recommendations to assure the quality, safety and efficacy ......who/ ipv_draft/ 2 december 2013...

WHO/IPV_DRAFT/2 December 2013

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WHO/IPV_DRAFT/2 December 2013 3

ENGLISH ONLY 4

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6

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Recommendations to Assure the Quality, Safety and Efficacy 8

of Poliomyelitis Vaccine (inactivated) 9

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Proposed replacement of: TRS 910, Annex 2 11

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NOTE: 14

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

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

Committee on Biological Standardization (ECBS). Publication of this early draft is to 18

provide information about the proposed WHO Recommendations to Assure the Quality, 19

Safety and Efficacy of Poliomyelitis Vaccine (inactivated) to a broad audience and to 20

improve transparency of the consultation process. 21

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The text in its present form does not necessarily represent an agreed formulation 24

of the Expert Committee. Written comments proposing modifications to this text 25

MUST be received by 20 January 2014 in the Comment Form available separately 26

and should be addressed to the World Health Organization, 1211 Geneva 27, 27

Switzerland, attention: Department of Essential Medicines and Health Products (EMP). 28

Comments may also be submitted electronically to the Responsible Officer: Dr TieQun 29

Zhou at email: [email protected]. 30

31

The outcome of the deliberations of the Expert Committee will be published in the 32

WHO Technical Report Series. The final agreed formulation of the document will be 33

edited to be in conformity with the "WHO style guide" (WHO/IMD/PUB/04.1). 34

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© World Health Organization 2013 37

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, 38 World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: 39 +41 22 791 4857; e-mail: [email protected]). Requests for permission to reproduce or translate WHO 40 publications – whether for sale or for non-commercial distribution – should be addressed to WHO Press, at 41 the above address (fax: +41 22 791 4806; e-mail: [email protected]). 42

The designations employed and the presentation of the material in this publication do not imply the 43 expression of any opinion whatsoever on the part of the World Health Organization concerning the legal 44 status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers 45

WHO/IPV_DRAFT/2 December 2013

or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full 1 agreement. 2 3 The mention of specific companies or of certain manufacturers’ products does not imply that they are 4 endorsed or recommended by the World Health Organization in preference to others of a similar nature 5 that are not mentioned. Errors and omissions excepted, the names of proprietary products are 6 distinguished by initial capital letters. 7 8 All reasonable precautions have been taken by the World Health Organization to verify the information 9 contained in this publication. However, the published material is being distributed without warranty of any 10 kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with 11 the reader. In no event shall the World Health Organization be liable for damages arising from its use. 12

13 The named authors [or editors as appropriate] alone are responsible for the views expressed in this 14 publication. 15 16

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Recommendations published by the WHO are intended to be scientific and advisory

in nature. Each of the following sections constitutes guidance for national regulatory

authorities (NRAs) and for manufacturers of biological products. If an NRA so

desires, these Recommendations may be adopted as definitive national requirements,

or modifications may be justified and made by the NRA. It is recommended that

modifications to these Recommendations be made only on condition that

modifications ensure that the vaccine is at least as safe and efficacious as that

prepared in accordance with the recommendations set out below. The parts of each

section printed in small type are comments or examples for additional guidance

intended for manufacturers and NRAs, which may benefit from those details.

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WHO/ IPV_DRAFT/ 2 December 2013

Table of contents 1

Introduction

General considerations

Part A. Manufacturing recommendations

A.1 Definitions

A.2 General manufacturing recommendations

A.3 Control of source materials

A.4 Control of vaccine production

A.5 Filling and containers

A.6 Control tests on the final lot

A.7 Records

A.8 Retained samples

A.9 Labelling

A.10 Distribution and shipping

A.11 Stability, storage and expiry date

Part B. Nonclinical evaluation of poliomyelitis vaccines (inactivated)

B.1 Characterization of poliovirus seed lots derived from attenuated strains (Sabin

strains and strains derived by recombinant DNA technology)

B.2 Antigenic profile

B.3 D-antigen content of IPV derived from attenuated strains (Sabin strains and

strains derived by recombinant DNA technology)

B.4 Evaluation of immunogenicity in animal models

B.5 Nonclinical safety studies

Part C. Clinical evaluation of poliomyelitis vaccines (inactivated)

C.1 General considerations

C.2 Immunogenicity studies

C.3 Concomitant administration with other vaccines

C.4 Pre-licensure safety data

C.5 Post-marketing studies and surveillance

Part D. Recommendations for national regulatory authorities

D.1 General

D.2 Official release and certification

Authors and Acknowledgements

References

Appendix 1

Overview of the virus seeds used in IPV production

Appendix2

In vivo Potency assay of IPV

Appendix 3


1

2

Introduction 3

The requirements for inactivated poliomyelitis vaccine (IPV) were first formulated in 1959 4

(1) and revised in 1965 (2). Following several advances in technology in vaccine 5

production, the requirements were further updated in 1981 (3) and amended in 1985 (4). At 6

that time, the introduction of continuous cells for manufacture of IPV was a novel 7

development and when the regulatory control of products manufactured in continuous cells 8

had been standardized, the requirements were again updated in 2000 (5). An addendum 9

was added in 2003 (6) which specified the measures to be taken to minimize the accidental 10

risk of reintroducing wild type poliovirus from a vaccine manufacturing facility into the 11

community after global certification of polio eradication. 12

13

Since the Recommendations for IPV were last revised in 2000 (5) and in 2003 (6), there 14

have been several changes in vaccine production, including the use of seed viruses derived 15

from Sabin strains, which make a further revision of the Recommendations necessary. To 16

facilitate this process, a discussion on international specifications for IPV attended by 17

experts from academia, National Regulatory Authorities (NRA)/National Control 18

Laboratories (NCL) and industry involved in the research, manufacture, authorization and 19

testing/release of IPV from countries around the world was convened by WHO on 29 March 20

2012. During the discussions, critical issues for the quality control (QC) and evaluation of 21

IPV (including Sabin-based IPV, sIPV) were considered, and for the revision of the current 22

recommendations WHO TRS 910 (Annex 2) (5) were identified. WHO convened a 23

Technical Working Group Meeting on 14- 15 May 2013 in Geneva, which was attended by 24

experts from academia, National Regulatory Authorities (NRA)/National Control 25

Laboratories (NCL) and industry involved in the development, manufacture, authorization 26

and testing/release of IPV including sIPV and other new developments of novel IPV from 27

countries around the world, to further discuss and reach consensus on critical issues relevant 28

to the revision of the TRS 910 (Annex 2) (Reference: meeting report when published). 29

30

Model summary protocol for manufacturing and control of poliomyelitis vaccine

(inactivated)

Appendix 4

Model certificate for the release of poliomyelitis vaccine (inactivated) by national

regulatory authorities


Major issues addressed in this revision include: 1

− an update on General considerations and other sections to reflect the future trend of 2

IPV use in line with the global programmatic need, e.g. use of Sabin strains and 3

strains derived by recombinant DNA technology; 4

− an update on the history of the different virus seed strains used by manufacturers 5

for IPV production with inclusion of a new Appendix 1; 6

− an update of the section on international standards and reference preparations; 7

− an update of the section on general manufacturing recommendations and control 8

tests; 9

− an update on terminology; 10

− the inclusion of specific tests for sIPV and IPV made from strains derived by 11

recombinant DNA technology 12

− an update on appendices; 13

− the inclusion of new sections on nonclinical and clinical evaluation of IPV. 14

15

Additional changes have been made to bring the document into line with other WHO 16

recommendations published since the last revision. 17

18

Scope 19

The scope of the present Recommendations encompasses inactivated poliomyelitis vaccines 20

derived from 1) the wild type strains that have been used in manufacture of IPV for many 21

years; 2) the attenuated Sabin strains that have been used in the manufacture of oral 22

poliomyelitis vaccine (OPV); and 3) new alternative poliovirus strains with improved 23

biosafety characteristics that are under development in some countries, including those 24

derived by recombinant DNA technology. 25

26

This document does not cover vaccines which are based on virus-like particles (VLPs) and 27

replicons. However, some aspects of the current document may be relevant and should be 28

taken into consideration during the vaccine development using these types of seeds. 29

30

This document should be read in conjunction with the relevant WHO guidelines such as 31

those on nonclinical (7) and clinical evaluation (8) of vaccines. 32

33


Among the most significant changes in production, there has been the increasing use of IPV 1

in combination with other vaccines and this introduces considerations that do not apply 2

when IPV is used as a stand-alone product, such as interaction of the poliovirus antigens 3

with other antigens and/or adjuvants. These considerations are dealt with in a separate WHO 4

document (9) but not in the present Recommendations. However, to provide further 5

guidance for control of the vaccine, key tests that may be influenced by other antigens and/ 6

or adjuvants in combined vaccines are identified. 7

8

General considerations 9

Poliomyelitis is an acute communicable disease of humans caused by three distinct 10

poliovirus serotypes, types 1, 2 and 3, distinguished by neutralization test (10). Poliovirus is 11

classified as a species C human enterovirus of the Picornaviridae family and is composed of 12

a single-stranded, positive-sense RNA genome and a protein capsid. 13

14

Where sanitation is poor, these viruses are believed to spread mainly by faecal-to-oral 15

transmission, whereas the oral-to-oral mode of transmission probably dominates in settings 16

with a higher standard of sanitation. However, in most settings, mixed patterns of 17

transmission are likely to occur. In the pre-vaccine era, roughly one out of 200 susceptible 18

individuals infected by polioviruses developed paralytic poliomyelitis (10). 19

20

Progress in polio control (and, since 1988, polio eradication) has been due mainly to 21

widespread use of vaccines. An inactivated poliomyelitis vaccine (IPV Salk vaccine) was 22

first licensed in 1955; live, attenuated oral poliomyelitis vaccine (Sabin vaccine) was 23

licensed in the US as monovalent OPV in 1961 and as trivalent OPV in 1963. Most 24

countries, that initially introduced vaccination with IPV, later changed to OPV because it 25

provided many advantages including ease of administration, suitability for mass 26

vaccination campaigns, superior induction of intestinal mucosal immunity and lower 27

production costs. In May 1988, the World Health Assembly resolved to eradicate 28

poliomyelitis globally by the year 2000. By 2013, four of the six WHO Regions had been 29

certified as free of wild type polioviruses, and wild type 2 had not been detected worldwide 30

since 1999 (10). The last case of wild type poliomyelitis in India occurred in January 2011 31

and although India has not yet completed the poliomyelitis-free status for the three years 32

which is needed to achieve certified status, it seems likely that it will be certified in due 33


course, leaving only three countries that still have endemic poliovirus. However, there 1

have been frequent sporadic outbreaks in previously poliomyelitis-free countries where the 2

virus has been introduced from endemic countries. Given the progress towards polio 3

eradication, countries have increasingly switched from using OPV to IPV in routine 4

immunization programmes, primarily to eliminate the burden of vaccine-associated 5

paralytic poliomyelitis (VAPP), a rare adverse event associated with OPV. The sole use of 6

IPV has successfully eradicated polio in a few countries, notably in Scandinavia and the 7

Netherlands. In most of the countries that have introduced IPV as the only poliomyelitis 8

vaccine over the last decade, there has been no evidence of continued circulation of 9

poliovirus strains, indicating that IPV is able to inhibit community transmission of 10

poliovirus. However, evidence of circulation and geographical spread of wild type virus in 11

Israel was found in environmental samples in 2013 where the vaccination has involved 12

IPV alone since 2006 and coverage is very high. 13

14

In addition to VAPP, the live polio strains in OPV can occasionally revert to a transmissible 15

form termed circulating vaccine derived polio virus (cVDPV) which is essentially the same 16

as the wild type in causing poliomyelitis. This is an obvious threat to the eradication 17

program which the use of IPV does not pose. 18

19

The Global Polio Eradication Initiative (GPEI) of the WHO, in conjunction with its partners, 20

developed the comprehensive Eradication and Endgame Strategic Plan 2013 -2018 with the 21

goal to deliver a polio-free world by 2018 (11). This plan involves poliovirus detection and 22

interruption of spread, immunization strengthening and OPV withdrawal, containment and 23

certification and legacy planning and gives a timetable of events following the identification 24

of the last wild type poliovirus. Three of the key features of the Strategic Plan are the 25

withdrawal of the type 2 OPV strain from trivalent OPV and the introduction of bivalent 26

(types 1 and 3) OPV (bOPV), the introduction of routine use of IPV for managing long-term 27

poliovirus risks including type 2 cVDPV and the cessation of all OPV use four years after 28

the isolation of the last wild type poliovirus. 29

30

Although the last type 2 wild poliovirus was detected in 1999, 90% of outbreaks of cVDPV 31

between 2000 - 2011 were as a result of type 2 poliovirus. There are 250 – 500 VAPP cases 32

per year and 40% of those were due to type 2 Sabin poliovirus. The need to synchronise 33

OPV cessation was identified in 2008 and withdrawal of the use of type 2 OPV and 34


introduction of bOPV began in 2012 (11). In 2012 the WHO Strategic Advisory Group of 1

Experts on Immunization (SAGE) recommended that all countries that use OPV should 2

introduce at least one dose of IPV in their routine immunisation programmes to mitigate 3

risks of withdrawal of OPV 2 (12). One of the prerequisites for OPV 2 cessation is the 4

availability of anaffordable IPV option for all OPV-using countries. This may include full 5

dose sIPV, fractional dose, adjuvanted IPV, and the intradermal use of IPV in addition to 6

intramuscular/subcutaneous administration. 7

8

When poliomyelitis due to wild type polioviruses is eradicated (13), laboratories and 9

manufacturers that use wild type polioviruses will become an important potential source of 10

accidental reintroduction of such viruses into a community. To minimize this risk, WHO 11

has developed a Global Action Plan that requires increased biocontainment for handling 12

live polioviruses (14). This plan specifies three levels of risk management where poliovirus 13

is used, including for IPV production and its control. Primary safeguards which involve the 14

design and use of the production facility are detailed in the WHO Guidelines for the Safe 15

Production and Quality Control of IPV manufactured from wild polioviruses (6). 16

Secondary safeguards relate to the epidemiological circumstances in the country where 17

manufacturing operations are taking place and specify that vaccine coverage should be 18

greater than 90%. Tertiary safeguards relate to public hygiene including sewage treatment 19

that should take place in the communities living around manufacturing facilities. It is 20

recommended that the introduction of these safeguards for wild type poliovirus 21

manufacturing operations begins one year after the detection and identification of the last 22

wild type isolate. 23

24

To mitigate biosafety and biosecurity concerns associated with virulent wild type viruses 25

used in manufacture of IPV, the use of attenuated strains for IPV production has been 26

proposed (15). Production of IPV from live-attenuated Sabin poliovirus seed viruses has 27

been shown to be technically feasible (16, 17, 18, 19, 20), and the first Sabin IPVs have 28

been licensed in Japan in the form of two combination vaccines. New manufacturers are 29

encouraged to explore the production of IPV from live-attenuated Sabin strains and IPV 30

manufacturers that currently use wild type strains are encouraged to evaluate the potential 31

offered by a Sabin-based IPV versus upgrading their production and control facilities to 32

meet the enhanced biosafety requirements (14) if not yet compliant. 33

34


After global certification of polio eradication and vaccination using OPV ceases 1

completely, increased containment levels, in line with recommendations set up by GAP 2

(14), will apply to all laboratories and industrial facilities that work with any live 3

polioviruses, including live-attenuated OPV strains. Until that time, the attenuated Sabin 4

vaccine strains of poliovirus will not require increased laboratory biocontainment levels 5

and they are therefore a more suitable choice for producing IPV in the immediate future 6

(14). However VAPPs and cVDPVs are hazards associated with the use of the Sabin 7

strains and the characteristics of the virus bulks before inactivation must be considered 8

should the production of IPV from the Sabin strains continue after OPV cessation. 9

10

Wild type polioviruses are both transmissible and virulent. They must be grown under high 11

containment when used to produce inactivated poliomyelitis vaccine once the virus is 12

eliminated in nature. The Sabin vaccine strains are attenuated and transmission from 13

recipients is limited so that they are thought to be a safer option for IPV production. 14

