Guidelines on the quality, safety and efficacy of Ebola vaccinesVer_5...16 undertaken in developing live attenuated Ebola vaccines although in theory EBOV could be 17 inactivated,

1

WHO/DRAFT6/25 JANUARY 2016 2 ENGLISH ONLY 3

4

Guidelines on the quality, safety and efficacy of Ebola vaccines 5

(Proposed new guidelines) 6

7

NOTE: 8

This document has been prepared for the purpose of inviting comments and suggestions on the proposals 9 contained therein, which will then be considered by the Expert Committee on Biological Standardization 10 (ECBS). Publication of this early draft is to provide information about the proposed Guidelines on the 11 quality, safety and efficacy of Ebola vaccines to a broad audience and to improve transparency of the 12 consultation process. 13

14

The text in its present form does not necessarily represent an agreed formulation of the Expert 15 Committee on Biological Standardization. Written comments proposing modifications to this text 16 MUST be received by 15 March 2016 in the Comment Form available separately and should be 17 addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: Department of 18 Essential Medicines and Health Products (EMP). Comments may also be submitted electronically to the 19 Responsible Officer: Dr TieQun Zhou at email: [email protected]. 20 21 The outcome of the deliberations of the Expert Committee on Biological Standardization will be published 22 in the WHO Technical Report Series. The final agreed formulation of the document will be edited to be in 23 conformity with the "WHO style guide" (WHO/IMD/PUB/04.1). 24

© World Health Organization 2016 25

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health 26 Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: 27 [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for non-28 commercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-mail: 29 [email protected]). 30 The designations employed and the presentation of the material in this publication do not imply the expression of any opinion 31 whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its 32 authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines 33 for which there may not yet be full agreement. 34 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended 35 by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions 36 excepted, the names of proprietary products are distinguished by initial capital letters. 37 All reasonable precautions have been taken by the World Health Organization to verify the information contained in this 38 publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The 39 responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization 40 be liable for damages arising from its use. 41 The named authors [or editors as appropriate] alone are responsible for the views expressed in this publication. 42

WHO/DRAFT6/25 JANUARY 2016

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Contents 1

2

Introduction 3

Scope 4

Background 5

Terminology 6

Part A. Guidelines on development, manufacturing and control of Ebola vaccines 7

A.1 General manufacturing guidelines 8

A.2 Control of source materials 9

A.3 Control of Ebola vaccine production 10

A.4 Filling and containers 11

A.5 Control tests on final lot 12

A.6 Records 13

A.7 Retained samples 14

A.8 Labelling 15

A.9 Distribution and transport 16

A.10 Stability testing, storage and expiry date 17

Part B. Nonclinical evaluation of Ebola vaccines 18

B.1.General remarks 19

B.2 Product characterization and process development 20

B.3 Pharmacodynamic studies 21

B.4 Nonclinical safety studies (toxicity testing) 22

B.5 Pharmacokinetic (Biodistribution) studies 23

B.6 Environmental risk 24

Part C. Clinical evaluation of Ebola vaccines 25

C.1 General considerations 26

C.2 Phases of clinical development and trial design 27

C.3 Statistical considerations 28

C.4 Serological and diagnostic assays 29

C.5 Special populations 30

C.6 Postmarketing surveillance 31

Part D. Guidelines for NRAs 32

D.1 General 33

D.2 Release and certification 34

35

Authors and Acknowledgement 36

References 37

Appendix 1. Overview of current candidate Ebola vaccines in clinical trial (2014/15) 38

Appendix 2. Provisional summary protocol for manufacturing and control of viral vectored 39

Ebola vaccine 40

WHO/ DRAFT6/25 JANUARY 2016

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Appendix 3. Model certificate for the release of viral vectored Ebola vaccine by NRAs 1

2

3

4

5

This document provides information and guidance on the development, production, quality 6

control and evaluation of the safety and efficacy of candidate Ebola vaccines. It is written in the 7

form of WHO Guidelines instead of Recommendations since there is at present no licensed 8

Ebola vaccine and is intended to facilitate progress towards the eventual licensure of such a 9

vaccine. Guidelines allow greater flexibility than Recommendations with respect to future 10

developments in the field. The parts printed in small type and indented text are comments or 11

examples that are intended to provide additional guidance for manufacturers and NRAs which 12

may benefit from these details. To facilitate the international distribution of vaccines produced in 13

accordance with these Guidelines, a summary protocol for recording test results has been 14

provided in Appendix 2. 15 16


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Introduction 1

The unprecedented scale and severity of the Ebola virus disease (EVD) epidemic in West Africa 2

in 2014-2015 led to calls for the urgent development and licensing of an Ebola vaccine (1,2). 3

Considerable work is now underway and there have been several international consultations on 4

public health issues and on Ebola vaccine development, evaluation and licensing (2-4). As part 5

of WHO’s ongoing measures to support the development of Ebola vaccines, guidance has been 6

prepared on the scientific and regulatory considerations concerning their quality, safety and 7

efficacy. 8

9

WHO convened an informal consultation at its headquarters in Geneva on 18-19 March 2015 – 10

attended by scientific experts, regulatory professionals and other stakeholders involved in Ebola 11

vaccine development, production, evaluation and licensure – to review draft Guidelines prepared 12

by a drafting group and to seek consensus on key technical and regulatory issues (5). The 13

following text was developed following these discussions and comments received from many 14

reviewers during subsequent consultations. It is written in the form of guidelines instead of 15

recommendations since guidelines allow greater flexibility than recommendations with respect to 16

the expected future of Ebola vaccine development, production, quality control and evaluation. 17

18

Scope 19

This doceument provides scientific and regulatory guidance for national regulatory authorities 20

(NRAs) and vaccine manufacturers on the quality, nonclinical and clinical aspects of Ebola 21

vaccines, particularly those based on viral vectors that are currently at the most advanced stage 22

of development. 23

24

In the past ten years, the WHO has convened two consultations to consider the development, 25

production and evaluation of viral vectored vaccines in general and the reports of these meetings 26

provide useful discussions and opinions on the quality, safety and efficacy aspects of such 27

vaccines (6,7). There is also a regional guideline available for live recombinant viral vectored 28

vaccines (8). 29


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Although recombinant viral vector based Ebola vaccines are by far the most advanced 1

candidates, other approaches to the development of Ebola vaccines are also being investigated. 2

These include different production platforms, such as recombinant DNA vaccines expressing an 3

Ebola virus (EBOV) antigen produced in Escherichia coli (9), Ebola Virus-Like Particles (VLP) 4

expressed from recombinant baculovirus in insect cells, and other forms of subunit vaccines. 5

6

General guidance on many of non-viral vector production technologies has already been 7

published by WHO and other sources (see list below for examples of references) and may 8

provide useful information for the development and manufacture of such Ebola vaccines. 9

a. Inactivated vaccines (10-12) 10

b. Protein antigens produced by recombinant DNA technology (13-16) 11

c. DNA vaccines (17,18) 12

13

Most developmental approaches to Ebola vaccine involve recombinant DNA technology. 14

Because EBOV has such a high case fatality rate, little or no work appears to have been 15

undertaken in developing live attenuated Ebola vaccines although in theory EBOV could be 16

inactivated, as is done for rabies vaccine (10). 17

18

Part A of this document focuses on the developmantal, manufacturing and quality control issues 19

relevant to viral vectored vaccines against EBOV. Although the key principles related to 20

nonclinical (Part B) and clinical development (Part C) may apply to vaccine approaches other 21

than those based on viral vectors, special considerations and guidance would be required for such 22

products and are not elaborated in this document. Examples of vaccines mentioned in this 23

document are provided for information only and should not be considered as endorsements of 24

any particular candidate vaccine. 25

26

This document should be read in conjunction with other relevant WHO guidelines such as those 27

on nonclinical (19,20) and clinical evaluation (21) of vaccines, as well as relevant documents 28

that describe the minimum requirement for an effective National Pharmacovigilance System 29

(22). Other WHO guidance, such as that for the evaluation of animal cell cultures as substrates 30


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for the manufacture of biological medicinal products and for the characterization of cell banks 1

