1 2 3 WHO/BS/2020.2388 4 ENGLISH ONLY 5 6 Recommendations to assure the quality, safety and efficacy of enterovirus 7 71 vaccines (inactivated) 8 9 10 Proposed new Recommendations 11 12 13 14 NOTE: 15 16 This draft document has been prepared for the purpose of inviting comments and suggestions 17 on the proposals contained therein, which will then be considered by the Expert Committee 18 on Biological Standardization (ECBS). The distribution of this document is intended to 19 provide information on the proposed WHO Recommendations to assure the quality, safety 20 and efficacy of enterovirus 71 vaccines (inactivated) to a broad audience and to ensure the 21 transparency of the consultation process. 22 23 These WHO Recommendations were developed based on the outcomes and consensus of a 24 WHO working group meeting and informal consultation convened in 2019 and 2020 25 respectively, involving participants from national regulatory authorities, national control 26 laboratories, vaccine manufacturers and research institutes. 27 28 The text in its present form does not necessarily represent the agreed formulation of the 29 ECBS. Written comments proposing modifications to this text MUST be received by 30 2 October 2020 in the separately available comment form and should be addressed to the 31 Department of Health Products Policy and Standards, World Health Organization, 20 Avenue 32 Appia, 1211 Geneva 27, Switzerland. 33 34 Comments may also be submitted electronically to the Responsible Officer: Dr Dianliang Lei 35 at: [email protected]. 36 37 The outcome of the deliberations of the ECBS will be published in the WHO Technical 38 Report Series. The final agreed formulation of the document will be edited to be in 39 conformity with the second edition of the WHO style guide (KMS/WHP/13.1). 40 WHO/BS/2020.2388 Page 2 1 2 © World Health Organization 2020 3 4 All rights reserved. 5 6 The designations employed and the presentation of the material in this draft document do not imply the 7 expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status 8 of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or 9 boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full 10 agreement. 11 12 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed 13 or recommended by the World Health Organization in preference to others of a similar nature that are not 14 mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital 15 letters. 16 17 All reasonable precautions have been taken by the World Health Organization to verify the information 18 contained in this draft document. However, the printed material is being distributed without warranty of any 19 kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the 20 reader. In no event shall the World Health Organization be liable for damages arising from its use. 21 22 This draft document does not necessarily represent the decisions or the stated policy of the World Health 23 Organization. The named authors [or editors as appropriate] alone are responsible for the views expressed 24 herein. 25 WHO/BS/2020.2388 Page 3

1 Recommendations to assure the quality, safety and efficacy of enterovirus 2 71 vaccines (inactivated) 3 4 5 6 Introduction 7 7 8 Purpose and scope 8 9 10 Terminology 8 11 12 General considerations 10 13 14 International reference materials 12 15 16 Part A. Manufacturing recommendations 13 17 18 A.1 Definitions 13 19 A.2 General manufacturing recommendations 14 20 A.3 Control of source materials 14 21 A.4 Control of vaccine production 18 22 A.5 Filling and containers 28 23 A.6 Control tests on the final lot 29 24 A.7 Records 31 25 A.8 Retained samples 31 26 A.9 Labelling 31 27 A.10 Distribution and transport 32 28 A.11 Stability testing, storage and expiry date 32 29 30 Part B. Nonclinical evaluation of enterovirus 71 vaccines (inactivated) 33 31 32 B.1 Product characterization and process development 33 33 B.2 Nonclinical immunogenicity and protection studies 34 34 B.3 Nonclinical safety studies 35 35 36 Part C. Clinical evaluation of enterovirus 71 vaccines (inactivated) 35 37 38 C.1 Introduction 35 39 C.2 Assays 36 40 C.3 Immunogenicity 37 41 C.4 Efficacy 38 42 C.5 Safety 42 43 WHO/BS/2020.2388 Page 4 1 Part D. Recommendations for NRAs 42 2 3 D.1 General recommendations 42 4 D.2 Official release and certification 43 5 6 Authors and acknowledgements 43 7 8 References 44 9 10 Appendix 1 Model summary protocol for the manufacturing and control of 11 enterovirus 71 vaccines (inactivated) 54 12 13 Appendix 2 Model NRA Lot Release Certificate for enterovirus 71 vaccines 14 (inactivated) 64 15 16 WHO/BS/2020.2388 Page 5 1 Recommendations published by the World Health Organization (WHO) are intended to be 2 scientific and advisory in nature. Each of the following sections constitutes recommendations 3 for national regulatory authorities (NRAs) and for manufacturers of biological products. If an 4 NRA so desires, these WHO Recommendations may be adopted as definitive national 5 requirements, or modifications may be justified and made by the NRA. It is recommended 6 that modifications to these WHO Recommendations are made only on condition that such 7 modifications ensure that the product is at least as safe and efficacious as that prepared in 8 accordance with the recommendations set out below. The parts of each section printed in 9 small type are comments or examples intended to provide additional guidance to 10 manufacturers and NRAs. 11 12 WHO/BS/2020.2388 Page 6

1 Abbreviations 2 3 4 BPL beta-propiolactone 5 ELISA enzyme-linked immunosorbent assay 6 EV71 enterovirus 71 7 HFMD hand, foot and mouth disease 8 ICP immune correlate of protection 9 Ig immunoglobulin 10 IPV inactivated poliomyelitis vaccine 11 MCB master cell bank 12 NAT nucleic acid amplification technique 13 NCL national control laboratory 14 NIBSC National Institute for Biological Standards and Control 15 NIFDC National Institutes for Food and Drug Control 16 NRA national regulatory authority 17 PCR polymerase chain reaction 18 PSGL-1 P-selectin glycoprotein ligand-1 19 RD rhabdomyosarcoma 20 SCARB2 scavenger receptor class B member 2 21 SCARB2 scavenger receptor class B member 2 (gene) 22 WCB working cell bank 23 WHO/BS/2020.2388 Page 7

1 Introduction 2 3 Enterovirus 71 (EV71) was first isolated from the faeces of a female suffering from 4 encephalitis in 1969 in California (1). However, a retrospective study conducted in the 5 Netherlands indicated that the virus could have emerged as early as 1963 (2); a finding 6 consistent with reports of possible worldwide EV71 epidemics in the late twentieth century 7 (3). EV71 has since been associated with a range of diseases, including hand, foot and mouth 8 disease (HFMD), throughout the world and with epidemics in Asia, Europe and the 9 Americas. The virus causes a range of effects from asymptomatic infection and mild HFMD 10 to neurological disease with severe central nervous system complications and 11 cardiopulmonary failure. In severe cases mortality rates can be high, especially in children 12 aged 5 years and younger. EV71 is considered to be the most severe neurotoxic enterovirus 13 and severe EV71 disease has become a major public health problem in China. In 2011, the 14 WHO Regional Office for the Western Pacific issued A guide to clinical management and 15 public health response for hand, foot and mouth disease (HFMD) (4) to support the 16 treatment, prevention and control of HFMD. 17 18 Several vaccines against EV71 are currently under development and three inactivated EV71 19 vaccines have been licensed in China (5–10). The WHO Expert Committee on Biological 20 Standardization discussed the EV71 situation at its 67th meeting in 2016 and considered it to 21 be of major regional significance (11). The Committee noted that the joint efforts of the 22 National Institutes for Food and Drug Control (NIFDC) and the National Institute for 23 Biological Standards and Control (NIBSC) had resulted in the development of the First WHO 24 International Standard for anti-EV71 serum (human), and recommended that consideration 25 also be given to the development of a WHO written standard for EV71 vaccines. In addition, 26 the First WHO International Standard for EV71 inactivated vaccine was established by the 27 Committee in October 2019 following a collaborative study led by NIBSC and NIFDC (12, 28 13). National standards for antigen content and neutralizing antibody responses for evaluating 29 EV71 vaccines are also available in China (14) where they have supported the development 30 and clinical assessment of such vaccines. In 2018, the Committee also endorsed a proposal to 31 develop WHO international standards for enterovirus RNA for nucleic acid amplification 32 technique (NAT)-based assays for EV71 (15, 16). 33 34 Following requests from regulators and other stakeholders for WHO to develop 35 recommendations to assure the quality, safety and efficacy of EV71 vaccines, a series of 36 meetings was convened by WHO to review the current status of their development and 37 licensure (17). These meetings were attended by experts from around the world involved in 38 the research, manufacture, regulatory assessment and approval, and control testing and 39 release of EV71 vaccines. Participants were drawn from academia, national regulatory 40 authorities (NRAs), national control laboratories (NCLs) and industry. The recommendations 41 provided in the current document for the production, quality control and evaluation of 42 inactivated EV71 vaccines have been based upon the experiences gained during the 43 development and production of the first three licensed EV71 vaccines in China, the candidate WHO/BS/2020.2388 Page 8 1 EV71 vaccines now under development (5–8, 18–22) and other inactivated viral vaccines, 2 such as inactivated poliomyelitis vaccines (IPVs) and hepatitis A vaccines (23, 24). 3 4 Purpose and scope 5 6 These WHO Recommendations provide guidance to NRAs and manufacturers on the 7 manufacturing processes, quality control and nonclinical and clinical evaluations needed to 8 assure the quality, safety and efficacy of inactivated EV71 vaccines. 9 10 The guidance applies to EV71 vaccines prepared by the inactivation of whole EV71 virus for 11 prophylactic use, grown in mammalian cells in culture, and using formaldehyde or other 12 chemical inactivation procedures. 13 14 The document does not cover recombinant and other forms of subunit vaccines, vectored 15 vaccines, virus-like particle vaccines or bivalent EV71-CA16 vaccines, which are at an early 16 stage of development. However, some aspects outlined in this document may be relevant and 17 may be taken into consideration during the development of such vaccines. 18 19 These WHO Recommendations should be read in conjunction with current WHO guidance 20 documents on the nonclinical (25) and clinical (26) evaluation of vaccines, good 21 manufacturing practices for biological products (27), characterization of cell banks (28), 22 nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines (29) and lot release (30). 23 24 Manufacturers and regulators should also take note of the decision of the Committee in 2018 25 to discontinue the inclusion of the innocuity test (also referred to as the abnormal toxicity test 26 or general safety test) in routine lot release testing requirements for all vaccines in WHO 27 Recommendations, Guidelines and other guidance documents for biological products (31, 28 32). As this test is no longer recommended, it is not included in the routine testing 29 requirements for EV71 vaccines provided in the current document. 30 31 Terminology 32 33 The definitions given below apply to the terms as used in these WHO Recommendations. 34 These terms may have different meanings in other contexts. 35 36 Adjuvant: a vaccine adjuvant is a substance, or combination of substances, that is 37 used in conjunction with a vaccine antigen to enhance (for example, increase, accelerate, 38 prolong and/or possibly target) the specific immune response to the vaccine antigen and the 39 clinical effectiveness of the vaccine. 40 Adventitious agents: contaminating microorganisms of the cell culture, or source 41 materials used in its culture, that may include bacteria, fungi, mycoplasmas, transmissible 42 spongiform encephalopathy agents and endogenous and exogenous viruses that have been 43 unintentionally introduced into the manufacturing process. WHO/BS/2020.2388 Page 9 1 Cell bank: a collection of vials of cells of uniform composition (though not 2 necessarily clonal) derived from a single tissue or cell and used for the production of a 3 vaccine, either directly or via a cell bank system. 4 Cell seed: a quantity of well-characterized cells derived from a single tissue or cell of 5 human or animal origin and stored frozen in liquid nitrogen in aliquots of uniform 6 composition, one or more of which may be used for the production of a master cell bank. 7 EV71 antigen: the virus-specific antigen produced in infected cell cultures and 8 purified from such cultures. It can be assayed by methods such as enzyme-linked 9 immunosorbent assay (ELISA) using EV71-specific antibodies. The antigen may consist of 10 empty or full virus particles or both. The full and empty particles differ in their antigenic 11 reactivity and both may be present in the final vaccine. 12 Final bulk: a formulated vaccine preparation from which the final containers are 13 filled. The final bulk may be prepared from one or more purified antigen bulks formulated to 14 contain all excipients and homogenous with respect to composition. 15 Final lot: a collection of sealed final containers of finished vaccine that is 16 homogeneous with respect to the risk of contamination during the filling process. A final lot 17 must therefore have been filled from a formulated bulk in one continuous working session. 18 Immunogenicity: the capacity of a vaccine to induce antibody-mediated and/or cell- 19 mediated immunity and/or immunological memory. 20 Master cell bank (MCB): a quantity of well-characterized cells of human or animal 21 origin derived from a cell seed at a specific population doubling level or passage level, 22 dispensed into multiple containers, cryopreserved and stored frozen under defined conditions 23 (such as the vapour or liquid phase of liquid nitrogen) in aliquots of uniform composition. 24 The MCB is prepared from a single homogeneously mixed pool of cells and is used to derive 25 all working cell banks. The testing performed on a replacement MCB (derived from the 26 same clone or from an existing master or working cell bank) is the same as for the initial 27 MCB, unless a justified exception is made. 28 Master seed lot: a quantity of virus suspension that has been processed at the same 29 time to ensure a uniform composition, and passaged for a specific number of times that does 30 not exceed the maximum approved by the NRA. It is characterized to the extent necessary to 31 support development of the working seed lot. 32 Purified inactivated bulk: a purified pool of virus harvests in which the virus has 33 been inactivated through the use of a validated method either before or after purification. It 34 may be prepared from one single harvest or a number of single harvests and may yield one 35 or more final bulks. 