided. 5

Continuous evaluation of protective vaccine efficacy post licensure is encouraged due to the 6

evolution of new EV71 strains or a rapid change of subgenogroups in different countries and 7

regions, which may result in outbreaks. Should new subgenogroups emerge in a country in 8

which the vaccine is licensed and used, further clinical investigation of potential cross-9

protection may be needed. 10

11

C.2 Assays 12

13

General guidance on the use and validation of assays for the evaluation of immune responses 14

is provided in section 5.3.3 of the WHO Guidelines on clinical evaluation of vaccines: 15

regulatory expectations (26). 16

17

Sections C.2.1 and C.2.2 provide specific guidance on the following assays relevant to the 18

investigation of immune responses to inactivated human EV71 vaccines in clinical trials and 19

to the confirmation of vaccine efficacy in pivotal studies respectively: 20

21

▪ serological assays for establishing the baseline serostatus of trial subjects and 22

evaluating the humoral immune response to vaccination (see also section C.3); and 23

▪ detection assays for laboratory confirmation of HFMD and herpangina caused by 24

EV71 infection, in vaccine efficacy trials (see also section C.4). 25

26

C.2.1 Serological assays 27

28

C.2.1.1 Functional antibody 29

30

The direct measurement of anti-EV71 neutralizing antibody is well established. Neutralizing 31

antibody has been estimated using methods such as plaque reduction neutralization assays 32

employing either isolated virus strains or pseudoviruses (5–7, 64, 65). Sponsors are 33

encouraged to develop high-throughput assays for anti-EV17 neutralizing antibody. These 34

assays should be standardized using the First WHO International Standard for anti-EV71 35

serum (human) and the First WHO International Reference Reagent for EV71 neutralization 36

assays (see International reference materials above). In addition, a reference neutralizing 37

antibody panel for the evaluation of neutralizing antibody responses has been established in 38

China (14). 39

40

C.2.2 Virus detection assays 41

42

WHO/BS/2020.2388

Since HFMD and herpangina can be caused by different human enteroviruses, including 1

EV71, Coxsackie and echoviruses, appropriate RNA or virus detection assays are required to 2

confirm the presence of EV71 in throat and vesicle swabs and/or stool samples (see section 3

C.4.2 below). International guidance on enterovirus diagnostics and characterization should 4

be taken into consideration (4). 5

6

For other enterovirus infections, laboratory confirmation of diagnosis based on cell culture, 7

virus isolation and virus identification remains a standard approach. The use of established 8

cell lines such as human RD or Vero cells is recommended for virus isolation. 9

10

Various quantitative polymerase chain reaction (PCR) assays are commercially available. 11

Although several EV71-specific PCR systems have been described, the ability of an assay to 12

reliably detect EV71 RNA from specific subgenogroups should be taken into account when 13

selecting the method to be used in clinical trials. In general, it is recommended that the 14

subgenogroup be determined based upon VP1 gene sequences. 15

16

Sponsors should provide full details of the methodology applied, and appropriate controls 17

should be used. 18

19

In addition, EV71 infection can be confirmed by the use of anti-EV71 immunoglobulin M 20

(IgM) assays. 21

22

C.3 Immunogenicity 23

24

C.3.1 Formulation, dose and regimen 25

26

C.3.1.1 Primary series 27

28

EV71 vaccines will be used mainly or exclusively in regions with relatively high rates of 29

clinically apparent infections. In naturally primed individuals the first dose of EV71 vaccine 30

may elicit large increments in antibody due to an anamnestic response. In contrast, multiple 31

doses of the same vaccine may be required to achieve similar antibody levels in EV71-naive 32

subjects. Since pre-vaccination testing for EV71 serostatus will not be practical in routine 33

use, it is important that the primary series should be selected on the basis of the immune 34

responses observed in subjects who were seronegative prior to vaccination. 35

36

In the absence of an internationally established ICP for EV71 – that is, one meeting the WHO 37

definition of an ICP (26) – the selection of the vaccine dose and regimen may be based on the 38

reaching of a plateau antibody response unless this is precluded by concerns over 39

reactogenicity. It is desirable that immunogenicity studies should explore the minimum 40

number of doses and the shortest dose interval(s) required to achieve a plateau immune 41

response. 42

43

WHO/BS/2020.2388

C.3.1.2 Cross-protection 1

2

The ability of a candidate EV71 vaccine to protect against a range of wild-type strains 3

covering the main EV71 genogroups may vary according to the vaccine strain used. For 4

example, lower cross-neutralization against an atypical C2-like strain was observed in 5

naturally infected EV71 patients (68) and in clinical trials using B4-based vaccine strains 6

(22). 7

8

In clinical trials in which vaccine-elicited antibody is determined against the antigen in the 9

vaccine, it is recommended that neutralizing activity is also measured using antigens derived 10

from a range of circulating wild-type EV71 strains from different (sub)genogroups. If marked 11

differences are observed in measured antibody levels using vaccine versus non-vaccine 12

strains, and/or by EV71 subgenogroup, it would be of particular interest to assess whether a 13

similar effect is observed for functional antibody levels in naturally infected individuals. 14

15

C.4 Efficacy 16

17

C.4.1 Requirement for a demonstration of vaccine efficacy 18

19

It is currently recommended that licensure of a candidate EV71 vaccine should be based on 20

evidence of its protective efficacy against clinically apparent HFMD and herpangina. The 21

following considerations apply: 22

23

▪ At the time of preparing these WHO Recommendations, three vaccines against 24

human EV71 had been licensed in one country (see General considerations above) 25

(4, 9, 17). 26

▪ These licensed vaccines are not yet widely used internationally. As a result, the use 27

of a control group that does not receive vaccination against EV71 is possible. 28