(23), should also be consulted as appropriate. 2

3

It should be noted that there are currently knowledge gaps in the scientific understanding of EVD 4

and Ebola vaccines, which are being addressed by ongoing research and development. This 5

document is therefore developed in the light of knowledge available so far, particularly of the 6

current most advanced Ebola vaccine candidates and will need to be updated as new data become 7

available from additional studies. 8

9

Background 10

Ebola virus, Ebola virus disease and epidemiology 11

Ebola virus (EBOV) belongs to the Filoviridae family of filamentous, negative-stranded RNA, 12

enveloped viruses consisting of three genera: Ebola virus, Marburg virus and Cueva virus the 13

latter being a pathogen of bats in Spain (24). There are five distinct species of EBOV: Ebola 14

virus Zaire (ZEBOV), Sudan Ebola virus (SUDV), Tai Forest Ebola virus (TAFV), Reston Ebola 15

virus (RESTV) and Bundibugyo Ebola virus (BDBV) (24,25). Marburg virus appears to be 16

antigenically stable and at present there is only a single species, Marburg virus (MARV). The 17

first recognized MARV outbreak in humans was in 1967 and linked to infected monkeys 18

imported from Uganda that infected laboratory workers in Marburg and Belgrade (26). Bats are 19

believed to be the natural reservoir of all filoviruses. EBOV and MARV cause severe 20

haemorragic fever in humans and non-human primates alike, with high morbidity and mortality 21

rates (27,28) . Outbreaks of infection with Ebola filoviruses have been noted since 1976 mainly 22

in Central Africa and recur at intervals. Prior to the 2014 -2015 EVD outbreak in West Africa 23

there had not previously been such a large scale epidemic nor had the disease been previously 24

recorded in West Africa, apart from a single infection with TAFV. The incubation period 25

following infection by EBOV prior to the onset of symptoms is believed to be approximately 2-26

21 days with initial symptoms being similar to diseases such as influenza or malaria (29,30). 27

Patients then progress rapidly to a life-threatening disease (31). Infected individuals seem to 28

become infective only once symptoms appear but those who do survive remain infective until the 29

virus is cleared from their blood and other body fluids. It has been reported that viable EBOV 30

can persist in ocular fluid for at least 9 weeks following clearance of virema (32). EBOV has also 31


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been detected in the semen of males for months following recovery from EVD, consistent with 1

the possible persistece of the virus within immune-privileged tissue sites in the body (33,34). 2

Presumptive sexual transmission of EBOV from recovered individuals has also been reported 3

(35,36). There are currently no licensed vaccines or therapeutics to prevent or treat Filovirus 4

infections. Nevertheless, individuals suffering from EVD have been treated aggressively with 5

oral and intravenous fluids, including electrolyte replacements, to combat severe diarrhoea and 6

dehydration, sometimes successfully surviving the infection (31). 7

8

Filoviruses are high risk agents and classified as Biosafety level (BSL)-4 pathogens. They 9

consist of a non-segmented RNA genome of approximately 19 kb containing 7 genes encoding 10

viral proteins VP24, VP30, VP35, VP40, a nucleoprotein, a glycoprotein (GP) and a polymerase 11

(37). The GP is a type-1 transmembrane glycoprotein that is cleaved into disulphide linked GP1 12

and GP2 subunits. The mature GP forms homotrimers that are presented as spikes on the surface 13

of infected cells and virions and is responsible for receptor binding, viral entry and most likely 14

immunity (38,39). Most of the vaccines under current development are based on the EBOV GP 15

and have been shown to confer protection from lethal EBOV challenge in animal models 16

including, importantly, non-human primates (40,41). 17

18

Natural immune responses to Ebola viruses 19

Filovirus infection in humans elicits both cellular and humoral responses. IgM and IgG 20

antibodies have been reported to develop early in infected patients who survive whereas fatal 21

cases are associated with immune dysregulation and high viraemia (42). Cellular responses can 22

also be detected. The generation of neutralizing antibodies during Filovirus infection and the 23

passive transfer of neutralizing monoclonal antibodies or monkey convalescent immunoglobulin 24

preparations have been shown to sometimes protect non-human primates against lethal Filovirus 25

challenge but overall the data are somewhat conflicting (42). It is suggested that antibodies play 26

a significant part in protection against Filovirus infection, however correlates of protection have 27

not been established nor has a role for cellular immunity been demonstrated. 28

29

Ebola vaccines currently under development 30


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A large number of candidate Ebola vaccines are under development. Some of these vaccines had 1

already been in pre-clinical development before the 2014-2015 epidemic began and are 2

substantially more advanced than the others. An overview of candidate Ebola vaccines which 3

are currently at advanced stages of development is provided in Appendix 1. 4

5

The most advanced candidate vaccines are those based on live recombinant virus vector 6

platforms (Appendix 1). Such candidate vaccines have been developed in Canada, the USA, 7

Europe, China and Russia. Four of the most advanced platforms used to engineer these vaccines 8

are - recombinant Vesicular Stomatitis Virus (rVSV) (43), Chimpanzee Adenovirus (ChAd) (44), 9

human Adenovirus 26 (Ad26) (45) and the Modified Vaccinia Virus Ankara strain (MVA) (46). 10

Monovalent candidate vaccines have been constructed to express the EBOV GP of one EBOV 11

strain, such as the Zaire strain responsible for most of the epidemic in West Africa, Others have 12

been developed as multivalent vaccines expressing the GP of two EBOV strains and MARV, as 13

well as, in one case, the EBOV nucleoprotein. These candidates are currently under study in non-14

human primates as well as in humans either as single vaccines or for use in heterologous prime-15

boost vaccine schedules, where priming is done with one vaccine and boosting with another. 16

17

The viral vectored vaccines under development include those that are replication-incompetent in 18

the human host or in human cells, except if the human cells have been engineered to allow their 19

replication, as well as those that are replication-competent but likely to be highly attenuated due 20

to their recombinant gene inserts and cell culture passage (see Appendix 1). Replication-21

incompetent vectors include adenoviral vectors, both those derived from human adenoviruses 22

(such as Ad26) and those derived from non-human primate adenoviruses (such as ChAd3), as 23

well as MVA. MVA is a highly attenuated vaccinia strain, derived by more than 500 passages in 24

hens’ eggs. The non-recombinant MVA was used as a human smallpox vaccine in Germany in 25

the 1970’s and was licensed in Canada and the EU. Replication-competent, but attenuated, 26

vectors include recombinant VSV, a negative –stranded RNA virus animal pathogen, where 27

attenuation is due to the insertion of a recombinant heterologous gene, for example the EBOV 28

GP, in place of the VSV glycoprotein. These viral vector platforms have been used to produce 29

other investigational products, including gene therapy products and both prophylactic and 30


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therapeutic vaccines, and data from their quality, non-clinical and clinical evaluation provide 1

supporting safety data for their use as Ebola vaccines (43,47, 48 ). 2

3

The need for careful clinical studies with candidate vaccines in the target population will be of 4

paramount importance and a proposed Target Product Profile, setting out optimal and minimal 5

criteria for Ebola vaccines for use in epidemic or endemic settings, has been developed by the 6

Wellcome Group (3). WHO has developed a document, Ebola Virus Disease (EVD) Vaccine 7