36 Seed lot: a preparation of live viruses constituting the starting material for the vaccine 37 antigen. A seed lot is of uniform composition (though not necessarily clonal), is derived from 38 a single culture process and is aliquoted into appropriate storage containers, from which all 39 future vaccine production will be derived either directly or via a seed lot system. 40 Single harvest: a quantity or suspension derived from a batch of production cells 41 inoculated with the same seed lot and processed together in a single production run. 42 Working cell bank (WCB): a quantity of cells of uniform composition derived from 43 one or more ampoules of the MCB at a finite passage level, stored frozen at −70 °C or below WHO/BS/2020.2388 Page 10 1 in aliquots, one or more of which are used for vaccine production. All containers are treated 2 identically and once removed from storage are not returned to the stock. 3 Working seed lot: a quantity of virus of uniform composition derived from the 4 master seed lot used at a passage level approved by the NRA for the manufacturing of 5 vaccine. 6 7 General considerations 8 9 HFMD was first reported in New Zealand in 1957 (33) and occurs mostly in young children, 10 with a peak incidence at about 2 years of age. The common mild disease involves lesions on 11 the mucosal surfaces of the mouth and spots on the palms of the hands and soles of the feet 12 which resolve in a few days; this is not life threatening. However, a more severe and 13 potentially fatal form of the disease was reported in 1969 (1) which is now recognized to 14 encompass meningitis/encephalitis, autonomic nervous system dysregulation, cardiovascular 15 collapse and pulmonary oedema. The overall mortality rate of HFMD is of the order of one 16 per 1000 to 10 000 cases. 17 18 The frequency of reported HFMD cases is geographically highly variable with most cases 19 occurring in East Asian countries, particularly China but including Viet Nam, Thailand, 20 Singapore, Malaysia and the Republic of Korea. Normally, only few cases of severe disease 21 are reported in Europe or the USA with reports of mild HFMD also being less common – 22 though underreporting of the latter is very likely. Typically, the total combined number of 23 cases in Europe and the USA is of the order of several hundred per year, whereas in 2008 24 there were 488 955 cases and 128 deaths in China alone (4, 34, 35). HFMD is now a 25 reportable disease in China (in contrast to Europe and the USA) and during the period 2013– 26 2018 between 300 000 and 400 000 cases occurred in May–June each year, with a small 27 number of deaths (4, 34, 35). The reason for the differences in disease burden in different 28 geographical areas is not yet clear. 29 30 The causative agents of HFMD are picornaviruses, most often of human enterovirus species 31 A. The picornaviruses belong to the Picornaviridae, a family of small non-enveloped viruses 32 with a single-stranded positive-sense RNA genome of about 7500 nucleotides. The 33 enteroviruses are a recognized taxon by the International Committee on Taxonomy of Viruses 34 and are sub-classified into numerous species including the four human enteroviruses A, B, C 35 and D (36). The archetypal human picornavirus is poliovirus (belonging to species C) but the 36 most common cause of HFMD is species A enteroviruses, chiefly the Coxsackie A viruses 37 and EV71. The relative frequency of different enterovirus species found varies from year to 38 year when clinical isolates or environmental samples such as sewage are examined. 39 40 In recent years, Coxsackie viruses such as A16, A10 and A6 have caused most of the HFMD 41 cases in Asia. However, other human enteroviruses of the Picornaviridae family have also 42 being implicated and as the different serotypes are antigenically distinct, the development of 43 a vaccine based on a single serotype to protect against all mild HFMD cases is unlikely using 44 existing technologies. There is therefore interest in the development of combination vaccines WHO/BS/2020.2388 Page 11 1 containing several serotypes. However, while mild HFMD is caused by many strains of 2 enterovirus, the great majority of severe disease in recent years has been caused by EV71 3 which accounts for 70% of severe HFMD cases and 90% of HFMD-related deaths in China 4 (35). As a result, EV71 has been the main focus of vaccine development. 5 6 EV71 isolates can be clustered according to their genomic sequence into at least eight 7 genogroups (A–H) (36, 37) but belong to one serotype. Genogroups B and C have been of 8 greatest interest because of their frequency of isolation and implication in disease in Eastern 9 Asia, and can each be sub-classified into five subgenogroups (C1–C5 and B1–B5). C4 is by 10 far the major genogroup circulating in China, while B4, B5 and C5 are found in other Asian 11 countries. In contrast, strains of genogroups C1 and C2 are predominantly found in Europe 12 where severe disease is uncommon. It is possible that this has some effect on the disease 13 burden, with C4 being particularly virulent. However, an outbreak of HFMD with severe 14 disease caused by a C1 genogroup strain occurred in Spain in 2016 (38, 39). 15 16 A valid animal model would be useful in vaccine development to measure protective efficacy 17 and potency, as well as to resolve issues related to virulence. As yet, the available models are 18 imperfect. Neonatal mice are susceptible to EV71 by intracerebral inoculation and neonatal 19 (but not adult) rhesus monkeys develop symptoms of HFMD on infection. Adult or infant 20 mice are not susceptible to infection. Infant rhesus monkeys have been demonstrated to 21 develop HFMD symptoms upon inoculation with the virus and could therefore be used as a 22 model of protection (40, 41). The neurovirulence of EV71 was demonstrated in cynomolgus 23 monkeys and this model would be useful for challenge-protection studies for candidate EV71 24 vaccines (42, 43). Picornaviruses are believed to use specific receptors to infect human cells. 25 Human P-selectin glycoprotein ligand-1 (PSGL-1) is expressed in leukocytes and involved in 26 their binding to endothelial cells in the early stages of inflammation, and has been identified 27 as a receptor for EV71. However, the disease produced by clinical EV71 strains in transgenic 28 mice carrying PSGL-1 was not enhanced compared to non-transgenic strains. Human 29 scavenger receptor class B member 2 (SCARB2) has also been identified as a receptor for 30 EV71. Transgenic mice carrying the SCARB2 gene are more susceptible to infection and 31 disease than non-transgenic controls but the effect is not dramatic – two-week old transgenic 32 mice develop mild symptoms and then recover (44–47). 33 34 Three vaccines against EV71 have been licensed in China, all using C4 genogroup strains. 35 Candidate vaccines containing B4 and B5 genogroups are in development elsewhere but have 36 not yet reached the licensing stage. In addition, the development of vaccines against 37 Coxsackie A16, A6 and A10 is being considered with a view to developing 38 combination/multivalent vaccines. The efficacy of the three licensed EV71 vaccines after two 39 doses ranges from 90.0% to 97.4% after one year of surveillance (5–7) to 95.1% after two 40 year follow-up (8). 41 42 Licensed EV71 vaccines are produced from virus grown on mammalian cells and inactivated 43 by validated methods – similar to the approach used for IPVs and hepatitis A vaccines. Tissue 44 culture grown virus harvests comprise two types of particle forms; one containing the RNA WHO/BS/2020.2388 Page 12 1 genome (full) and one that does not (empty). In the case of both polioviruses and EV71 2 viruses, the two particle forms have different antigenic and immunogenic properties; 3 poliomyelitis vaccines are purified so that they contain little if any empty virus particles but 4 EV71 vaccines contain both types; potentially complicating potency assays. The atomic 5 structures of both full and empty particles of EV71 viruses and polioviruses have been 6 resolved by X-ray crystallography and cryogenic electron microscopy (48, 49). 7 8 Specific issues in the development of inactivated EV71 vaccines include: 9 10 ▪ The degree to which a vaccine based on one genogroup will protect against the others 11 is not established. Although there is good cross-neutralization between genogroups, 12 including by sera induced by vaccination (50, 51), it has not been established that this 13 translates into good cross-protection in humans. One recent collaborative study 14 indicated that assays of antigen content work acceptably on all genogroups tested. 15 However clinical cross-protection has not been demonstrated. Thus, the C4 16 genogroup vaccines may or may not protect against other genogroups. 17 ▪ There is a lack of a convenient and convincing animal model, with the model most 18 accurately reflecting human disease at present being infant rhesus monkeys. This 19 makes the study of protective efficacy and immunogenic potency difficult other than 20 by clinical trial. Neonatal mice are susceptible to disease and transgenic mice 21 carrying the SCARB2 gene which encodes EV71 receptors have been developed and 22 can prove useful without fully imitating human pathogenesis. 23 ▪ Virological issues include the existence of full and empty particle forms in the 24 licensed products. These two particle forms differ in their antigenic and 25 immunogenic properties, thus complicating potency assays. It is not clear whether 26 current national and international vaccine reference standards and antigen potency 27 assays are suitable for specifically detecting one or other of the particle forms (52). 28 29 International reference materials 30 31 Subsequent sections of this document refer to WHO reference materials that may be used in 32 laboratory or clinical evaluations. The following key standards are currently used in the 33 control of EV71 vaccines. 34 35 ▪ A WHO international standard for anti-EV71 serum is available for the 36 standardization of diagnostic tests for use in seroprevalence studies and for assessing 37 immunity. This standard was established by the WHO Expert Committee on 38 Biological Standardization in 2015 as the First WHO International Standard for anti- 39 EV71 serum (human) (NIBSC code 14/140) with an assigned value of 40 1000 IU/ampoule (53, 54). The preparation is held and distributed by NIBSC and 41 NIFDC. 42 ▪ A WHO international reference reagent for EV71 neutralization assays (low-titre) is 43 also available for use in the standardization of virus neutralization assays. This WHO/BS/2020.2388 Page 13 1 reference reagent was established by the Committee in 2015 as the First WHO 2 International Reference Reagent for EV71 neutralization assays (NIBSC code 3 13/238) with an assigned value of 300 IU/ampoule (53, 54). The preparation is held 4 and distributed by NIBSC and NIFDC. 5 ▪ In 2019, the First WHO International Standard for EV71 inactivated vaccine (NIBSC 6 code 18/116) was established by the Committee with an assigned unitage of 7 3625 IU/ampoule (12, 13). In addition, WHO international reference reagents for 8 EV71 genogroups C4 and B4 inactivated vaccine (NIBSC codes 18/120 and 18/156 9 respectively) were established with assigned unitages of 300 and 250 IU/ampoule 10 respectively (12, 13). These preparations are held and distributed by NIBSC. The 11 First WHO International Standard for EV71 inactivated vaccine is intended for use in 12 in vitro assays to measure the antigen content of vaccine products through the 13 calibration of secondary reference preparations. However, it is known that full and 14 empty virus particles (known to be present in EV71 vaccine preparations from all 15 manufacturers) differ in their antigenicity and immunogenicity. The proportion of 16 empty/full virus particles in the WHO international standard is not known. It is also 17 not known whether this matters for the overall assessment of vaccines or if the 18 current international standard is suitable for accurately measuring antigen content 19 across manufacturers. International standards and reference reagents for the control 20 of in vivo potency assays are under investigation (13). 21 ▪ Product-specific national standards for EV71 neutralizing antibody and EV71 antigen 22 were established by NIFDC following collaborative studies conducted by NIFDC and 23 the three main vaccine manufacturers in China (14). This has helped to ensure the 24 accuracy, comparability and repeatability of anti-EV71 neutralizing antibody and 25 EV71 antigen detection assays, and hence improve EV71 vaccine standardization. In 26 addition, NIFDC has also developed a new national reference for in vivo vaccine 27 potency. These preparations are held and distributed by NIFDC. 28 29 Part A. Manufacturing recommendations 30 31 A.1 Definitions 32 33 A.1.1 International name and proper name 34 35 The international name of the vaccine should be “enterovirus 71 vaccine (inactivated)”. The 36 proper name should be the equivalent of the international name in the language of the country 37 of origin. 38 39 The use of the international name should be limited to vaccines that meet the specifications 40 given below. 41 42 A.1.2 Descriptive definition 43 WHO/BS/2020.2388 Page 14 1 An EV71 vaccine (inactivated) consists of a sterile suspension of EV71 grown in cell 2 cultures, concentrated, purified and inactivated. The antigen may be formulated for delivery 3 with a suitable adjuvant. The preparation should meet all of the specifications given below. 4 5 A.2 General manufacturing recommendations 6 7 The general guidance provided in WHO good manufacturing practices for pharmaceutical 8 products: main principles (55) and WHO good manufacturing practices for biological 9 products (27) should be applied to the design, establishment, operation, control and 10 maintenance of manufacturing facilities for EV71 vaccine. 11 12 Staff involved in the production and quality control of inactivated EV71 vaccine should be 13 shown to be immune to EV71. 14 15 A.3 Control of source materials 16 17 A.3.1 Virus strains and seed lot system 18 19 A.3.1.1 Virus strains 20 21 Strains of EV71 used in the production of EV71 vaccine should be identified by historical 22 records, which should include information on their origin and subsequent manipulation or 23 passage (for example, the genogroup and subgenogroup of EV71). Strain identity should be 24 determined by infectivity tests and immunological methods, and by full or partial genomic 25 sequencing. 26 27 Only virus strains that are approved by the NRA and that yield a vaccine complying with the 28 recommendations set out in these WHO Recommendations should be used. 29 30 A.3.1.2 Virus seed lot system 31 32 Vaccine production should be based on the virus seed lot system. Unless otherwise justified 33 and authorized, the virus in the final vaccine should not have undergone more passages from 34 the virus master seed lot than were used to prepare a vaccine shown to be satisfactory with 35 respect to safety and efficacy. 36 37 Virus master and working seed lots should be stored in dedicated temperature-monitored 38 freezers at a temperature that ensures stability during storage (for example, at or below 39 −60 °C). 