▪ In jurisdictions in which a licensed vaccine is available, it is possible that individual 29

NRAs may consider that licensure can be based on a trial that evaluates the efficacy 30

of the candidate vaccine relative to that of the licensed vaccine in a population 31

similar to that in which the efficacy of the licensed vaccine was established. 32

▪ The lack of an established ICP against EV71 does not rule out immunobridging a 33

candidate vaccine to a licensed vaccine that has been shown to be efficacious. 34

However, this approach is only possible if both vaccines contain the same antigen(s) 35

so that anti-EV71 neutralizing antibody immune responses can be compared directly. 36

In addition, the demonstration of efficacy of all three licensed vaccines was confined 37

to EV71 subgenogroup C4 and it is not known whether protective efficacy may vary 38

between genogroups circulating in different regions. 39

40

Taking these considerations into account, the focus of the following sections is on clinical 41

development programmes that include vaccine efficacy trials in which the control group does 42

not receive vaccination against EV71. However, most of the recommendations are also 43

WHO/BS/2020.2388

applicable to trials in which the control group receives a licensed vaccine against EV71. 1

Clinical programmes leading to licensure based on immunobridging are not addressed in the 2

following guidance. The general principles to be considered are discussed in sections 5.6.2 3

and 6.3.3 of the WHO Guidelines on clinical evaluation of vaccines: regulatory expectations 4

(26). 5

6

C.4.2 Considerations for efficacy trial design 7

8

C.4.2.1 Primary objective 9

10

The primary objective will be to demonstrate that the candidate vaccine protects against 11

clinically apparent (that is, symptomatic) HFMD and herpangina caused by EV71 infection 12

regardless of the genogroup (see section C.4.2.4). 13

14

▪ It is not required for efficacy to be shown against asymptomatic EV71 infection as 15

such infections are of no clinical significance. 16

▪ It is not required for vaccine efficacy trials to be powered to demonstrate genogroup-17

specific efficacy (see section C.4.2.2). 18

19

C.4.2.2 Trial sites 20

21

Efficacy trials will be conducted in endemic areas in which the estimated attack rate for 22

HFMD and herpangina due to EV71 infection is sufficient to complete enrolment into an 23

adequately powered vaccine efficacy trial within a reasonable time frame. Sites may be 24

chosen on the basis of available public health disease-surveillance data and/or pre-trial 25

evaluations of epidemiology conducted by the sponsor. In three prior efficacy trials (6–9) the 26

EV71 viruses that caused clinically apparent HFMD and herpangina were limited to strains of 27

the C4 subgenogroup circulating at the trial sites in the years in which they were conducted. 28

Sponsors are encouraged to consider selecting sites in a range of geographical areas in which 29

strains of different genogroups are circulating and/or to conduct separate vaccine efficacy 30

trials in regions with different genogroup distributions. 31

32

C.4.2.3 Subject selection criteria 33

34

Because of the age-dependent incidence and severity of EV71 infections it is likely that 35

vaccine efficacy trials will target infants and children. An upper age limit may be set 36

depending on the age-specific attack rates. 37

38

C.4.2.4 Primary end-point 39

40

In accordance with the recommended primary objective above, the primary end-point should 41

be clinically apparent HFMD or herpangina that is confirmed to be due to EV71 infection. 42

Sponsors could consider appointing an independent data-monitoring committee to review the 43

WHO/BS/2020.2388

data and determine which subjects meet the case definition to be counted in the primary 1

analysis. 2

3

C.4.2.4.1 Clinical features for the case definition 4

5

The clinical features that cause subjects to present to study site staff or to a local designated 6

health care facility for laboratory investigations for acute HFMD or herpangina should be 7

identified with the aim of capturing as many cases as possible while limiting unnecessary 8

investigations. On this basis, it is reasonable to define a possible case of HFMD or 9

herpangina requiring laboratory investigation as an illness presenting with febrile illness 10

accompanied by a papular or vesicular rash in the characteristic distribution on the oral 11

mucosa, hands, feet or buttocks. A severe case of HFMD should be defined as associated 12

with neurological, respiratory or circulatory complications as described by WHO (4). 13

14

C.4.2.4.2 Laboratory confirmation of HFMD or herpangina caused by EV71 infection 15

16

It is recommended that laboratory confirmation of HFMD and herpangina cases should be 17

conducted in a designated qualified central laboratory. If more than one central laboratory is 18

necessary for practical reasons, it is essential that all of the laboratories use identical 19

methodologies, and consideration should be given to testing a randomly selected subset of 20

samples at each laboratory to assess concordance. The laboratory methods used should be 21

validated. 22

23

The confirmation of EV71 as causative of the clinical picture should be based on any of the 24

following: 25

26

▪ detection of EV71 RNA in vesicle/throat swabs or in stool; 27

▪ virus isolation and analysis of VP1 sequence; 28

▪ detection of IgM against EV71 – which is often detectable at the time of onset of 29

clinical symptoms but which may peak after 1–2 weeks. 30

31

To avoid cases being missed, protocols should plan for appropriately timed repeat specimens 32

to be collected from individuals with a first negative test for EV71 RNA (for example, 3–7 33

days after the first sample). 34

35

Samples obtained at first presentation and repeat specimens should also be tested to detect 36

infection with other enteroviruses, such as Coxsackie and echoviruses, that can also cause 37