However they are unstable on passage in cell culture and the human gut and can revert to 15

give circulating vaccine derived polio viruses (cVDPVs). Animal and molecular tests for 16

attenuation are therefore applied to all batches of oral (Sabin) vaccine produced. However in 17

vitro or animal models (in vivo) of transmissibility are not available. 18

19

The escape of live virus from a production facility is likely to be rare, accidental and local, 20

in contrast to virus exposure in OPV vaccination programmes. Thus the sparse data on 21

transmissibility derived from vaccination may not be applicable to accidental escapes 22

depending, on the epidemiological circumstances and gut and humoral immunity in the 23

exposed population. 24

25

The bulk virus before inactivation will be to some extent transmissible and to some extent of 26

virulent potential. If the escapes are sufficiently rare and the virus sufficiently attenuated and 27

non transmissible, the use of the Sabin strains could ensure safety despite their genetic 28

instability. Nevertheless what this means practically is unknown. 29

30

Given these uncertainties, some assurance of the characteristics of the live virus before 31

inactivation is required if the containment of production is to be relaxed. The following are 32

possible approaches: 33

34


1) Produce and test the bulk virus before inactivation as for OPV. This would ensure 1

that the phenotype was as for OPV. This would not assure safety but might be the 2

best that could be done. It is very demanding of expertise and resource including 3

animal testing and is probably not a practical option. 4

2) Produce the bulk virus as for OPV and apply a limited range of tests, such as 5

MAPREC on every bulk to ensure consistency. 6

3) Produce the bulk virus under conditions suitable for OPV production having 7

established and validated the process by demonstrating that the bulk virus meets the 8

OPV specifications. Do no routine tests on the bulk virus other than to assess bulks 9

from time to time to ensure that the conditions have been kept constant. 10

4) Produce as needed to maximise yield on the assumption that the strains retain 11

sufficient of the OPV properties to make them safer. There is no evidence that this 12

will be the case and this is unacceptable because it will inevitably lead to live virus 13

of higher virulence. 14

15

It is assumed that the transmissibility and genetic evolution of the virus to a more 16

transmissible phenotype are linked to the attenuated phenotype. Given this assumption either 17

option 2 or 3 may be acceptable. It is not clear how much reassurance they will provide and 18

production will still have to be adequately contained. 19

20

In addition to the Sabin strains that are used in the manufacture of OPV, a number of 21

alternative attenuation methods utilising recombinant DNA technology are being 22

investigated (21, 22, 23, 24, 25). Strains derived by such methodology may have properties 23

specifically designed to be suitable for the safe production of vaccine, e.g. unable to 24

replicate in the human gut, and should also be considered as they become available and 25

might require specific characterization. Biocontainment requirements for such strains will 26

need to be determined on a case by case basis. Only virus strains that are approved by the 27

national regulatory authority should be used. 28

29

An overview of the history of virus seeds that are currently used in IPV production is given 30

in Appendix 1. 31

32

The in vivo potency assay in rats has been standardized and shown to have advantages over 33

previously described in vivo tests for IPV (26). The assay in rats is described in detail in 34


this document. The in vivo assay should be used to characterize the vaccine after changes 1

in the manufacturing process that may influence the quality of the vaccine, for stability 2

studies of the vaccine, and to establish consistency of vaccine production. When the in vivo 3

assay is required for regular production batches, it should be performed at the level of the 4

final bulk. The in vivo potency test described in these Recommendations requires the assay 5

of neutralizing antibodies to each of the three poliovirus types. This test requires the use of 6

live poliovirus and, for historical reasons, many laboratories use wild type strains of 7

poliovirus. The attenuated Sabin strains of poliovirus have been shown to be suitable for 8

the assay of neutralizing antibodies in the in vivo test in principle by a collaborative study 9

and should be used (26), but validation of the use of the Sabin strains by each manufacturer 10

should be provided. 11

12

Immunization with OPV will cease at some point in the future, once the disease has been 13

eradicated. After that time, the containment levels for use of the Sabin strains for laboratory 14

work will be reviewed. Laboratories are thus encouraged to investigate the use of 15

alternatives to live viruses for assay of poliovirus neutralizing antibodies in order to comply 16

with future biocontainment requirements. 17

18

The development of transgenic mice that express the human poliovirus receptor (TgPVR 19

mice) (27, 28) has led to the development of an immunization/challenge model (29, 30) that 20

may be useful for assessment of vaccine efficacy for new poliovirus strains. This test is not 21

proposed for lot release. Any work with transgenic mice should comply with WHO 22

guidelines (31). 23

24

The manufacturer of the final lot must be responsible for ensuring conformity with all the 25

recommendations applicable to the final vaccine (Part A, sections A.5−A.11) even where 26

manufacturing involves only the formulation of the final bulk with vaccine obtained in bulk 27

form from another manufacturing establishment and/or filling of final containers. The 28

manufacturer of the final lot must also be responsible for any production and control tests 29

performed by an external contract laboratory, if applicable, with the approval of the NRA. 30

31

Part A. Manufacturing recommendations 32

A.1 Definitions 33


A.1.1 International name and proper name 1

The international name should be poliomyelitis vaccine (inactivated). The proper name 2

should be equivalent to the international name in the language of the country of origin. 3

4

The use of the international name should be limited to vaccines that satisfy the 5

recommendations formulated below. 6

7

A.1.2 Descriptive definition 8

Poliomyelitis vaccine (inactivated) should consist of a sterile aqueous suspension of 9

poliovirus types 1, 2 and 3 grown in cell cultures, concentrated, purified and inactivated. 10

The antigen may be formulated with a suitable adjuvant. The preparation should satisfy all 11

the recommendations formulated below. 12

13

A.1.3 International reference materials 14

An International Standard of IPV for use in in vitro assays to measure the D-Ag content of 15

IPV is available. It is stored frozen in ampoules containing 1ml of trivalent inactivated 16

poliomyelitis vaccine. This material is for use in the calibration of secondary reference 17

preparations of IPV, which are included in each potency test so that potencies in D-Antigen 18

units may be calculated. International standards and reference reagents for the control of in 19

vivo potency assays for IPV are not available. 20

21

An International Standard for anti-poliovirus types 1, 2 and 3 antibodies (human) is 22

available for the standardization of neutralizing antibody tests for poliovirus (32). 23

24

The International Standards listed above are available from the National 25

Institute for Biological Standards and Control, Potters Bar, United 26

Kingdom. 27

28

A.1.4 Terminology 29

The definitions given below apply to the terms as used in these recommendations. They may 30

have different meanings in other contexts. 31

32

Adjuvant: A vaccine adjuvant is a substances or combination of substances that are used in 33

conjunction with a vaccine antigen to enhance (e.g., increase, accelerate, prolong and/or 34


possibly target) the specific immune response to the vaccine antigen and the clinical 1

effectiveness of the vaccine. 2

3

Adventitious agents: Contaminating microorganisms of the cell culture or source materials 4

used in its culture, that may include bacteria, fungi, mycoplasmas, and endogenous and 5

exogenous viruses that have been unintentionally introduced into the manufacturing process. 6

7

Cell-culture infective dose 50% (CCID50): The quantity of a virus suspension that will infect 8

50% of cell cultures. 9

10

Cell bank: A cell bank is a collection of appropriate containers whose contents are of 11

uniform composition stored under defined conditions. Each container represents an aliquot 12

of a single pool of cells. 13

The individual containers (e.g., ampoules, vials) should be 14

representative of the pool of cells from which they are taken and 15

should be frozen on the same day by following the same procedure 16

and by using the same equipment and reagents 17

18

Cell seed: A quantity of well-characterized cells derived from a single tissue or cell of 19

human or animal origin and stored frozen in liquid nitrogen in aliquots of uniform 20

composition, one or more of which may be used for the production of a master cell bank. 21

22

D-antigen: The term refers to the antigen found in sucrose gradient fraction that contains 23

native virus particles, which are the target of neutralising antibodies (33). D-antigen units 24

were originally defined based on an agar precipitin test performed with D-antigen specific 25

polyclonal sera. A vaccine preparation that produced precipitin line at the distance of 25 26

millimetres from the centre was arbitrarily assigned a value of 600 D-antigen units using a 27

particular antibody at a particular concentration. This test was used in the initial calibration 28

of reference materials. The D-antigen content of IPV is currently determined by an ELISA 29

test. 30

31

Final bulk: The finished vaccine present in the container from which the final containers are 32

filled. The final bulk may be prepared from one or more trivalent bulks. 33

34


Final lot: A collection of sealed final containers of finished vaccine that is homogeneous 1

with respect to the risk of contamination during the filling process. All of the final 2

containers must therefore have been filled from a single vessel of final bulk in one working 3

session. 4

5

Master cell bank (MCB): A quantity of well characterized cells of human or animal origin 6

derived from a cell seed at a specific population doubling level (PDL) or passage level, 7

dispensed into 8

multiple containers, cryopreserved, and stored frozen under defined conditions, such as the 9

vapour or liquid phase of liquid nitrogen in aliquots of uniform composition. The master cell 10

bank is prepared from a single homogeneously mixed pool of cells and is used to derive all 11

working cell banks (WCB). The testing performed on a replacement master cell bank 12

(derived from the same clone or from an existing master or working cell bank) is the same as 13

for the initial master cell bank, unless a justified exception is made. 14

15

Monovalent pool: A pool of a number of single harvests of the same virus type processed at 16

the same time. 17

18

Production cell culture: A collection of cell cultures derived from one or more ampoules of 19

the WCB used for the production of IPV. 20

21

Purified monovalent pool: A concentrated and purified pool of a number of single harvests 22

of the same virus type processed at the same time. 23

24

Single harvest: A quantity of virus suspension of one virus type harvested from cell cultures 25

derived from the same WCB and prepared from a single production run. 26

27

Trivalent bulk: A pool of a number of inactivated purified monovalent pools processed at the 28

same time and containing all three virus types, blended to achieve a defined D- antigen 29

content for each type. 30

31

Virus master seed lot: A quantity of virus suspension that has been processed at the same 32

time to assure a uniform composition and passaged for a specific number of times that does 33


not exceed the maximum approved by the NRA. It has been characterized to the extent 1

necessary to support development of the virus working seed lot. 2

3

Virus working seed lot: A quantity of virus of uniform composition derived from the virus 4

master seed lot made at the multiplicity of infection, ensuring that cytopathic effect 5

develops within an appropriate timeframe and at a passage level approved by the NRA. 6

7

Working cell bank (WCB): A quantity of cells of uniform composition derived from one or 8

more ampoules of the MCB at a finite passage level, stored frozen at –70°C or below in 9

aliquots, one or more of which would be used for vaccine production. All containers are 10

treated identically and once removed from storage are not returned to the stock. 11

12

A.2 General manufacturing recommendations 13

The general manufacturing requirements contained in Good manufacturing practices for 14

pharmaceutical products: main principles (34) and Good Manufacturing Practices for 15

Biological Products (35) should apply to establishments manufacturing IPV. In addition, 16

establishments that manufacture IPV should comply with the current global 17

recommendations for poliovirus containment appropriate to the particular poliovirus strains 18

used for production in both the production and quality control departments (14). 19

20

For vaccines prepared using wild type poliovirus, the current version of WHO Guidelines for 21

the Safe Production and Quality Control of IPV manufactured from wild polioviruses should 22

also be implemented (6). 23

24

The guidelines state that one year after the last wild type poliovirus is detected increased 25

biocontainment requirements will be introduced for the use of wild type polioviruses in the 26

laboratory, both at the level of production of vaccines using wild type strains and for the 27

control of such vaccines. The guidelines also mention that before the certification of the 28

eradication of wild type poliovirus and the cessation of usage of OPV, attenuated 29

poliovirus strains (such as Sabin strains) that have been approved by the national 30

regulatory authority in the country of manufacture for manufacturing an OPV, when used 31

for manufacturing IPV, do not require containment in BSL-3/IPV facilities provided they 32

are produced under conditions that would make them suitable for oral vaccine use (6). 33


After global certification of eradication and cessation of usage of OPV, these containment 1

conditions will need to be revised (14). 2

3

When attenuated strains derived by recombinant DNA technology are used in IPV 4

production, a number of issues should be considered in order to determine the stringency of 5

biocontainment requirements for virus growth and virus manipulation in the laboratory. 6

Different strains might require different assessments but they should include production 7

conditions that ensure an acceptable level of phenotypic stability and a phenotype that 8

justifies the containment proposed. The strain should not be readily transmissible from 9

person to person. 10

11

In any case, any biocontainment arrangement should comply with GAP requirements at the 12

time of production (14). 13

14

The staff involved in the production and quality control of IPV should be shown to have 15

immunity to all three types of polioviruses as assessed by neutralization assay. 16

17

A.3 Control of source materials 18

A.3.1 Virus strains and seed lot system 19

A.3.1.1 Virus strains 20

Strains of poliovirus used in the production of IPV shall be identified by historical records, 21

which should include information on their origin and subsequent manipulation. The strain 22

identity should be determined by infectivity tests and immunological methods. In addition, 23

Sabin strains and strains derived by recombinant DNA technology should be identified by 24

nucleotide sequence analysis. Only virus strains that are approved by the NRA and that yield 25

a vaccine meeting the Recommendations set out in the present document should be used. 26

27

A.3.1.2 Virus seed lot system 28

Vaccine production should be based on the virus seed lot system. Unless otherwise 29

justified and authorised, the virus in the final vaccine shall not have undergone more 30

passages from the virus master seed lot than were used to prepare the vaccine shown to be 31

satisfactory with respect to safety and efficacy and biocontainment requirements. 32

33


If Sabin virus master seeds are supplied by WHO, a virus sub-master 1

seed lot should be prepared by a single passage from the WHO 2

master seed at a multiplicity of infection that ensures the 3

development of cytopathic effect within an appropriate timeframe, 4

and that has been processed at the same time to assure a uniform 5

composition. The virus sub-master seed lot should be characterized 6

to the extent necessary to support the development of the virus 7

working seed lot. The characterized virus sub-master seed lot is used 8

for the preparation of virus working seed lots, (see section A.3.2.2 9

and Part B of the Recommendations to Assure the Quality, Safety 10

and Efficacy of Live Attenuated Poliomyelitis Vaccine (oral) Revised 11

2012 (36)). The virus sub-master seed lot should be subjected to the 12

same tests as a virus master seed lot. 13

14

Virus master and working seed lots should be stored in dedicated temperature-monitored 15

freezers at a temperature that ensures stability on storage e.g. ≤ -60º. 16

17

A.3.1.3 Tests on virus master and working seed lots 18

Each virus master and working seed lot used for the production of vaccine batches should be 19

subjected to the tests listed in this section and certain tests applicable to single harvests listed 20

in sections A.4.3 (A.4.3.1 Sterility test for bacteria fungi and mycoplasma, A.4.3.2 Virus 21

titration and A.4.3.3 Identity test.) 22

23

Each virus master working seed lot should have been derived from materials that comply 24

with the Recommendations made in sections A.3.2 and A.3.3 and should be approved by the 25

NRA. 26

27

A.3.1.3.1 Tests in rabbit kidney cell cultures (only for virus master seeds derived from 28

strains which have previously been passaged on primary monkey kidney cells) 29

Virus master seeds that have previously been passaged on primary monkey kidney cells 30

should be tested for the presence of herpes B virus and other viruses in rabbit kidney cell 31

cultures. A sample of at least 10 ml of virus seeds should be tested. Serum used in the 32

nutrient medium of the cultures should have been shown to be free from B virus inhibitors 33

using herpes simplex virus as an indicator virus. The pooled fluid should be inoculated into 34


bottles of these cell cultures in such a way that the dilution of the pooled fluid in the 1

nutrient medium does not exceed 1 in 4. The area of the cell sheet should be at least 3 cm2 2

per ml of pooled fluid. At least one bottle of each kind of cell culture should remain 3

uninoculated and should serve as a control. 4

5

The inoculated and control cultures should be incubated at a temperature of 37 °C and 6

should be observed for a period of at least 2 weeks. 7

8

For the test to be valid, not more than 20% of the culture vessels should have been 9

discarded for nonspecific, accidental reasons by the end of the test period. The sensitivity 10

of each batch of rabbit kidney cells should be demonstrated by challenge with a validated 11

amount of herpes simplex virus. The challenge test should be approved by the NRA. 12

13

A.3.1.3.2 Tests for extraneous viruses and freedom from detectable SV40 sequences 14