Target Product Profile, providing guidance on WHO’s preferences for Ebola vaccines of two 8

categories (for reactive use and prophylactic use) (49). Ecouraging results on the 9

immunogenicicty and safety, as well as on clinical efficacy based on disease end-points, have 10

already been generated (30) and their evaluation in larger phase 2 and 3 trials are ongoing.The 11

results of one human clinical study (50) using the ChAd3-vectored vaccine encoding the Zaire 12

strain GP shows no serious safety concerns at the doses used but immune responses were less 13

than those reported for the same vaccine in non-human primates protected against Filovirus 14

challenge, even at the top dose. This is not surprising as higher immune responses at the same 15

dose of vaccine in non-human primates (NHP) than in humans has been seen with other vectors 16

and other immunogens. However, the ChAd3-vectored vaccine is reported to have induced in 17

some cases transient fever at the top dose used in humans although no other unexpected serious 18

adverse reaactions were found. The therapeutic window for non – replicating viral vectored 19

vaccines is as yet unclear and it may be that in order to achieve optimal immunogenicity, doses 20

may be too reactogenic to be acceptable. This issue might be overcome by using the prime-boost 21

approach mentioned above (50). Various two dose schedules of ChAd3/MVA and Ad26/MVA 22

have been evaluated in the clinic and this approach may also be relevant where long term 23

protection is required. Data from phase 1 and 1b studies of the rVSV-ZEBOV vaccine in several 24

locations, including Canada, Gabon, Germany, Kenya, Switzerland and the USA show that pain 25

at the injection site was common as were systemic symptoms such as fever and malaise, 26

generally lasting up to three days. Administration of rVSV vaccine resulted in viremia that is 27

detectable by polymerase chain reaction (PCR) during the first and sometimes second week after 28

vaccination. The vaccine virus was also detected by PCR in the urine and saliva of a minority of 29

the recipients. No serious vaccine related adverse reactions have been reported so far for rVSV 30

vaccine from these studies although an unexpected safety signal was detected. Mild to moderate 31


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and generally short lived arthritis or arthalgia developed during the second week following 1

immunization in a minority (20% ) of recipients in one site in particular (51). Less of these 2

adverse events (5%) occurred at other immunization sites. Although general rVSV vaccine 3

reactogenicity was found to be dose related, the arthritis/athralgia events were not linked with 4

dose; they occurred at similar frequency in recipients receiving the lowest and highest doses.This 5

is believed to be due to the chimeric VSV vaccine virus, like EBOV itself, showing a tropism for 6

joints. On-going phase 2/3 clinical stuides are expected to provide further safety data especially 7

in African settings. Preliminary data from adaptive trials design clinical studies (ring vaccination 8

) using the rVSV vaccine have shown encouraging efficacy (52). However, the epidemiological 9

situation has now changed significantly. Using strict infection control and public health 10

measures, the EBOV epidemic has been eliminated. In December 2015, Guinea was declared by 11

WHO to be free of Ebola transmission, bringing to an end the Ebola outbreak in the three main 12

African countries affected, Guinea, Sierra Leone and Liberia (53). The asseessment of EBOV 13

vaccine efficacy will therefore now be more challenging. 14

15

Terminology 16

The definitions given below apply to the terms used in these guidelines. They may have different 17

meanings in other contexts. 18

19

Adventitious agents: contaminating microorganisms of a cell culture or source materials 20

including bacteria, fungi, mycoplasmas/spiroplasmas, mycobacteria, Rickettsia, protozoa, 21

parasites, transmissible spongiform encephalopathy (TSE) agents, and viruses that have been 22

unintentionally introduced into the manufacturing process of a biological product. 23

24

Adverse event of special interest: an adverse event of special interest (serious or non-serious) is 25

one of scientific and medical concern specific to the sponsor’s product or program, for which 26

ongoing monitoring and rapid communication by the investigator to the sponsor can be 27

appropriate. Such an event might warrant further investigation in order to characterize and 28

understand it. Depending on the nature of the event, rapid communication by the trial sponsor to 29

other parties (e.g., regulators) might also be warranted. 30


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1

Benefit-risk assessment: a decision-making process on whether or not the benefits of a given 2

medicinal product outweigh the risks. Benefits and risks need to be identified from all parts of a 3

dossier, i.e. the quality, non-clinical and clinical parts, to be integrated into the overall 4

assessment. 5

6

Candidate vaccine: an investigational vaccine which is in the research and clinical development 7

stages and has not been granted marketing authorization or licensure by a regulatory agency. 8

9

Cell bank: a collection of appropriate containers whose contents are of uniform composition, 10

stored under defined conditions. Each container represents an aliquot of a single pool of cells. 11

12

Cell substrate: cells used to manufacture a biological product. 13

14

Expression construct: the expression vector containing the coding sequence of the recombinant 15

protein. 16

17

Expression system: the host cell containing the expression construct and the cell culture process 18

that is capable of expressing protein encoded by the expression construct. 19

20

Final bulk: the formulated vaccine preparation from which the final containers are filled. If 21

applicable, the final bulk may be prepared from one or more monovalent antigen bulks and in 22

this case, mixing should result in a uniform preparation to ensure final containers are 23

homogenous. 24

25

Final lot: a collection of sealed final containers of formulated vaccine that is homogeneous with 26

respect to the risk of contamination during the filling process. A final lot must therefore have 27

been filled from a single vessel of final bulk or prepared in one working session. 28

29

Heterologous gene: transgene from the disease-causing organism that is integrated into the 30

genomic sequence of the viral vector. 31


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1

Immune correlate of protection (ICP):an immunological response that correlates with vaccine 2

induced protection from disease and is considered predictive of clinical efficacy. The ICP may 3

be mechanistic (i.e., causative for protection) or it may be non-mechanistic (I.e., non-causative, 4

an immune response present in persons protected by vaccination, but not the cause of protection). 5

6

Immunogenicity: the capacity of a vaccine to elicit a measurable immune response. 7

8

Marketing authorization (MA): a formal authorization for a medicine to be marketed. Once an 9

NRA approves a market authorization application for a new medicine, the medicine may be 10

marketed and may be available for physicians to prescribe. Also referred to as product license or 11

product authorization. 12

13

Master cell bank (MCB): a quantity of well-characterized cells of animal or other origin, derived 14

from a cell seed at a specific population doubling level (PDL) or passage level, dispensed into 15

multiple containers, cryopreserved, and stored frozen under defined conditions, such as the 16

vapour or liquid phase of liquid nitrogen in aliquots of uniform composition. The MCB is 17

prepared from a single homogeneously mixed pool of cells. In some cases, such as genetically 18

engineered cells, the MCB may be prepared from a selected cell clone established under defined 19

conditions. Frequently, however, the MCB is not clonal. It is considered best practice for the 20

MCB to be used to derive working cell banks. 21

22

Master seed lot: a collection of appropriate containers whose contents are of uniform 23

composition, stored under defined conditions. Each container represents an aliquot of a single 24

pool of virus particles of defined passage and from which the working seed lots are derived. 25

26

Platform technology: a production technology with which different viral vectored vaccines are 27

produced by incorporating heterologous genes for different proteins into an identical viral vector 28

backbone. 29

30

Pooled virus harvest: a homogeneous pool of several single virus harvests. 31


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1

Public health emergency of international concern1: means an extraordinary event that is 2

determined, as provided in the WHO International Health Regulations (54): 3

(i) to constitute a public health risk to other States through the international spread of disease 4

and (ii) to potentially require a coordinated international response. 5

6

Seed lot: a seed lot system is a system according to which successive batches of viral vector 7

vaccine are derived from the same master seed lot of viral vector at a given passage level. For 8

routine production, a working seed lot is prepared from the master seed lot. The final product is 9

derived from the working seed lot and has not undergone more passages from the master seed lot 10

than the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. 11

The origin and the passage history of the master seed lot and the working seed lot are recorded. 12

13

Single virus harvest: viral vector from a single culture, after separation from production cells but 14

before purification. 15

16

Vaccine efficacy: an estimate of the reduction in the chance or odds ratio of developing clinical 17

disease after vaccination relative to the chance or odds ratio when not vaccinated against the 18

disease to be prevented. Vaccine efficacy measures direct protection (i.e., protection induced by 19

vaccination in the vaccinated population sample). 20

21

Vaccine effectiveness: an estimate of the protection conferred by vaccination in a specified 22

population. Vaccine effectiveness measure both direct and indirect protection (e.g., the estimate 23

may reflect in part protection of non-vaccinated persons secondary to the effect of the vaccine in 24

the vaccinated population). Vaccine effectiveness may also be inferred from a vaccine-induce 25

immune response (e.g., pre-specified antibody threshold induced by the vaccine in vaccinated 26

persons.) 27

28

1 Public health emergency of national concern may also be considered in this document.


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Working seed lot: a collection of appropriate containers whose contents are of uniform 1

composition, stored under defined conditions. Each container represents an aliquot of a single 2

pool of virus particles of defined passage directly derived from the master seed lot and which is 3

the starting material for individual manufacturing batches of viral vectored vaccine product. 4