HFMD and which regularly co-circulate with EV71 in affected countries. 38

39

C.4.2.5 Primary, secondary and other analyses 40

41

In a vaccine efficacy trial, it may be permissible for the primary analysis to include only 42

confirmed cases of HFMD and herpangina caused by EV71 as follows: 43

44

WHO/BS/2020.2388

▪ in subjects who completed the vaccination series within predetermined visit 1

windows, if more than one dose is required; and 2

▪ with symptom onset occurring more than a defined period after the only or final dose 3

of the series, taking into account what is known about the timing of the post-dose 4

anti-EV71 IgG peak. 5

6

This approach gives the most optimistic estimation of vaccine efficacy. 7

8

If the primary analysis is confined to cases counted as described above, it is essential that 9

predefined secondary analyses are carried out to estimate vaccine efficacy based on 10

confirmed cases of clinically apparent HFMD and herpangina caused by EV71 infection, 11

defined and counted as follows: 12

13

▪ all cases in subjects who received at least one assigned dose as randomized, and 14

regardless of adherence to study visit windows; 15

▪ cases that occurred at any time after the last dose received (that is, counted from the 16

day of dosing) in those who completed the assigned number of doses; 17

▪ cases that occurred after each sequential dose, depending on the number of doses in 18

the series and counted from the day of dosing. 19

20

Vaccine efficacy should be explored according to EV71 genogroup if this is feasible, 21

depending on the numbers of cases that occur due to individual genogroups. 22

23

It is recommended that an additional analysis should explore any differences in clinical or 24

laboratory features (including severity) between cases that occur in the candidate vaccine 25

group and the control group (whether the control group receives placebo or a licensed vaccine 26

against EV71). The analysis should take into account whether the severity observed in 27

individual subjects could reflect coinfection with other enteroviruses. 28

29

C.4.2.6 Case ascertainment 30

31

It is recommended that an active case-ascertainment strategy is used throughout the time 32

frame of a vaccine efficacy trial. This is essential at least up to the time of the primary 33

analysis, which may be conducted after a specific number of total cases has been 34

accumulated or after a predefined period in which a sufficient number of cases are expected 35

to occur to estimate vaccine efficacy. 36

37

C.4.2.7 Duration of protection 38

39

While the primary analysis may lead to licensure, it is recommended that trials continue to 40

use active case ascertainment to follow up subjects for several years to provide data on 41

waning vaccine protection, without un-blinding of treatment assignment at the level of the 42

individual. These data can then be reported at some point after licensure of the vaccine and 43

WHO/BS/2020.2388

may point to a need for further doses to be administered at intervals to maintain protection, or 1

a need to change the vaccine strains used. 2

3

C.4.2.8 Vaccine effectiveness 4

5

The need for vaccine effectiveness studies should be established at the time of licensure. 6

7

If longer-term follow-up within a pre-licensure trial is not considered to be feasible, the 8

duration of vaccine protection should be investigated within a vaccine effectiveness study 9

and/or as part of routine disease surveillance conducted by public health authorities. 10

Furthermore, the efficacy of the vaccine against individual subgenogroups should be explored 11

as part of a vaccine effectiveness study and/or during routine disease surveillance. 12

13

C.5 Safety 14

15

Evaluation of the safety of candidate EV71 vaccines should be undertaken in accordance with 16

the recommendations made in section 7 of the WHO Guidelines on clinical evaluation of 17

vaccines: regulatory expectations (26). If the primary series consists of several vaccine doses 18

it is important to document whether reactogenicity increases with sequential doses. 19

Additionally, the safety of post-primary doses should be evaluated. There may be special 20

considerations for vaccine safety depending on the vaccine construct and the intended target 21

population. 22

23

If a candidate vaccine is evaluated in a large pre-licensure trial, and if the safety profile 24

documented during immunogenicity trials did not give rise to any major concerns, it may be 25

acceptable for a full assessment of safety (that is, including detailed documentation of local 26

and systemic reactogenicity, as well as all unsolicited adverse events) to be confined to a 27

randomized subset of the total subjects. Any serious adverse event occurring in any subject 28

enrolled at any of the trial sites should be documented. 29

30

Part D. Recommendations for NRAs 31

32

D.1 General recommendations 33

34

The guidance for NRAs and NCLs given in the WHO Guidelines for national authorities on 35

quality assurance for biological products (69) and WHO Guidelines for independent lot 36

release of vaccines by regulatory authorities (30) should be followed. These guidelines 37

specify that no new biological product should be released until consistency of lot 38

manufacturing and quality has been established and demonstrated by the manufacturer. 39

40

The detailed production and control procedures, as well as any significant changes in them 41

that may affect the quality, safety or efficacy of inactivated EV71 vaccines (70), should be 42

discussed with and approved by the NRA. For control purposes, the relevant international 43

WHO/BS/2020.2388

reference preparations currently in force should be obtained for the purpose of calibrating 1

national, regional and working standards as appropriate (71). The NRA may obtain from the 2

manufacturer the product-specific or working reference to be used for lot release. 3

4

Consistency of production has been recognized as an essential component in the quality 5

assurance of inactivated EV71 vaccines. The NRA should carefully monitor production 6

records and quality control test results for clinical lots, as well as for a series of consecutive 7

lots of the final product. 8

9

D.2 Official release and certification 10

11

A vaccine lot should be released only if it fulfils all national requirements and/or satisfies 12

Part A of these WHO Recommendations (30). 13

14

A summary protocol for the manufacturing and control of enterovirus 71 vaccines 15

(inactivated), based on the model summary protocol provided below in Appendix 1 and 16

signed by the responsible official of the manufacturing establishment, should be prepared and 17

submitted to the NRA in support of a request for the release of a vaccine for use. 18

19

A lot release certificate signed by the appropriate NRA official should then be provided if 20

requested by the manufacturing establishment, and should certify that the lot of vaccine meets 21

all national requirements and/or Part A of these WHO Recommendations. The certificate 22

should provide sufficient information on the vaccine lot, including the basis of the release 23

decision (by summary protocol review or independent laboratory testing). The purpose of this 24

official national lot release certificate is to facilitate the exchange of vaccines between 25

countries and should be provided to importers of the vaccines. A model NRA Lot Release 26