The virus master and working seed lot used for the production of vaccine batches should be 15

free from extraneous viruses in cell culture assays or using molecular techniques (e.g. 16

nucleic acid amplification (NAT)) (37). 17

18

A sample of at least 40 ml of each virus master and working seed lot should be tested for the 19

presence adventitious agents. The sample should be neutralized by a high-titred antiserum 20

against the specific type of poliovirus. 21

22

The Sabin strains may be used as immunizing antigen. The 23

immunizing antigen used for the preparation of the antiserum should 24

not be the same as the production seed. 25

26

The immunizing antigen should be shown to be free from extraneous 27

agents and grown in cell cultures free from extraneous microbial 28

agents that might elicit antibodies that could inhibit the growth of any 29

adventitious agents present in the single harvest. 30

31

The sample should be tested in primary Cercopithecus kidney cell cultures and in human 32

diploid cells. The tissue cultures should be incubated at 37 °C and observed for 2 weeks. At 33

the end of this observation period, at least one subculture of supernatant fluid should be 34


made in the same tissue culture system. The sample should be inoculated in such a way that 1

the dilution of the supernatant fluid in the nutrient medium does not exceed 1 in 4. The area 2

of the cell sheet should be at least 3 cm2 per ml of supernatant fluid. At least one bottle of 3

the cell cultures should remain uninoculated and should serve as a control. 4

5

The inoculated and control cultures should be incubated at 37 °C and observed for an 6

additional 2 weeks. 7

8

If necessary, serum may be added to the primary cultures at this 9

stage, provided that the serum does not contain SV40 antibody or 10

other inhibitors. 11

12

The virus master and working seed virus passes the test if there is no evidence of the 13

presence adventitious agents. For the test to be valid, not more than 20% of the culture 14

vessels should have been discarded for nonspecific, accidental reasons by the end of the 15

observation period. 16

17

New molecular methods with broad detection capabilities are being 18

developed for detection of adventitious agents. These methods include 19

degenerate NAT for whole virus families with analysis of the 20

amplicons by hybridization, sequencing or mass spectrometry; NAT 21

with random primers followed by analysis of the amplicons on large 22

oligonucleotide micro-arrays of conserved viral sequencing or digital 23

subtraction of expressed sequences; and high throughput sequencing. 24

These methods might be used in the future to supplement existing 25

methods or as alternative methods to both in vivo and in vitro tests 26

after appropriate validation and approval of the NRA (37). 27

28

The theoretical risk of the presence of potential human, simian, 29

bovine or porcine extraneous agents in the seed lots which may be 30

derived from the use of bovine serum or porcine trypsin should be 31

assessed. If necessary, viruses such as bovine polyoma virus, porcine 32

parvovirus or porcine circovirus (PCV) may be screened by using 33


specific assays, such as molecular techniques e.g., nucleic acid 1

amplification (NAT) (37). 2

3

The virus master seed lot should also be free from detectable SV40 sequences by using 4

specific validated assays which are approved by the NRA, such as molecular techniques 5

(e.g., nucleic acid amplification (NAT)) (37). 6

7

DNA of SV40 is widely used as molecular biological reagent, and 8

contamination of polymerase chain reaction (PCR) assays is 9

potentially a major problem. One approach is to identify separate 10

genomic regions of SV40 for amplification, and to use one region for 11

screening purposes and the other for the confirmation of repeatedly 12

positive samples. It is useful if the second genomic region used for 13

confirmation varies between isolates from different sources, as it is 14

then possible to show that it has a unique sequence and that positive 15

results are not due to contamination with laboratory strains of SV40. 16

The sensitivity of the PCR assays for the genomic regions used 17

should be established. 18

19

A.3.1.3.3 Additional tests on seeds from Sabin strains and other attenuated strains derived 20

by recombinant DNA technology 21

If live-attenuated Sabin strains are used for vaccine production, established master seeds 22

should be selected and additional tests should be performed. The virus master seed lots used 23

for the production of vaccine batches should be tested to monitor virus molecular 24

characteristics e.g.by MAPREC and meet the specifications established in agreement with 25

the NRA. Specifications for OPV based on Sabin strains are described in the 26

Recommendations to Assure the Quality, Safety and Efficacy of Live Attenuated 27

Poliomyelitis Vaccine (oral) Revised 2012 (36) section A.3.2.4 (Tests to monitor virus 28

molecular characteristics). These may include in vitro tests (A.3.2.4.1) and in vivo 29

neurovirulence tests (A.3.2.4.2). (See also Section A.4.4.2.7 of this document). 30

31

Suitable in vitro tests should be performed on the master seed from attenuated strains 32

derived by recombinant DNA technology. The tests may include full genome 33

characterization by nucleotide sequencing or deep sequencing techniques and 34


demonstration of genetic and phenotypic stability on passage under production conditions. 1

Such tests should be scientifically validated and approved by the national regulatory 2

authority. 3

4

The need for testing virus master seed lots of attenuated strains derived by recombinant 5

DNA technology in in vivo neurovirulence tests should be considered and scientifically 6

justified. 7

8

Any new virus working seed derived from an established master seed, including Sabin 9

strains and other attenuated strains derived by recombinant DNA technology, and at least 10

three consecutive monovalent pools should be analyzed in tests to monitor virus molecular 11

characteristics such as MAPREC, as relevant, see tests in Section A.4.4.2.7.1. 12

13

A.3.2 Cell lines 14

The general production precautions as formulated in Good Manufacturing Practices for 15

Biological Products (35) should apply to the manufacture of poliomyelitis vaccine 16

(inactivated), with the addition that, during production, only one type of cell should be 17

introduced or handled in the production area at any one time. Vaccines may be produced in 18

a human diploid cell line or in a continuous cell line. 19

20

A.3.2.1 Master cell bank (MCB) and working cell bank (WCB) 21

The use of a cell line for the manufacture of IPV should be based on the cell bank system. 22

The cell seed and cell banks should conform to the Recommendations for the evaluation of 23

animal cell cultures as substrates for the manufacture of biological medicinal products and 24

for the characterization of cell banks (37). The MCB should be approved by the NRA. The 25

maximum number of passages (or population doublings) by which the WCB is derived from 26

the MCB and the maximum number of passages of the production cultures should be 27

established by the manufacturer and approved by the NRA. Additional tests may include, 28

but are not limited to: examination for the presence of retrovirus and tumorigenicity in an 29

animal test system (37) and propagation of the MCB or WCB cells to or beyond the 30

maximum in vitro age for production. 31

32

The WHO Vero reference cell bank 10-87 is considered suitable for 33

use as a cell seed for generating an MCB (38) and is available to 34


manufacturers on application to the Coordinator, Technologies 1

Standards and Norms Team, Essential Medicines and Health 2

Products (EMP) Department, Health Systems and Innovation (HIS) 3

Cluster World Health Organization, Geneva, Switzerland. 4

5

A.3.2.2 Identity test 6

Identity tests on the master (MCB) and working cell banks (WCB) are performed in 7

accordance with WHO’s Recommendations for the evaluation of animal cell cultures as 8

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

of cell banks (37) and should be approved by the NRA. 10

11

The WCB should be identified by means of tests such as biochemical tests (e.g. isoenzyme 12

analysis), immunological tests, cytogenetic marker tests and DNA fingerprinting or 13

sequencing. The tests should be approved by the NRA. 14

15

A.3.3 Cell culture medium 16

Serum used for the propagation of cells should be tested to demonstrate freedom from 17

bacterial, fungal and mycoplasma contamination by appropriate tests as specified in Part A, 18

sections 5.2 (39) and 5.3 (40) of the General requirements for the sterility of biological 19

substances, and freedom from infectious viruses. Suitable tests for detecting viruses in 20

bovine serum are given in Appendix 1 of the WHO Recommendations for the evaluation of 21

animal cell cultures as substrates for the manufacture of biological medicinal products and 22

for the characterization of cell banks (37). 23

24

Validated molecular tests for bovine viruses may replace the cell culture tests of bovine sera 25

if approved by the NRA. As an additional monitor of quality, sera may be examined for 26

freedom from bacteriophage and endotoxin. Gamma-irradiation may be used to inactivate 27

potential contaminant viruses, recognizing that some viruses are relatively resistant to 28

gamma-irradiation. 29

30

The source(s) of animal components used in the culture medium should be approved by the 31

NRA. The components should comply with the current WHO guidelines on transmissible 32

spongiform encephalopathies in relation to biological and pharmaceutical products (41). 33


The serum protein concentration should be reduced by rinsing the cell cultures with serum-1

free medium and/or purification of the virus harvests. 2

3

In some countries, control tests are carried out to detect the residual 4

animal serum content in the final vaccine (see section A.6.6). 5

6

Human serum should not be used. If human serum albumin is used at any stage of product 7

manufacture, the NRA should be consulted regarding the requirements, as these may differ 8

from country to country. As a minimum, it should meet the Requirements for the Collection, 9

Processing and Quality Control of Blood, Blood Components and Plasma Derivatives (42). 10

In addition, human albumin and materials of animal origin should comply with current 11

WHO guidelines on transmissible spongiform encephalopathies in relation to biological and 12

pharmaceutical products (41). 13

14

Manufacturers are encouraged to explore the possibilities of using 15

serum-free media for production of IPV. 16

17

Penicillin and other beta-lactams should not be used at any stage of manufacture because of 18

their nature as highly sensitizing substances in humans. 19

20

Other antibiotics may be used at any stage of manufacture, provided 21

that the quantity present in the final product is acceptable to the NRA. 22

23

Nontoxic pH indicators may be added, e.g. phenol red at a 24

concentration of 0.002%. 25

26

Only substances that have been approved by the NRA may be added. 27

28

Bovine or porcine trypsin used for preparing cell cultures should be tested and found free of 29

cultivable bacteria, fungi, mycoplasmas and infectious viruses, as appropriate (37). The 30

methods used to ensure this should be approved by the NRA. 31

32

In some countries, irradiation is used to inactivate potential 33

contaminant viruses. If irradiation is used, it is important to ensure 34


that a reproducible dose is delivered to all batches and to the 1

component units of each batch. The irradiation dose must be low 2

enough for the biological properties of the reagents to be retained 3

but also high enough to reduce virological risk. Therefore, 4

irradiation cannot be considered a sterilizing process (37). 5

6

Recombinant trypsin is available and should be considered; however 7

it should not be assumed to be free from risk of contamination and 8

should be subject to the usual considerations for any reagent of 9

biological origin (37). 10

11

The source(s) of trypsin of bovine origin, if used, should be approved by the NRA and 12

should comply with current WHO guidelines on transmissible spongiform encephalopathies 13

in relation to biological and pharmaceutical products (41). 14

15

A.4 Control of vaccine production 16

A.4.1 Control cell cultures 17

When human diploid or continuous cell lines are used to prepare cultures for the production 18

of vaccine, a fraction equivalent to at least 5 % of the total or 500 ml of cell suspension, or 19

100 million cells, at the concentration and cell passage level employed for seeding vaccine 20

production cultures, should be used to prepare control cultures. 21

22

If bioreactor technology is used, the NRA should determine the size and treatment of the cell 23

sample to be examined. 24

25

A.4.1.1 Tests of control cell cultures 26

The treatment of the cells set aside as control material should be similar to that of the 27

production cell cultures, but they should remain uninoculated for use as control cultures for 28

the detection of any adventitious agents. 29

30

These control cell cultures should be incubated under conditions as similar as possible to the 31

inoculated cultures for at least two weeks, and should be tested for the presence of 32

adventitious agents as described below. For the test to be valid, not more than 20% of the 33

control cell cultures should have been discarded for nonspecific, accidental reasons. 34


1

At the end of the observation period, the control cell cultures should be examined for 2

evidence of degeneration caused by an extraneous agent. If this examination, or any of the 3

tests specified in this section, shows evidence of the presence in the control culture of any 4

adventitious agent, the poliovirus grown in the corresponding inoculated cultures should not 5

be used for vaccine production. 6

7

Samples if not tested immediately should be stored at -60°C or below. 8

9

A.4.1.2 Tests for haemadsorbing viruses 10

At the end of the observation period, at least 25% of the control cells should be tested for the 11

presence of haemadsorbing viruses using guinea-pig red blood cells. If the latter cells have 12

been stored, the duration of storage should not have exceeded seven days and the storage 13

temperature should have been in the range of 2–8 °C. In tests for haemadsorbing viruses, 14

calcium and magnesium ions should be absent from the medium. 15

16

Some NRAs require, as an additional test for haemadsorbing viruses, 17

that other types of red cells, including cells from humans (blood 18

group IV O), monkeys and chickens (or other avian species), should 19

be used in addition to guinea-pig cells. 20

21

A reading should be taken after incubation at 2-8 °C for 30 minutes and again after a further 22

incubation for 30 minutes at 20–25 °C. 23

24

If a test with monkey red cells is performed, readings should also be 25

taken after a final incubation for 30 minutes at 34–37 °C. 26

27

In some countries the sensitivity of each new batch of red blood 28

cells is demonstrated by titration against a haemagglutin antigen 29

before use in the test for haemadsorbing viruses. 30

31

A.4.1.3 Tests for other adventitious agents in cell supernatant fluid 32

At the end of the observation period a sample of the pooled supernatant fluid from each 33

group of control cultures should be tested for other adventitious agents. For this purpose, 10 34


ml of each pool should be tested in the same cells, but not the same batch of cells, as those 1

used for the production of vaccine. 2

3

A second indicator cell line should be used to test an additional 10 ml sample of each pool. 4

When a human diploid cell line is used for production, a simian kidney cell line should be 5

used as the second indicator cell line. When a simian kidney cell line is used for production, 6

a human diploid cell line should be used as the second indicator cell line (37). 7

8

The pooled fluid should be inoculated into bottles of these cell cultures in such a way that 9

the dilution of the pooled fluid in the nutrient medium does not exceed 1 part in 4. The area 10

of the cell sheet should be at least 3 cm2 per ml of pooled fluid. At least one bottle of each 11

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

13

The inoculated cultures should be incubated at a temperature of 35–37 °C and should be 14

observed for a period of at least 14 days. 15

16

Some NRAs require that, at the end of this observation period, a 17

subculture is made in the same culture system and observed for at 18

least an additional 14 days. Furthermore, some NRAs require that 19

these cells should be tested for the presence of haemadsorbing 20

viruses. 21

22

For the tests to be valid, not more than 20% of the culture vessels should have been 23

discarded for nonspecific, accidental reasons by the end of the test period. 24

25

If any cytopathic changes due to adventitious agents occur in any of the cultures, the virus 26

harvests produced from the batch of cells from which the control cells were taken should be 27

discarded. 28

29

Some selected viruses may be screened by using specific validated assays which are 30

approved by the NRA, such as molecular techniques (e.g. nucleic acid amplification) (37). 31

32

If these tests are not performed immediately, the samples should be kept at a temperature of 33

−60 °C or below. 34


1

A.4.1.4 Identity tests 2

At the production level, the control cells should be identified by means of tests approved by 3

the NRA. 4

5

Suitable methods are, but are not limited to, biochemical tests (e.g. isoenzyme analyses), 6

immunological tests, cytogenetic tests (e.g. for chromosomal markers) and tests for genetic 7

markers (e.g. DNA fingerprinting or sequencing). 8

9

A.4.2 Control of vaccine production 10

A.4.2.1 Cell cultures for vaccine production 11

A.4.2.1.1 Observation of cultures for adventitious agents 12

On the day of inoculation with the virus working seed lot, each cell culture or a sample from 13

each culture vessel should be examined visually for degeneration caused by infective agents. 14

If such examination shows evidence of the presence in a cell culture of any adventitious 15

agent, the culture should not be used for vaccine production. 16

17

If animal serum is used for cell cultures before the inoculation of virus, the medium should 18

be removed and replaced with serum-free maintenance medium after the cells have been 19

washed with serum-free medium, if appropriate. 20

21

A.4.3 Control of single harvests 22

After inoculation of the production cells with virus, the culture conditions of inoculated and 23

control cell cultures culture conditions should be standardised and kept within limits agreed 24

with the NRA. 25

26

Samples required for the testing of single harvests should be taken immediately on 27

harvesting. 28

29

In some countries, samples are taken after storage and filtration after 30

agreement of the NRA 31

32

A.4.3.1 Sterility test for bacteria, fungi and mycoplasma 33


A volume of at least 10 ml of each virus master and working seed lot (see A.3.1.3) and 1

single harvest should be tested for bacterial, fungal, and mycoplasmal contamination by 2

appropriate tests, as specified in Part A, sections 5.2 (39) and 5.3 (40) of the General 3

requirements for the sterility of biological substances, or by a method approved by the 4

NRA. If this test is done outside the production facilities, adequate containment procedures 5

(14) should be used according to the virus strain used for production and the GAP 6

recommendations applicable at the time (14). 7

8

Nucleic Acid Amplification Techniques (NAT) alone or in 9

combination with cell culture, with an appropriate detection 10

method, might be used as an alternative to one or both of the 11

compendial mycoplasma detection methods after suitable 12

validation and agreement from NRA (37). 13

14

In some countries this test is performed on the purified monovalent 15

harvest instead of the single harvest. 16

17

A.4.3.2 Virus titration 18

The virus concentration of each virus master and working seed lot (see A.3.1.3.) and single 19

harvest should be determined by titration of infectious virus using tissue culture methods. 20