5

Part A. Guidelines on development, manufacturing and control 6

of Ebola vaccines 7

8

At the time of writing this document, no WHO guidance for viral vectored vaccines is available, 9

therefore this section focuses on the relevant issues relating to development, manufacturing and 10

quality control leading to the licensing of this type of vaccine developed to protect against EVD. 11

12

Sections in the main text of Part A consider in general terms the full quality requirements for 13

viral vectored vaccines for a license submission. Additionally, the principles which may be 14

applied to product development, manufacturing and control during a public health emergency to 15

allow the rapid introduction of an Ebola vaccine are considered. Where development may be 16

accelerated during a public health emergency, context-specific examples are given in small print 17

and indented within the main text of Part A. Considerations of the quality requirements at 18

different stages of clinical development are discussed in the sections entitled “Special 19

considerations”. 20

21

The replication abilities of the lead viral vectored vaccines are summarized in Appendix 1. The 22

relevance of aspects of the guidance provided in this document should be considered with respect 23

to the replication status of the products. For example, tests for reversion to competency apply to 24

replication incompetent viral vectors which have had specific genetic elements removed and 25

which rely on these elements being supplied by the production cell line (i.e. the adenovirus 26

vectors). On the other hand, for replication competent viral vectored vaccines, the level of 27

attenuation of the parent and recombinant viral vectors should be considered. 28

29

30


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Accelerated availability of vaccines during a public health emergency – general principles 1

The quality of a vaccine must always be taken into account during the process of evaluating 2

whether the benefit derived from its administration is greater than any risks which might be 3

associated with its use. This is a principle by which all pharmaceuticals, whether they are 4

chemical or biological, medicine or vaccine, are evaluated to decide whether they should be 5

made available for use or not. It applies equally whether the product is intended for use in a 6

clinical trial, as a licensed product, or where the product is made available through emergency 7

procedures. In addition, there is an obligation to provide full assurance that the vaccine will not 8

cause harm to the recipient due to a failure of manufacture and control resulting in contamination 9

of the product with unwanted components such as microorganisms, viruses, or chemicals. This 10

requirement is absolute regardless of the stage of development of the product or the urgency of 11

the need for its availability. 12

13

Beyond this, the process and product characterization requirements will depend on the prevailing 14

clinical situation and the urgency of the need for product. However it is generally accepted that 15

to gain a marketing authorization for the product, the usual standards for quality development, 16

manufacturing and control will apply. During the assessment of a marketing authorization 17

application, the benefit vs the risk of the product to the intended population is taken into 18

consideration and must be found to be positive if the product is to be granted marketing 19

approval. The specific findings related to the assessment of product quality are taken into 20

account in this benefit-risk assessment. 21

22

It is not possible to provide a “road map” of the minimum process and product characterization 23

and control requirements for a viral vectored vaccine against EVD, or any other disease with the 24

potential to cause a public health emergency since the requirements will be partially dependent 25

on the ongoing epidemic situation in the affected countries. 26

27

In the case of viral vectored vaccines, many of the opportunities to accelerate development and 28

product availability during a public health emergency are likely to be due to exploiting the 29

knowledge gained from similar products manufactured with the same vector backbone (i.e. 30

platform technology). Where it can be reasonably argued and shown with data (where available) 31


Page 16

that the manufacture and control of the Ebola vaccine-specific virus vector behaves similarly 1

with respect to process and product characterization to a previously developed product based on 2

the same platform technology, then several aspects of manufacture and control could be based on 3

the more fully developed product with only confirmatory information required for the EBOV 4

specific insert. This principle is especially applicable during the clinical trial phase of 5

development. For licensure, product-specific data will be required but such supportive platform-6

derived data may decrease the requirement for some product data if it can be shown that the 7

benefit-risk assessment remains positive. Such an approach should be discussed with the NRA in 8

advance of the license submission. 9

10

During product development, it might be possible to defer certain tests and development 11

procedures provided it can be justified that their deferral does not affect product safety, and if it 12

can also be argued that performing the tests or development procedures hinder the availability of 13

product, for example by being on the critical path for product availability, or by using large 14

quantities of scarce material that is required for clinical purposes. These deferrals would be 15

identified on a case-by-case basis and should be discussed with the NRA. 16

17

However, even if the nature of a public health emergency affected the benefit/risk balance in 18

such a way as to justify accelerated development and licensing of a vaccine, the Marketing 19

Authorization Holder would retain an obligation to complete full development work and submit 20

the full data to the NRA as soon as they become available, even if this is after product approval 21

through an accelerated mechanism. 22

23

A.1 General manufacturing guidelines 24

The general manufacturing requirements, contained in the WHO Good manufacturing practices 25

for pharmaceutical products:main principles (55) and Good manufacturing practices for 26

biological products (56), should apply to the design, establishment, operation, control, and 27

maintenance of manufacturing facilities for recombinant Ebola vaccines. 28

29

Quality control during the manufacturing process relies on the implementation of quality 30

systems, such as good manufacturing practice (GMP), to ensure the production of consistent 31


Page 17

vaccine lots with characteristics similar to those of lots shown to be safe and effective in clinical 1

trials. Throughout the process, a number of in-process control tests should be established (with 2

acceptable limits) to allow quality to be monitored for each lot from the beginning to the end of 3

production. It is important to note that most release specifications are product-specific, and 4

should be agreed with the NRA as part of the clinical trial or marketing authorization. 5

6

Manufacturers should present a risk assessment regarding the BSL of their manufacturing 7

facility. The principles presented in the WHO Laboratory Biosafety Manual (57) should be 8

followed to justify this classification. Approval for the classification should be sought from the 9

relevant authority in the country/region where the manufacturing facility is located. 10

11

A.1.1 International reference materials 12

Plasma from a recovered repatriated patient who contracted Ebola in West Africa has been 13

established by the Expert Committee on Biological Standardization (ECBS) as the first Anti-14

EBOV plasma, human WHO Reference Reagent. The highly pathogenic nature of EBOV raises 15

particular concerns for the preparation of International Reference materials as they must be safe 16

in use but also resemble the natural material to be analysed very closely if they are to be 17

commutable. The plasma was from a natural infection and therefore is likely to have all relevant 18

specificities and is considered of acceptable safety for three reasons. The patient was fully 19

recovered clinically, the plasma was negative for EBOV nucleic acid in a range of PCR assays 20

and finally it was treated with solvent/detergent, an established method for the inactivation of 21

enveloped viruses used in the blood products industry for decades. 22

23

Two EBOV RNA preparations have been established by the ECBS for the standardisation of 24

nucleic acid assays. The first, EBOV RNA NP-VP35-GP WHO Reference Reagent consists of 25

the RNA encoding the NP,VP35 and GP genes and is to be used to standardize assays directed at 26

these genes only. The second, EBOV RNA VP40-L WHO Reference Reagent consists of the 27

RNA encoding the VP40 and L genes and is intended to standardize assays directed at these 28

genes only. Both preparations are packaged in non-replicating HIV vectors; the EBOV genes 29

also include mutations to make them inactive. 30

31


Page 18

The reference materials listed above are available from the National Institute for Biological 1

Standards and Control, Potters Bar, United Kingdom. The WHO catalogue of international 2

biological standards should be consulted for the latest list of appropriate WHO International 3

Standards and reference materials (58). 4

5

A.2 Control of source materials 6

A.2.1 Viral vector 7

A.2.1.1 Master and working virus seeds 8

The use of any viral vectors should be based on a seed lot system, analogous to the cell banking 9

system used for production cells described below. 10

11

The rationale behind the development of the viral vectored vaccine should be described. The 12

origin of all genetic components of the vaccine, and their function should be specified, and 13

overall this should allow a clear understanding of the functionality of the vaccine and how it is 14

attenuated or made replication incompetent, where this is the result of genetic engineering. All 15

intended and unintended genetic modifications such as site-specific mutations, insertions, 16

deletions and/or rearrangements to any component as compared with their natural counterparts 17

should be detailed. For a vaccine construct that incorporates genetic elements to control the 18

expression of a transgene in, for example, a temporal or tissue-specific manner, evidence should 19

be provided on product characterization and control to demonstrate such specificity. RNA 20

editing should be discussed if relevant. 21

22

All of the steps from derivation of material that ultimately resulted in the candidate vaccine to 23

the master seed level should be described. A diagrammatic description of the components used 24

during vaccine development should be provided and annotated. Cloning of the viral vectored 25

vaccine should be described and the final construct should be genetically characterized according 26

to the principles discussed in this section. 27

28

The use of stably integrated clones may avoid the need for selective markers (e.g. antibiotics) 29

during production. Therefore, cloning strategies such as transfection (e.g. lipofection), 30

electroporation or TA cloning which do not rely on selection markers are preferred. If they are 31