40 41 A.3.1.3 Tests on virus master and working seed lots 42 WHO/BS/2020.2388 Page 15 1 Each virus master and working seed lot used for the production of vaccine lots should be 2 subjected to the tests listed in this section. 3 4 Each virus master and working seed lot should have been derived from materials that comply 5 with the recommendations made in sections A.3.2 and A.3.3 below and should be approved 6 by the NRA. 7 8 A.3.1.3.1 Tests for adventitious agents 9 10 The virus master and working seed lots used for the production of vaccine lots should be free 11 from adventitious agents. 12 13 A sample of at least 20 mL of each virus master and working seed lot should be tested for the 14 presence of adventitious agents. The sample should be neutralized by a high-titred antiserum 15 against EV71. 16 17 If polyclonal antisera are used, the immunizing antigen used for the preparation of the antiserum should 18 not be the same as the production seed. 19 20 The immunizing antigen should be shown to be free from adventitious agents and should be grown in 21 cell cultures free from adventitious microbial agents that might elicit antibodies that could inhibit the 22 growth of any adventitious agents present. 23 24 The sample should be tested in susceptible cells such as Vero, human rhabdomyosarcoma 25 (RD) or human diploid cells. The tissue cultures should be incubated at 37 °C and observed 26 for 2 weeks. Within this observation period, at least one subculture of supernatant fluid 27 should be made in the same tissue culture system. The sample should be inoculated in such a 28 way that the dilution of the supernatant fluid in the nutrient medium does not exceed 1 in 4. 29 The area of the cell sheet should be at least 3 cm2 per mL of supernatant fluid. At least one 30 culture vessel of the cell cultures should remain uninoculated and should serve as a control. 31 The cells inoculated with the supernatant fluid and the uninoculated control cultures should 32 be incubated at 37 °C and observed at appropriate intervals for an additional 2 weeks. 33 34 The virus master and working seed lots pass the test if there is no evidence of the presence of 35 adventitious agents. For the test to be valid, not more than 20% of the culture vessels should 36 have been discarded for any reason by the end of the observation period. 37 38 New molecular methods with broad detection capabilities are being developed for the detection of 39 adventitious agents. These methods include: (a) degenerate NAT for whole virus families, with analysis 40 of the amplicons by hybridization, sequencing or mass spectrometry; (b) NAT with random primers 41 followed by analysis of the amplicons on large oligonucleotide micro-arrays of conserved viral 42 sequencing, or digital subtraction of expressed sequences; and (c) high-throughput sequencing. These 43 methods might be used in the future to supplement existing methods or as alternative methods to both 44 in vivo and in vitro tests after appropriate validation and with the approval of the NRA (28). 45 WHO/BS/2020.2388 Page 16

1 The theoretical risk of the presence of potential human, simian, bovine or porcine adventitious agents in 2 the seed lots, which may be derived from the use of bovine serum or porcine trypsin, should be 3 assessed. If necessary, viruses such as bovine polyomavirus, porcine parvovirus or porcine circovirus 4 may be screened for using specific assays, such as molecular NAT-based assays (28). 5 6 A.3.1.3.2 Identity test 7 8 The strain identity of the seed lot should be determined by infectivity tests. The test for 9 antigen content described in section A.4.4.2.2 below can be used to identify the seed lot. 10 11 A.3.1.3.3 Virus titration 12 13 The virus concentration of the seed lot should be determined by titration of infectious virus 14 using validated tissue culture methods. This titration should be carried out in not more than 15 10-fold dilution steps using 10 cultures per dilution, or by any other arrangement yielding 16 equal precision. 17 18 The use of human RD, human diploid or Vero cells in microtitre plates is suitable for this purpose (28). 19 20 A.3.2 Cell lines 21 22 The general production precautions, as formulated in WHO good manufacturing practices for 23 biological products (27), should apply to the manufacture of EV71 vaccine, with the 24 additional requirement that, during production, only one type of cell should be introduced or 25 handled in the production area at any one time. Vaccines may be produced in a human 26 diploid cell line or in a continuous cell line. 27 28 A.3.2.1 Master cell bank (MCB) and working cell bank (WCB) 29 30 The use of a cell line for the manufacture of EV71 vaccine should be based on the cell bank 31 system. The cell seed and cell banks should conform to WHO Recommendations for the 32 evaluation of animal cell cultures as substrates for the manufacture of biological medicinal 33 products and for the characterization of cell banks (28). The MCB should be approved by the 34 NRA. The maximum number of passages (or population doublings) by which the WCB is 35 derived from the MCB and the maximum number of passages of the production cultures 36 should be established by the manufacturer and approved by the NRA. 37 38 The WHO Vero reference cell bank 10-87 is considered suitable for use as a cell seed for generating an 39 MCB (56) and is available to manufacturers on application to the Group Lead, Norms and Standards 40 for Biologicals, Technical Specifications and Standards, Department of Health Product Policy and 41 Standards, Access to Medicines and Health Products Division, World Health Organization, Geneva, 42 Switzerland. 43 44 A.3.2.2 Identity test 45 WHO/BS/2020.2388 Page 17 1 Identity tests on the MCB and WCB should be performed in accordance with WHO 2 Recommendations for the evaluation of animal cell cultures as substrates for the manufacture 3 of biological medicinal products and for the characterization of cell banks (28) and should be 4 approved by the NRA. 5 6 The cell banks should be identified by means of tests such as biochemical tests (for example, 7 isoenzyme analysis), immunological tests, cytogenetic marker tests and DNA fingerprinting 8 or sequencing. The tests used should be approved by the NRA. 9 10 A.3.3 Cell culture medium 11 12 Where serum is used for the propagation of cells it should be tested to demonstrate freedom 13 from bacterial, fungal and mycoplasmal contamination – as specified in Part A, sections 5.2 14 (57) and 5.3 (58) of the WHO General requirements for the sterility of biological substances 15 – as well as freedom from infectious viruses. Suitable tests for detecting viruses in bovine 16 serum are given in Appendix 1 of the WHO Recommendations for the evaluation of animal 17 cell cultures as substrates for the manufacture of biological medicinal products and for the 18 characterization of cell banks (28). 19 20 Validated molecular tests for bovine viruses may replace the cell culture tests of bovine sera 21 if approved by the NRA. As an additional monitor of quality, sera may be examined for 22 freedom from bacteriophage and endotoxin. Gamma irradiation may be used to inactivate 23 potential contaminant viruses, while recognizing that some viruses are relatively resistant to 24 gamma irradiation. 25 26 The source(s) of animal components used in the culture medium should be approved by the 27 NRA. The components should comply with the current WHO guidelines on transmissible 28 spongiform encephalopathies in relation to biological and pharmaceutical products (59). The 29 serum protein concentration should be reduced by rinsing the cell cultures with serum-free 30 medium and/or by purification of the virus harvests. 31 32 In some countries, control tests are carried out to detect the residual animal serum content in the final 33 vaccine. 34 35 Human serum should not be used. If human serum albumin is used at any stage of product 36 manufacture, the NRA should be consulted regarding the requirements, as these may differ 37 from country to country. At a minimum, it should meet the WHO Requirements for the 38 collection, processing and quality control of blood, blood components and plasma derivatives 39 (60). In addition, human albumin and materials of animal origin should comply with the 40 current WHO guidelines on transmissible spongiform encephalopathies in relation to 41 biological and pharmaceutical products (59). 42 43 Manufacturers are encouraged to explore the possibilities of using serum-free media for the production 44 of EV71 vaccine. WHO/BS/2020.2388 Page 18 1 2 Penicillin and other beta-lactams should not be used at any stage of manufacture because they 3 are highly sensitizing substances in humans. Other antibiotics may be used during early 4 stages of production. In this case, the use of antibiotics should be well justified, and they 5 should be cleared from the manufacturing process at the stage specified in the marketing 6 authorization. Acceptable residual antibiotic levels should be approved by the NRA. 7 8 Bovine or porcine trypsin used for preparing cell cultures should be tested and found to be 9 free of cultivatable bacteria, fungi, mycoplasmas and infectious viruses, as appropriate (28). 10 The methods used to ensure this should be approved by the NRA. 11 12 In some countries, irradiation is used to inactivate potential contaminant viruses. If irradiation is used, 13 it is important to ensure that a reproducible dose is delivered to all lots and to the component units of 14 each lot. The irradiation dose must be low enough for the biological properties of the reagents to be 15 retained but also high enough to reduce virological risk. Therefore, irradiation cannot be considered a 16 sterilizing process (28). 17 18 Recombinant trypsin is available and should be considered; however, it should not be assumed to be 19 free from risk of contamination and should be subject to the usual considerations for any reagent of 20 biological origin (28). 21 22 The source(s) of trypsin of bovine origin, if used, should be approved by the NRA and should 23 comply with the current WHO guidelines on transmissible spongiform encephalopathies in 24 relation to biological and pharmaceutical products (59). 25 26 A.4 Control of vaccine production 27 28 A.4.1 Control cell cultures 29 30 A fraction of the production cell culture equivalent to at least 5% of the total or 500 mL of 31 cell suspension, or 100 million cells, at the concentration and cell passage level employed for 32 seeding vaccine production cultures, should be used to prepare control cultures. 33 34 If bioreactor technology is used, the NRA should determine the size and treatment of the cell 35 sample to be examined. 36 37 A.4.1.1 Tests of control cell cultures 38 39 The treatment of the cells set aside as control material should be similar to that of the 40 production cell cultures but they should remain uninoculated for use as control cultures for 41 the detection of any adventitious agents. 42 43 These control cell cultures should be incubated under conditions as similar as possible to the 44 inoculated cultures for at least 2 weeks, and should be tested for the presence of adventitious WHO/BS/2020.2388 Page 19 1 agents as described below. For the test to be valid, not more than 20% of the control cell 2 cultures should have been discarded for any reason by the end of the test period. 3 4 At the end of the observation period, the control cell cultures should be examined for 5 evidence of degeneration caused by an adventitious agent. If this examination or any of the 6 tests specified in this section shows evidence of the presence of any adventitious agent in the 7 control culture, the EV71 grown in the corresponding inoculated cultures should not be used 8 for vaccine production. 9 10 If not tested immediately, samples should be stored at −60 °C or below. 11 12 A.4.1.2 Tests for haemadsorbing viruses 13 14 At the end of the observation period, at least 25% of the control cells should be tested for the 15 presence of haemadsorbing viruses using guinea-pig red blood cells. If the latter cells have 16 been stored, the duration of storage should not have exceeded 7 days and the storage 17 temperature should have been in the range of 2–8 °C. In tests for haemadsorbing viruses, 18 calcium and magnesium ions should be absent from the medium. 19 20 Some NRAs require, as a test for haemadsorbing viruses, that other types of red blood cells, including 21 cells from humans, monkeys and chickens (or other avian species), are also used instead of guinea-pig 22 cells alone. 23 24 A reading should be taken after incubation at 2–8 °C for 30 minutes, and again after a further 25 incubation for 30 minutes at 20–25 °C. 26 27 If a test with monkey red blood cells is performed, readings should also be taken after a final incubation 28 for 30 minutes at 34–37 °C. 29 30 In some countries the sensitivity of each new lot of red blood cells is demonstrated by titration against a 31 haemagglutinin antigen before use in the test for haemadsorbing viruses. 32 33 A.4.1.3 Tests for other adventitious agents in cell supernatant fluid 34 35 At the end of the observation period, a sample of the pooled supernatant fluid from each 36 group of control cultures should be tested for adventitious agents. For this purpose, 10 mL of 37 each pool should be tested in the same cells, but not the same lot of cells, as those used for 38 the production of vaccine. 39 40 A second indicator cell line should be used to test an additional 10 mL sample of each pool. 41 When a human diploid cell line is used for production, a simian kidney cell line should be 42 used as the second indicator cell line. When a simian kidney cell line is used for production, a 43 human diploid cell line should be used as the second indicator cell line (28). 44 WHO/BS/2020.2388 Page 20 1 The pooled fluid should be inoculated into culture vessels of these cell cultures in such a way 2 that the dilution of the pooled fluid in the nutrient medium does not exceed 1 part in 4. The 3 area of the cell sheet should be at least 3 cm2 per mL of pooled fluid. At least one culture 4 vessel of each kind of cell culture should remain uninoculated and should serve as a control. 5 6 The inoculated cultures should be incubated at the same temperature as that of the production 7 of virus antigen and observed at appropriate intervals for a period of at least 14 days. 8 9 Some NRAs require that, at the end of this observation period, a subculture is made in the same culture 10 system and observed for at least an additional 14 days. Furthermore, some NRAs require that these 11 cells should be tested for the presence of haemadsorbing viruses. 12 13 For the tests to be valid, not more than 20% of the culture vessels should have been discarded 14 for any reason by the end of the test period. 15 16 If any cytopathic changes due to adventitious agents occur in any of the cultures, the virus 17 harvests produced from the lot of cells from which the control cells were taken should be 18 discarded. 19 20 Some selected viruses may be screened for using specific validated assays which are 21 approved by the NRA, such as molecular NAT-based assays (28). 22 23 If these tests are not performed immediately, the samples should be kept at a temperature of 24 −60 °C or below. 