This titration should be carried out in not more than 10-fold dilution steps and using 10 21

cultures per dilution, or any other arrangement yielding equal precision. 22

23

The use of Hep-2C or Vero cells in microtitre plates is suitable for 24

this purpose (36). The same cells should be used for virus titrations 25

before and after the inactivation process. 26

27

Information on virus titre will help selecting samples that can be 28

expected to meet potency requirements after inactivation. 29

30

In some countries the test for virus concentration may be carried 31

out on the purified, pooled monovalent harvest after demonstration 32

of consistency of production at the stage of the single harvest. 33

34


A.4.3.3 Identity test 1

The poliovirus in each virus master and working seed lot (see A.3.1.3) and single harvest 2

should be tested for serotype and strain identity by neutralization with specific antiserum or 3

molecular methods approved by the NRA. 4

5

Care should be taken to ensure that the sera used are monospecific by 6

titrating them against homotypic and heterotypic viruses of known 7

virus titre. Monoclonal antibodies may be useful in this test. 8

9

The strain identity of each of the three serotypes may be determined 10

by standard or deep nucleotide sequence analysis or a suitable 11

molecular technique. 12

13

In some countries this test is performed on the purified monovalent 14

harvest instead of the single harvest. 15

16

A.4.4 Control of purified monovalent pools 17

A.4.4.1 Purification of monovalent pools 18

Each monovalent pool of virus, consisting of several single harvests of the same serotype, 19

should be purified before inactivation. 20

21

An acceptable method is to clarify the virus suspension by filtration, 22

to concentrate the virus by ultrafiltration and, thereafter, collect the 23

virus peak after passing it through a gel-filtration column. Further 24

purification is achieved by passing the virus through an ion-exchange 25

column. Other purification procedures, such as passing the 26

preparation through an immobilized DNase column, may be used. 27

28

A.4.4.2 Tests on purified monovalent pools 29

A.4.4.2.1 Residual cellular DNA 30

For viruses grown in continuous cells the purified monovalent pools should be tested for 31

residual cellular DNA. By calculation the purification process should be shown to reduce 32

consistently the level of cellular DNA to less than 10ng per human dose. This test may be 33


omitted from routine testing, with the agreement of the NRA, if the manufacturing process is 1

validated to achieve this specification (37). 2

3

If assessed, the size distribution of the DNA may be considered as a 4

characterization test, taking into account the amount of DNA 5

detectable using state-of-the-art methods, as approved by the NRA. 6

7

In some countries this test is performed on the trivalent bulk 8

following validation and agreement of the NRA 9

10

A.4.4.2.2 Virus titration 11

The virus concentration of each purified monovalent pool should be determined by titration 12

of infectious virus using tissue culture methods. This titration should be carried out in not 13

more than 10-fold dilution steps and using 10 cultures per dilution, or any other arrangement 14

yielding equal precision. 15

16

The use of Hep-2C or Vero cells in microtitre plates is suitable for this 17

purpose (37). The same cells should be used for virus titrations before 18

and after the inactivation process. 19

20

Information on virus titre will help selecting purified monovalent 21

pools that can be expected to meet potency requirements after 22

inactivation. 23

24

A.4.4.2.3 Identity test 25

The poliovirus in each purified monovalent pool should be tested for serotype and strain 26

identity by neutralization with specific antiserum or molecular methods approved by the 27

NRA. 28

29

Care should be taken to ensure that the sera used are monospecific by titrating them against 30

homotypic and heterotypic viruses of known virus titre. Monoclonal antibodies may be 31

useful in this test. 32

33


The strain identity of each of the three serotypes may be determined by nucleotide sequence 1

analysis or a suitable molecular technique. 2

3

A.4.4.2.4 D-antigen content 4

The D-antigen content of each purified monovalent pool should be determined using a 5

validated immunochemical method and calculated using a reference vaccine calibrated 6

against the WHO International Standard (see section A.1.3). 7

8

A.4.4.2.5 Protein content 9

The purified monovalent pool should be shown to contain no more than 0.1µg of protein per 10

D-antigen unit of poliovirus or within the limits approved for that particular product by the 11

NRA. 12

13

A.4.4.2.6 Filtration before inactivation 14

Each purified monovalent pool should be filtered before inactivation. 15

16

Satisfactory results have been reported with several filter types but a 17

final filtration using a 0.22-µm filter should be used. 18

19

Filters containing asbestos should not be used. 20

21

Inactivation should be initiated as soon as possible and not later than 72 h after filtration. 22

23

It is preferable to start inactivation within 24 h of filtration. Since the 24

purpose of the filtration step is to remove particulate matter and 25

other interfering substances that may diminish the effectiveness of 26

the inactivation process, and since aggregates tend to increase on 27

standing after filtration, efforts should be made to keep within this 28

time limit. 29

30

A sample of the filtered purified monovalent pool should be retained and its virus titre 31

determined as described in A.4.4.2.2. 32

33


The main purpose of determining the titre of filtered virus pools 1

destined for inactivation is to provide the starting titre to monitor the 2

kinetics of inactivation. 3

4

A.4.4.2.7 Additional tests for purified monovalent pools produced from Sabin vaccine seeds 5

or from other attenuated seeds derived by recombinant DNA technology 6

The quality of the virus in the purified monovalent pools before inactivation must be 7

suitable. Two acceptable strategies may be followed with the approval of the NRA. Firstly, 8

purified monovalent pools may be produced by a validated process shown to give rise to 9

viruses suitable for use as an OPV and a limited range of tests, such as MAPREC applied 10

to every purified monovalent pool to ensure consistency. 11

12

Alternatively the bulk may be produced by a validated process shown to give rise to a bulk 13

suitable for use as an OPV without further routine tests, but assessing batches from time to 14

time by in vitro and in vivo tests to ensure that the conditions have been kept constant. In 15

vitro tests to monitor virus molecular characteristics (consistency) and in vivo 16

neurovirulence tests which could be used for this purpose are described in A.4.4.2.7.1 and 17

A.4.4.2.7.2 respectively. 18

19

Suitable in vitro tests should be performed on other attenuated strains derived by 20

recombinant DNA technology. Tests may include full genome characterization by 21

nucleotide sequencing or deep sequencing techniques and demonstration of genetic and 22

phenotypic stability on passage under production conditions. Such tests should be 23

scientifically validated and approved by the national regulatory authority. 24

25

An in vitro test (described above) for the molecular consistency of 26

production may be performed on single harvests before preparing the 27

monovalent pool. If performed, the acceptance/rejection criteria 28

should be updated periodically and approved by the national 29

regulatory authority. 30

31

32

A.4.4.2.7.1 Tests to monitor virus molecular characteristics (consistency) 33


The poliovirus in the purified monovalent pool (before inactivation), prepared as described 1

in section A.4.4.1 (Purification of monovalent pools), should be tested to ensure that the 2

vaccine virus has not undergone changes during its multiplication in the production cell 3

culture. Where production is based on the Sabin strains, the virus bulk should be tested for 4

a marker of neurovirulence by at least one in vitro test e.g. MAPREC, and should meet the 5

specifications for the test used (36). 6

7

Results from MAPREC tests should be expressed as ratios relative to the relevant type-8

specific International Standard for MAPREC analysis of poliovirus (Sabin). The acceptable 9

variation of mutant content from batch to batch should be agreed with the NRA in the light 10

of production and testing experience. 11

12

For type 3 (472-C), a batch should be rejected if the level of mutations is above 1.0% when 13

normalized against the International Standard. The limits for types 1 and 2 should be 14

approved by the NRA. 15

16

Levels of mutations obtained by manufacturers who have 17

implemented the test for types 1 and 2 virus have been less than 18

2.0% for type 1 Sabin (for the sum of both mutations 480-A, 525-C) 19

and 1.5% for type 2 Sabin (481-G) (36, 43). 20

21

The test(s) used should be approved by the national regulatory authority. The MAPREC 22

assay provides a sensitive and quantitative measure for consistency purposes for OPV seeds. 23

24

A.4.4.2.7.2 Neurovirulence tests 25

An appropriate in vivo test may be used to evaluate the phenotype of virus monovalent 26

pools produced from the Sabin vaccine strains as described in section A.4.4.7.2 of the 27

Recommendations to Assure the Quality, Safety and Efficacy of Live Attenuated 28

Poliomyelitis Vaccine (oral), Revised 2012 (36). 29

30

For other attenuated strains derived by recombinant DNA technology, the need for testing 31

virus purified monovalent pools in in vivo neurovirulence tests should be considered and 32

scientifically justified. 33

34


A.4.5 Control of inactivated purified monovalent pools 1

A.4.5.1 Inactivation procedure 2

The virus in the filtered purified monovalent pools should be inactivated through the use of a 3

method approved by the NRA. 4

5

Most manufacturers during the last 40-50 years have used 6

formaldehyde as the method for inactivation 7

8

The method of inactivation should be shown to give consistent inactivation for the 9

production of acceptable vaccine. A record of consistency (effective inactivation and kinetic 10

of inactivation) should be established by the production of at least five consecutive lots and 11

if broken, a root cause analysis should be performed and a further five consecutive filtered 12

purified monovalent pools should be prepared and shown to be satisfactory for re-13

establishing this record. 14

15

The progress of inactivation should be followed by suitably spaced determinations of virus 16

titres. The inactivation period should exceed the time taken to reduce the titre of live virus to 17

undetectable amounts by a factor of at least 2. 18

19

A second filtration during the process of inactivation should be made. 20

21

This step is made after the virus titre has fallen below detectable 22

levels but before the first sample for the safety test is taken. 23

24

A.4.5.2 Test for effective inactivation 25

Two samples of a volume equivalent to at least 1500 human doses of each inactivated 26

purified monovalent pool should be taken, one at the end of the inactivation period and the 27

other not later than three-quarters of the way through this period. After removal or 28

neutralization of the inactivating agent, the samples should be tested by inoculation into 29

tissue cultures for the absence of infective poliovirus. Kidney cells from some monkey 30

species, for instance those of the genera Macaca, Cercopithecus and Papio, appear to be 31

more sensitive than others. If other tissue culture systems, including continuous cell lines 32

(e.g. L20B), are used, they should have been shown to possess at least the same sensitivity 33

to poliovirus as those specified above. When primary monkey kidney cells are used for this 34


test, the two samples should be inoculated into bottles of tissue cultures derived from 1

different batches of cells. 2

3

The dilution of the sample in the nutrient fluid should not exceed 1 in 4 and the area of the 4

cell sheet should be at least 3 cm2

per ml of sample. One or more bottles of each batch of 5

cultures should be set aside to serve as uninoculated control bottles with the same medium. 6

7

The formaldehyde in samples of vaccine for tissue culture tests is 8

generally neutralized at the time of sampling by the addition of 9

bisulfite. Usually, the samples are subsequently dialysed. 10

11

It is possible to conduct tissue culture tests on nondialysed material; 12

however, this is often found to be toxic to cells, even with a dilution 13

of 1 in 4. If in such tests nonspecific degeneration of cells occurs, or 14

if the sensitivity of the tissue culture system is reduced, the test 15

should be repeated on dialysed material. The virus D-antigen content 16

after dialysis should be determined to discard any virus loss during 17

the dialysis process. 18

19

The tissue culture bottles should be observed for at least three weeks. Not less than two 20

subcultures should be made from each original bottle, one at the end of the observation 21

period and the other one week earlier. The subcultures should be observed for at least two 22

weeks. 23

24

If infectious poliovirus is isolated, the inactivated purified monovalent pool should not be 25

used for further processing. The isolation of live poliovirus from an inactivated purified 26

monovalent pool must be regarded as a break in the manufacturing consistency record. 27

28

If primary monkey kidney cells are used in this test, they may contain adventitious agents 29

that could interfere with the test result. It is important to demonstrate that each test retains 30

sensitivity to detect partially inactivated polioviruses. 31

32

At the end of the observation period, the cell culture used for the detection of residual live 33

virus should be challenged with a validated amount of live Sabin virus of the same type as 34


that of the inactivated purified monovalent pool. The details of the challenge procedure 1

should be approved by the NRA. 2

If continuous cell lines are used, the ability to detect infectious 3

virus should be checked concurrently for each test by introducing a 4

positive control at the beginning of each test. Positive control flasks 5

should be inoculated with a low quantity of virus close to the 6

detection limit of the method. 7

8

The problem of detecting residual active poliovirus in a vaccine is 9

not the same as that of measuring infective virus in untreated 10

suspensions. Poliovirus that has been exposed to the action of 11

formaldehyde without becoming inactivated has been shown to 12

require a much longer time to produce cytopathogenic changes than 13

does untreated virus. For this reason it is desirable that tissue 14

cultures in tests for the presence of residual active virus be observed 15

for as long a time as is technically possible. A satisfactory tissue 16

culture system for this purpose therefore depends not only on the 17

sensitivity of the cells used for the preparation of the cultures but 18

also on the nutrient fluid. 19

20

The serum added to the nutrient fluid should be tested for inhibitors 21

to poliovirus at serum concentrations up to 50%. Only serum free 22

from inhibitors to all three types of poliovirus should be used. 23

24

Maintenance of the cultures in good condition may require frequent 25

changes of culture medium. However, it should be borne in mind 26

that by early changes of fluid unabsorbed virus might be removed 27

and the validity of the test thus impaired; therefore, the fluid should 28

be changed no earlier than 5–7 days after inoculation. 29

30

A.4.5.3 Kinetics of inactivation 31

The kinetics of inactivation should be established by each manufacturer and approved by the 32

NRA. Adequate data on inactivation kinetics should be obtained and consistency of the 33

inactivation process should be monitored. For this purpose, the virus titre of each filtered 34


purified monovalent pool before, during and at the end of inactivation should also be 1

determined as specified in A.4.4.2.6. 2

3

A.4.5.4 Sterility test for bacteria and fungi 4

Each inactivated purified monovalent pool should be tested for bacterial and fungal 5

sterility, as specified in Part A, section 5.2 of the General requirements for the sterility of 6

biological substances (39), or by methods approved by the NRA. 7

8

A.4.5.5 D-antigen content 9

The D-antigen content of each inactivated purified monovalent pool should be determined 10

using a validated immunochemical method and calculated using a reference vaccine 11

calibrated against the WHO International Standard (see section A.1.3). The results obtained 12

should be within the required limits established by the NRA. 13

14

A.4.6 Control of trivalent bulk 15

Only those inactivated purified monovalent pools that have been shown to be satisfactory 16

should be blended to form a trivalent bulk. 17

18

A.4.6.1 Test for absence of infective poliovirus 19

A sample of at least 1500 ml or, if purified and concentrated vaccine is prepared, the 20

equivalent of at least 1500 doses of each trivalent bulk should be tested in cell cultures for 21

the absence of infective poliovirus by the procedure described in section A.4.5.2 of these 22

Recommendations. If infective poliovirus is isolated, this trivalent bulk, or product derived 23

from it, should not be used. 24

25

In some countries this test may be omitted, following a review of 26

manufacturing records, subject to approval by the NRA. 27

28

When a trivalent bulk is supplied by one manufacturer to another, 29

the validation of inactivation may rely on the inactivation tests 30

performed by the bulk supplier. 31

32



The trivalent bulk should be tested for bacterial and fungal sterility, as specified in Part A, 1

section 5.2 of the General requirements for the sterility of biological substances (39), or by 2

the methods approved by the NRA. 3

4

A.4.6.3 Residual formaldehyde 5

The content of free residual formaldehyde in the trivalent bulk should be determined by a 6

method approved by the NRA. The limits should be approved by the NRA. 7

8

A.4.6.4 D-antigen content 9

The D-antigen content of each trivalent bulk should be determined using a validated 10

immunochemical method and calculated using a reference vaccine calibrated against the 11

WHO International Standard (see section A.1.3). The results obtained should be within the 12

required limits established by the NRA. 13

14

A.4.7 Control of final bulk 15

Preservatives, excipients or other substances that might be added to or combined with the 16

trivalent bulk to form the final bulk should have been shown to have no deleterious effect on 17

the immunizing potency and the safety profile of the poliovirus antigens. 18

19

The operations necessary for preparing the final bulk from trivalent bulk should be 20

conducted in such a manner as to avoid contamination of the product. In preparing the final 21

vaccine bulk, any substances such as diluents, stabilizers or adjuvants that are added to the 22

product should have been shown to the satisfaction of the NRA not to impair the safety and 23

efficacy of the vaccine in the concentration used. Until the final bulk is filled into containers, 24

the final vaccine bulk suspension should be stored under conditions shown by the 25

manufacturer to retain the desired biological activity. 26

27


The final bulk should be tested for bacterial and fungal sterility, as specified in Part A, 29