Page 19

used, the impact of selection markers used during screening and development and remaining in 1

the final product should be carefully evaluated. In this respect, the presence of antibiotic 2

resistance genes in the vaccine vector should be avoided if possible. 3

4

A full description of the biological characteristics of the recombinant viral vectors should be 5

given. The culture conditions used to promote the growth of the cloned vector in the production 6

cells should be described in detail. The description should include the construction , genetics and 7

structure of the viral vector; and the origin and identification of the vector and gene that is being 8

cloned. 9

10

The nucleotide sequence of the gene insert and of adjacent segments of the vector, and 11

restriction-enzyme mapping of the vector containing the gene insert, should be provided. 12

Genetic stability of the vector with the recombinant construct should be demonstrated. The 13

stability of a recombinant vector should be assessed by comparing the structure or sequence of 14

the vector at the level of a pre-seed or master seed to its structure or sequence at or preferably 15

beyond the anticipated end-of-production passage level. They should ensure that no changes 16

occur to regions involved in attenuation (where known) or replication deficiency. Any 17

modifications to the sequence of the heterologous insert should be investigated and demonstrated 18

to have no impact on the resulting amino acid sequence (i.e., be a conservative change) or to the 19

antigenic characteristics of the vaccine. 20

21

A.2.1.2 Tests on viral vector master viral seed (MVS) and working viral seed (WVS) 22

The MVS should be characterized as fully as possible. If this characterisation is limited (for 23

example due to limited quantities of material), then the WVS should be fully characterised in 24

addition to the limited characterisation of the MVS. It should be noted that it would not be 25

feasible to manufacture from the MVS in these circumstances. 26

27

MVS characterization will include a description of the genetic and phenotypic properties of the 28

vaccine vector. This should include a comparison with the parental vector and is particularly 29

important where vector modification might affect attenuation or replication competency, 30


Page 20

pathogenicity, and tissue tropism or species specificity of the vaccine vector compared with the 1

parental vector. 2

3

Genetic characterization will involve nucleotide sequence analysis of the vaccine vector. 4

Restriction mapping, southern blotting, polymerase chain reaction (PCR) analysis, or DNA 5

fingerprinting will also be useful adjuncts. Individual elements involved in expression of the 6

heterologous gene(s) (including relevant junction regions) should be described and delineated. 7

8

Genetic stability of the vaccine seed to a passage level comparable to final virus bulk and 9

preferably beyond the end of production cells should be demonstrated. 10

11

Phenotypic characterization should focus on the markers for attenuation/modification and 12

expression of the heterologous antigen(s), and should generally be performed in vitro under 13

conditions allowing detection of revertants (including the emergence of replication competent 14

vectors from replication incompetent vectors during passage). However, other studies including 15

antigenic analysis, infectivity titre and in vitro yield should form part of the characterization. For 16

replicating vectors, in vivo growth characteristics in a suitable animal model may also be 17

informative and should be performed if justified. For some vectors, (e.g. adenoviral vectors) 18

particle number should be measured in addition to infectivity titre. 19

20

A subset of the above studies should be applied to the working seed lot and justification for the 21

chosen subset should be provided. 22

23

During a public health emergency, it is anticipated that the majority of the above 24

information should be available and submitted in full for evaluation since it is essential 25

to demonstrate the suitability and safety of the product. 26

27

It may be justified to initiate clinical trials using a product which is manufactured prior 28

to the seed banking system having been established. In such a case, the safety of the 29

product, especially with regards to adventitious agents (23), replication-competence, 30

attenuation and other phenotypes, stability and suitable genetic sequence must be 31

established prior to its use. 32


Page 21

1

A.2.2 Cell substrates 2

The cell substrate for the manufacture of Ebola vaccine should be based on controlled primary 3

cells or a cell banking system. 4

5

A.2.2.1.1 Master and working cell banks 6

The cell banks should conform to the Recommendations for the evaluation of animal cell 7

cultures as substrates for the manufacture of biological medicinal products and for the 8

characterization of cell banks (23). 9

10

An appropriate history of the cell bank should be provided. This should include the original, 11

identification, development manipulations and characteristics for the purposes of the vaccine. 12

For packaging cell lines, full details of their construction should be given, including the nature 13

and identity of the helper viral nucleic acid and its encoded proteins/functions. If available, 14

information on the chromosomal location of the helper viral nucleic acid should also be 15

provided. Information should be given on the testing for adventitious agents and on gene 16

homogeneity for the MCB and WCB. 17

18

Genetic stability of the cell lines should be demonstrated. The stability of a production cell line 19

should be assessed by comparing the critical regions of the cell line (and flanking regions) at the 20

level of a pre-cell or master cell to its structure or sequence at or beyond the anticipated end-of-21

production passage level. Stability studies should also be performed to confirm cell viability after 22

retrieval from storage, maintenance of the expression system, etc. These studies may be 23

performed as part of the routine use in production or may include samples taken specifically for 24

this purpose. 25

26

With regard to cell cultures, the maximum number of passages (or population doublings) 27

allowable from the MCB, through the WCB, and through the production in cells should be 28

defined, based on the stability data generated above, and approved by the NRA. 29

30

A.2.2.1.2 Primary cells 31


Page 22

Primary cells are used within the first passage after establishment from the original tissue and so 1

it is not possible to carry out extensive characterisation of the cells prior to their use. Therefore 2

additional emphasis is placed on the origin of the tissues from which the cell line is derived. 3

Tissue should be derived from healthy birds/animals subjected to veterinary and laboratory 4

monitoring to certify the absence of pathogenic agents. Whenever possible, donor birds/animals 5

should be obtained from closed, specific pathogen-free colonies or flocks. Animals used as tissue 6

donors should not have been used previously for experimental studies. Birds/animals should be 7

adequately quarantined for an appropriate period of time prior to use for the preparation of cells. 8

Information on materials and components used for the preparation of primary cell substrates 9

should be provided, including the identity and source of all reagents of human or animal origin. 10

A description of testing performed on components of animal origin to certify the absence of 11

detectable contaminants and adventitious agents should be included. 12

13

The methods used for the isolation of cells from tissue, establishment of primary cell cultures 14

and maintenance of cultures should be described. 15

16

A.2.2.2.1 Tests on cell substrates MCB and WCB 17

MCBs and WCBs should be tested for the absence of bacterial, fungal, and mycoplasmal and 18

viral contamination by appropriate tests, as specified in Recommendations for the evaluation of 19

animal cell cultures as substrates for the manufacture of biological medicinal products and for 20

the characterization of cell banks (23), or by a method approved by the NRA to demonstrate that 21

the MCB and WCB are not contaminated with adventitious agents. 22

23

Rapid sterility methods to demonstrate freedom from bacteria and fungi as well as nucleic acid 24

amplification techniques (NAT) alone or in combination with cell culture, with an appropriate 25

detection method, might be used as an alternative to one or both of the compendial 26

mycoplasmal detection methods after suitable validation and agreement from NRA (23). 27

28

The cell bank should be tested for tumourigenicity if it is of mammalian origin, as described in 29

Section B of the Recommendations for the evaluation of animal cell cultures as substrates for the 30

manufacture of biological medicinal products and for the characterization of cell banks (23). 31


Page 23

The tumorigenic potential of the cell banks(s) should be described and strategies to mitigate risks 1

that might be associated with this biological property should be described and justified. 2