25 26 A.4.1.4 Identity tests 27 28 At the production level, the control cells should be identified using tests approved by the 29 NRA. 30 31 Suitable methods include, but are not limited to, biochemical tests (for example, isoenzyme 32 analyses), immunological tests, cytogenetic tests (for example, for chromosomal markers), 33 morphological identification and tests for genetic markers (for example, DNA fingerprinting 34 or sequencing). 35 36 A.4.2 Cell cultures for vaccine production 37 38 A.4.2.1 Observation of cultures for adventitious agents 39 40 On the day of inoculation with the virus working seed lot, each cell culture or a sample from 41 each culture vessel should be examined visually for degeneration caused by infective agents. 42 If this examination shows evidence of the presence of any adventitious agent in a cell culture 43 then the culture should not be used for vaccine production. 44 WHO/BS/2020.2388 Page 21 1 A.4.3 Control of single harvests 2 3 After inoculation of the production cells with virus, the culture conditions of inoculated and 4 control cell cultures should be standardized and kept within limits agreed with the NRA. 5 6 Samples required for the testing of single harvests should be taken immediately on 7 harvesting. 8 9 Samples may be taken after storage and filtration with the agreement of the NRA. 10 11 A.4.3.1 Identity test 12 13 The strain identity of the single harvest should be determined by infectivity tests. The test for 14 antigen content described in section A.4.4.2.2 below can be used to identify the single 15 harvest. 16 17 A.4.3.2 Sterility tests for bacteria, fungi and mycoplasmas 18 19 A volume of at least 10 mL of each single harvest should be tested for bacterial, fungal and 20 mycoplasmal contamination using appropriate tests, as specified in Part A, sections 5.2 (57) 21 and 5.3 (58) of the WHO General requirements for the sterility of biological substances, or by 22 methods approved by the NRA. 23 24 NAT alone or in combination with cell culture, with an appropriate detection method, may be used as 25 an alternative to one or both of the compendial mycoplasma detection methods following suitable 26 validation and the agreement of the NRA (28). 27 28 In some countries this test is performed on the purified virus harvest instead of on the single harvest. 29 30 A.4.3.3 Virus titration 31 32 The virus concentration of each single harvest should be determined by titration of infectious 33 virus using validated tissue culture methods to monitor production consistency and as a 34 starting point for monitoring the inactivation curve. This titration should be carried out in not 35 more than 10-fold dilution steps using 10 cultures per dilution, or by any other arrangement 36 yielding equal precision. 37 38 The use of human RD, human diploid or Vero cells in microtitre plates is suitable for this purpose (28). 39 The same cells should be used for virus titrations throughout the production process. 40 41 Information on virus titre will help in selecting single harvests that can be expected to meet potency 42 requirements after inactivation. 43 44 The virus titration may be carried out on the pooled harvest after demonstration of consistency of 45 production at the stage of the single harvest. 46 WHO/BS/2020.2388 Page 22 1 A.4.4 Control of virus pools 2 3 Several single harvests may be mixed to prepare a pool of virus before inactivation. The order 4 in which the purification, filtration and inactivation of virus pools is conducted should be 5 carefully established by the manufacturer to ensure consistent full virus inactivation and 6 absence of residual infectivity. Based on experience of the production of IPVs, the WHO 7 Recommendations to assure the quality, safety and efficacy of poliomyelitis vaccines 8 (inactivated) (23) recommends purification, filtration and inactivation steps in that order. 9 10 The requirement for filtration before and during inactivation was introduced into the IPV production 11 process following the Cutter incident during which a number of paralytic polio cases occurred in 12 children following vaccination with a defective IPV (23). The vaccine used was later found to contain 13 aggregates which led to incomplete virus inactivation likely due to formaldehyde not accessing some 14 virus particles inside the aggregates. 15 16 Any deviation from the production sequence shown to be acceptable for IPV for a vaccine 17 against a virus similar to poliovirus (such as EV71) should be fully validated and justified in 18 terms of yielding a product of equivalent safety. 19 20 Inactivation of virus may be performed before or after purification according to current 21 approved procedures for the production of licensed vaccines. The method of purification and 22 inactivation as well as the agent used for inactivation should be appropriately validated and 23 should be approved by the NRA. 24 25 If inactivation of the virus pool is conducted after purification, please see section A.4.4.1 26 below. If inactivation of the virus pool is conducted before purification please see section 27 A.4.4.2 below. 28 29 A.4.4.1 Purification of virus pools 30 31 Each pool of virus should be purified. Removal of host cell protein should be assessed during 32 process validation (28). 33 34 An acceptable method is to clarify the virus suspension by filtration, to concentrate the virus by 35 ultrafiltration and, thereafter, to collect the virus peak after passing it through a gel-filtration column. 36 Further purification is achieved by passing the virus through an ion-exchange column. Other 37 purification procedures resulting in acceptable release criteria may be used. 38 39 A.4.4.2 Tests on virus pools (purified or not) before inactivation 40 41 A.4.4.2.1 Virus titration 42 43 The virus concentration of each virus pool should be determined by titration of infectious 44 virus using validated tissue culture methods. This titration should be carried out in not more WHO/BS/2020.2388 Page 23 1 than 10-fold dilution steps using 10 cultures per dilution, or by any other arrangement 2 yielding equal precision. 3 4 The use of human RD, human diploid or Vero cells in microtitre plates is suitable for this purpose (28). 5 The same cells should be used for virus titrations throughout the production process. 6 7 Information on virus titre will help in selecting pools that can be expected to meet potency 8 requirements following inactivation. 9 10 A.4.4.2.2 Virus antigen content 11 12 The antigen content of each virus pool should be determined using a validated 13 immunochemical method and should be calculated using a reference vaccine calibrated 14 against the First WHO International Standard for EV71 inactivated vaccine (see 15 International reference materials above) and expressed in IU. 16 17 A.4.4.2.3 Specific activity 18 19 The ratio of virus concentration or antigen content to the total protein content (specific 20 activity) of the virus pool before inactivation should be within the limits of material shown to 21 be safe and effective in clinical trials and approved by the NRA. This would ensure a 22 consistent ratio of chemical agent to the viral protein, and thus a consistent inactivation 23 process. 24 25 A. 4.4.3 Filtration before inactivation 26 27 In order to avoid interference with the inactivation process, virus aggregation should be 28 prevented or aggregates should be removed immediately before and during the inactivation 29 process. For this reason, each virus pool should be filtered before inactivation. 30 31 Satisfactory results have been reported with several filter types but a final filtration using a 0.22 µm 32 filter should be used. 33 34 Inactivation should be initiated as soon as possible and, in any case, not later than 72 hours 35 after filtration. 36 37 It is preferable to start inactivation within 24 hours of filtration. Since the purpose of the filtration step 38 is to remove particulate matter and other interfering substances that may diminish the effectiveness of 39 the inactivation process and since aggregates tend to increase on standing after filtration, efforts should 40 be made to keep within this time limit. 41 42 A sample of the filtered virus pool should be retained and its virus titre determined as 43 described in section A.4.4.2.1 above. 44 45 The main purpose of determining the titre of filtered virus pools destined for inactivation is to provide 46 the starting titre to monitor the kinetics of inactivation. WHO/BS/2020.2388 Page 24 1 2 A.4.5 Control of purified inactivated bulk 3 4 A.4.5.1 Inactivation procedure 5 6 The virus in the filtered pools should be inactivated using a validated method approved by the 7 NRA. Prior to inactivation, the concentration of the filtered pool (based on viral titre, virus 8 antigen or protein content) should be adjusted to the acceptable range established during the 9 process validation. 10 11 Most manufacturers currently use formaldehyde as the method for inactivation but at least one 12 manufacturer is using other inactivating agents such as beta-propiolactone (BPL). 13 14 The method of inactivation should have been shown to consistently inactivate EV71 virus 15 without destroying the antigenic and immunogenic activity. Inactivation of the virus pool 16 may take place before or after purification depending on the approved production process. 17 The progress of inactivation should be monitored by suitably spaced determinations of virus 18 titres. The inactivation period should usually exceed the time taken to reduce the titre of live 19 virus to undetectable amounts by a factor of at least 2 and be agreed to by the NRA. 20 21 A second filtration should be made during the process of inactivation. This step is made after 22 the virus titre has fallen below detectable levels but before the first sample for the safety test 23 is taken. 24 25 The kinetics of viral inactivation should be established by each manufacturer and approved 26 by the NRA. During the validation studies, an inactivation curve should be established with at 27 least four time points showing the decrease in live virus concentration with time. The 28 consistency of the inactivation process should be monitored; the virus titre and antigen 29 content of each pool before, during and at the end of inactivation should be determined. 30 31 A record of consistency in the effectiveness and kinetics of inactivation should be established 32 by the production of at least five consecutive lots and, if broken, a root-cause analysis should 33 be performed and a further five consecutive filtered purified virus pools should be prepared 34 and shown to be satisfactory for establishing this record. 35 36 A.4.5.2 Purification of inactivated virus pool 37 38 If inactivation is conducted using a non-purified virus pool, the inactivated pool should be 39 purified as described in section A.4.4.1 above. 40 41 A.4.5.3 Tests on purified inactivated bulk 42 43 A.4.5.3.1 Test for effective inactivation 44 WHO/BS/2020.2388 Page 25 1 After removal or neutralization (as appropriate) of the inactivating agent, the absence of 2 residual live EV71 virus should be verified by inoculating a quantity of the inactivated virus 3 bulk equivalent to 5% of the lot (or not less than 1000 doses of vaccine) into sensitive cell 4 cultures of the same type as those used for vaccine production. 5 6 The virus sample should be incubated for no less than 21 days making no fewer than two cell 7 passages during that period. The dilution of the sample in the nutrient fluid should not exceed 8 1 in 4 and the area of the cell sheet should be at least 3 cm2 per mL of sample. One or more 9 culture vessels of each lot of cultures should be set aside to serve as uninoculated control 10 culture vessels with the same medium. The sensitivity of the assay should be demonstrated. 11 12 If formaldehyde has been used as the inactivating agent, samples of vaccine for tissue culture tests are 13 generally neutralized at the time of sampling by the addition of bisulfite. Usually, the samples are 14 subsequently dialysed or another validated method used. 15 16 It is possible to conduct tissue culture tests on non-dialysed material. However, this is often found to be 17 toxic to cells, even with a dilution of 1 in 4. If in such tests, nonspecific degeneration of cells occurs, or 18 if the sensitivity of the tissue culture system is reduced, the test should be repeated on dialysed 19 material. The virus antigen content after dialysis should be determined to ascertain whether the viral 20 antigen was lost during the dialysis process. 21 22 If infectious virus is detected, the bulk should not be used for further processing. The 23 isolation of live virus from an inactivated bulk should be regarded as a break in the 24 manufacturing consistency record and a production process review and revalidation should be 25 undertaken. 26 27 It is important to demonstrate that each test retains sensitivity to detect partially inactivated 28 EV71 virus. At the end of the observation period, the cell culture used for the detection of 29 residual live virus should be challenged with a validated amount of live EV71 virus of the 30 same strain as that of the inactivated virus bulk. The details of the challenge procedure should 31 be approved by the NRA. The ability to detect infectious virus should be checked 32 concurrently for each test by including a positive control at the beginning of each test. 33 Positive control flasks should be inoculated with a low quantity of virus close to the detection 34 limit of the method. Alternatively, if no positive control is used, a challenge test should be 35 performed as described above. 36 37 The problem of detecting residual active virus in an inactivated vaccine is not the same as 38 that of measuring infective virus in untreated suspensions. Other similar viruses that have 39 been exposed to the action of formaldehyde without becoming inactivated have been shown 40 to require a much longer period to produce cytopathic changes than untreated virus. For this 41 reason, it is desirable that tissue cultures in tests for the presence of residual active virus are 42 observed for as long a time as is technically possible. A satisfactory tissue culture system for 43 this purpose depends, therefore, not only on the sensitivity of the cells used for the 44 preparation of the cultures but also on the nutrient fluid. 45 WHO/BS/2020.2388 Page 26