32

A.4.7.2 Potency tests 33


Each final bulk should be tested in an in vivo assay for immunogenicity by tests approved by 1

the NRA. An in vivo potency assay in rats has been standardized and shown to be a suitable 2

in vivo test for IPV (See Appendix 2). Product-specific reference preparations may be used 3

in these tests (see Appendix 2). 4

5

The D-antigen content of each final bulk should be determined using a validated 6

immunochemical method and calculated using a reference vaccine calibrated against the 7

WHO International Standard (see section A.1.3). The results obtained should be within the 8

required limits established by the NRA. 9

10

When consistency of production has been established on a suitable number of consecutive 11

final bulks, the in vivo assay may be omitted with the agreement of the NRA. This can occur 12

once it has been demonstrated that the acceptance criteria for the D-antigen determination 13

are such that it yields a comparable result to the in vivo assay in terms of acceptance or 14

rejection of a batch. This demonstration must include testing of subpotent batches, produced 15

experimentally if necessary, for example by heat treatment or other means of diminishing 16

the immunogenic activity. 17

18

Where there is a significant change in the manufacturing process of the antigens or their 19

formulation, the in vivo test should be performed to demonstrate the comparability of the 20

manufacturing process, i.e previous versus new. If the process change impacts the in vivo 21

test, the need for revalidation should be considered and clinical data may be required for the 22

approval by the NRA. 23

24

The in vitro assay that has been found most suitable for measuring the antigen content is 25

the D-antigen enzyme-linked immunosorbent assay (ELISA). Although this assay is 26

widely used, particular attention is required for its standardization. Some NRAs accept the 27

use of polyclonal antisera whereas others accept the use of monoclonal antibodies in the 28

test. The use of different antibodies may give different results. The D-antigen specificity of 29

the antibodies should be demonstrated. Whichever types of antisera are used, the validation 30

studies should show that the assay can determine consistency of production. For D-antigen 31

ELISAs to be valid, they should comply with specified criteria of linearity and parallelism. 32

The effect of a change in the method of calculation of the D-antigen content on registered 33

specifications should also be taken into account. 34


1

Other validated tests such as multiplex antibody test or plasmon 2

resonance technology (e.g. Biacore) (44, 45) may be used subject to 3

the approval of the NRA. 4

5

If the use of an adjuvant in the final bulk interferes with the assay, a 6

desorption or treatment step may be necessary before performing the 7

D-antigen ELISA. 8

9

If the final bulk is formulated with poliovirus trivalent bulk and with other antigens into a 10

combination vaccine, then the suitability of performing the D-antigen ELISA on the final 11

bulk will have to be determined. If the D-antigen ELISA is not suitable for a particular 12

combination, an in vivo assay should be used. 13

14

The potency of the final bulk for each virus type should be approved by the NRA. 15

16

A.4.7.3 Preservative content 17

If preservative is added, the content in the final bulk should be determined by a method 18

approved by the NRA. The preservative used and content permitted should be approved by 19

the NRA. This test may be omitted on the final bulk if conducted on the final lot. 20

21

A.4.7.4 Endotoxin content 22

The endotoxin content in the final bulk should be determined by a method approved by the 23

NRA. Endotoxin content or pyrogenic activity should be consistent with levels found to be 24

acceptable in vaccine lots used in pre-licensure clinical trials and approved by the NRA. 25

26

The test is conducted routinely until consistency of production is demonstrated to the 27

satisfaction of the NRA. This test may be omitted on the final bulk if conducted on the 28

final lot. 29

30

A.4.7.5 Adjuvant (if applicable) 31

Each final vaccine bulk should be assayed for the content of adjuvant. This test may be 32

omitted if performed on the final lot. Where aluminium compounds are used, the content of 33

aluminium should not be greater than 1.25 mg per single human dose. 34


1

A.5 Filling and containers 2

The requirements concerning filling and containers given in Good Manufacturing Practices 3

for Biological Products (35) should apply to vaccine filled in the final form. 4

5

Single- and multiple-dose containers may be used. 6

7

A.6 Control tests on the final lot 8

Samples should be taken from each final lot for the tests described in the following sections. 9

The following tests should be performed on each final lot of vaccine (i.e. in the final 10

containers). Unless otherwise justified and authorized, the tests should be performed on 11

labeled containers from each final lot by means of validated methods approved by the NRA. 12

The permissible limits for the different parameters listed under this section, unless otherwise 13

specified, should be approved by the NRA. 14

15

A.6.1 Inspection of final containers 16

Every container in each final lot shall be inspected visually or mechanically, and those 17

showing abnormalities shall be discarded and recorded for each relevant abnormality. A 18

limit for percentage of rejection should be established. 19

20

A.6.1.1 Appearance 21

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

23

A.6.2 Identity test 24

An identity test should be done on at least one labelled container from each final lot by an 25

appropriate method. 26

The potency test described in section A.6.5 of these 27

Recommendations may serve as the identity test. 28

29

A.6.3 Sterility test for bacteria and fungi 30

Each final lot should be tested for bacterial and fungal sterility, as specified in Part A, 31



34


A.6.4 General safety test 1

Each final lot should be tested for the absence of abnormal toxicity in mice or guinea pigs 2

using test approved by the NRA. This test may be omitted for routine release once 3

consistency of production has been established to the satisfaction of the NRA. 4

5

A.6.5 Potency test 6

Each final lot should be tested by a validated immunochemical method for D-antigen 7

content (see section A.4.5.5 and A.4.7.2) and calculated using a reference vaccine 8

calibrated against the WHO International Standard (see section A.1.3).. 9

In some countries, this test is omitted provided that the 10

determination of the D-antigen content has been carried out with 11

satisfactory results on the final bulk product and provided that a 12

validation has been performed to demonstrate that there is no loss of 13

potency between the final bulk product and the final lot, subject to 14

approval by the NRA, 15

16

If the use of an adjuvant in the final bulk interferes with the assay, a desorption or treatment 17

step may be necessary before performing the D-antigen ELISA. If a treatment/desorption is 18

not possible, the interference of the adjuvant should be documented and an in vivo assay 19

should be performed (see A.4.7.2 and Appendix 2). 20

21

In general, vaccines manufactured from wild type poliovirus strains 22

that have been formulated to contain 40, 8 and 32 D-antigen units or 23

more per dose for types 1, 2 and 3, respectively, are effective (46). 24

Vaccines with lower D-antigen contents may be acceptable, if 25

supported by clinical data. Vaccines in which adjuvants are used or 26

vaccines produced from other seed viruses (e.g. Sabin viruses) may 27

also be licensed with a different antigenic composition, if supported 28

by clinical data. 29

30

If the final bulk is formulated from a trivalent bulk and other 31

antigens into a combination vaccine, then the suitability of 32

performing the D-antigen ELISA on the final lot will have to be 33

determined. If the D-antigen ELISA is not suitable for a particular 34


combination, an in vivo assay such as that described in Appendix 2 1

should be used. 2

3

The potency of the vaccines for each virus type should be approved by the NRA. 4

5

A.6.6 Protein content 6

Poliomyelitis vaccine (inactivated) should not contain more than 10µg of protein per human 7

dose. This test may be omitted for routine lot release once consistency of production has 8

been established to the satisfaction of the NRA. 9

10

If animal serum is used for the growth of cell cultures, the serum protein concentration 11

(bovine serum albumin) in the final lot should be no more than 50ng per human dose. 12

13

A.6.7 Preservative content 14

Where appropriate, the preservative content of each final lot should be determined by a 15

method approved by the NRA. The method used and content permitted should be approved 16

by the NRA. 17

18

A.6.8 Endotoxin content 19

The endotoxin content of each final lot should be determined by a method approved by the 20

NRA. Levels should be consistent with levels found to be acceptable in vaccine lots used in 21

pre-licensure clinical trials and approved by the NRA. 22

23

A.6.9 Test for residual formaldehyde 24

The content of free residual formaldehyde in each final lot should be determined by a 25

method approved by the NRA, The limit should be approved by the NRA. This test may be 26

omitted if performed on the trivalent bulk. 27

28

A.6.10 Test for pH 29

The pH of each final lot should be determined and be within limits approved by the NRA. 30

31

A.6.11 Adjuvant and degree of adsorption (if applicable) 32

If an adjuvant is used in the formulation, each final lot should be assayed for the content of 33

adjuvant. Where aluminium compounds are used, the content of aluminium should not be 34


greater than 1.25 mg per single human dose. This test may be omitted on the final lot if 1

performed on the final bulk. 2

3

The degree of adsorption of the antigen to the aluminium compounds (aluminium hydroxide 4

or hydrated aluminium phosphate) in each final vaccine lot should be assessed. 5

A.6.12 Residual antibiotics (if applicable) 6

If any antibiotics are added in the vaccine production, the content of the residual antibiotics 7

should be determined and be within limits approved by the NRA. This test may be omitted 8

for routine lot release once consistency of production has been established to the satisfaction 9

of the NRA. 10

11

A.7 Records 12

The requirements given in Good Manufacturing Practices for Biological Products (35) 13

should apply. 14

15

A.8 Retained samples 16


should apply. 18

19

A.9 Labelling 20


should apply, with the addition of the following. The label on the container or package 22

should include the following information: 23

− the designation(s) of the strain(s) of poliovirus contained in the vaccine; 24

− the cell substrate used for the preparation of vaccine; 25

− the D-antigen content of each poliovirus type; 26

− the method and inactivating agent used to inactivate the virus; 27

− the nature and amount of any stabilizer and preservative present in the vaccine. 28

− the nature and amount of adjuvant, if applicable 29

30

It is desirable for the label to carry the names both of the producer 31

and of the source of the bulk material if the producer of the final 32

vaccine did not prepare it. The nature and amount of the antibiotics 33

present in the vaccine, if any, may be included. 34


1

A.10 Distribution and shipping 2


should apply. Further guidance is provided in the WHO Model guidance for the storage 4

and transport of time and temperature–sensitive pharmaceutical products (47). 5

6

A.11 Stability testing, storage and expiry date 7

A.11.1 Stability testing 8

Adequate stability studies form an essential part of vaccine development. Current guidance 9

on evaluation of vaccine stability is provided in the WHO guidelines on stability evaluation 10

of vaccines (48). Stability testing should be performed at different stages of production 11

when intermediate product is stored, namely on single harvests, inactivated purified 12

monovalent pool, trivalent bulk, final bulk, final lot. Stability-indicating parameters should 13

be defined or selected appropriately according to the stage of production. A shelf-life 14

should be assigned to all in-process materials during vaccine production, in particular 15

intermediates such as single harvests, inactivated purified monovalent pool, trivalent bulk 16

and final bulk. 17

18

The stability of the vaccine in its final containers, maintained at the recommended storage 19

temperature up to the expiry date, should be demonstrated to the satisfaction of the NRA. 20

As a guide, containers from at least three consecutive final lots, and derived from different 21

monovalent pools and different trivalent bulks, may be tested. 22

23

Where manufacturing involves only formulation of the final bulk from trivalent bulks 24

supplied by another manufacturing establishment and the filling of final containers, 25

stability data should be generated by the manufacturer on the trivalent bulk in their storage 26

conditions and the shelf life established until use. 27

28

The formulation of vaccine should be stable throughout its shelf-life. Acceptable limits for 29

stability should be agreed with the NRA. Following licensure, ongoing monitoring of 30

vaccine stability is recommended to support shelf-life specifications and to refine the 31

stability profile (48). Data should be provided to the NRA as per local regulatory 32

requirements. 33

34


The efficacy of the preservative should be confirmed at the end of shelf life. In case of 1

multi-dose presentations, efficacy of the preservative should be confirmed for the duration 2

during which the vial can be open, in compliance with the multi-dose open vial policy. 3

4

The final stability testing program should be approved by the NRA and should include an 5

agreed set of stability indicating parameters, procedures for the ongoing collection and 6

sharing of stability data and criteria to reject vaccine(s). 7

8

A.11.2 Storage conditions 9

Poliomyelitis vaccine (inactivated) should be stored at all times at a temperature between 10

2°C and 8 °C. For novel vaccines, appropriate storage conditions should be validated and 11

approved by the NRA. 12

13

A.11.3 Expiry date 14

The expiry date should be defined on the basis of shelf-life and supported by the stability 15

studies with the approval of the NRA and should relate to the date of blending of final 16

bulk, date of filling or the date of the first potency test on the final lot, performed in an 17

assay as described in Appendix 2. 18

19

Where an in vivo potency test is used, the date of the potency test is 20

the date on which the test animals were inoculated with the final 21

bulk. 22

23

Part B. Nonclinical evaluation of poliomyelitis vaccines (inactivated) 24

25

The nonclinical evaluation of candidate inactivated poliomyelitis vaccines should be based 26

on WHO guidelines on nonclinical evaluation of vaccines (7). The following specific 27

issues are intended for new IPV candidates and should also be referred to when a 28

significant change in manufacturing process or vaccine formulation is made to a licensed 29

IPV. 30

31

B.1 Characterization of poliovirus seed lots derived from attenuated strains (Sabin 32

strains and strains derived by recombinant DNA technology) 33


The virus master and working seed lots derived from attenuated strains (Sabin strains and 1

strains derived by recombinant DNA technology) that are used to manufacture a candidate 2

IPV should be extensively characterized. Ideally, the characterization studies should be 3

performed on seed lots used to prepare the vaccine lots tested in preclinical and clinical 4

studies. 5

6

When Sabin strains are used, the complete nucleotide sequence of the virus master and 7

working seed lots for each poliovirus type should be determined and shown to be 8

consistent with known sequence characteristics of poliovirus. 9

10

When attenuated poliovirus strains derived by recombinant DNA technology are used to 11

prepare a candidate IPV, the mutations responsible for attenuation should be identified 12

along with the mutations that can revert to partial or full virulence phenotype. The rate of 13

such reversions should be evaluated and shown to be not higher than the reversion rate 14

observed for conventional Sabin strains manipulated in similar conditions. The 15

neurovirulence of these seeds should be assessed. In addition, the genetic stability of the 16

strains derived by recombinant DNA technology should be confirmed at the passage level 17

used to prepare the vaccine or beyond. Efforts should also be made to develop an in vitro 18

test to detect reversion to partial and full virulent virus. 19

20

B.2 Antigenic profile 21

The available evidence suggests that there might be significant differences in the antigenic 22

composition of various IPV products developed independently (49, 50), in particular when 23

comparing sIPV to inactivated poliomyelitis vaccine derived from wild type strains 24

(wIPV1). It is likely that antigenic profiles of IPV are influenced by the different virus 25

strains, cell substrates and process parameters used in the manufacture. The antigenic 26

structure of a candidate IPV might be established using monoclonal antibodies (50, 51) of 27

known specificity at the early stage of product development and used as a characterization 28

tool for investigating vaccine stability and demonstrating manufacturing consistency 29

during product development. 30

1 In this document the use of the abbreviation IPV refers to inactivated poliomyelitis

vaccine derived from any strain. wIPV indicates inactivated poliomyelitis vaccine derived

from wild type strains only and sIPV stands for IPV derived from Sabin strains only.