Certificate for enterovirus 71 vaccines (inactivated) is provided below in Appendix 2. 27

28

Authors and acknowledgements 29

30

The first draft of these WHO Recommendations was prepared by a WHO drafting group 31

comprising Dr E. Griffiths, Kingston upon Thames, the United Kingdom; Dr P. Minor, St 32

Albans, the United Kingdom; Dr Y. Sun, Paul-Ehrlich-Institut, Germany; Dr H. Meyer, Paul-33

Ehrlich-Institut, Germany; Dr J. Wang, National Institutes for Food and Drug Control, China; 34

Dr J. Martin, National Institute for Biological Standards and Control, the United Kingdom; 35

and Dr D. Lei, World Health Organization, Switzerland, taking into consideration the 36

discussions and consensus reached during a WHO working group meeting to develop WHO 37

Recommendations to assure the quality, safety and efficacy of enterovirus 71 vaccines 38

(inactivated), held in Shanghai, China, 11–12 September 2019 and attended by: Dr X. Chen 39

and Dr Z. Wang, Wuhan Institute of Biological Products Co. Ltd, China; Mr Z. Fu, Dr F. 40

Gao, Dr P. He, Ms Z. Jiang, Dr Q. Mao, Dr J. Wang, Dr Y. Wang and Dr M. Xu, National 41

Institutes for Food and Drug Control, China; Dr E. Griffiths, Kingston upon Thames, the 42

United Kingdom; Dr Y. Hu and Dr W. Meng, Sinovac Biotech Co., Ltd, China; Mrs T. 43

WHO/BS/2020.2388

Jivapaisarnpong, King Mongkut’s University of Technology Thonburi, Thailand; Dr E. Jung, 1

CJ HealthCare R&D Biomedicine, Republic of Korea; Dr J. Lee, Korea Centers for Disease 2

Control and Prevention, Republic of Korea; Dr Q. Li and Mr L. Yi, Institute of Medical 3

Biology Chinese Academy of Medical Sciences, China; Dr X. Li Shanghai Institute of 4

Biological Products Co. Ltd, China; Dr Z. Li and Mr J. Liu, Beijing Minhai Biotechnology 5

Co., Ltd, China; Dr J. Martin, National Institute for Biological Standards and Control, the 6

United Kingdom; Dr H. Meyer and Dr Y. Sun, Paul-Ehrlich-Institut, Germany; Dr P. Minor, 7

St Albans, the United Kingdom; Dr S. Phumiamorn, Institute of Biological Products, 8

Thailand; Dr J. Shin, World Health Organization, Regional Office for the Western Pacific, 9

Philippines; Mr Y. Tang, World Health Organization China Office, China; Dr H. Wang and 10

Dr H. Yang, Center for Drug Evaluation of the National Medical Products Administration, 11

China; and Dr D. Lei, World Health Organization, Switzerland. 12

13

The second draft of this document was prepared by Dr J. Martin, National Institute for 14

Biological Standards and Control, the United Kingdom; Dr H. Meyer, Paul-Ehrlich-Institut, 15

Germany; Dr E. Griffiths, Kingston upon Thames, the United Kingdom; Dr Y. Sun, Paul-16

Ehrlich-Institut, Germany; Dr J. Wang, National Institutes for Food and Drug Control, China; 17

and Dr D. Lei, World Health Organization, Switzerland, incorporating as appropriate 18

comments received from regulators, manufacturers and academia following public 19

consultation, and taking into consideration the discussions and consensus reached during a 20

WHO informal consultation on WHO Recommendations to assure the quality, safety and 21

efficacy of enterovirus 71 vaccines (inactivated), held via WebEx, 8–10 June 2020 and 22

attended by: Dr X. Chen, Mr C. Guo, Ms L. Li, Ms Q. Li, Ms W. Lv and Dr J. Shi, Wuhan 23

Institute of Biological Products Co. Ltd, China; Dr E. Griffiths, Kingston upon Thames, the 24

United Kingdom; Ms Y. Hu, Sinovac Biotech Co., Ltd, China; Mrs T. Jivapaisarnpong, King 25

Mongkut’s University of Technology Thonburi, Thailand; Dr E. Jung, CJ HealthCare R&D 26

Biomedicine, Republic of Korea; Dr H. Langar, World Health Organization, Regional Office 27

for the Eastern Mediterranean, Egypt; Dr Q. Li, Mr L. Yi and Ms J. Zou, Institute of Medical 28

Biology Chinese Academy of Medical Sciences, China; Dr Z. Li and Mr J. Liu, Beijing 29

Minhai Biotechnology Co., Ltd, China; Dr Z. Liang, Dr Q. Mao, Dr J. Wang, Dr Y. Wang 30

and Dr M. Xu, National Institutes for Food and Drug Control, China; Dr J. Martin, National 31

Institute for Biological Standards and Control, the United Kingdom; Dr S. Phumiamorn, 32

Institute of Biological Products, Thailand; Dr H. Shimizu, National Institute of Infectious 33

Diseases, Japan; Dr J. Shin, World Health Organization, Regional Office for the Western 34

Pacific, Philippines; Dr Y. Sun, Paul-Ehrlich-Institut, Germany; Mr Y. Tang, World Health 35

Organization China Office, China; and Dr I Knezevic and Dr D. Lei, World Health 36

Organization, Switzerland. 37

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components and plasma derivatives (Requirements for Biological Substances No. 27, 34

revised 1992). In: WHO Expert Committee on Biological Standardization: forty-third 35