1 Serum added to the nutrient fluid should be tested for inhibitors to EV71 at serum concentrations up to 2 50%. Only serum free from inhibitors should be used. 3 4 Maintenance of the cultures in good condition may require frequent changes of culture medium. 5 However, it should be borne in mind that early changes of fluid may result in unadsorbed virus being 6 removed and the validity of the test would thus be impaired. Therefore, the fluid should be changed no 7 earlier than 5–7 days after inoculation. 8 9 A.4.5.3.2 Sterility tests for bacteria and fungi 10 11 Each purified inactivated bulk should be tested for bacterial and fungal sterility as specified 12 in Part A, section 5.2 of the WHO General requirements for the sterility of biological 13 substances (57), or by methods approved by the NRA. 14 15 A.4.5.3.3 Antigen content 16 17 The EV71 antigen content of each purified inactivated bulk should be determined by the use 18 of a validated immunochemical method and should be calculated using a reference vaccine 19 calibrated against the First WHO International Standard for EV71 inactivated vaccine (see 20 International reference materials above). The results obtained should be expressed in IU 21 and be within the required limits established by the NRA. 22 23 A.4.5.3.4 Residual inactivating agent 24 25 The concentration of free residual formaldehyde or any other chemical used for inactivating 26 the virus should be determined by a method approved by the NRA. The maximum acceptable 27 limit should be approved by the NRA. 28 29 If BPL is used then its hydrolysation kinetics should be determined during the validation of the 30 inactivation process to ensure that no residual BPL is present in the inactivated bulk at the end of the 31 inactivation step. 32 33 A.4.5.3.5 Residual cellular DNA 34 35 If continuous cell lines are used for production, the purification procedure should have been 36 shown to consistently reduce the level of residual host cell DNA (28). The amount and size of 37 residual host cell DNA should not exceed the maximum levels agreed with the NRA, taking 38 into consideration issues such as those discussed in the WHO Recommendations for the 39 evaluation of animal cell cultures as substrates for the manufacture of biological medicinal 40 products and for the characterization of cell banks (28). Human diploid cell lines have been 41 used successfully for many years for the production of viral vaccines and the residual cellular 42 DNA deriving from these cells is not considered to pose any significant risk (28). 43 44 This test can be performed on the purified virus bulk and may be omitted from routine testing, with the 45 agreement of the NRA, if the manufacturing process is validated to achieve this specification (28). 46 WHO/BS/2020.2388 Page 27

1 If assessed, the size distribution of the DNA may be considered as a characterization test, taking into 2 account the amount of DNA detectable using appropriate methods, as approved by the NRA (28). 3 4 A.4.5.3.6 Residual chemicals 5 6 If chemical substances are used during the purification process, tests for these substances 7 should be carried out. Their concentration should not exceed the limits approved for the 8 particular product. 9 10 A.4.6 Control of final bulk 11 12 Preservatives, excipients or other substances that might be added to form the final bulk 13 should have been shown, to the satisfaction of the NRA, to have no deleterious effect on the 14 immunizing potency and safety profile of the EV71 antigens. Preservative efficacy should be 15 demonstrated during product development using a method approved by the NRA. 16 17 The operations necessary for preparing the final bulk from the purified inactivated bulk 18 should be conducted in such a manner as to avoid contamination of the product. In preparing 19 the final vaccine bulk, any substances that are added to the product (such as diluents, 20 stabilizers or adjuvants) should have been shown, to the satisfaction of the NRA, not to 21 impair the safety and efficacy of the vaccine in the concentrations used. Until the final bulk is 22 filled into containers, the final vaccine bulk suspension should be stored under conditions 23 shown by the manufacturer to retain the desired biological activity. 24 25 A.4.6.1 Sterility tests for bacteria and fungi 26 27 The final bulk should be tested for bacterial and fungal sterility as specified in Part A, section 28 5.2 of the WHO General requirements for the sterility of biological substances (57), or by 29 methods approved by the NRA. 30 31 A.4.6.2 Potency tests 32 33 Each final bulk should be tested using an in vivo assay for immunogenicity and in vitro 34 antigen content assay approved by the NRA, unless this is to be performed on final product. 35 Product-specific reference preparations may be used in these tests. 36 37 The EV71 antigen content of each final bulk should be determined using a validated 38 immunochemical method and calculated using a reference vaccine calibrated against the First 39 WHO International Standard for EV71 inactivated vaccine (see International reference 40 materials above). The in vitro assay found to be the most suitable for measuring the antigen 41 content is the EV71 antigen ELISA. The results obtained should be within the required limits 42 established by the NRA. 43 WHO/BS/2020.2388 Page 28 1 Once consistency of production has been established for a suitable number of consecutive 2 final bulks, the in vivo assay may be omitted for the purpose of routine lot release, with the 3 agreement of the NRA. This can occur once it has been demonstrated that the acceptance 4 criteria for the EV71 antigen determination are such that the in vitro test yields a comparable 5 result to the in vivo assay in terms of acceptance or rejection of a lot. This demonstration 6 should include testing of sub-potent lots, produced experimentally if necessary by heat 7 treatment or other means of diminishing the immunogenic activity. 8 9 If an adjuvant is used in the final bulk, a desorption or treatment step may be necessary 10 before performing the EV71 antigen ELISA. 11 12 If the final bulk is formulated with other antigens into a combination vaccine, the suitability 13 of performing the EV71 antigen ELISA on the final bulk will have to be determined. If the 14 EV71 antigen ELISA is not suitable for a particular combination, an in vivo assay should be 15 used. 16 17 The potency of the final bulk should be approved by the NRA. 18 19 A.4.6.3 Preservative content (if applicable) 20 21 If preservative is added, its concentration in the final bulk should be determined by a method 22 approved by the NRA. The preservative used and concentration permitted should be 23 approved by the NRA. The preservative should not adversely affect the quality of the 24 antigens. 25 26 A.4.6.4 Adjuvant (if applicable) 27 28 Each final vaccine bulk should be assayed for adjuvant content. This test may be omitted if it 29 is to be performed on the final lot. Where aluminium compounds are used, the aluminium 30 content should not exceed 1.25 mg per single human dose. 31 32 A.5 Filling and containers 33 34 The requirements concerning filling and containers given in WHO good manufacturing 35 practices for pharmaceutical products: main principles (55) and WHO good manufacturing 36 practices for biological products (27) should apply to vaccine filled in the final form. Single- 37 and multi-dose containers may be used. 38 39 Care should be taken to ensure that the materials of which the container and (if applicable) 40 the transference devices and closure are made do not adversely affect the quality of the 41 vaccine. 42 WHO/BS/2020.2388 Page 29 1 Manufacturers should provide the NRA with adequate data to prove the stability of the 2 product under appropriate conditions of storage and transport. 3 4 A.6 Control tests on the final lot 5 6 Samples should be taken from each final lot for the tests described in the following sections. 7 The following tests should be performed on each final lot of vaccine (that is, in the final 8 containers). Unless otherwise justified and authorized, the tests should be performed on 9 labelled containers from each final lot by means of validated methods approved by the NRA. 10 All tests and specifications, including methods used and permitted concentrations, should be 11 approved by the NRA. 12 13 A.6.1 Inspection of final containers 14 15 Every container in each final lot should be inspected visually or mechanically, and those 16 showing abnormalities should be discarded and recorded for each relevant abnormality. A 17 limit should be established for the percentage of rejections permitted before triggering an 18 investigation of the cause, potentially resulting in lot failure. 19 20 A.6.1.1 Appearance 21 22 The appearance of the vaccine should be described with respect to its form and colour. 23 24 A.6.2 Identity test 25 26 An identity test should be performed on at least one labelled container from each final lot 27 using an appropriate method. The potency test described in section A.6.4 below may serve as 28 the identity test. 29 30 A.6.3 Sterility tests for bacteria and fungi 31 32 Each final lot should be tested for bacterial and fungal sterility as specified in Part A, section 33 5.2 of the WHO General requirements for the sterility of biological substances (57), or by 34 methods approved by the NRA. 35 36 A.6.4 Potency test 37 38 The potency of each final lot should be determined using an in vivo assay and a validated in 39 vitro antigen content assay (see section A.4.6.2 above) if such a test has not been performed 40 on the final bulk. Potency should be calculated using a reference vaccine calibrated against 41 the First WHO International Standard for EV71 inactivated vaccine (see International 42 reference materials above) or other reference preparation. 43 WHO/BS/2020.2388 Page 30 1 If the use of an adjuvant in the final bulk interferes with the assay, a desorption or treatment 2 step may be necessary. If treatment/desorption is not possible, the interference of the adjuvant 3 should be documented and an in vivo assay should be performed (see section A.4.6.2 above). 4 5 The potency of the vaccine should be approved by the NRA. 6 7 A.6.5 Preservative content 8 9 Where appropriate, the preservative content of each final lot should be determined. The 10 method used and content permitted should be approved by the NRA. This test may be omitted 11 if conducted on the final bulk. 12 13 A.6.6 Endotoxin content 14 15 The endotoxin content of each final lot should be determined using a method approved by the 16 NRA. Endotoxin levels should be consistent with levels found to be acceptable in vaccine 17 lots used in pre-licensure clinical trials and should be approved by the NRA. 18 19 A.6.7 Residual formaldehyde 20 21 The concentration of free residual formaldehyde in each final lot should be determined using 22 a method approved by the NRA. The acceptable maximum limit should be approved by the 23 NRA. This test may be omitted if performed on the final bulk. 24 25 A.6.8 pH 26 27 The pH of each final lot should be determined and should be within limits approved by the 28 NRA. 29 30 A.6.9 Adjuvant and degree of adsorption (if applicable) 31 32 If an adjuvant is used in the formulation, each final lot should be assayed for adjuvant 33 content. Where aluminium compounds are used, the aluminium content should not exceed 34 1.25 mg per single human dose. 35 36 The degree of adsorption of the antigen to the aluminium compounds (aluminium hydroxide 37 or hydrated aluminium phosphate) in each final lot should be assessed. 38 39 Both tests may be omitted for routine lot release upon demonstration of consistency of 40 production, subject to the agreement of the NRA. 41 42 A.6.10 Residual antibiotics (if applicable) 43 WHO/BS/2020.2388 Page 31 1 If any antibiotics are added during vaccine production, the residual antibiotic content should 2 be determined and should be within limits approved by the NRA. This test may be omitted 3 for routine lot release once consistency of production has been established to the satisfaction 4 of the NRA. 5 6 If aluminium adsorption has an impact on the test, then testing for antibiotic content may be done at the 7 purified inactivated bulk stage. 8 9 A.6.11 Extractable volume 10 11 For vaccines filled into single-dose containers, the extractable content should be checked and 12 shown to be not less than the intended dose. 13 14 For vaccines filled into multi-dose containers, the extractable content should be checked and 15 should be shown to be sufficient for the intended number of doses. 16 17 A.7 Records 18 19 The requirements given in WHO good manufacturing practices for pharmaceutical products: 20 main principles (55) and WHO good manufacturing practices for biological products (27) 21 should apply. 22 23 A.8 Retained samples 24 25 The requirements given in WHO good manufacturing practices for pharmaceutical products: 26 main principles (55) and WHO good manufacturing practices for biological products (27) 27 should apply. 28 29 A.9 Labelling 30 31 The requirements given in WHO good manufacturing practices for pharmaceutical products: 32 main principles (55) and WHO good manufacturing practices for biological products (27) 33 should apply, and additionally the label on the container or package, or the leaflet 34 accompanying each container, should include the following information: 35 36 ▪ the designation(s) of the strain(s) of EV71 contained in the vaccine 37 ▪ the cell substrate used for the preparation of vaccine 38 ▪ the antigen content 39 ▪ the method and inactivating agent used to inactivate the virus 40 ▪ the nature and amount of any stabilizer and preservative present in the vaccine 41 ▪ the nature and amount of adjuvant, if applicable. 42 WHO/BS/2020.2388 Page 32

1 It is desirable for the label to carry the names both of the producer and of the source of the bulk 2 material if the producer of the final vaccine did not prepare it. The nature and amount of antibiotics 3 present in the vaccine, if any, may also be included. 4 5 A.10 Distribution and transport 6 7 The requirements given in WHO good manufacturing practices for pharmaceutical products: 8 main principles (55) and WHO good manufacturing practices for biological products (27) 9 should apply. Further guidance is provided in the WHO Model guidance for the storage and 10 transport of time- and temperature-sensitive pharmaceutical products (61). 11 12 A.11 Stability testing, storage and expiry date 13 14 A.11.1 Stability testing 15 16 Adequate stability studies form an essential part of vaccine development. Current guidance 17 on the evaluation of vaccine stability is provided in the WHO Guidelines on stability 18 evaluation of vaccines (62). Stability testing should be performed at different stages of 19 production when intermediate product is stored, namely on single harvests, purified 20 inactivated bulk, final bulk and final lot. Stability-indicating parameters should be defined or 21 selected appropriately according to the stage of production. During vaccine production a 22 shelf-life should be assigned to all in-process materials – particularly intermediates such as 23 single harvests, purified inactivated bulk and final bulk. 24 25 The stability of the vaccine in its final containers, maintained at the recommended storage 26 temperature up to the expiry date, should be demonstrated to the satisfaction of the NRA. As 27 a guide, containers from at least three consecutive final lots derived from different bulks may 28 be tested. 29 30 Accelerated stability tests may be undertaken to provide additional information on the overall 31 characteristics of the vaccine and may also aid in assessing comparability should the 32 manufacturer decide to change any aspect of manufacturing. 33 34 The formulation of the vaccine should be stable throughout its shelf-life. Acceptable limits 35 for stability should be agreed with the NRA. Following licensure, ongoing monitoring of 36 vaccine stability is recommended to support shelf-life specifications and to refine the stability 37 profile (62). Data should be provided to the NRA in accordance with local regulatory 38 requirements. 