1

B.3 D-antigen content of IPV derived from attenuated strains (Sabin strains and 2

strains derived by recombinant DNA technology) 3

The type-specific antigen content of current licensed wIPV is measured using various 4

enzyme-linked immunosorbent assay (ELISA) procedures (51) and reported as D-antigen 5

units relative to a reference preparation traceable to the International Standard. When 6

attenuated strains (e.g. Sabin strains and strains derived by recombinant DNA technology) 7

are used to prepare the IPV candidate, an in-house ELISA should be developed and 8

implemented to determine type-specific D-antigen content. An in-house reference standard 9

should be established at the early stage of product development and calibrated to the 10

International Standard. A monitoring program should be put in place to ensure the stability 11

of the in-house reference standard and the comparability of its subsequent replacement. In 12

addition, the ratio between virus titre (per mL) and D-antigen content (per mL) of purified 13

monovalent pools, prior to inactivation, should also be established for each polio type 14

during product development and monitored during commercial production. This provides 15

further assurance that the D-antigen content of commercial lots, throughout product life 16

cycle, is comparable to lots shown to be safe and immunogenic in clinical studies. 17

18

Most licensed wIPV products have been formulated to contain 40, 8 and 32 D-antigen 19

units per human dose. However, the D-antigen unit is not well defined particularly with 20

respect to the strain of virus used in the manufacture and it is known to be influenced by 21

the specificity of the antibodies used as ELISA reagents. Therefore, it is not possible to 22

directly compare the D-antigen content of various IPV (sIPV versus IPV) measured using 23

different monoclonal antibody based ELISA procedures (52). It is recognized that IPV 24

derived from attenuated strains or adjuvanted IPV will require different D-antigen content 25

to induce adequate immune responses in humans. 26

27

B.4 Evaluation of immunogenicity in animal models 28

Prior to initiating clinical trials, the immunogenic properties of a candidate IPV should be 29

studied in suitable animal models (e.g. rats). Proof of concept nonclinical studies should 30

include the comparison of immunogenicity between a candidate IPV and a current licensed 31

wIPV based on type-specific serum neutralizing antibody titres against both Sabin and wild 32

type strains. Those studies may also assist in the selection of D-antigen content to be tested 33

in the dose-finding studies in human. However, it is important to note that immunogenicity 34


data in animals do not reliably predict the antigen content that might be appropriate to be 1

included as a single human dose in the final vaccine formulation. Alternatively, the assay 2

using transgenic mice may be performed to compare the immune response and protection 3

against virulent challenge induced by a candidate IPV to that induced by a licensed wIPV 4

against virus challenge (27, 28). The in vivo tests are also important tools to be used as 5

characterization tests to demonstrate comparable manufacturing process when major 6

changes are introduced. 7

8

When an adjuvant is included in the formulation, manufacturers should provide a rationale 9

and immunogenicity data to support the use of an adjuvant in their vaccine (53). 10

11

B.5 Nonclinical safety studies 12

If a candidate IPV is formulated with a novel adjuvant or excipient (e.g. stabilizer), 13

nonclinical safety studies should be conducted as appropriate for the final vaccine 14

formulation (53). The use of a delivery device as well as alternative administration routes 15

(e.g. intradermal) may affect vaccine potency/immunogenicity, tolerability, toxicity and 16

long term safety, and the design of nonclinical safety studies should follow special 17

considerations outlined in section 5 of WHO guidelines on nonclinical evaluation of 18

vaccines (7). 19

20

Part C. Clinical evaluation of poliomyelitis vaccine (inactivated) 21

22

Clinical trials should adhere to the principles described in the WHO guidelines for good 23

clinical practice (GCP) for trials on pharmaceutical products (54) and to the WHO 24

guidelines on clinical evaluation of vaccines: regulatory expectations (8). All clinical trials 25

should be approved by the relevant NRAs before initiation. 26

27

Some of the issues that are specific to the clinical evaluation of inactivated poliomyelitis 28

vaccines are discussed in the following sections, which are applicable to IPV derived from 29

wild type strains as well as attenuated strains (e.g. Sabin strains and strains derived by 30

recombinant DNA technology). When relevant and applicable, specific requirements 31

applicable to sIPV will be identified. 32

33


C.1 General considerations 1

The global poliomyelitis eradication initiative following the World Health Assembly 2

resolution in 1988 has led to the dramatic decrease in poliomyelitis cases globally (10). 3

Therefore, clinical efficacy studies to support the licensure of all candidate IPV are no 4

longer feasible, and the clinical evaluation should be based on the comparative assessment 5

of safety and immunogenicity of a candidate vaccine with a licensed vaccine (comparator 6

vaccine). The assessment of seroconversion should be based on the elicitation of serum 7

neutralizing antibodies, which have been established to be the basis of protection (10). The 8

licensure of a candidate IPV should be based on a clear demonstration of non-inferiority in 9

terms of immunogenicity when compared to a comparator vaccine. 10

11

C.2 Immunogenicity studies 12

C.2.1 Assessment of the immune response 13

A serum neutralizing antibody titre of 1/4–1/8 is considered to be a marker of protection 14

against poliovirus (55). The demonstration of an immune response to IPV vaccination 15

should be based on the measurement of neutralizing antibody titres at pre- and post-16

vaccination timepoints. Seroconversion for polio antigen is defined as: 17

- For subjects seronegative at the pre-vaccination time point: antibody titres post-18

vaccination above the cut-off (titre 1/4 -1/8). 19

- For subjects seropositive at pre-vaccination time point: a ≥ 4-fold rise in antibody 20

titres post-vaccination. In the event that the pre-vaccination titre is due to maternal 21

antibodies, a 4-fold rise above the expected titre of maternal antibodies based on 22

the pre-vaccination titre declining with a half-life of 28 days indicates 23

seroconversion. 24

- In populations with high pre-vaccination antibody titres, a change from below the 25

highest dilution tested (<8192) to above the highest dilution tested (>8192) will 26

also indicate seroconversion. 27

28

It is recommended that the assay used to assess serum neutralizing antibodies be 29

standardized as described in WHO Manual for Virological Investigation of Poliomyelitis 30

(56), in particular with respect to the use of appropriate cell lines, International Standards 31

of anti-poliovirus sera and other important reagents. The level of neutralizing antibody 32

present in a serum sample is expressed as a titre, which is the reciprocal of the highest 33

serum dilution that inhibit the viral cytopathic effect in 50% of cell culture. 34


1

For the evaluation of sIPV performance, serum neutralizing antibody titres against both 2

Sabin and wild type poliovirus should also be determined, to ensure that the conclusions of 3

clinical studies are applicable to both types of strains. The use of recently isolated wild 4

type strains and of cVDPVs or immunodeficiency-associated vaccine-derived polioviruses 5

(iVDPVs) which show extensive antigenic changes should also be considered for these 6

tests 7

8

The presence of neutralizing antibody against polioviruses is considered a reliable correlate 9

of protection against poliomyelitis. However, immunity induced by one serotype does not 10

provide protection against the other two serotypes. 11

12

C.2.2 Immunogenicity studies 13

A candidate IPV should be directly compared with a licensed IPV prepared from wild 14

poliovirus strains in prospectively controlled studies. In the event that none of the wIPV 15

products are licensed in the country where the clinical studies are conducted, the use of 16

OPV as a comparator may be acceptable. The comparator vaccine(s) should have been in 17

use for some years so that some effectiveness data as well as a substantial safety database 18

are available. Non-inferiority studies to evaluate immunogenicity after completion of 19

primary vaccination series in target population, naïve infants, are required for regulatory 20

approval of a candidate IPV. Persistency of the serum neutralizing antibodies after the 21

primary series should also be investigated to recommend whether and when a booster dose 22

is required. However, data concerning long term antibody persistency might not be 23

available prior to regulatory approval. Waning of antibodies over time is inevitable and 24

should not be interpreted per se to indicate the need for a booster dose. It is important that 25

longer-term antibody titres are viewed in conjunction with effectiveness data to assess the 26

potential need for additional doses later in life to maintain protection. 27

28

C.2.3 Study population and region 29

In general, the first clinical study (Phase I) of a candidate IPV should be performed in 30

healthy adults to assess vaccine safety. Due to wide use of IPV and OPV, the 31

immunogenicity of a candidate IPV can only be reliably evaluated in the naive target 32

population, infants. 33

34


Exposure of study subjects to circulating wild or OPV derived poliovirus may enhance the 1

immune response induced by IPV, and in turn, compromise study outcome (false 2

conclusions. Therefore, clinical trials to evaluate the immunogenicity of a candidate IPV, 3

including dose-finding studies and non-inferiority studies, should be performed in regions 4

where IPV is used exclusively or in countries such as Cuba, where OPV is given nationally 5

on specified dates and where naïve children can be identified. Alternatively, special 6

measures should be taken to minimize the potential of exposure among study participants. 7

8

C.2.4 Endpoints and analyses 9

The primary study analysis should be based on the rate of seroconversion (as described in 10

section C.2.1) measured at approximately 4 weeks following completion of the primary 11

infant immunization series against both Sabin and wild type strains. The primary study 12

objectives should be based on the demonstration of the non-inferiority of the 13

seroconversion achieved with the candidate IPV versus the comparator vaccine. Non-14

inferiority should be defined by taking a 5 percentage points as the maximum acceptable 15

clinical margin in two-sided comparisons. 16

17

The use of recently isolated wild type strains and cVDPVs or iVDPVs which show 18

extensive antigenic changes should also be considered for these non-inferiority hypothesis 19

testing. The pre-defined clinical margins of non-inferiority should be justified, and the 20

calculations of the proposed sample size required should be clearly explained in study 21

protocol. Further details on demonstrating non-inferiority are described in the WHO 22

guidelines on clinical evaluation of vaccines: regulatory expectations (8). Consideration 23

should be given to the overall statistical power of these multiple comparisons, and efforts 24

should be made to maintain sufficient overall power for the studies. 25

26

Comparison of geometric mean titres (GMTs) and reverse cumulative distributions (RCD) 27

of individual titres against both Sabin and wild type poliovirus should also be provided as 28

exploratory analyses. It may be that the GMT(s) for one or more poliovirus types induced 29

by the candidate IPV derived from attenuated strains is lower than that induced by the 30

licensed wIPV. However, a lower GMT may not be clinically significant, as the available 31

data suggest that persistent immune memory is sufficient to protect against poliomyelitis 32

(57, 58). 33

34


The minimal D-antigen content present in the candidate vaccine at the end of shelf-life 1

should be based on the D-antigen content of the clinical lots which were shown to induce 2

effective immune responses and to have an acceptable safety profile in clinical studies (e.g. 3

lots used in dose-finding study). 4

5

C.2.5 Immunization schedule 6

Different immunization schedules are used in different regions or countries for licensed 7

wIPV. It is common that IPV is administered using the same schedule as DTP containing 8

vaccines to achieve high compliance rate. Clinical trial data have shown that the immune 9

response induced by licensed wIPV varies according to the immunization schedule used. In 10

general, longer intervals in the primary immunisation series (e.g. 2, 4 and 6 months) induce 11

higher neutralization titres and better seroconversion rate (59, 60). Immunization schedules 12

should be defined for the targeted countries or regions, wherever possible and dose-finding 13

and non-inferiority studies for a candidate IPV should be conducted with the immunization 14

schedule anticipated to be the lowest immunogenic. However, it is not feasible to study a 15

candidate vaccine using every possible schedule in all target regions. Manufacturers should 16

justify the relevance of the clinical data provided to each country in which approval is 17

sought and should discuss the basis for extrapolation of the findings. For example, 18

satisfactory immune responses using a schedule with short interval (e.g. 2, 3 and 4 months) 19

supports an expectation that satisfactory immune responses would also be observed using a 20

schedule with longer interval (e.g. 2, 4 and 6 months). However, the local and systemic 21

reactogenicity associated with a candidate IPV may also be different between schedules 22

within a specific population so there is still a need to collect some safety data, prior to 23

regulatory approval, for the proposed schedules (e.g. 2, 4 and 6 months). 24

25

An immunization schedule combining both IPV and OPV could potentially achieve both 26

the high serum antibody levels and the intestinal protection. Clinical studies designed to 27

establish such a combination (sequential) schedule should also examine patterns of virus 28

excretion following poliovirus challenge (with OPV), other than serum neutralizing 29

antibodies, in different sequential schedules. 30

31

C.3 Concomitant administration with other vaccines 32

IPV is commonly co-administered with other infant and toddler vaccines. Therefore, it is 33

essential to evaluate the immune responses to a candidate IPV as well as to all other 34


antigens co-administered in all the co-administration situations claimed. Due to the large 1

number of licensed vaccines that may need to be co-administered with IPV in infants and 2

toddlers using a variety of schedules, it is not feasible for manufacturers to study every 3

possible combination. The data on the effects of co-administration that are available at the 4

time of initial licensure may be limited and should be expanded in post-approval studies. If 5

study results indicate that immune responses are lower on co-administration with other 6

vaccine(s), the NRAs will need to consider the potential clinical consequences on a case by 7

case basis. 8

9

C.4 Pre-licensure safety data 10

The general approach to safety assessment of a candidate IPV during pre-licensure clinical 11

studies should be in accordance with the WHO guidelines on clinical evaluation of 12

vaccines: regulatory expectations (8). The safety profile of a candidate IPV derived from 13

attenuated strains is expected to be very similar to that of current licensed wIPV, which is 14

very well tolerated. The NRAs may decide that large safety studies are not required. 15

However, if a candidate IPV includes novel adjuvants and/or excipients, uses a delivery 16

device or is administered using an alternate route, a safety database similar in size to what 17

is requested for any new vaccine entity might be needed. This should be discussed and 18

approved by the NRAs on a case-by-case basis. In addition, it is likely that adverse events 19

at the injection site are more frequent if a candidate IPV contains adjuvant. This may be 20

acceptable given that the incidence of adverse events is comparable to that observed for 21

other licensed adjuvanted vaccines and the benefit clearly outweighs risks. 22

23

An appropriate pharmacovigilance plan should be developed and approved by the NRAs 24

prior to licensure. 25

26

C.5 Post-marketing studies and surveillance 27

Post-marketing surveillance should be undertaken during the initial post-approval years in 28

collaboration with the NRAs. Manufacturers and health authorities should work in 29

collaboration with the global polio surveillance laboratory network to monitor new 30

vaccines once introduced in immunization programs. The enhanced safety surveillance is 31

particularly important for vaccines which include novel adjuvants and/or excipients. Due to 32

the possibility that the sIPV may induce lower GMT for one or more poliovirus type, the 33

persistence of antibody and the need for booster dose should be studied post-marketing. 34


1

The total duration of enhanced surveillance should be regularly reviewed by the NRAs. If 2

particular issues arise during pre-licensure studies or during post-licensure safety 3

surveillance then it may be necessary to conduct specific post-licensure safety studies. 4

5

Part D. Recommendations for national regulatory authorities 6

D.1 General 7

The general recommendations for national regulatory authorities and national control 8

laboratories given in the Guidelines for national authorities on quality assurance for 9

biological products (61) and Guidelines for Independent Lot Release of Vaccines by 10

Regulatory Authorities (62) should apply. 11

12

The detailed production and control procedures as well as any significant changes in them 13

that may affect quality, safety and efficacy of poliomyelitis vaccine (inactivated) should be 14

discussed with and approved by the NRA. 15

16

For control purpose, the currently in force International Standards should be obtained for 17

the purpose of calibration of the national/regional/working standards (63). The NRA may 18

obtain the product specific/working reference from the manufacturer to be used for lot 19

release until international/national standard preparation is established. 20

21

Consistency of the production has been recognized as an essential component in the quality 22

assurance of poliomyelitis vaccine (inactivated). In particular, the NRA should carefully 23

monitor production records as well as quality control test results for clinical lots as well as 24

a series of consecutive lots of the vaccine. 25

26

D.2 Official release and certification 27

A vaccine lot should be released only if it fulfils the national requirements and/or satisfies 28

Part A of the present Recommendations (62). 29

30

A protocol based on the model given in Appendix 3, signed by the responsible official of 31

the manufacturing establishment, should be prepared and submitted to the NRA in support 32

of a request for release of vaccine for use. 33


1

A statement signed by the appropriate official of the NRA should be provided to the 2

manufacturing establishment and should certify that the lot of vaccine in question meets all 3

national requirements, as well as Part A of these Recommendations. The certificate should 4

provide sufficient information on the vaccine lot. A model certificate is given in Appendix 5

4. The official national release certificate should be provided to importers of the vaccines. 6

The purpose of the certificate is to facilitate the exchange of vaccines between countries. 7


1

Authors and Acknowledgements 2

3

A pre-draft 1 of this document was prepared by Dr M. Lennon (Ferguson), Horning, UK; 4

Dr C Li, National Institutes for Food and Drug Control (NIFDC), Beijing, P.R.China; Dr J. 5

Martin, National Institute for Biological Standards and Control, Medicines and Healthcare 6

Products Regulatory Agency, Potters Bar, England; Dr P. Minor, National Institute for 7

Biological Standards and Control, Medicines and Healthcare Products Regulatory Agency, 8

Potters Bar, England; Dr K. Chumakov, Center for Biologics Evaluation and Research, 9

Food & Drug Administration, Maryland, USA; Dr T. Wu, Health Canada, Ottawa, Canada; 10

with support from the WHO Secretariat: Dr T.Q. Zhou, Dr J. Fournier-Caruana, Dr I. 11

Knezevic, Dr D.J. Wood, Essential Medicines and Health Products (EMP) 12

Department/Health Systems and Innovation (HIS) Cluster, World Health Organization 13

(WHO), Geneva, Switzerland; Dr H Okayasu, Dr R. Sutter, Research, Policy and Product 14

Development, PEC/POL/RAP),World Health Organization, Geneva, Switzerland taking 15

into considerations the discussions at a Working Group meeting on Technical 16

Specifications for Manufacturing and Evaluating the WHO Recommendations for IPV: 17