3

During a public health emergency, it is anticipated that the majority of the above 4

information should be available and submitted for evaluation since it is essential to 5

demonstrate the suitability and safety of the product. However, it may be justified to 6

initiate clinical trials using a product which is manufactured prior to the cell banking 7

system having been established. In such a case, the suitability and safety of the 8

product, especially with regards to adventitious agents (9), must be established prior to 9

its use. 10

11

A.2.2.2.2 Tests on primary cells 12

The nature of primary cells precludes extensive testing and characterisation before use. Testing 13

to demonstrate the absence of adventitious agents (bacteria, fungi, mycoplasma and viruses) is 14

therefore conducted concurrently and should include, where relevant, the observation of control 15

(uninfected) cultures during parallel fermentations to the production runs. The inoculation of 16

culture fluid from production and (where available) control cultures into various susceptible 17

indicator cell cultures capable of detecting a wide range of relevant viruses followed by 18

examination for cytopathic changes and testing for the presence of heamadsorbing viruses, 19

should also be routinely performed for batch release, in addition to pharmacopoeial testing for 20

bacterial, fungi and mycoplasma in the control (if relevant) and production cultures. Mycoplasma 21

and specific viruses of notable concern may also be tested for by additional methods such as 22

PCR. 23

24

In the specific case of Chick Embryo Fibroblasts (CEFs), the tissue should be sourced from SPF 25

eggs. After preparation, the CEF cells should be tested for bacterial, fungal and mycoplasma 26

contamination, for viral adventitious agents by in vitro assay using 3 cells lines, including avian 27

and human cells, such as CEF, MRC-5 and Vero cells, and for viral adventitious agents by in 28

vivo assay using mice and embryonated eggs, for Avian Leucosis Virus contamination and for 29

presence of retroviruses by measuring the RT-activity. 30

31


Page 24

A.2.3 Cell culture medium 1

If serum is used for the propagation of cells, it should be tested to demonstrate freedom from 2

bacteria, fungi and mycoplasmas, as specified in the requirements given in Part A, sections 5.2 3

(59) and 5.3 (60) of the WHO General requirements for the sterility of biological substances and 4

freedom from adventitious viruses. 5

6

Detailed guidelines for detecting bovine viruses in serum for establishing MCB and WCB are 7

given in Appendix 1 of the WHO Recommendations for the evaluation of animal cell cultures as 8

substrates for the manufacture of biological medicinal products and for the characterization of 9

cell banks (23) and should be applied as appropriate. The guidelines for detecting bovine viruses 10

in serum for establishing the cell banks may also be applicable to production cell cultures. As an 11

additional monitor of quality, sera may be examined for endotoxin. Gamma-irradiation may be 12

used to inactivate potential contaminant viruses, recognizing that some viruses are relatively 13

resistant to gamma-irradiation.Whatever the process used, the validation study has to determine 14

the consistency and effectiveness of the viral inactivation process while maintaining serum 15

performance. The use of non-inactivated serum should be justified and is not advised without 16

strong justification. The non-inactivated serum must meet the same criteria as the inactivated 17

serum when tested for sterility and absence of mycoplasma and viral contaminants. 18

19

The source(s) of animal components used in culture medium should be approved by the NRA. 20

These components should comply with the current WHO guidelines on transmissible spongiform 21

encephalopathies in relation to biological and pharmaceutical products (61). 22

23

Bovine or porcine trypsin used for preparing cell cultures should be tested and found free of 24

bacteria, fungi, mycoplasmas and adventitious viruses, as appropriate. The methods used to 25

ensure this should be approved by the NRA. The source(s) of trypsin of bovine origin, if used, 26

should be approved by the NRA and should comply with the current WHO guidelines on 27

transmissible spongiform encephalopathies in relation to biological and pharmaceutical 28

products (61). 29

30


Page 25

In some countries, irradiation is used to inactivate potential contaminant viruses in trypsin. If 1

irradiation is used, it is important to ensure that a reproducible dose is delivered to all batches 2

and the component units of each batch. The irradiation dose must be low enough so that the 3

biological properties of the reagents are retained while being high enough to reduce virological 4

risk. Therefore, irradiation cannot be considered a sterilizing process (23). The irradiation 5

method should be validated and approved by the NRA. 6

7

Recombinant trypsin is available and should be considered; however it should not be assumed to 8

be free of risk of contamination and should be subject to the usual considerations for any reagent 9

of biological origin (23). 10

11

Human serum should not be used. 12

13

If human serum albumin is used at any stage of product manufacture, the NRA should be 14

consulted regarding the requirements, as these may differ from country to country. As a 15

minimum, it should meet the WHO Requirements for the Collection, Processing and Quality 16

Control of Blood, Blood Components and Plasma Derivatives (62). In addition, human albumin 17

and materials of animal origin should comply with the current WHO guidelines on transmissible 18

spongiform encephalopathies in relation to biological and pharmaceutical products (61). 19

20

Penicillin and other beta-lactams should not be used at any stage of the manufacture because 21

they are highly sensitizing substances. 22

23

Other antibiotics may be used in the manufacture provided that the quantity present in the final 24

lot is acceptable to the NRA. 25

26

Non-toxic pH indicators may be added, e.g., phenol red at a concentration of 0.002%. Only 27

substances that have been approved by the NRA may be added. 28

29

A.2.4 Special considerations for the development and testing of the viral vector and 30

production cell lines 31


Page 26

Early phase clinical and non-clinical studies are generally supplied with product for which 1

knowledge of the manufacture and control is expected to be fairly rudimentary since few batches 2

will have been manufactured, and analytical methods will be in early stages of development. The 3

material is required for the provision of early safety and proof-of-concept studies, as well as to 4

begin the dose-finding evaluation. Product will be tested initially in animals and then in a small 5

number of human subjects in a well-controlled environment. This is the normal situation 6

occurring when there is no public health emergency and in these circumstances guidance on the 7

quality requirements for investigational medicinal products in clinical trials is available (19,20). 8

9

The majority of the data to be provided to the NRA before the human studies can begin will be 10

concerning the derivation and safety of the viral vector product and the production cell line, to 11

ensure that the product and production system are well designed, the function of each genetic 12

element is known and its inclusion in the product or cell line is justified. It should be confirmed 13

that the expected elements are present in the product and cell line and that the final structure of 14

the product is as predicted. A full description of the origin and construction of the genetic 15

components of the viral vector and cell line should be provided, and the genetic stability to or 16

preferably beyond the anticipated passage level in manufacture should be given. Ideally, a 17

MVS/WVS for the viral vector and MCB/WCB for the production cell line should be prepared 18

early on in the development of the product but it is acknowledged that this may not be practical 19

in the initial stages. Testing of the seed lots and cell banks, at the time of their establishment, 20

should confirm comparability to the parental material. Any starting material (viral seeds and 21

production cell lines) used in the production of product for clinical use must be fully tested to 22

ensure the absence of bacteria, fungi, mycoplasmas, and adventitious viruses. Where applicable, 23

freedom from TSEs must also be addressed (61). The potential for tumourigenicity of the cell 24

line should also be tested and meet current regulatory standards if it is of mammalian origin. All 25

reagents used in the manufacture of the seed or cell lines (including cell culture solutions) should 26

be tested and characterized to be of adequate quality, particularly regarding freedom from 27

adventitious agents. 28

29

A.3 Control of Ebola vaccine production 30

A.3.1 Manufacture and purification 31


Page 27

The manufacture of vaccine vectors starts with the amplification of the vaccine vector seed stock 1

in a suitable cell line. The number of passages between the working seed lot and viral vectored 2

vaccine product should be kept to a minimum and should not exceed that used for production of 3

the vaccine shown in clinical studies to be satisfactory, unless otherwise justified and authorized. 4