39 40 The final stability testing programme should be approved by the NRA and should include an 41 agreed set of stability-indicating parameters, procedures for the ongoing collection and 42 sharing of stability data, and criteria for rejecting vaccine(s). 43 44 A.11.2 Storage conditions WHO/BS/2020.2388 Page 33 1 2 EV71 vaccine (inactivated) should be stored at all times at a temperature of 2–8 °C. 3 4 If a vaccine has been shown to be stable at temperature ranges higher than the approved 2– 5 8 °C range, it may be stored under extended controlled temperature conditions for a defined 6 period, subject to the agreement of the NRA (63). 7 8 A.11.3 Expiry date 9 10 The expiry date should be based on the shelf-life as supported by stability studies and 11 approved by the NRA. The start of the dating period should be based on the date of blending 12 of the final bulk, the date of filling or the date of the first valid potency test on the final lot, 13 and should be agreed with the NRA. 14 15 Where an in vivo potency test is used, the date of the potency test is the date on which the test animals 16 were inoculated with the final bulk. 17 18 Part B. Nonclinical evaluation of enterovirus 71 vaccines (inactivated) 19 20 Nonclinical evaluation of a new EV71 vaccine should follow the principles outlined in the 21 WHO guidelines on nonclinical evaluation of vaccines (25) which provide details on the 22 design, conducting, analysis and evaluation of nonclinical studies. Further guidance on the 23 general principles for the nonclinical evaluation of vaccine adjuvants and adjuvanted 24 vaccines can be found in separate WHO Guidelines (29). The following sections B.1–B.3 25 provide specific guidance on addressing important issues related to the nonclinical 26 development of a new inactivated whole EV71 virus vaccine. 27 28 B.1 Product characterization and process development 29 30 The vaccine lots used in nonclinical studies should be adequately characterized as described 31 in Part A of these WHO Recommendations, taking into consideration the stage of product 32 development. Both the antigen(s) of the vaccine and the end product need to be clearly 33 defined, and the manufacturing process carefully monitored for all crucial steps so as to 34 confirm the consistency of production. It is essential that sufficient data are generated to 35 confirm the full inactivation of vaccine virus and the absence of virulent virus in the end 36 product. Furthermore, sufficient vaccine stability data are necessary to support its suitability 37 for use in the nonclinical studies. 38 39 It is crucially important that vaccine manufacturing processes are appropriately standardized 40 and controlled to ensure consistency of production. The extent of product characterization 41 may vary according to the stage of development. To support their validity, nonclinical studies 42 should be carried out on vaccine lots that are adequately representative of the concurrent 43 clinical lots in terms of their physicochemical data, stability, qualitative and quantitative 44 impurity profiles, and formulation. WHO/BS/2020.2388 Page 34 1 2 B.2 Nonclinical immunogenicity and protection studies 3 4 B.2.1 Evaluation of immunogenicity in animal models 5 6 Unless otherwise justified, the immunogenicity of any new EV71 vaccine needs to be 7 characterized in relevant animal models (for example, mice, rats or rabbits) before proceeding 8 to human trials. These proof-of-concept nonclinical studies should reflect the clinically 9 proposed use of the vaccine, including the administration route, and should include an 10 evaluation of serum neutralizing antibody response against isolated virus strains or 11 pseudoviruses (5–7, 64, 65), and dose-range testing of the antigen. The immune response to 12 the candidate vaccine should ideally be assessed after each dose of vaccine, and whenever 13 possible against a licensed EV71 vaccine used as an active control. Data on cross-neutralizing 14 antibodies should be obtained from nonclinical immunogenicity studies using a range of 15 isolated heterologous viruses or pseudoviruses of different subgenogroups. These data may 16 guide selection of the doses, dosing regimen and administration route to be evaluated in 17 clinical trials. 18 19 When a candidate EV71 vaccine is formulated with a new adjuvant, a rationale for the 20 selection of the adjuvant should be provided and the benefit of its inclusion in the vaccine 21 formulation should be demonstrated by the immunogenicity data. 22 23 The immunogenicity studies in animals may additionally be considered, when appropriate, as 24 part of a comparability exercise to demonstrate the reproducibility of the manufacturing 25 process whenever major changes are introduced during the different stages of process 26 development or during the validation phase of a new candidate EV71 vaccine manufacturing 27 process. 28 29 B.2.2 Challenge-protection studies 30 31 Current evidence suggests that serological immune responses play an essential role in 32 mediating protection by formalin-inactivated whole EV71 virus vaccines. Animal studies 33 conducted in newborn mice, transgenic mice and nonhuman primates have demonstrated that 34 vaccination with inactivated EV71 vaccines induces protective immunity against EV71 and 35 that protection in challenged animals is primarily mediated by neutralizing antibodies (41, 47, 36 50, 66). Importantly, human efficacy trials conducted with several formalin-inactivated EV71 37 vaccines have shown a strong correlation between vaccine-induced serum neutralizing 38 antibodies and protection against EV71-associated diseases (5–7). Based on these 39 observations, it is considered that, for a similarly manufactured candidate EV71 vaccine, no 40 further challenge-protection studies in animal models need to be performed. 41 42 However, challenge-protection studies may be useful for any candidate EV71 vaccine based 43 on a novel production process or intended to have novel mechanisms of action. WHO/BS/2020.2388 Page 35 1 2 Since evidence from epidemiological studies and clinical trials for cross-protection against 3 EV71 disease has thus far been limited, any claims of cross-protection should in general be 4 supported by appropriate animal data. Specifically, challenge-protection studies should be 5 conducted in appropriate animal models to evaluate the potential for protection against 6 heterologous viruses of different genogroups, as this could indicate the breadth of protection. 7 8 B.3 Nonclinical safety studies 9 10 For a new EV71 vaccine based on inactivated whole EV71 virus, a repeat-dose toxicity study 11 in a relevant animal species is generally needed to assess potential local and systemic toxicity 12 and any other undesirable effects. Omission of standalone local tolerance and single-dose 13 toxicity studies is possible if assessment of acute toxic effects and local tolerance has been 14 incorporated into the repeat-dose toxicity study. 15 16 If the candidate vaccine contains a novel adjuvant, the principles set out in the WHO 17 Guidelines on the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines (29) 18 should be followed. For example, consideration should be given to assessing the toxicity of 19 the adjuvant alone. 20 21 For candidate EV71 vaccines manufactured using novel cell substrates, efforts should be 22 made during the nonclinical safety study to explore biomarkers indicative of potential allergic 23 reactions – for example, by measuring type 2 CD4+ T-helper cell responses. 24 25 Part C. Clinical evaluation of enterovirus 71 vaccines (inactivated) 26 27 C.1 Introduction 28 29 Clinical trials should adhere to the principles described in the WHO Guidelines for good 30 clinical practice (GCP) for trials on pharmaceutical products (67) and the WHO Guidelines 31 on clinical evaluation of vaccines: regulatory expectations (26). This section focuses only on 32 issues relevant or specific to the clinical development of inactivated EV71 vaccines. 33 34 At present, no efficacy data on cross-protection are available from completed clinical trials 35 with inactivated EV71 vaccines due to the distinct regional circulation of specific 36 subgenogroups. In addition, no internationally recognized immune correlate of protection 37 (ICP) or surrogate marker of protection has been established. Although immunogenicity 38 results from clinical trials suggest that a neutralizing antibody titre of 1:16 to 1:32 might be 39 related to protection, further analysis based upon the use of a scaled logit model has indicated 40 that significantly higher levels of neutralizing antibodies might be needed to achieve 41 protection (5–7, 51, 64). 42 WHO/BS/2020.2388 Page 36 1 If cross-protection against heterologous EV71 viruses is to be claimed then appropriate 2 nonclinical and/or clinical studies should be conducted to evaluate the potential for such 3 cross-protection (see section B.2.1 above). In addition, data demonstrating the ability of 4 antibodies obtained from vaccinated individuals to neutralize various subgenogroups of EV71 5 viruses in vitro, including recently circulating isolates, are expected to be provided. 6 Continuous evaluation of protective vaccine efficacy post licensure is encouraged due to the 7 evolution of new EV71 strains or a rapid change of subgenogroups in different countries and 8 regions, which may result in outbreaks. Should new subgenogroups emerge in a country in 9 which the vaccine is licensed and used, further clinical investigation of potential cross- 10 protection may be needed. 11 12 C.2 Assays 13 14 General guidance on the use and validation of assays for the evaluation of immune responses 15 is provided in section 5.3.3 of the WHO Guidelines on clinical evaluation of vaccines: 16 regulatory expectations (26). 17 18 Sections C.2.1 and C.2.2 provide specific guidance on the following assays relevant to the 19 investigation of immune responses to inactivated human EV71 vaccines in clinical trials and 20 to the confirmation of vaccine efficacy in pivotal studies respectively: 21 22 ▪ serological assays for establishing the baseline serostatus of trial subjects and 23 evaluating the humoral immune response to vaccination (see also section C.3); and 24 ▪ detection assays for laboratory confirmation of HFMD and herpangina caused by 25 EV71 infection, in vaccine efficacy trials (see also section C.4). 26 27 C.2.1 Serological assays 28 29 C.2.1.1 Functional antibody 30 31 The direct measurement of anti-EV71 neutralizing antibody is well established. Neutralizing 32 antibody has been estimated using methods such as plaque reduction neutralization assays 33 employing either isolated virus strains or pseudoviruses (5–7, 64, 65). Sponsors are 34 encouraged to develop high-throughput assays for anti-EV17 neutralizing antibody. These 35 assays should be standardized using the First WHO International Standard for anti-EV71 36 serum (human) and the First WHO International Reference Reagent for EV71 neutralization 37 assays (see International reference materials above). In addition, a reference neutralizing 38 antibody panel for the evaluation of neutralizing antibody responses has been established in 39 China (14). 40 41 C.2.2 Virus detection assays 42 WHO/BS/2020.2388 Page 37 1 Since HFMD and herpangina can be caused by different human enteroviruses, including 2 EV71, Coxsackie and echoviruses, appropriate RNA or virus detection assays are required to 3 confirm the presence of EV71 in throat and vesicle swabs and/or stool samples (see section 4 C.4.2 below). International guidance on enterovirus diagnostics and characterization should 5 be taken into consideration (4). 6 7 For other enterovirus infections, laboratory confirmation of diagnosis based on cell culture, 8 virus isolation and virus identification remains a standard approach. The use of established 9 cell lines such as human RD or Vero cells is recommended for virus isolation. 10 11 Various quantitative polymerase chain reaction (PCR) assays are commercially available. 12 Although several EV71-specific PCR systems have been described, the ability of an assay to 13 reliably detect EV71 RNA from specific subgenogroups should be taken into account when 14 selecting the method to be used in clinical trials. In general, it is recommended that the 15 subgenogroup be determined based upon VP1 gene sequences. 16 17 Sponsors should provide full details of the methodology applied, and appropriate controls 18 should be used. 19 20 In addition, EV71 infection can be confirmed by the use of anti-EV71 immunoglobulin M 21 (IgM) assays. 22 23 C.3 Immunogenicity 24 25 C.3.1 Formulation, dose and regimen 26 27 C.3.1.1 Primary series 28 29 EV71 vaccines will be used mainly or exclusively in regions with relatively high rates of 30 clinically apparent infections. In naturally primed individuals the first dose of EV71 vaccine 31 may elicit large increments in antibody due to an anamnestic response. In contrast, multiple 32 doses of the same vaccine may be required to achieve similar antibody levels in EV71-naive 33 subjects. Since pre-vaccination testing for EV71 serostatus will not be practical in routine 34 use, it is important that the primary series should be selected on the basis of the immune 35 responses observed in subjects who were seronegative prior to vaccination. 36 37 In the absence of an internationally established ICP for EV71 – that is, one meeting the WHO 38 definition of an ICP (26) – the selection of the vaccine dose and regimen may be based on the 39 reaching of a plateau antibody response unless this is precluded by concerns over 40 reactogenicity. It is desirable that immunogenicity studies should explore the minimum 41 number of doses and the shortest dose interval(s) required to achieve a plateau immune 42 response. 43 WHO/BS/2020.2388 Page 38 1 C.3.1.2 Cross-protection 2 3 The ability of a candidate EV71 vaccine to protect against a range of wild-type strains 4 covering the main EV71 genogroups may vary according to the vaccine strain used. For 5 example, lower cross-neutralization against an atypical C2-like strain was observed in 6 naturally infected EV71 patients (68) and in clinical trials using B4-based vaccine strains 7 (22). 8 9 In clinical trials in which vaccine-elicited antibody is determined against the antigen in the 10 vaccine, it is recommended that neutralizing activity is also measured using antigens derived 11 from a range of circulating wild-type EV71 strains from different (sub)genogroups. If marked 12 differences are observed in measured antibody levels using vaccine versus non-vaccine 13 strains, and/or by EV71 subgenogroup, it would be of particular interest to assess whether a 14 similar effect is observed for functional antibody levels in naturally infected individuals. 15 16 C.4 Efficacy 17 18 C.4.1 Requirement for a demonstration of vaccine efficacy 19 20 It is currently recommended that licensure of a candidate EV71 vaccine should be based on 21 evidence of its protective efficacy against clinically apparent HFMD and herpangina. The 22 following considerations apply: 23 24 ▪ At the time of preparing these WHO Recommendations, three vaccines against 25 human EV71 had been licensed in one country (see General considerations above) 26 (4, 9, 17). 27 ▪ These licensed vaccines are not yet widely used internationally. As a result, the use 28 of a control group that does not receive vaccination against EV71 is possible. 