TRS No. 910 in Geneva, Switzerland from 27-29 March 2012 attended by; Dr M. Baca-18

Estrada, Health Canada, Ottawa, Canada; Dr W. A.M. Bakker, National Institute for Public 19

Health and the Environment, Bilthoven, The Netherlands; Dr J. Fournier-Caruana, 20

Essential Medicines and Health Products (EMP) Department/Health Systems and 21

Innovation (HIS) Cluster, World Health Organization (WHO), Geneva, Switzerland; Mr 22

B.S. Chauhan, Bharat Biotech International Limited, Hyderabad, India; Dr K. Chumakov, 23

Center for Biologics Evaluation and Research, Food & Drug Administration, Bethesda, 24

USA; Dr E. Coppens, Sanofi Pasteur, France; Dr M. Duchêne, GSK Biologicals, Wavre, 25

Belgium; Ms G. Dunn, National Institute for Biological Standards and Control, Medicines 26

and Healthcare Products Regulatory Agency, United Kingdom, England ; Dr D. Felnerova, 27

Crucell, Berne, Switzerland; Dr M. Ferguson, Horning, United Kingdom; Dr L. Fiore, 28

Istituto Superiore di Sanita, Roma, Italy; Mr J.B. González, Laboratorios de Biológicos y 29

Reactivos de México S.A. de C.V. México D.F., Mexico; Dr M.A. Gonzalez, Federal 30

Commission for the Protection from Sanitary Risks (COFEPRIS), Ministry of Health, 31

Mexico; Prof V. Grachev, Russian Academy of Medical Sciences (RAMS), Moscow 32

Region, Russian Federation; Ms H. Wang , Tiantan Biological Products Co., Ltd , Beijing, 33


China; Mrs T. Jivapaisarnpong, Department of Medical Sciences, Ministry of Public 1

Health, Bangkok, Thailand; Dr I. Knezevic, Essential Medicines and Health Products 2

(EMP) Department/Health Systems and Innovation (HIS) Cluster, World Health 3

Organization (WHO), Geneva, Switzerland; Dr H. Okayasu, Research, Policy and Product 4

Development, World Health Organization, Geneva, Switzerland; Dr D. Kusmiaty, National 5

Quality Control Laboratory of Drug and Food, Ministry of Health, Jakarta, Indonesia; Dr 6

K. Katayama, National Institute of Infectious Diseases, Tokyo, Japan; Dr C Li, National 7

Institutes for Food and Drug Control, Beijing, P.R.China; Dr J. Martin, National Institute 8

for Biological Standards and Control, South Mimms, England; Dr C. Milne, European 9

Directorate for the Quality of Medicines and HealthCare (EDQM), Strasbourg, France; Dr 10

R. Modi, Cadila Pharmaceuticals Limited, Ahmedabad, India; Ms E. Niogret, Sanofi 11

Pasteur, Marcy L'Etoile, France; Dr L.V. Phung, National Institute for Control of Vaccine 12

and Biologicals, Hanoi, Vietnam; Dr V. Pithon, Agence Nationale de Sécurité du 13

Médicament et des Produits de Santé, Lyon, France; Dr A. Sinyugina, Federal State 14

Unitary Enterprise of Chumakov Institute of Poliomyelitis and Viral Encephalitides, 15

Russian Academy of Medical Sciences (RAMS), Moscow Region, Russian Federation; Dr 16

R. Sutter, Research, Policy and Product Development, World Health Organization, 17

Geneva, Switzerland; Mr D. Ugiyadi, BioFarma, Bandung, Indonesia; Dr G. Waeterloos, 18

Scientific Institute of Public Health, Brussels, Belgium; Dr S. Yamazaki, Japan 19

Poliomyelitis Research Institute, Tokyo, Japan; Dr D.J. Wood, Essential Medicines and 20

Health Products (EMP) Department/Health Systems and Innovation (HIS) Cluster, World 21

Health Organization (WHO), Geneva, Switzerland; Mr L. Yi, Institute of Medical Biology, 22

Kunming, P.R. China; Dr T.Q. Zhou, Essential Medicines and Health Products (EMP) 23


(WHO), Geneva, Switzerland. 25

26

Draft 1 was prepared by Dr M. Lennon (Ferguson), Horning, Norfolk, UK; Dr C Li, 27

National Institutes for Food and Drug Control (NIFDC), Beijing, P.R.China; Dr J Martin, 28

National Institute for Biological Standards and Control, Medicines and Healthcare 29

Products Regulatory Agency, Potters Bar, England; Dr P. Minor, National Institute for 30

Biological Standards and Control, Medicines and Healthcare Products Regulatory Agency, 31

Potters Bar, England; Dr K. Chumakov, Center for Biologics Evaluation and Research, 32

Food & Drug Administration, Maryland, USA; Dr T. Wu, Health Canada, Ottawa, Canada; 33

with support from the WHO Secretariat: Dr T.Q. Zhou, Dr J. Fournier-Caruana, Dr I. 34


Knezevic, Dr D.J. Wood , Essential Medicines and Health Products (EMP) 1


(WHO), Geneva, Switzerland; Dr Hiromasa Okayasu, Dr H.S Jafari, Policy and Product 3

Development, World Health Organization, Geneva, Switzerland; taking into considerations 4

the discussions at a Working Group meeting on Technical Specifications for 5

Manufacturing and Evaluating the WHO Recommendations for IPV: TRS No. 910 in 6

Geneva, Switzerland from 14-15 May 2013 attended by Ms P Agsiri, Department of 7

Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand; Dr W A.M. Bakker, 8

Institute for Translational Vaccinology (Intravacc), Bilthoven, The Netherlands; Dr K. 9

Chumakov, Center for Biologics Evaluation and Research, Food & Drug Administration, 10

Maryland, USA; Dr J. Fournier-Caruana, Essential Medicines and Health Products (EMP) 11


(WHO), Geneva, Switzerland; Dr E Griffiths, Kingston upon Thames, Surrey, UK; Dr I 13

Hansenne, Scientific Institute of Public Health, Brussels, Belgium; Dr K Katayama, 14

Department of Virology II, National Institute of Infectious Diseases (NIID), Tokyo, Japan; 15

Dr J Korimbocus, Agence nationale de sécurité du médicament et des produits de santé 16

(ANSM), Lyon, France; Dr M Lennon, Horning, Norfolk, UK; Dr C Li, National Institutes 17

for Food and Drug Control (NIFDC), No.2, Tiantan Xili, Beijing, People's Republic of 18

China; Dr J Martin, National Institute for Biological Standards and Control, Medicines and 19

Healthcare Products Regulatory Agency,, Potters Bar, England; Dr P. Minor, National 20

Institute for Biological Standards and Control, Medicines and Healthcare Products 21

Regulatory Agency, Potters Bar, England; Mr M Mitsuki, Office of Vaccines and Blood 22

Products, Pharmaceuticals and Medical Devices Agency (PMDA), Tokyo, Japan; Dr P 23

Neels, Federal Agency for Medicinal and Health Products, Brussels, Belgium; Dr M Van 24

Oijen, Institute for Translational Vaccinology (Intravacc), Bilthoven, The Netherlands; Dr 25

V.G. Somani, Central Drugs Standard Control Organization (CDSCO), Food and Drug 26

Administration (FDA), New Delhi, India; Dr T Wu, Bacterial and Combination Vaccines 27

Division, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Canada; 28

Professor H Yin, Deputy Director, Center for Drug Evaluation (CDE), State Food and 29

Drug Administration, Beijing, People's Republic of China; Dr N Benno Chukilizo, 30

Tanzania Food and Drugs Authority, Dar-es-Salaam, United Republic of Tanzania, Mrs D 31

Darko, Food and Drugs Board, Accra, Ghana; Dr K Mahmood, Vaccine Development 32

Global Program, PATH, Seattle, USA; Dr C Milne, Department of Biological 33

Standardisation, European Directorate for the Quality of Medicines & HealthCare 34


(EDQM), Strasbourg, France; Dr D-Y Choi, Lucky Goldstar Life Sciences, Seoul, Korea; 1

Dr R Dhere, Serum Institute of India Ltd., Pune, India; Dr S Konda, Panacea Biotec Ltd, 2

New Delhi, India; Ms X Li, National Vaccine and Serum Institute(NVSI), Beijing, People's 3

Republic of China; Mr D Ugiyadi, PT Biofarma, Bandung, Indonesia; Ms H Wang, 4

Tiantan Biological Products, Beijing, People's Republic of China; Dr S De Walque, 5

GlaxoSmithKline Vaccines, Wavre, Belgium; Dr T de Rosa, Crucell Switzerland Ltd, 6

Berne, Switzerland; Dr Emmanuel Vidor, Sanofi Pasteur, Lyon, France; Dr Y Kino, The 7

Chemo-Sero-Therapeutic Research Institute (Kaketsken), Kumamoto, Japan; Prof Q Li, 8

Director, Kunming Institute of Medical Biology, Kunming, People's Republic of China; Dr 9

G Stawski, Statens Serum Institut (SSI), Copenhagen, Denmark; Dr K Wakabayashi, Japan 10

Poliomyelitis Research Institute (JPRI), Tokyo, Japan; Dr D.J Wood, Essential Medicines 11

and Health Products (EMP) Department/Health Systems and Innovation (HIS) Cluster, 12

World Health Organization (WHO), Geneva, Switzerland. 13

14

Draft 2 was prepared by Dr M. Lennon (Ferguson), Horning, Norfolk, UK and Dr T.Q. 15

Zhou, Essential Medicines and Health Products (EMP) Department/Health Systems and 16

Innovation (HIS) Cluster, World Health Organization (WHO), Geneva, Switzerland taking 17

into consideration comments from Ms P Agsiri, Department of Medical Sciences, Ministry 18

of Public Health, Nonthaburi, Thailand; Dr W A.M. Bakker, Institute for Translational 19

Vaccinology (Intravacc), Bilthoven, The Netherlands; Dr J. Fournier-Caruana, Essential 20

Medicines and Health Products (EMP) Department/Health Systems and Innovation (HIS) 21

Cluster, World Health Organization (WHO), Geneva, Switzerland; Dr J Korimbocus, 22

Agence nationale de sécurité du médicament et des produits de santé (ANSM), Lyon, 23

France; Dr C Li, National Institutes for Food and Drug Control (NIFDC), No.2, Tiantan 24

Xili, Beijing, People's Republic of China; Dr J Martin, National Institute for Biological 25

Standards and Control, Medicines and Healthcare Products Regulatory Agency, Potters 26

Bar, England; Dr P. Minor, National Institute for Biological Standards and Control, 27

Medicines and Healthcare Products Regulatory Agency, Potters Bar, England; Dr Monique 28

Van Oijen, Institute for Translational Vaccinology (Intravacc), Bilthoven, The 29

Netherlands; Dr Tong Wu, Bacterial and Combination Vaccines Division, Biologics and 30

Genetic Therapies Directorate, Health Canada, Ottawa, Canada; Dr K Mahmood, Vaccine 31

Development Global Program, PATH, Seattle, USA; Dr Catherine Milne, Department of 32

Biological Standardisation, European Directorate for the Quality of Medicines & 33

HealthCare (EDQM), Strasbourg, France; Dr S Konda, Panacea Biotec Ltd, New Delhi, 34


India; Dr E Vidor, Sanofi Pasteur, Lyon, France; Dr Y Kino, The Chemo-Sero-Therapeutic 1

Research Institute (Kaketsken), Kumamoto, Japan; Prof Q Li, Director, Kunming Institute 2

of Medical Biology, Kunming, People's Republic of China; Dr K Wakabayashi, Japan 3

Poliomyelitis Research Institute (JPRI), Tokyo, Japan. 4

5

The following experts provided responses to a WHO survey on IPV seeds and quality 6

control information conducted during 2012-2013: 7

S Yamazaki and S Abe, Japan Poliomyelitis Research Institute, Tokyo, Japan; R M Dhere, 8

L.R. Yeolekar, and S Gairola, Serum Institute of India Ltd., India; M.B Sun, GY Liao and 9

Y Li, Institute of Medical Biology Chinese Academy of Medical Sciences, KunMing, 10

China; H Wang, Beijing TianTan Biological Products Co., limited (TianTan Bio.), China; 11

G Stawski, Statens Serum Institut, Denmark; M. van Oijen and W Bakker, Institute for 12

Translational Vaccinology (Intravacc), Bilthoven, The Netherlands; D Ugiyadi, PT Bio 13

Farma (Persero), Indonesia; R.K.Suri, H.S Mali and D Rastogi, Panacea Biotec Ltd, India; 14

E Niogret, Sanofi Pasteur, France; X Bouwstra, Bilthoven Biologicals B.V., The 15

Netherlands; M Duchêne and C Saillez, GlaxoSmithKline Vaccines, Belgium; Ds Rudert-16

Dolby, Sanofi Pasteur Limited, Canada. 17

18


1

References 2

3

1. Requirements for Poliomyelitis Vaccine (Inactivated) (Requirements for Biological 4

Substances No. 2). In Requirements for Biological Substances Report of a study 5

group, 1959. (WHO Technical Report Series, No. 178). 6


Substances No. 2). In Requirements for Biological Substances Report of a study 8

group, 1965. (WHO Technical Report Series, No. 323). 9


Substances No. 2, revised 1981). In: WHO Expert Committee on Biological 11

Standardization. Thirty-second report. Geneva, World Health Organization, 1982, 12

Annex 2 (WHO Technical Report Series, No. 673). 13

4. Requirements for Poliomyelitis Vaccine (Inactivated) (Addendum 1985). In: WHO 14

Expert Committee on Biological Standardization. Thirty-sixth report. Geneva, World 15

Health Organization, 1987, Annex 4 (WHO Technical Report Series, No. 745) 16

5. Recommendations for the production and control of poliomyelitis vaccine 17

(inactivated). (Requirements for Biological Substances No. 2, revised 2000) WHO 18

Technical Report Series, No. 910, 2002 Annex 2 19

6. Guidelines for the safe production and quality control of inactivated poliomyelitis 20

vaccine manufactured from wild polioviruses (Addendum, 2003, to the 21

Recommendations for the Production and Quality Control of Poliomyelitis Vaccine 22

(Inactivated)). WHO Technical Report Series, No. 926, Annex 2 23

7. WHO guidelines on nonclinical evaluation of vaccines. Annex 1 in: WHO Expert 24

Committee on Biological Standardization. Fifty-fourth report. Geneva, World Health 25

Organization, 2005 (WHO Technical Report Series, No. 927) 26

(http://www.who.int/biologicals/publications/trs/en/, accessed 16 January 2013). 27

8. Guidelines on clinical evaluation of vaccines: regulatory expectations. Annex 1 in: 28

WHO Expert Committee on Biological Standardization. Fifty-second report. Geneva, 29

World Health Organization, 2004 (WHO Technical Report Series No. 924) 30

(http://www.who.int/biologicals/publications/trs/en/, accessed 16 January 2013) 31

9. Recommendations to Assure the Quality, Safety and Efficacy of DT-based Combined 32

Vaccines, Proposed replacement of TRS 800 Annex 2. (ECBS 2012) 33

10. Polio vaccines and polio immunization in the pre-eradication era: WHO position 34

paper. Weekly Epidemiological Record, 2010, 85, 213–228. 35


11. Polio Eradication and Endgame Strategic Plan 2013-2018. – Global Polio Eradication 1

Initiative Draft 14 April 2013 2

http://www.polioeradication.org/Portals/0/Document/Resources/StrategyWork/EndGa3

meStratPlan_WHAversion.pdf 4

12. Weekly Epidemiological Record 2013, 88, 1–16. Meeting of the Strategic Advisory 5

Group of Experts on Immunization, November 2012– conclusions and 6

recommendations. To be reviewed and updated before the ECBS 2014 7

13. Aylward RB, Hull HF, Cochi SL, Sutter RW, Olive JM, Melgaard B. Disease 8

eradication as a public health strategy: a case study of poliomyelitis eradication. 9

Bulletin of the World Health Organization, 2000, 78:285–297. 10

14. WHO Global Action Plan for Laboratory Containment of Wild Polioviruses. Geneva, 11

World Health Organization, WHO/V&B/03.11 (To be updated if GAP III approved 12

and published before ECBS 2014) 13

15. Dowdle W, van der Avoort H, de Gourville E, Delpeyroux F, Desphande J, Hovi T, 14

Martin J, Pallansch M, Kew O, and Wolff C. 2006. Containment of polioviruses after 15

eradication and OPV cessation: characterizing risks to improve management. Risk 16