Series, No. 840; 37

https://apps.who.int/iris/bitstream/handle/10665/39048/WHO_TRS_840_(part1).pdf?s38

equence=1, accessed 23 July 2020). 39

40

61. Model guidance for the storage and transport of time- and temperature-sensitive 41

pharmaceutical products. In: WHO Expert Committee on Specifications for 42

Pharmaceutical Preparations: forty-fifth report. Geneva: World Health Organization; 43


http://www.who.int/medicines/areas/quality_safety/quality_assurance/ModelGuidance45

ForStorageTransportTRS961Annex9.pdf, accessed 23 July 2020). 46

47

62. Guidelines on stability evaluation of vaccines. In: WHO Expert Committee on 48

Biological Standardization: fifty-seventh report. Geneva: World Health Organization; 49


WHO/BS/2020.2388

http://www.who.int/biologicals/vaccines/Annex_3_WHO_TRS_962-3.pdf?ua=1, 1


3

63. Guidelines on the stability evaluation of vaccines for use under extended controlled 4

temperature conditions. In: WHO Expert Committee on Biological Standardization: 5

sixty-sixth report. Geneva: World Health Organization; 2016: Annex 5 (WHO 6

Technical Report Series, No. 999; 7

http://www.who.int/biologicals/areas/vaccines/Annex_5_Guidelines_on_Stability_eva8

luation_vaccines_ECTC.pdf?ua=1, accessed 23 July 2020). 9

10

64. Gu W, Li J, Zhu F. Immunologic surrogate of protection for inactivated enterovirus 11

71 vaccines. J Public Health Emerg. 2017;1(2):27 12

(http://jphe.amegroups.com/article/view/3779/4568, accessed 23 July 2020). 13

14

65. Zhang H, An D, Liu W, Mao Q, Jin J, Xu L et al. Analysis of cross-reactive 15

neutralizing antibodies in human HFMD serum with an EV71 pseudovirus-based 16

assay. PLoS One. 2014;9(6):e100545 17


19

66. Dong C, Liu L, Zhao H, Wang J, Liao Y, Zhang X et al. Immunoprotection elicited by 20

an enterovirus type 71 experimental inactivated vaccine in mice and rhesus monkeys. 21

Vaccine. 2011;29(37):6269–75 (abstract: 22

https://www.sciencedirect.com/science/article/pii/S0264410X1100908X, accessed 23 23

July 2020). 24

25

67. Guidelines for good clinical practice (GCP) for trials on pharmaceutical products. In: 26

WHO Expert Committee on the Use of Essential Drugs: sixth report. Geneva: World 27

Health Organization; 1995: Annex 3 (WHO Technical Report Series, No. 850; 28

https://apps.who.int/iris/bitstream/handle/10665/37340/WHO_TRS_850.pdf?sequenc29

e=1, accessed 23 July 2020). 30

31

68. Huang Y, Lin T, Lin T, Wu H. Antigenic and genetic diversity of human enterovirus 32

71 from 2009 to 2012, Taiwan. PLoS One. 2013;8(11):e80942 33


35

69. Guidelines for national authorities on quality assurance for biological products. In: 36

WHO Expert Committee on Biological Standardization: forty-second report. Geneva: 37

World Health Organization; 1992: Annex 2 (WHO Technical Report Series, No. 822; 38

http://www.who.int/biologicals/publications/trs/areas/biological_products/WHO_TRS39

_822_A2.pdf, accessed 23 July 2020). 40

41

70. Guidelines on procedures and data requirements for changes to approved vaccines. In: 42

WHO Expert Committee on Biological Standardization: sixty-fifth report. Geneva: 43

World Health Organization; 2015: Annex 4 (WHO Technical Report Series, 993; 44

http://www.who.int/biologicals/vaccines/Annex4_Guidelines_changes_to_approved_45

vaccines_eng.pdf?ua=1, accessed 23 July 2020). 46

47

71. Recommendations for the preparation, characterization and establishment of 48

international and other biological reference standards (revised 2004). In: WHO Expert 49

Committee on Biological Standardization: fifty-fifth report. Geneva: World Health 50

WHO/BS/2020.2388


http://www.who.int/immunization_standards/vaccine_reference_preparations/TRS9322

Annex%202_Inter%20_biol%20ef%20standards%20rev2004.pdf?ua=1, accessed 23 3

July 2020). 4

5

6

WHO/BS/2020.2388

Appendix 1 1

2

Model summary protocol for the manufacturing and control of enterovirus 3


5

The following protocol is intended for guidance and indicates the minimum information that 6

should be provided by the manufacturer to the NRA. Information and tests may be added or 7

omitted as necessary with the approval of the NRA. 8

9

It is possible that a protocol for a specific product may differ in detail from the model 10

provided. The essential point is that all relevant details demonstrating compliance with the 11

licence and with the relevant WHO recommendations for a particular product should be 12

provided in the protocol submitted. 13

14

The section concerning the final product should be accompanied by a sample of the label and 15

a copy of the leaflet (package insert) that accompanies the vaccine container. If the protocol 16

is being submitted in support of a request to permit importation, it should also be 17

accompanied by a certificate for the lot release of enterovirus 71 vaccines (inactivated) (see 18

Appendix 2) from the NRA of the country in which the vaccine was produced or released, 19

stating that the product meets national requirements as well as the recommendations in Part A 20

of this document. 21

22

1. Summary information on final lot 23

24

International name of product: ________________________________________________ 25

Commercial/trade name: _____________________________________________________ 26

Product licence (marketing authorization) number: ________________________________ 27

Country: _________________________________________________________________ 28

Name and address of manufacturer: ____________________________________________ 29

Name and address of licence holder, if different: __________________________________ 30

Final packaging lot number: __________________________________________________ 31

Type of container: __________________________________________________________ 32

Number of containers in this final lot: __________________________________________ 33

Final container lot number: ___________________________________________________ 34

Nature of final product (adsorbed):_____________________________________________ 35