29 ▪ In jurisdictions in which a licensed vaccine is available, it is possible that individual 30 NRAs may consider that licensure can be based on a trial that evaluates the efficacy 31 of the candidate vaccine relative to that of the licensed vaccine in a population 32 similar to that in which the efficacy of the licensed vaccine was established. 33 ▪ The lack of an established ICP against EV71 does not rule out immunobridging a 34 candidate vaccine to a licensed vaccine that has been shown to be efficacious. 35 However, this approach is only possible if both vaccines contain the same antigen(s) 36 so that anti-EV71 neutralizing antibody immune responses can be compared directly. 37 In addition, the demonstration of efficacy of all three licensed vaccines was confined 38 to EV71 subgenogroup C4 and it is not known whether protective efficacy may vary 39 between genogroups circulating in different regions. 40 41 Taking these considerations into account, the focus of the following sections is on clinical 42 development programmes that include vaccine efficacy trials in which the control group does 43 not receive vaccination against EV71. However, most of the recommendations are also WHO/BS/2020.2388 Page 39 1 applicable to trials in which the control group receives a licensed vaccine against EV71. 2 Clinical programmes leading to licensure based on immunobridging are not addressed in the 3 following guidance. The general principles to be considered are discussed in sections 5.6.2 4 and 6.3.3 of the WHO Guidelines on clinical evaluation of vaccines: regulatory expectations 5 (26). 6 7 C.4.2 Considerations for efficacy trial design 8 9 C.4.2.1 Primary objective 10 11 The primary objective will be to demonstrate that the candidate vaccine protects against 12 clinically apparent (that is, symptomatic) HFMD and herpangina caused by EV71 infection 13 regardless of the genogroup (see section C.4.2.4). 14 15 ▪ It is not required for efficacy to be shown against asymptomatic EV71 infection as 16 such infections are of no clinical significance. 17 ▪ It is not required for vaccine efficacy trials to be powered to demonstrate genogroup- 18 specific efficacy (see section C.4.2.2). 19 20 C.4.2.2 Trial sites 21 22 Efficacy trials will be conducted in endemic areas in which the estimated attack rate for 23 HFMD and herpangina due to EV71 infection is sufficient to complete enrolment into an 24 adequately powered vaccine efficacy trial within a reasonable time frame. Sites may be 25 chosen on the basis of available public health disease-surveillance data and/or pre-trial 26 evaluations of epidemiology conducted by the sponsor. In three prior efficacy trials (6–9) the 27 EV71 viruses that caused clinically apparent HFMD and herpangina were limited to strains of 28 the C4 subgenogroup circulating at the trial sites in the years in which they were conducted. 29 Sponsors are encouraged to consider selecting sites in a range of geographical areas in which 30 strains of different genogroups are circulating and/or to conduct separate vaccine efficacy 31 trials in regions with different genogroup distributions. 32 33 C.4.2.3 Subject selection criteria 34 35 Because of the age-dependent incidence and severity of EV71 infections it is likely that 36 vaccine efficacy trials will target infants and children. An upper age limit may be set 37 depending on the age-specific attack rates. 38 39 C.4.2.4 Primary end-point 40 41 In accordance with the recommended primary objective above, the primary end-point should 42 be clinically apparent HFMD or herpangina that is confirmed to be due to EV71 infection. 43 Sponsors could consider appointing an independent data-monitoring committee to review the WHO/BS/2020.2388 Page 40 1 data and determine which subjects meet the case definition to be counted in the primary 2 analysis. 3 4 C.4.2.4.1 Clinical features for the case definition 5 6 The clinical features that cause subjects to present to study site staff or to a local designated 7 health care facility for laboratory investigations for acute HFMD or herpangina should be 8 identified with the aim of capturing as many cases as possible while limiting unnecessary 9 investigations. On this basis, it is reasonable to define a possible case of HFMD or 10 herpangina requiring laboratory investigation as an illness presenting with febrile illness 11 accompanied by a papular or vesicular rash in the characteristic distribution on the oral 12 mucosa, hands, feet or buttocks. A severe case of HFMD should be defined as associated 13 with neurological, respiratory or circulatory complications as described by WHO (4). 14 15 C.4.2.4.2 Laboratory confirmation of HFMD or herpangina caused by EV71 infection 16 17 It is recommended that laboratory confirmation of HFMD and herpangina cases should be 18 conducted in a designated qualified central laboratory. If more than one central laboratory is 19 necessary for practical reasons, it is essential that all of the laboratories use identical 20 methodologies, and consideration should be given to testing a randomly selected subset of 21 samples at each laboratory to assess concordance. The laboratory methods used should be 22 validated. 23 24 The confirmation of EV71 as causative of the clinical picture should be based on any of the 25 following: 26 27 ▪ detection of EV71 RNA in vesicle/throat swabs or in stool; 28 ▪ virus isolation and analysis of VP1 sequence; 29 ▪ detection of IgM against EV71 – which is often detectable at the time of onset of 30 clinical symptoms but which may peak after 1–2 weeks. 31 32 To avoid cases being missed, protocols should plan for appropriately timed repeat specimens 33 to be collected from individuals with a first negative test for EV71 RNA (for example, 3–7 34 days after the first sample). 35 36 Samples obtained at first presentation and repeat specimens should also be tested to detect 37 infection with other enteroviruses, such as Coxsackie and echoviruses, that can also cause 38 HFMD and which regularly co-circulate with EV71 in affected countries. 39 40 C.4.2.5 Primary, secondary and other analyses 41 42 In a vaccine efficacy trial, it may be permissible for the primary analysis to include only 43 confirmed cases of HFMD and herpangina caused by EV71 as follows: 44 WHO/BS/2020.2388 Page 41 1 ▪ in subjects who completed the vaccination series within predetermined visit 2 windows, if more than one dose is required; and 3 ▪ with symptom onset occurring more than a defined period after the only or final dose 4 of the series, taking into account what is known about the timing of the post-dose 5 anti-EV71 IgG peak. 6 7 This approach gives the most optimistic estimation of vaccine efficacy. 8 9 If the primary analysis is confined to cases counted as described above, it is essential that 10 predefined secondary analyses are carried out to estimate vaccine efficacy based on 11 confirmed cases of clinically apparent HFMD and herpangina caused by EV71 infection, 12 defined and counted as follows: 13 14 ▪ all cases in subjects who received at least one assigned dose as randomized, and 15 regardless of adherence to study visit windows; 16 ▪ cases that occurred at any time after the last dose received (that is, counted from the 17 day of dosing) in those who completed the assigned number of doses; 18 ▪ cases that occurred after each sequential dose, depending on the number of doses in 19 the series and counted from the day of dosing. 20 21 Vaccine efficacy should be explored according to EV71 genogroup if this is feasible, 22 depending on the numbers of cases that occur due to individual genogroups. 23 24 It is recommended that an additional analysis should explore any differences in clinical or 25 laboratory features (including severity) between cases that occur in the candidate vaccine 26 group and the control group (whether the control group receives placebo or a licensed vaccine 27 against EV71). The analysis should take into account whether the severity observed in 28 individual subjects could reflect coinfection with other enteroviruses. 29 30 C.4.2.6 Case ascertainment 31 32 It is recommended that an active case-ascertainment strategy is used throughout the time 33 frame of a vaccine efficacy trial. This is essential at least up to the time of the primary 34 analysis, which may be conducted after a specific number of total cases has been 35 accumulated or after a predefined period in which a sufficient number of cases are expected 36 to occur to estimate vaccine efficacy. 37 38 C.4.2.7 Duration of protection 39 40 While the primary analysis may lead to licensure, it is recommended that trials continue to 41 use active case ascertainment to follow up subjects for several years to provide data on 42 waning vaccine protection, without un-blinding of treatment assignment at the level of the 43 individual. These data can then be reported at some point after licensure of the vaccine and WHO/BS/2020.2388 Page 42 1 may point to a need for further doses to be administered at intervals to maintain protection, or 2 a need to change the vaccine strains used. 3 4 C.4.2.8 Vaccine effectiveness 5 6 The need for vaccine effectiveness studies should be established at the time of licensure. 7 8 If longer-term follow-up within a pre-licensure trial is not considered to be feasible, the 9 duration of vaccine protection should be investigated within a vaccine effectiveness study 10 and/or as part of routine disease surveillance conducted by public health authorities. 11 Furthermore, the efficacy of the vaccine against individual subgenogroups should be explored 12 as part of a vaccine effectiveness study and/or during routine disease surveillance. 13 14 C.5 Safety 15 16 Evaluation of the safety of candidate EV71 vaccines should be undertaken in accordance with 17 the recommendations made in section 7 of the WHO Guidelines on clinical evaluation of 18 vaccines: regulatory expectations (26). If the primary series consists of several vaccine doses 19 it is important to document whether reactogenicity increases with sequential doses. 20 Additionally, the safety of post-primary doses should be evaluated. There may be special 21 considerations for vaccine safety depending on the vaccine construct and the intended target 22 population. 23 24 If a candidate vaccine is evaluated in a large pre-licensure trial, and if the safety profile 25 documented during immunogenicity trials did not give rise to any major concerns, it may be 26 acceptable for a full assessment of safety (that is, including detailed documentation of local 27 and systemic reactogenicity, as well as all unsolicited adverse events) to be confined to a 28 randomized subset of the total subjects. Any serious adverse event occurring in any subject 29 enrolled at any of the trial sites should be documented. 30 31 Part D. Recommendations for NRAs 32 33 D.1 General recommendations 34 35 The guidance for NRAs and NCLs given in the WHO Guidelines for national authorities on 36 quality assurance for biological products (69) and WHO Guidelines for independent lot 37 release of vaccines by regulatory authorities (30) should be followed. These guidelines 38 specify that no new biological product should be released until consistency of lot 39 manufacturing and quality has been established and demonstrated by the manufacturer. 40 41 The detailed production and control procedures, as well as any significant changes in them 42 that may affect the quality, safety or efficacy of inactivated EV71 vaccines (70), should be 43 discussed with and approved by the NRA. For control purposes, the relevant international WHO/BS/2020.2388 Page 43 1 reference preparations currently in force should be obtained for the purpose of calibrating 2 national, regional and working standards as appropriate (71). The NRA may obtain from the 3 manufacturer the product-specific or working reference to be used for lot release. 4 5 Consistency of production has been recognized as an essential component in the quality 6 assurance of inactivated EV71 vaccines. The NRA should carefully monitor production 7 records and quality control test results for clinical lots, as well as for a series of consecutive 8 lots of the final product. 9 10 D.2 Official release and certification 11 12 A vaccine lot should be released only if it fulfils all national requirements and/or satisfies 13 Part A of these WHO Recommendations (30). 14 15 A summary protocol for the manufacturing and control of enterovirus 71 vaccines 16 (inactivated), based on the model summary protocol provided below in Appendix 1 and 17 signed by the responsible official of the manufacturing establishment, should be prepared and 18 submitted to the NRA in support of a request for the release of a vaccine for use. 19 20 A lot release certificate signed by the appropriate NRA official should then be provided if 21 requested by the manufacturing establishment, and should certify that the lot of vaccine meets 22 all national requirements and/or Part A of these WHO Recommendations. The certificate 23 should provide sufficient information on the vaccine lot, including the basis of the release 24 decision (by summary protocol review or independent laboratory testing). The purpose of this 25 official national lot release certificate is to facilitate the exchange of vaccines between 26 countries and should be provided to importers of the vaccines. A model NRA Lot Release 27 Certificate for enterovirus 71 vaccines (inactivated) is provided below in Appendix 2. 28 29 Authors and acknowledgements 30 31 The first draft of these WHO Recommendations was prepared by a WHO drafting group 32 comprising Dr E. Griffiths, Kingston upon Thames, the United Kingdom; Dr P. Minor, St 33 Albans, the United Kingdom; Dr Y. Sun, Paul-Ehrlich-Institut, Germany; Dr H. Meyer, Paul- 34 Ehrlich-Institut, Germany; Dr J. Wang, National Institutes for Food and Drug Control, China; 35 Dr J. Martin, National Institute for Biological Standards and Control, the United Kingdom; 36 and Dr D. Lei, World Health Organization, Switzerland, taking into consideration the 37 discussions and consensus reached during a WHO working group meeting to develop WHO 38 Recommendations to assure the quality, safety and efficacy of enterovirus 71 vaccines 39 (inactivated), held in Shanghai, China, 11–12 September 2019 and attended by: Dr X. Chen 40 and Dr Z. Wang, Wuhan Institute of Biological Products Co. Ltd, China; Mr Z. Fu, Dr F. 41 Gao, Dr P. He, Ms Z. Jiang, Dr Q. Mao, Dr J. Wang, Dr Y. Wang and Dr M. Xu, National 42 Institutes for Food and Drug Control, China; Dr E. Griffiths, Kingston upon Thames, the 43 United Kingdom; Dr Y. Hu and Dr W. Meng, Sinovac Biotech Co., Ltd, China; Mrs T. WHO/BS/2020.2388 Page 44 1 Jivapaisarnpong, King Mongkut’s University of Technology Thonburi, Thailand; Dr E. Jung, 2 CJ HealthCare R&D Biomedicine, Republic of Korea; Dr J. Lee, Korea Centers for Disease 3 Control and Prevention, Republic of Korea; Dr Q. Li and Mr L. Yi, Institute of Medical 4 Biology Chinese Academy of Medical Sciences, China; Dr X. Li Shanghai Institute of 5 Biological Products Co. Ltd, China; Dr Z. Li and Mr J. Liu, Beijing Minhai Biotechnology 6 Co., Ltd, China; Dr J. Martin, National Institute for Biological Standards and Control, the 7 United Kingdom; Dr H. Meyer and Dr Y. Sun, Paul-Ehrlich-Institut, Germany; Dr P. Minor, 8 St Albans, the United Kingdom; Dr S. Phumiamorn, Institute of Biological Products, 9 Thailand; Dr J. Shin, World Health Organization, Regional Office for the Western Pacific, 10 Philippines; Mr Y. Tang, World Health Organization China Office, China; Dr H. Wang and 11 Dr H. Yang, Center for Drug Evaluation of the National Medical Products Administration, 12 China; and Dr D. Lei, World Health Organization, Switzerland. 