5

If applicable to the vector platform, a control cell culture should be maintained simultaneously 6

and in parallel to the production cell culture. Cells should be derived from the same expansion 7

series but no virus vector should be added to the control cells. All other manipulations should be 8

as similar as possible. 9

10

After harvesting of the culture product, the purification procedure can be applied to a single 11

harvest or to a pool of single harvests.The maximum number of single harvests that may be 12

pooled should be defined on the basis of validation studies. During early development, 13

validation may not have been completed and so the number of harvests pooled should be defined 14

based on other criteria such as production requirements. 15

16

By the time of submitting a Marketing Authorization Application, the manufacturing process 17

should be adequately validated by demonstrating that a sufficient number of commercial scale 18

batches can be routinely manufactured under a state of control, by meeting pre-determined in-19

process controls, critical process parameters and lot release specifications. Any materials added 20

during the purification process should be documented, and their removal should be adequately 21

validated or residual amounts tested for as appropriate. Validation should also demonstrate that 22

the manufacturing facility and equipment have been qualified, cleaning of product contact 23

surfaces is adequate and critical process steps such as sterile filtrations and aseptic operations 24

have been validated. 25

26

The purified viral vector bulk and intermediates should be maintained under conditions shown 27

by the manufacturer to ensure they retain the desired biological activity. Hold times should be 28

defined. 29

30


Page 28

During early clinical trials, it is unlikely that there will be data from sufficient batches to 1

validate/qualify product manufacture. However, as development progresses, data from 2

subsequent manufacture should be gained and used in support of an eventual application for 3

commercial supply of the product. 4

5

During a public health emergency, on a case-by-case basis, some requirements of 6

process validation may be abbreviated provided it can be demonstrated the product 7

will remain safe and well-controlled. For example if platform-specific data have 8

demonstrated that scale-up for a vector is independent of the specific heterologous 9

insert, then this information may be used to justify fewer full-scale batches with the 10

EVD gene insert and a greater reliance on engineering and pilot- plant-scale batches. 11

Validation data from the manufacture of platform-related products may provide 12

useful supportive information, particularly in the identification of critical parameters. 13

14

Since it is likely that there will initially be insufficient time to generate full 15

validation data during an emergency situation, as much information as possible 16

regarding the control of each batch should be presented to the NRA as supporting 17

evidence that batch manufacture is sufficiently controlled. However, the 18

manufacturers should agree the strategy with the NRA before relying on platform 19

specific validation data. 20

21

In addition to control during manufacture, the products should be adequately characterized, 22

relevant to the stage of development. These attributes serve to understand the biology of the 23

candidate vaccine and to assess the impact of any changes in manufacturing that are introduced 24

as development advances or in a post-licensure setting. The immunogenicity of product, when 25

relevant and available, should also be included in the characterization programme, for example 26

as part of the nonclinical pharmacodynamic evaluation. 27

28

A.3.1.1 Tests on control cell cultures (if applicable) 29

Where the NRA requires the use of control cells, the following procedures should be followed. 30

From the cells used to prepare cultures for production of vaccine, a fraction equivalent to at least 31


Page 29

5% of the total or 500 ml of cell suspension, or 100 million cells, should be used to prepare 1

uninfected control cell cultures. 2

3

These control cultures should be observed microscopically for cytopathic and morphological 4

changes attributable to the presence of adventitious agents for at least 14 days at a temperature of 5

35–37 °C after the day of inoculation of the production cultures, or until the time of final virus 6

harvest, whichever comes last. At the end of the observation period, supernatant fluids collected 7

from the control culture should be tested for the presence of adventitious agents as described 8

below. Samples that are not tested immediately should be stored at –60 °C or lower, until such 9

tests can be conducted. 10

11

If adventitious agent testing of control cultures yields a positive result, the harvest of virus from 12

the parallel vaccine virus-infected cultures should not be used for production. 13

14

For the test to be valid, not more than 20% of the control culture flasks should have been 15

discarded, for any reason, by the end of the test period. 16

17

A.3.1.1.1 Tests for haemadsorbing viruses 18

At the end of the observation period, a fraction of control cells comprising not less than 25% of 19

the total should be tested for the presence of haemadsorbing viruses, using guinea-pig red blood 20

cells. If the red blood cells have been stored prior to use in the haemadsorption assay, the 21

duration of storage should not have exceeded 7 days, and the temperature of storage should have 22

been in the range of 2–8 °C. 23

24

In some countries, the NRA requires that additional tests for haemadsorbing viruses are to be 25

performed using red blood cells of other species including from humans (blood group O), 26

monkeys and/or chickens (or other avian species). All haemadsorption tests should be read after 27

incubation for 30 minutes at 0–4 °C, and again after a further incubation for 30 minutes at 20–25 28

°C. The test with monkey red cells should be read once more after additional incubation for 30 29

minutes at 34–37 °C. 30

31


Page 30

For the tests to be valid, not more than 20% of the culture vessels should have been discarded for 1

any reason by the end of the test period. 2

3

A.3.1.1.2 Tests for other adventitious agents 4

At the end of the observation period, a sample of the pooled fluid and/or cell lysate from each 5

group of control cell cultures should be tested for adventitious agents. For this purpose, an 6

aliquot of each pool should be tested in cells of the same species as used for the production of 7

virus, but not cultures derived directly from the production cell expansion series for the batch 8

which is subject to test. If primary cells are used for production, then a separate batch of that 9

primary cell type should be used for the test than was used for production. Samples of each pool 10

should also be tested in human cells and in a simian kidney cell line. At least one culture vessel 11

of each kind of cell culture should remain uninoculated as a control. 12

13

The inoculated cultures should be incubated at the appropriate growth temperature and should be 14

observed for cytopathic effects for a period of at least 14 days. 15

16

Some NRAs require that, at the end of this observation period, a subculture is made in the same 17

culture system and observed for at least an additional seven days. Furthermore, some NRAs 18

require that these cells should be tested for the presence of haemadsorbing viruses. 19

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

any reason by the end of the test period.. 21

22

A.3.2 Single virus harvest 23

The method of harvesting the vaccine vector should be described and the titre of virus 24

ascertained. A reference preparation should be included to validate the titration assay. Minimum 25

acceptable titres should be established for single virus harvest or pooled single harvests. 26

27

The integrity of the integrated heterologous gene should be confirmed. An expression assay 28

method should be described and performed on production harvest material or downstream (e.g., 29

purified drug substance). For example, a Western blot analysis or other method to confirm that 30

the integrated gene is present and expressed should be included in the testing of every batch. 31


Page 31

1

A.3.2.1 Control tests on single virus harvest 2

Tests for adventitious agents should be performed on each single harvest according to relevant 3

parts of section B.11 of WHO’s Recommendations for the evaluation of animal cells as 4

substrates for the manufacture of biological medicinal products and for the characterization of 5

cell banks (23). Additional testing for adventitious viruses may be performed using validated 6

nucleic acid amplification techniques. 7

8

New molecular methods with broad detection capabilities are being developed for adventitious 9

agent detection. These methods include: (i) degenerate nucleic acid amplification techniques for 10

whole virus families with analysis of the amplicons by hybridization, sequencing or mass 11

spectrometry; (ii) nucleic acid amplification techniques with random primers followed by 12

analysis of the amplicons on large oligonucleotide micro-arrays of conserved viral sequencing or 13

digital subtraction of expressed sequences; and (iii) high throughput sequencing. These methods 14

may be used in the future to supplement existing methods or as alternative methods to both in 15

vivo and in vitro tests after appropriate validation and agreement from the NRA. 16

17

Single or pooled virus harvests should be tested to demonstrate freedom from bacteria, fungi and 18

mycoplasmas, as specified in the requirements given in Part A, sections 5.2 (59) and 5.3 (60) of 19

the WHO General requirements for the sterility of biological substances. 20

21

For viral vectored vaccines, due to the very high titers of the single harvests, alternatives to the 22

classical testing for adventitious agents may be applied with the approval of the NRA. 23

24

Tests for replication competent viruses performed on a suitable sensitive cell line(s) may be 25

necessary for certain replication defective vectors, e.g. replication defective adenovirus vectored 26

vaccines. 27

Provided cell banks and viral seed stocks have been comprehensively tested and 28

released, demonstrating they are free of adventitious agents, it may be discussed and 29

should be agreed with the NRA, to evaluate the possibility to delay in vitro testing for 30

extraneous agents (viral pathogens and mycoplasmas) at the cell harvest or bulk 31


Page 32

substance stages, or replace with validated PCR tests.The method of production should 1

be taken into account when deciding the nature of any specified viruses being sought 2

3

Additional considerations for this approach are that no animal derived raw materials 4

are used during manufacture, and that the manufacturing facility operates under a 5