Preservative and nominal concentration: ________________________________________ 36

Volume of each single human dose: ____________________________________________ 37

Number of doses per final container: ___________________________________________ 38

WHO/BS/2020.2388

Virus strain: _______________________________________________________________ 1

Cell substrate used for production: _____________________________________________ 2

3

Summary of composition (include a summary of the qualitative and quantitative composition 4

of the vaccine per single human dose, including any adjuvant used and other excipients): 5

_________________________________________________________________________ 6

_________________________________________________________________________ 7

8

Shelf-life approved (months): _________________________________________________ 9

Date of manufacture: ________________________________________________________ 10

Expiry date: _______________________________________________________________ 11

Storage condition: __________________________________________________________ 12

13

2. Detailed information on manufacture and control 14

15

The following sections are intended for reporting the results of the tests performed during the 16

production of the vaccine, so that the complete document will provide evidence of consistency 17

of production. If any test had to be repeated, this must be indicated. Any abnormal results 18

must be recorded on a separate sheet. 19

20

Summary of source materials 21

22

Identity of seed lot strain used for vaccine production 23

Reference number of seed lot: ________________________________________________ 24

Date(s) of reconstitution (or opening) of seed lot container(s): _______________________ 25

26

Test for adventitious agents 27

Methods: _________________________________________________________________ 28

Result: ___________________________________________________________________ 29

Date: ____________________________________________________________________ 30

31

Identity of cell bank used for vaccine production 32

Reference number of cell bank: _______________________________________________ 33

Date(s) of reconstitution (or opening) of cell bank container(s): ______________________ 34

35

Identity test 36

Methods: _________________________________________________________________ 37

Result: ___________________________________________________________________ 38

Date: ____________________________________________________________________ 39

40

WHO/BS/2020.2388

Control of vaccine production 1

2

Control cell cultures 3

Tests on control cell cultures 4

Ratio of control to production cell cultures: ______________________________________ 5

Incubation conditions: _______________________________________________________ 6

Period of observation of cultures: ______________________________________________ 7

Dates observation started/ended: ______________________________________________ 8

Ratio or proportion of cultures discarded: _______________________________________ 9

Results of observation: ______________________________________________________ 10

11

Tests for haemadsorbing viruses 12

Quantity of cell tested: ______________________________________________________ 13

Method used: ______________________________________________________________ 14

Date of start of test-1: _______________________________________________________ 15

Date of end of test-1:________________________________________________________ 16

Results: __________________________________________________________________ 17

Date of start of test-2: _______________________________________________________ 18

Date of end of test-2:________________________________________________________ 19

Results: __________________________________________________________________ 20

21

Tests for adventitious agents on supernatant culture fluids 22

Method used: ______________________________________________________________ 23

Date of start of test: _________________________________________________________ 24

Date of end of test: _________________________________________________________ 25

Result: ___________________________________________________________________ 26

27

Identity test 28

Method used: ______________________________________________________________ 29



Result: ___________________________________________________________________ 32

33

34

Control of single harvests 35

Name of the culture medium: _________________________________________________ 36

Date of inoculation:_________________________________________________________ 37

Temperature of incubation: ___________________________________________________ 38

Microscopic observation _____________________________________________________ 39

Result: _____________________________________________________________ 40

Date: ______________________________________________________________ 41

Date of harvest: ____________________________________________________________ 42

Volume of harvest: _________________________________________________________ 43

WHO/BS/2020.2388

Yield (mg/mL): ____________________________________________________________ 1

2

Sterility tests for bacteria, fungi and mycoplasmas 3

Test for bacteria and fungi 4

Method: ____________________________________________________________ 5

Media: _____________________________________________________________ 6

Volume inoculated: ___________________________________________________ 7

Date of start of test: ___________________________________________________ 8

Date of end of test: ___________________________________________________ 9

Result: _____________________________________________________________ 10

11

Test for mycoplasmas (if applicable) 12

Method: ____________________________________________________________ 13

Volume inoculated: ___________________________________________________ 14

Date of start of test: ___________________________________________________ 15

Date of end of test: ___________________________________________________ 16

Result: _____________________________________________________________ 17

18

Virus titration 19

Method: ____________________________________________________________ 20

Reference lot no. _____________________________________________________ 21

Date: ______________________________________________________________ 22

Result: _____________________________________________________________ 23

24

25

Control of virus pool 26

Lot number of virus pool: ____________________________________________________ 27

Date of pooling: ___________________________________________________________ 28

Number of harvests: ________________________________________________________ 29

Volume(s), storage temperature, storage time and approved storage period: _____________ 30

_________________________________________________________________________ 31

32

Purification of virus pool (may be performed after inactivation) 33

Purification methods: _______________________________________________________ 34

Volume before purification: __________________________________________________ 35

Volume after purification:____________________________________________________ 36

Date: ____________________________________________________________________ 37

38

Tests on virus pool 39

Virus titration 40

Method: ____________________________________________________________ 41

Reference lot no. _____________________________________________________ 42

Date: ______________________________________________________________ 43

Result: _____________________________________________________________ 44

WHO/BS/2020.2388

1

Virus antigen content 2

Method: ____________________________________________________________ 3

Reference lot no. _____________________________________________________ 4

Date: ______________________________________________________________ 5

Result: _____________________________________________________________ 6

7

Specific activity 8

Virus antigen content: _________________________________________________ 9

Total protein content: _________________________________________________ 10

Specification: _______________________________________________________ 11

Date: ______________________________________________________________ 12

Result: _____________________________________________________________ 13

14

Inactivation of harvest pool (may be performed before purification) 15

Filtration before inactivation 16

Filtration method: ____________________________________________________ 17

Date: ______________________________________________________________ 18

Time of start of filtration: ______________________________________________ 19

Time of end of filtration:_______________________________________________ 20

21

Inactivation 22

Agent(s) and concentration of inactivation agent: ___________________________ 23

Temperature of inactivation: ____________________________________________ 24

Date of start of inactivation: ____________________________________________ 25

Virus antigen units at start of inactivation: _________________________________ 26