13 14 The second draft of this document was prepared by Dr J. Martin, National Institute for 15 Biological Standards and Control, the United Kingdom; Dr H. Meyer, Paul-Ehrlich-Institut, 16 Germany; Dr E. Griffiths, Kingston upon Thames, the United Kingdom; Dr Y. Sun, Paul- 17 Ehrlich-Institut, Germany; Dr J. Wang, National Institutes for Food and Drug Control, China; 18 and Dr D. Lei, World Health Organization, Switzerland, incorporating as appropriate 19 comments received from regulators, manufacturers and academia following public 20 consultation, and taking into consideration the discussions and consensus reached during a 21 WHO informal consultation on WHO Recommendations to assure the quality, safety and 22 efficacy of enterovirus 71 vaccines (inactivated), held via WebEx, 8–10 June 2020 and 23 attended by: Dr X. Chen, Mr C. Guo, Ms L. Li, Ms Q. Li, Ms W. Lv and Dr J. Shi, Wuhan 24 Institute of Biological Products Co. Ltd, China; Dr E. Griffiths, Kingston upon Thames, the 25 United Kingdom; Ms Y. Hu, Sinovac Biotech Co., Ltd, China; Mrs T. Jivapaisarnpong, King 26 Mongkut’s University of Technology Thonburi, Thailand; Dr E. 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1 Appendix 1 2 3 Model summary protocol for the manufacturing and control of enterovirus 4 71 vaccines (inactivated) 5 6 The following protocol is intended for guidance and indicates the minimum information that 7 should be provided by the manufacturer to the NRA. Information and tests may be added or 8 omitted as necessary with the approval of the NRA. 9 10 It is possible that a protocol for a specific product may differ in detail from the model 11 provided. The essential point is that all relevant details demonstrating compliance with the 12 licence and with the relevant WHO recommendations for a particular product should be 13 provided in the protocol submitted. 14 15 The section concerning the final product should be accompanied by a sample of the label and 16 a copy of the leaflet (package insert) that accompanies the vaccine container. If the protocol 17 is being submitted in support of a request to permit importation, it should also be 18 accompanied by a certificate for the lot release of enterovirus 71 vaccines (inactivated) (see 19 Appendix 2) from the NRA of the country in which the vaccine was produced or released, 20 stating that the product meets national requirements as well as the recommendations in Part A 21 of this document. 22 23 1. Summary information on final lot 24 25 International name of product: ______26 Commercial/trade name: ______27 Product licence (marketing authorization) number: ______28 Country: ______29 Name and address of manufacturer: ______30 Name and address of licence holder, if different: ______31 Final packaging lot number: ______32 Type of container: ______33 Number of containers in this final lot: ______34 Final container lot number: ______35 Nature of final product (adsorbed):______36 Preservative and nominal concentration: ______37 Volume of each single human dose: ______38 Number of doses per final container: ______WHO/BS/2020.2388 Page 55 1 Virus strain: ______2 Cell substrate used for production: ______3 4 Summary of composition (include a summary of the qualitative and quantitative composition 5 of the vaccine per single human dose, including any adjuvant used and other excipients): 6 ______7 ______8 9 Shelf-life approved (months): ______10 Date of manufacture: ______11 Expiry date: ______12 Storage condition: ______13 14 2. Detailed information on manufacture and control 15 16 The following sections are intended for reporting the results of the tests performed during the 17 production of the vaccine, so that the complete document will provide evidence of consistency 18 of production. If any test had to be repeated, this must be indicated. Any abnormal results 19 must be recorded on a separate sheet. 20 21 Summary of source materials 22 23 Identity of seed lot strain used for vaccine production 24 Reference number of seed lot: ______25 Date(s) of reconstitution (or opening) of seed lot container(s): ______26 27 Test for adventitious agents 28 Methods: ______29 Result: ______30 Date: ______31 32 Identity of cell bank used for vaccine production 33 Reference number of cell bank: ______34 Date(s) of reconstitution (or opening) of cell bank container(s): ______35 36 Identity test 37 Methods: ______38 Result: ______39 Date: ______40 WHO/BS/2020.2388 Page 56

1 Control of vaccine production 2 3 Control cell cultures 4 Tests on control cell cultures 5 Ratio of control to production cell cultures: ______6 Incubation conditions: ______7 Period of observation of cultures: ______8 Dates observation started/ended: ______9 Ratio or proportion of cultures discarded: ______10 Results of observation: ______11 12 Tests for haemadsorbing viruses 13 Quantity of cell tested: ______14 Method used: ______15 Date of start of test-1: ______16 Date of end of test-1:______17 Results: ______18 Date of start of test-2: ______19 Date of end of test-2:______20 Results: ______21 22 Tests for adventitious agents on supernatant culture fluids 23 Method used: ______24 Date of start of test: ______25 Date of end of test: ______26 Result: ______27 28 Identity test 29 Method used: ______30 Date of start of test: ______31 Date of end of test: ______32 Result: ______33 34 35 Control of single harvests 36 Name of the culture medium: ______37 Date of inoculation:______38 Temperature of incubation: ______39 Microscopic observation ______40 Result: ______41 Date: ______42 Date of harvest: ______43 Volume of harvest: ______WHO/BS/2020.2388 Page 57 1 Yield (mg/mL): ______2 3 Sterility tests for bacteria, fungi and mycoplasmas 4 Test for bacteria and fungi 5 Method: ______6 Media: ______7 Volume inoculated: ______8 Date of start of test: ______9 Date of end of test: ______10 Result: ______11 12 Test for mycoplasmas (if applicable) 13 Method: ______14 Volume inoculated: ______15 Date of start of test: ______16 Date of end of test: ______17 Result: ______18 19 Virus titration 20 Method: ______21 Reference lot no. ______22 Date: ______23 Result: ______24 25 26 Control of virus pool 27 Lot number of virus pool: ______28 Date of pooling: ______29 Number of harvests: ______30 Volume(s), storage temperature, storage time and approved storage period: ______31 ______32 33 Purification of virus pool (may be performed after inactivation) 34 Purification methods: ______35 Volume before purification: ______36 Volume after purification:______37 Date: ______38 39 Tests on virus pool 40 Virus titration 41 Method: ______42 Reference lot no. ______43 Date: ______44 Result: ______WHO/BS/2020.2388 Page 58 1 2 Virus antigen content 3 Method: ______4 Reference lot no. ______5 Date: ______6 Result: ______7 8 Specific activity 9 Virus antigen content: ______10 Total protein content: ______11 Specification: ______12 Date: ______13 Result: ______14 15 Inactivation of harvest pool (may be performed before purification) 16 Filtration before inactivation 17 Filtration method: ______18 Date: ______19 Time of start of filtration: ______20 Time of end of filtration:______21 22 Inactivation 23 Agent(s) and concentration of inactivation agent: ______24 Temperature of inactivation: ______25 Date of start of inactivation: ______26 Virus antigen units at start of inactivation: ______27 Date of taking first samples: ______28 Date of completion of inactivation: ______29 Virus antigen units at end of inactivation: ______30 31 Filtration during inactivation 32 Filtration method: ______33 Date: ______34 Time of start of filtration: ______35 Time of end of filtration:______36 37 38 Control of purified inactivated bulk 39 Test for effective inactivation (after removal/neutralization of inactivating agent) 40 Sample size tested: ______41 Dates of sampling (1–4): ______42 Test method: ______43 Period of observation of cell cultures: ______44 Period of observation of subcultures: ______WHO/BS/2020.2388 Page 59 1 Period of observation of subcultures: ______2 Period of observation of subcultures: ______3 Result: ______4 5 Sterility tests for bacteria and fungi 6 Method: ______7 Media: ______8 Volume inoculated: ______9 Date of start of test: ______10 Date of end of test: ______11 Result: ______12 13 Antigen content 14 Method: ______15 Specification: ______16 Date: ______17 Result: ______18 19 Test for residual inactivating agent 20 Method: ______21 Specification: ______22 Date: ______23 Result: ______24 25 Test for residual host-cell DNA (if applicable) 26 Method: ______27 Specification: ______28 Date: ______29 Result: ______30 31 Test for residual chemicals (if applicable) 32 Method: ______33 Specification: ______34 Date: ______35 Result: ______36 37 38 Control of final bulk 39 Identification (lot number): ______40 Date of manufacture/blending: ______41 Volume(s), storage temperature, storage time and approved storage period: ______42 ______43 44 WHO/BS/2020.2388 Page 60 1 2 Blending: Prescription (per dose) Added 3 Virus antigen (IU or unit): ______4 Adjuvant: ______5 Preservative (specify): ______6 Others (chemicals): ______7 Final volume (mL): ______8 9 10 Sterility tests for bacteria and fungi 11 Method: ______12 Media: ______13 Volume inoculated: ______14 Date of start of test: ______15 Date of end of test: ______16 Result: ______17 18 Potency test 19 In vivo assay (may be performed at final bulk stage) 20 Species, strain, sex and weight specifications: ______21 Number of mice tested: ______22 Dates of vaccination, bleeding: ______23 Date of assay: ______24 Lot number of reference vaccine and assigned potency: ______25 Vaccine doses (dilutions) and number of animals responding at each dose: ______26 ______27 ED50 of reference and test vaccine: ______28 Potency of test vaccine (with 95% fiducial limits): ______29 30 If an in vitro assay is used 31 Method: ______32 Specification: ______33 Date: ______34 Result: ______35 36 Adjuvant content 37 Method: ______38 Specification: ______39 Date: ______40 Result: ______41 42 Preservative content (if applicable) 43 Method: ______44 Specification: ______WHO/BS/2020.2388 Page 61 1 Date: ______2 Result: ______3 4 5 Control of final lot 6 Lot number: ______7 Date of filling: ______8 Type of container: ______9 Filling volume: ______10 Number of containers after inspection: ______11 Number and percentage of containers rejected: ______12 13 Appearance 14 Method: ______15 Specification: ______16 Date: ______17 Result: ______18 19 Identity test 20 Method: ______21 Specification: ______22 Date: ______23 Result: ______24 25 Sterility tests for bacteria and fungi 26 Method: ______27 Media: ______28 Volume inoculated: ______29 Date of start of test: ______30 Date of end of test: ______31 Result: ______32 33 Potency test 34 In vivo assay (may be performed at final bulk stage) 35 Species, strain, sex and weight specifications: ______36 Number of mice tested: ______37 Dates of vaccination, bleeding: ______38 Date of assay: ______39 Lot number of reference vaccine and assigned potency: ______40 Vaccine doses (dilutions) and number of animals responding at each dose: ______41 ______42 ED50 of reference and test vaccine: ______43 Potency of test vaccine (with 95% fiducial limits): ______44 WHO/BS/2020.2388 Page 62 1 If an in vitro assay is used 2 Method: ______3 Lot number of reference and assigned potency: ______4 Specification: ______5 Date: ______6 Result: ______7 8 Preservative content (if applicable) 9 Method: ______10 Specification: ______11 Date: ______12 Result: ______13 14 Endotoxin content 15 Method: ______16 Specification: ______17 Date: ______18 Result: ______19 20 pH 21 Method: ______22 Specification: ______23 Date: ______24 Result: ______25 26 Adjuvant content 27 Method: ______28 Specification: ______29 Date: ______30 Result: ______31 32 Degree of adsorption 33 Method: ______34 Specification: ______35 Date: ______36 Result: ______37 38 Residual antibiotics (if applicable) 39 Method: ______40 Specification: ______41 Date: ______42 Result: ______43 44 Extractable volume WHO/BS/2020.2388 Page 63 1 Method: ______2 Specification: ______3 Date: ______4 Result: ______5 6 7 3. Certification by the manufacturer 8 9 Name of head of quality control (typed) ______10 11 Certification by the person from the control laboratory of the manufacturing company taking 12 overall responsibility for the production and quality control of the vaccine. 13 14 I certify that lot no. ______of enterovirus 71 vaccine (inactivated), 15 whose number appears on the label of the final containers, meets all national requirements 16 and/or satisfies Part A1 of the WHO Recommendations to assure the quality, safety and 17 efficacy of enterovirus 71 vaccines (inactivated).2 18 19 Signature ______20 Name (typed) ______21 Date ______22 23 24 4. Certification by the NRA 25 26 If the vaccine is to be exported, attach the model NRA Lot Release Certificate for enterovirus 27 71 vaccines (inactivated) (as shown in Appendix 2), a label from a final container and an 28 instruction leaflet for users. 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 Page 64

1 Appendix 2 2 3 Model NRA Lot Release Certificate for enterovirus 71 vaccines 4 (inactivated) 5 6 7 Certificate no. 8 9 10 The following lot(s) of enterovirus 71 vaccine (inactivated) produced by1 11 12 whose numbers appear on the labels of the final evaluated containers, complies with the 13 relevant marketing authorization, the national specifications and provisions for the release of 14 biological products2 and Part A3 of the WHO Recommendations to assure the quality, safety 15 and efficacy of enterovirus 71 vaccines (inactivated) (2021),4 and with corresponding WHO 16 recommendations for each of the vaccine’s individual components, as well as with WHO 17 good manufacturing practices for pharmaceutical products: main principles;5 WHO good 18 manufacturing practices for biological products;6 and the WHO Guidelines for independent 19 lot release of vaccines by regulatory authorities.7 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 Page 65 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 7 This batch has been examined using documented procedures which form part of a quality 8 system which is in accordance with the ISO/IEC 17025 standard. 9 10 The release decision is based on the elements described in paragraph 7.3 of the WHO 11 Guidelines for independent lot release of vaccines by regulatory authorities.3 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 Page 66 1 2 This batch has been found to be compliant with the above by the following institute, member 3 of the WHO National Control Laboratory Network for Biologicals (WHO-NNB). 4 5 Name (typed) 6 7 Institute 8 9 Position 10 11 Signature 12 13 Date 14 15 16 17 18 19 20 21 22 Stamp 23 24 25 26 27 Importing/requesting authority: 28 29 30 31 32 This certificate conforms to the format recommended by the World Health Organization. 33 34 35