GMP certificate with assurances that prevention of cross contamination is well 6

controlled in the facility. Samples should be retained for testing at a later date if 7

required. 8

9

A.3.3 Pooled virus harvests 10

Depending on the unit size of production, single virus harvests may be pooled and from these 11

virus pools, the final bulk vaccine will be prepared. The strategy for pooling of single virus 12

harvests should be described. All processing of the virus pool should be described in detail. 13

14

A.3.3.1 Control tests on pooled virus harvests. 15

Virus pools should be tested to demonstrate freedom from bacteria, fungi and mycoplasmas, as 16

specified in the requirements given in Part A, sections 5.2 (46) and 5.3 (60) of the WHO General 17

requirements for the sterility of biological substances. Alternatively, if single virus harvests 18

have been tested to demonstrate freedom from bacteria, fungi and mycoplasmas, these tests may 19

be omitted on the pooled virus harvests. 20

21

A.3.4 Final bulk vaccine 22

The final bulk vaccine can be prepared from one or several virus pools or it may be derived from 23

a single virus harvest. Substances such as diluents or stabilizers or any other excipients added 24

during preparation of the final bulk vaccine should have been shown not to impair the potency 25

and safety of the vaccine in the concentrations employed. 26

27

Penicillin and other beta-lactams should not be used at any stage of manufacture because of their 28

nature as highly sensitizing substances in humans. Other antibiotics may be used at any stage of 29

manufacture, provided that the quantity present in the final product is acceptable to the NRA 30

31


Page 33

For multi-dose preparations, the need for effective antimicrobial preservation should be 1

evaluated taking into account possible contamination during use and the maximum 2

recommended period of use after opening the container or reconstitution of the vaccine. If an 3

antimicrobial preservative is used, it should not impair the safety or potency of the vaccine and 4

the intended concentration of the preservative should be justified and its effectivness should be 5

validated. 6

7

A.3.4.1 Control tests on final bulk 8

The final bulk vaccine should be tested and consideration should be given to using the tests listed 9

below as appropriate for the individual products. Alternatively, if the final bulk will be held for a 10

short period of time, some of the tests listed below could be performed on the final lot instead. 11

All quality-control release tests, and specifications for purified bulk, should be validated and 12

shown to be suitable for the intended purpose. Assay validation or qualification should be 13

appropriate for the stage of the development life cycle. Additional tests on intermediates during 14

the purification process may be used to monitor the consistency and safety. 15

16

During the manufacture of products for early clinical trials, not all attributes tested may have 17

specification ranges set since insufficient batches may have been made to know what an 18

acceptable range is. Nor at this time, is a clinically meaningful range always known. However, as 19

the clinical programme continues and certainly by the initiation of phase 3 trials, specification 20

ranges should be set for each attribute. 21

22

During an emergency situation, it is anticipated that critical assays would be fully 23

validated. Specifications should also be given for each critical parameter. Qualification 24

or validation as well as specifications for some assays may be based on related products 25

(e.g. with the same vector backbone but differing in heterologous gene from the Ebola 26

glycoprotein) where it can be justified that the specific heterologous gene is unlikely to 27

impact the result. An example of this could be particle quantification by qPCR where 28

the probe is demonstrated to be a non-EBOV sequence in the vector. 29

30


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With appropriate justification, validation for non-critical assays could be completed 1

after product approval provided assay verification adequately demonstrates the assay is 2

fit for purpose and under control. 3

4

Similarly, if adequately justified, not all of the proposed assays may need to be 5

completed for early batch release. If it can be justified that product safety and potency 6

are not compromised, that completion of the tests delays product availability for use in 7

clinical trials, and/or that the test would use an unacceptably large volume of product 8

which is urgently required for clinical trials, it may be possible to omit or delay the test, 9

or replace it with one that is more acceptable to the overall aims of the clinical trials in 10

an emergency situation. 11

12

However, all of the approaches discussed above should be agreed with the NRA on a 13

case-by-case basis. 14

15

A.3.4.1.1 Purity 16

The degree of purity of each final bulk vaccine should be assessed using suitable methods. The 17

purity of the bulk should be ascertained for fragments, aggregates or empty particles of the 18

product, as well as for contamination by residual cellular proteins. Residual cellular DNA levels, 19

when non-primary cell substrates are used for production, should also be assessed. Process 20

additives should also be controlled. In particular, if any antibiotics are added during vaccine 21

production, the residual antibiotic content should be determined and should be within limits 22

approved by the NRA. 23

24

At the initial stages of development, testing may not be required to determine residual levels of 25

process contaminants (except DNA and proteins) if sufficient justification can be provided by 26

theoretical calculation. However, data to confirm the calculations should be provided prior to 27

license application. 28

In a public health emergency situation, theoretical calculations to determine residual 29

levels of process contaminants (except DNA and proteins) may be acceptable at the time 30

of licensure though data should be submitted as soon as possible post licensure. 31

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The amount of residual host cell DNA derived from non-primary cell lines should be determined 1

in each purified final bulk vaccine batch by suitably sensitive methods. The level of host cell 2

DNA should not exceed the maximum level agreed with the NRA taking into consideration 3

issues such as those discussed in the WHO Recommendations for the evaluation of animal cell 4

cultures as substrates for the manufacture of biological medicinal products and for the 5

characterization of cell banks (23). 6

7

These tests may be omitted for routine lot release upon demonstration that the process 8

consistently clears the residuals from the final bulk vaccine, subject to the agreement of the 9

NRA. 10

11

A.3.4.1.2 Potency 12

Each final bulk vaccine should be tested for potency measured by a combination of the following 13

methods. 14

15

A.3.4.1.2.1 Particle number 16

For relevant vectors (e.g., adenovirus vectors), the total number of virus particles per millilitre, 17

quantitated by a technique such as qPCR or HPLC should be provided for each bulk batch. 18

19

A.3.4.1.2.2 Infectivity 20

Infectious virus titre, as a measure of active product should be tested for each batch of final bulk. 21

Direct methods such as a plaque-forming assay, or indirect methods such as qPCR, if suitably 22

correlated with a direct measure of infectivity could be considered. For relevant vectors, the 23

particle to infectivity ratio should also be specified. 24

25

A.3.4.1.2.3 Expression of the heterologous antigen in vitro 26

The ability of the viral particles to express the heterologous gene should be demonstrated, e.g. by 27

the generation of immunoblots using antigen-specific antibodies, after growth of the vector in a 28

suitable cell line. 29

30

A.3.4.1.3 Identity 31


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Tests used for assessing other properties of the viral vector, such as antigen expression, 1

restriction analysis, PCR with a specific probe or sequencing will generally be suitable for 2

assessing the identity of the product. 3

4

A.3.4.1.4 Sterility or Bioburden tests for bacteria and fungi 5

Each final bulk should be tested for bacterial and fungal bioburden or sterility. Bioburden testing 6

should be justified in terms of product safety. Sterility testing should be as specified in Part A, 7

section 5.2 of the WHO General requirements for the sterility of biological substances (59), or 8

by methods approved by the NRA. 9

10

A.3.4.1.5 Bacterial endotoxins 11

Each final bulk should be tested for bacterial endotoxins. At the concentration of the final 12

formulation of the vaccine, the total amount of residual endotoxins should not exceed that found 13

in vaccine lots shown to be safe in clinical trials or in data from other lots used to support 14

licensing. The test may be omitted once production consistency has been demonstrated after 15

agreement from the NRA. 16

17

A.3.4.1.6 Reversion to replication competency or loss of attenuation 18

Viral vector Ebola vaccines under development are either replication incompetent in human 19

cells, or adequately attenuated to prevent disease symptoms related to the viral vector backbone. 20

Although manufacturers generally provide theoretical justifications for why reversion to 21

competency or virulence is unlikely to occur, low levels of replication competent particles have 22

been found in some adenovirus preparations from Per.C6 cell lines, formed by unconventional 23

recombination. It should also be taken into account that in the Ebola target population, many of 24

the subjects could be immunocompromised. Consequently, it should be shown that product is 25

still replication incompetent or fully attenua

Documents

Guidelines on the quality, safety and efficacy of Ebola vaccinesVer_5...16 undertaken in developing live attenuated Ebola vaccines although in theory EBOV could be 17 inactivated,