Date of taking first samples: ____________________________________________ 27

Date of completion of inactivation: ______________________________________ 28

Virus antigen units at end of inactivation: _________________________________ 29

30

Filtration during inactivation 31

Filtration method: ____________________________________________________ 32

Date: ______________________________________________________________ 33

Time of start of filtration: ______________________________________________ 34

Time of end of filtration:_______________________________________________ 35

36

37

Control of purified inactivated bulk 38

Test for effective inactivation (after removal/neutralization of inactivating agent) 39

Sample size tested: _________________________________________________________ 40

Dates of sampling (1–4): _____________________________________________________ 41

Test method: ______________________________________________________________ 42

Period of observation of cell cultures: __________________________________________ 43

Period of observation of subcultures: ___________________________________________ 44

WHO/BS/2020.2388



Result: ___________________________________________________________________ 3

4

Sterility tests for bacteria and fungi 5

Method: __________________________________________________________________ 6

Media: ___________________________________________________________________ 7

Volume inoculated: _________________________________________________________ 8



Result: ___________________________________________________________________ 11

12

Antigen content 13

Method: __________________________________________________________________ 14

Specification: _____________________________________________________________ 15

Date: ____________________________________________________________________ 16

Result: ___________________________________________________________________ 17

18

Test for residual inactivating agent 19

Method: __________________________________________________________________ 20

Specification: _____________________________________________________________ 21

Date: ____________________________________________________________________ 22

Result: ___________________________________________________________________ 23

24

Test for residual host-cell DNA (if applicable) 25

Method: __________________________________________________________________ 26

Specification: _____________________________________________________________ 27

Date: ____________________________________________________________________ 28

Result: ___________________________________________________________________ 29

30

Test for residual chemicals (if applicable) 31

Method: __________________________________________________________________ 32

Specification: _____________________________________________________________ 33

Date: ____________________________________________________________________ 34

Result: ___________________________________________________________________ 35

36

37

Control of final bulk 38

Identification (lot number): ___________________________________________________ 39

Date of manufacture/blending: ________________________________________________ 40

Volume(s), storage temperature, storage time and approved storage period: _____________ 41

_________________________________________________________________________ 42

43

44

WHO/BS/2020.2388

1

Blending: Prescription (per dose) Added 2

Virus antigen (IU or unit): ____________ ____________ 3

Adjuvant: ____________ ____________ 4

Preservative (specify): ____________ ____________ 5

Others (chemicals): ____________ ____________ 6

Final volume (mL): ____________ ____________ 7

8

9


Method: __________________________________________________________________ 11

Media: ___________________________________________________________________ 12




Result: ___________________________________________________________________ 16

17

Potency test 18

In vivo assay (may be performed at final bulk stage) 19

Species, strain, sex and weight specifications: ______________________________ 20

Number of mice tested: ________________________________________________ 21

Dates of vaccination, bleeding: __________________________________________ 22

Date of assay: _______________________________________________________ 23

Lot number of reference vaccine and assigned potency: ______________________ 24

Vaccine doses (dilutions) and number of animals responding at each dose: _______ 25

___________________________________________________________________ 26

ED50 of reference and test vaccine: _______________________________________ 27

Potency of test vaccine (with 95% fiducial limits): __________________________ 28

29

If an in vitro assay is used 30

Method: ____________________________________________________________ 31

Specification: _______________________________________________________ 32

Date: ______________________________________________________________ 33

Result: _____________________________________________________________ 34

35

Adjuvant content 36

Method: __________________________________________________________________ 37

Specification: _____________________________________________________________ 38

Date: ____________________________________________________________________ 39

Result: ___________________________________________________________________ 40

41

Preservative content (if applicable) 42

Method: __________________________________________________________________ 43

Specification: _____________________________________________________________ 44

WHO/BS/2020.2388

Date: ____________________________________________________________________ 1

Result: ___________________________________________________________________ 2

3

4

Control of final lot 5

Lot number: _______________________________________________________________ 6

Date of filling: _____________________________________________________________ 7

Type of container: __________________________________________________________ 8

Filling volume: ____________________________________________________________ 9

Number of containers after inspection: __________________________________________ 10

Number and percentage of containers rejected: ___________________________________ 11

12

Appearance 13

Method: __________________________________________________________________ 14

Specification: _____________________________________________________________ 15

Date: ____________________________________________________________________ 16

Result: ___________________________________________________________________ 17

18

Identity test 19

Method: __________________________________________________________________ 20

Specification: _____________________________________________________________ 21

Date: ____________________________________________________________________ 22

Result: ___________________________________________________________________ 23

24


Method: __________________________________________________________________ 26

Media: ___________________________________________________________________ 27




Result: ___________________________________________________________________ 31

32

Potency test 33

In vivo assay (may be performed at final bulk stage) 34

Species, strain, sex and weight specifications: ______________________________ 35

Number of mice tested: ________________________________________________ 36

Dates of vaccination, bleeding: __________________________________________ 37

Date of assay: _______________________________________________________ 38

Lot number of reference vaccine and assigned potency: ______________________ 39

Vaccine doses (dilutions) and number of animals responding at each dose: _______ 40

___________________________________________________________________ 41

ED50 of reference and test vaccine: _______________________________________ 42

Potency of test vaccine (with 95% fiducial limits): __________________________ 43

44

WHO/BS/2020.2388

If an in vitro assay is used 1

Method: ____________________________________________________________ 2

Lot number of reference and assigned potency: _____________________________ 3

Specification: _______________________________________________________ 4

Date: ______________________________________________________________ 5

Result: _____________________________________________________________ 6

7

Preservative content (if applicable) 8

Method: __________________________________________________________________ 9

Specification: _____________________________________________________________ 10

Date: ____________________________________________________________________ 11

Result: ___________________________________________________________________ 12

13

Endotoxin content 14

Method: __________________________________________________________________ 15

Specification: _____________________________________________________________ 16

Date: ____________________________________________________________________ 17

Result: ___________________________________________________________________ 18

19

pH 20

Method: __________________________________________________________________ 21

Specification: _____________________________________________________________ 22

Date: ____________________________________________________________________ 23

Result: ___________________________________________________________________ 24

25

Adjuvant content 26

Method: __________________________________________________________________ 27

Specification: _____________________________________________________________ 28

Date: ____________________________________________________________________ 29

Result: ___________________________________________________________________ 30

31

Degree of adsorption 32

Method: __________________________________________________________________ 33

Specification: _____________________________________________________________ 34

Date: ____________________________________________________________________ 35

Result: ___________________________________________________________________ 36

37

Residual antibiotics (if applicable) 38

Method: __________________________________________________________________ 39

Specification: _____________________________________________________________ 40

Date: ____________________________________________________________________ 41

Result: ___________________________________________________________________ 42

43

Extractable volume 44

WHO/BS/2020.2388

Method: __________________________________________________________________ 1

Specification: _____________________________________________________________ 2

Date: ____________________________________________________________________ 3

Result: ___________________________________________________________________ 4

5

6

3. Certification by the manufacturer 7

8

Name of head of quality control (typed) _________________________________________ 9

10

Certification by the person from the control laboratory of the manufacturing company taking 11

overall responsibility for the production and quality control of the vaccine. 12

13

I certify that lot no. ______________________ of enterovirus 71 vaccine (inactivated), 14

whose number appears on the label of the final containers, meets all national requirements 15

and/or satisfies Part A1 of the WHO Recommendations to assure the quality, safety and 16

efficacy of enterovirus 71 vaccines (inactivated).2 17

18

Signature _________________________________________________________________ 19

Name (typed) _____________________________________________________________ 20

Date _____________________________________________________________________ 21

22

23

4. Certification by the NRA 24

25

If the vaccine is to be exported, attach the model NRA Lot Release Certificate for enterovirus 26

71 vaccines (inactivated) (as shown in Appendix 2), a label from a final container and an 27

instruction leaflet for users. 28

29

1 With the exception of provisions on distribution and transport, which the NRA may not be in a position to

assess. 2 WHO Technical Report Series, No. XXXX, Annex X.

WHO/BS/2020.2388

Appendix 2 1

2

Model NRA Lot Release Certificate for enterovirus 71 vaccines 3

(inactivated) 4

5

6

Certificate no. 7

8

9

The following lot(s) of enterovirus 71 vaccine (inactivated) produced by1 10

11

whose numbers appear on the labels of the final evaluated containers, complies with the 12

relevant marketing authorization, the national specifications and provisions for the release of 13

biological products2 and Part A3 of the WHO Recommendations to assure the quality, safety 14

and efficacy of enterovirus 71 vaccines (inactivated) (2021),4 and with corresponding WHO 15

recommendations for each of the vaccine’s individual components, as well as with WHO 16

good manufacturing practices for pharmaceutical products: main principles;5 WHO good 17

manufacturing practices for biological products;6 and the WHO Guidelines for independent 18

lot release of vaccines by regulatory authorities.7 19

20

21

22

23

24

25

26

27

28

29

30

31

1 Name of manufacturer. 2 If any national requirements have not been met, specify which one(s) and indicate why the release of the lot(s)

has nevertheless been authorized by the NRA. 3 With the exception of provisions on distribution and transport, which the NRA may not be in a position to

assess. 4 WHO Technical Report Series, No. XXXX, Annex X. 5 WHO Technical Report Series, No. 986, Annex 2. 6 WHO Technical Report Series, No. 999, Annex 2. 7 WHO Technical Report Series, No. 978, Annex 2.

WHO/BS/2020.2388

1

2

3

4

Trade name:

International nonproprietary/common name:

Batch numbers appearing on package and

other identification numbers associated with

this batch:1

Type of container used:

Total number of containers or lot size:

Number of doses per container:

Date of start of period of validity

(e.g. manufacturing date):

Date of expiry (DD/MM/YYYY):

Storage conditions:2

Diluent lot number(s) (if applicable):

Diluent expiry date(s):

Marketing authorization number issued by

(country):

Name and address of manufacturer:

Site(s) of manufacturing:

Name and address of marketing

authorization holder if different:

5

6

This batch has been examined using documented procedures which form part of a quality 7

system which is in accordance with the ISO/IEC 17025 standard. 8

9

The release decision is based on the elements described in paragraph 7.3 of the WHO 10

Guidelines for independent lot release of vaccines by regulatory authorities.3 11

12

13

1Such as batch number of final bulk. 2 Some products may also have approved extended controlled temperature conditions at the end of use. 3 WHO Technical Report Series, No. 978, Annex 2.

WHO/BS/2020.2388

1

This batch has been found to be compliant with the above by the following institute, member 2

of the WHO National Control Laboratory Network for Biologicals (WHO-NNB). 3

4

Name (typed) 5

6

Institute 7

8

Position 9

10

Signature 11

12

Date 13

14

15

16

17

18

19

20

21

Stamp 22

23

24

25

26

Importing/requesting authority: 27

28

29

30

31

This certificate conforms to the format recommended by the World Health Organization. 32

33

34

35

who ev71 vaccine recommendations 2nd pc

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