Analysis. 2006 26: 1449–1469. 17

16. Doi Y, Abe S, Yamamoto H, Horie H, Ohyama H, Satoh K, Tano Y, Ota Y, Miyazawa 18

M, Wakabayashi K, Hashizume S Progress with inactivated poliovirus vaccines 19

derived from Sabin strains. Developments in Biologicals, 2001, 105:163–169. 20

17. Liao G, Li R, Li C, Sun M, Li Y, Chu J, Jiang S, Li Q. Safety and immunogenicity of 21

inactivated poliovirus vaccine made from Sabin strains: a phase II, randomized, 22

positive-controlled trial. J Infect Dis. 2012, 205: 237-43. 23

18. Bakker WA, Thomassen YE, van't Oever AG, Westdijk J, van Oijen MG, Sundermann 24

LC, van't Veld P, Sleeman E, van Nimwegen FW, Hamidi A, Kersten GF, van den 25

Heuvel N, Hendriks JT, van der Pol LA. Inactivated polio vaccine development for 26

technology transfer using attenuated Sabin poliovirus strains to shift from Salk-IPV to 27

Sabin-IPV. Vaccine. 2011, 29:7188-7196 28

19. Verdijk P, Rots NY, Bakker WA. Clinical development of a novel inactivated 29

poliomyelitis vaccine based on attenuated Sabin poliovirus strains. Expert Rev 30

Vaccines. 2011, 10:635-44. 31

20. Okada K, Miyazaki C, Kino Y, Ozaki T, Hirose M, Ueda K. Phase II and III Clinical 32

Studies of Diphtheria-Tetanus-Acellular Pertussis Vaccine Containing Inactivated 33


Polio Vaccine Derived from Sabin Strains (DTaP-sIPV). J Infect Dis. 2013. 208: 275-1

83. 2

21. Macadam AJ, Ferguson G, Stone, DM, Meredith J, Knowlson S, Auda G, Almond J, 3

Minor PD. Rational design of genetically stable, live-attenuated poliovirus vaccines of 4

all three serotypes: relevance to poliomyelitis eradication. 2006 J Virol.: 80: 8653–5

8663. 6

22. Burns CC, Campagnoli R, Shaw J, Vincent A, Jorba J, Kew O. Genetic inactivation of 7

poliovirus infectivity by increasing the frequencies of CpG and UpA dinucleotides 8

within and across synonymous capsid region codons. J. Virol. 83(19), 9957–9969 9

(2009). 10

23. Cello J, Paul AV, Wimmer E. Chemical synthesis of poliovirus cDNA: generation of 11

infectious virus in the absence of natural template. Science 297(5583), 1016–1018 12

(2002). 13

24. Coleman JR, Papamichail D, Skiena S, Futcher B, Wimmer E, Mueller S. Virus 14

attenuation by genome-scale changes in codon pair bias. Science 320(5884), 1784–15

1787 (2008). 16

25. Vignuzzi M, Wendt E, Andino R. Engineering attenuated virus vaccines by controlling 17

replication fidelity. Nat. Med. 14(2), 154–161 (2008). 18

26. Wood DJ, Heath AB. Collaborative study for the establishment of a rat bioassay for 19

inactivated poliovaccine. Pharmeuropa special issue BIO 2000–1:25–49. 20

27. Ren R, Costantini F, Gorgacz EI, Lee II, and Racaniello VR, Transgenic mice 21

expressing a human poliovirus receptor: a new model for poliomyelitis. Cell, 1990, 22

63:353–362. 23

28. Koike S, Taya C, Kurata T, Abe W, Ise I, Yonekawa H, Nomoto A. Transgenic mice 24

susceptible to polioviruses. Proceedings of the National Academy of Sciences of the 25

United States of America, 1991, 88:951–955. 26

29. Taffs RE, Chernokhvostova YV, Dragunsky EM, Nomura T, Hioki K, Beuvery EC, 27

Fitzgerald EA, Levenbook IS, Asher DM. Inactivated poliovirus vaccine protects 28

transgenic poliovirus receptor mice against type 3 poliovirus challenge. Journal of 29

Infectious Diseases, 1997, 175:441–444. 30

30. Martin J, Crossland G, Wood DJ, Minor PD. Characterisation of formaldehyde-31

inactivated poliovirus preparations made from live-attenuated strains. 2003. Journal of 32

General Virology. 84:1781-8. 33


31. Maintenance and distribution of transgenic mice susceptible to human viruses: 1

memorandum from a WHO meeting. Bulletin of the World Health Organization, 2

1993, 71:493–502. 3

32. Wood DJ, Heath AB. The second International Standard for anti-poliovirus sera 4

types 1, 2 and 3. Biologicals, 1992, 20:203–211. 5

33. Beale AJ, Mason PJ. The measurement of the D-antigen in poliovirus preparations. J. 6

Hyg., 1962: 60, 113 7

34. WHO good manufacturing practices for pharmaceutical products: main principles. 8

Annex 3 in: WHO Expert Committee on Specifications for Pharmaceutical 9

Preparations. Forty-fifth report. Geneva, World Health Organization, 2011 (WHO 10

Technical Report Series, No. 961) 11

(http://apps.who.int/medicinedocs/documents/s18679en/s18679en.pdf, accessed 16 12

January 2013) 13

35. Good manufacturing practices for biological products. Annex 1 in: WHO Expert 14

Committee on Biological Standardization. Forty-second report. Geneva, World 15

Health Organization, 1992 (WHO Technical Report Series, No. 822) 16

(http://www.who.int/biologicals/publications/trs/en/index.html, accessed 16 January 17

2013). 18

36. Recommendations to Assure the Quality, Safety and Efficacy of Live Attenuated 19

Poliomyelitis Vaccine (oral) Revised 2012. Annex x in WHO Expert Committee on 20

Biological Standardization. Sixty third report. Geneva, World Health Organization, 21

xxxx (WHO Technical Report Series, No. xxx) 22

http://www.who.int/biologicals/vaccines/BS2185_OPV_Post_ECBS_DB_TZ_DBFi23

nal12Feb2013.pdf , accessed 31 0ctober 2013 24

37. Recommendations for the evaluation of animal cell cultures as substrates for the 25

manufacture of biological medicinal products and for the characterization of cell 26

banks Annex 3 in: WHO Expert Committee on Biological Standardization. Sixty first 27

report. Geneva, World Health Organization, 2013 (WHO Technical Report Series, 28

No. 978) http://www.who.int/biologicals/vaccines/TRS_978_Annex_3.pdf, accessed 29

31 0ctober 2013 30

38. Requirements for the use of animal cells as in vitro substrates for the production of 31

biologicals (Addendum 2003). Annex 4 in: WHO Expert Committee on Biological 32

Standardization. Fifty-fourth report. Geneva, World Health Organization, 2005 33

(WHO Technical Report Series, No. 927) 34



2013). 2

39. General requirements for the sterility of biological substances (Requirements for 3

Biological Substances No. 6, revised 1973). Annex 4 in: WHO Expert Committee on 4

Biological Standardization. Twenty-fifth report. Geneva, World Health Organization, 5

1973 (WHO Technical Report Series, No. 530) 6


2013). 8

40. General requirements for the sterility of biological substances. (Requirements for 9

Biological Substances No. 6, revised 1973, amendment 1995). Annex 3 in: WHO 10

Expert Committee on Biological Standardization. Forty-sixth report. Geneva, World 11

Health Organization, 1998 (WHO Technical Report Series, No. 872) 12


2013) 14

41. WHO Guidelines on transmissible spongiform encephalopathies in relation to 15

biological and pharmaceutical products. World Health Organization, 2003 16

42. Requirements for the collection, processing and quality control of blood, blood 17

components and plasma derivatives (revised 1992). Annex 2 in: WHO Expert 18

Committee on Biological Standardization. Forty-third report. Geneva, World Health 19

Organization, 1994 (WHO Technical Report Series, No.840) 20


2013). 22

43. WHO Working Group Meeting to Discuss the Revision of the WHO 23

Recommendations for OPV: TRS No. 904 and 910. Geneva, Switzerland 20−22 July 24

2010. Geneva, World Health Organization, 2010 25

(http://www.who.int/biologicals/vaccines/OPV_Meeting_report_Final_Clean_13Ma26

y2011.pdf, accessed 16 January 2013). 27

44. Westdijk J, Brugmans D, Martin J, van't Oever A, Bakker WA, Levels L, Kersten G. 28

Characterization and standardization of Sabin based inactivated polio vaccine: 29

proposal for a new antigen unit for inactivated polio vaccines. Vaccine. 30

2011,29:3390-7. 31


45. Manin C, Naville S, Gueugnon M, Dupuy M, Bravo de Alba Y, Adam O. Method for 1

the simultaneous assay of the different poliovirus types using surface plasmon 2

resonance technology. Vaccine. 2013,31:1034-9. 3

46. Van Steenis A, van Wezel AL, Sekhuis VM.. Potency testing of killed polio vaccine 4

in rats. Developments in Biological Standardization, 1981, 47:119–128. 5

47. Model guidance for the storage and transport of time- and temperature-sensitive 6

pharmaceutical products. Annex 9 in: WHO Expert Committee on Specifications for 7

Pharmaceutical Preparations. Forty-fifth report. Geneva, World Health 8

Organization, 2011 (WHO Technical Report Series, No.961) 9

(http://www.who.int/medicines/publications/pharmprep/en/index.html, accessed 16 10

January 2013). 11

48. Guidelines on stability evaluation of vaccines. Annex 3 in: WHO Expert Committee 12

on Biological Standardization. Fifty-seventh report. Geneva, World Health 13

Organization, 2011 (WHO Technical Report Series, No. 962) 14

49. Ferguson M, Wood DJ, Minor PD. Antigenic structure of poliovirus in inactivated 15

vaccines. J Gen Virol, 1993, 74:685–90. 16

50. Rezapkin G, Martin J, Chumakov K. Analysis of antigenic profiles of inactivated 17

poliovirus vaccine and vaccine-derived polioviruses by block-ELISA method. 18

Biologicals, 2005, 33:29–39. 19

51. Sawyer,LA, Wood D, Ferguson M, Crainic R, Coen Beuvery E, JMcInnis J, Albrecht 20

P. Potency of Wild-Type or Sabin Trivalent Inactivated Poliovirus Vaccine, by 21

Enzyme-Linked Immunosorbent Assay using Monoclonal Antibodies Specific for 22

Each Antigenic Site Biologicals 1997, 25: 299–306. 23

52. Martín J, Cooper G, Stephens L, Hamill M, Heath AB, Minor P. Report on the WHO 24

pilot study to investigate the collaborative study to establish the 3rd

International 25

Standard (replacement) for inactivated polio vaccine (IPV). WHO/BS/2011.2162 26

53. WHO Guidelines on nonclinical evaluation of adjuvants and adjuvanted vaccines. 27

(in preparation, ECBS 2013) 28

54. Guidelines for good clinical practice (GCP) for trials on pharmaceutical products. In: 29

WHO Expert Committee on the Use of Essential Drugs. Sixth report. Geneva, World 30

Health Organization, 1995, Annex 3 (WHO Technical Report Series, No. 850 31

55. Vidor E, Plotkin SA. Poliovirus vaccine-inactivated. In: Plotkin SA, Orenstein WA, 32

Offit PA, eds. Vaccines. 6th ed. London, UK: Elsevier, 2012:573–97. 33


56. WHO Manual for Virological Investigation of Poliomyelitis 1

http://whqlibdoc.who.int/hq/1997/WHO_EPI_GEN_97.01.pdf 2

57. Salk J, Salk D. Control of influenza and poliomyelitis with killed virus vaccines. 3

Science, 1977, 195, 834–847. 4

58. Salk J. Immune response and minimum requirement for immunity to disease. Scand. 5

J. Infect. Dis. Suppl. 1982, 36:65–67. 6

59. Taranger J, Trollfors B, Knutsson N, Sundh V, Lagergard T, Ostergaard E. 7

Vaccination of infants with a four-dose and a three-dose vaccination schedule. 8

Vaccine, 2000, 18(9–10):884–891. 9

60. Salk J. One-dose immunization against paralytic poliomyelitis using a non-infectious 10

vaccine. Rev. Infect. Dis. 1984, 6(Suppl. 2):S444–S450. 11

61. Guidelines for national authorities on quality assurance for biological products. 12

Annex 2 in: WHO Expert Committee on Biological Standardization. Forty-second 13

report. Geneva, World Health Organization, 1992 (WHO Technical Report Series, 14

No. 822) (whqlibdoc.who.int/trs/WHO_TRS_822.pdf, accessed 16 January 2013). 15

62. Guidelines for independent lot release of vaccines by regulatory authorities. Annex 2 16

in: WHO Expert Committee on Biological Standardization. Sixty first report. Geneva, 17

World Health Organization, 2013 (WHO Technical Report Series, No. 978) 18

http://www.who.int/biologicals/TRS_978_Annex_2.pdf , accessed 31 October 2013 19

63. WHO manual for the establishment of national and other secondary standards for 20

vaccines. Geneva, World Health Organization, 2011 21

(http://whqlibdoc.who.int/hq/2011/WHO_IVB_11.03_eng.pdf, accessed 16 January 22

2013). 23

24


Appendix 1. Overview of the virus seeds used in IPV production 1

2

This appendix gives an overview of the history of virus seeds that are currently used in 3

production or may be used in the future. They include the wild strains used in current IPV 4

production and the attenuated Sabin strains which are considered to pose a lower risk and 5

are being developed as alternative seeds. Novel strains intended to be safer to use in 6

production are also in development. 7

8

1. IPV made from virulent strains 9

Both classical IPV which was developed by Jonas Salk et al. and licensed in 1955 and the 10

enhanced potency IPV which was introduced in the late 1980s are prepared from wild 11

(virulent) polioviruses of three serotypes. The strains selected by Salk were Mahoney, 12

MEF-1, and Saukett, representing types 1, 2, and 3, respectively. The Mahoney strain was 13

isolated in 1941 by Drs. Francis and Mack from the pooled faeces of three healthy children 14

in Cleveland, OH (1). It was subsequently passaged by Salk, including 14 times in living 15

monkeys and twice in monkey testicular cultures (2). The MEF-1 strain was isolated by 16

inoculation of monkeys in Egypt in 1940 (3) during a polio outbreak among allied troops 17

of the Mediterranean Expeditionary Force (hence the name MEF). It was adapted by 18

Schlessinger and Olitsky to growth in mice (4), and then transferred by Salk from the 19

spinal cord of a paralyzed mouse to tissue culture (2). The original Saukett strain was 20

isolated by Salk in 1950 by direct inoculation of tissue culture with a faecal specimen from 21

a paralyzed patient (2). Seed stocks of the viruses were provided by Salk to most 22

manufacturers and were used to establish their virus master seeds. An alternative strain of 23

type 1 poliovirus (Brunhilde) is used by the Staten Serum Institute in Denmark. The strain 24

was isolated in 1939 by David Bodian from a pool of stool specimens from 7 patients in 25

Maryland (5). The strain was provided to the laboratory of Dr. John Enders in Harvard 26

Medical School, and from there to Dr. Arne Svedmyr’s laboratory in Stockholm, Sweden, 27

who supplied SSI with the virus. Table 1 summarizes the history of isolation and early 28

passaging of these viruses. 29

30

Table 1 History of isolation and early passaging of wild polioviruses used in the 31

production of IPV 32

33

Strain

name

Source of isolation Location Yea

r

Referen

ce


Mahone

y

Stool of 3 healthy

children

Clevelan

d, OH

194

1

Francis

and

Mack

MEF-1 CNS of a paralyzed

patient

Egypt 194

1

Van

Rooyen,

1943

Saukett Stool of a paralyzed

patient

USA 195

0

Salk et

al., 1953

Brunhil

de

Stool of 7 patients Marylan

d

193

9

Bodian,

1941

1

Subsequent studies raised questions regarding these strains. The nucleotide sequence of 2

MEF-1 was found to be very close to the sequence of another type 2 strain (Lansing, 3

isolated in 1937 in Michigan), with only 17 nucleotide and 2 amino acid differences (6). 4

Since the strains were isolated 4 years apart in the middle-East and USA, it is unlikely that 5

the similarity represents their natural relatedness. MEF-1 from spinal cords of monkeys 6

was adapted to growth in mice (4) and was found in this early study to be indistinguishable 7

in pathogenicity and immunological properties from Lansing, which was also adapted to 8

growth in mice. A plausible explanation is that the Lansing strain used as a reference strain 9

in Schlessinger and Olitsky’s lab was inadvertently substituted for MEF-1, and all 10

subsequent stocks of MEF-1 are derivatives of Lansing strain. In addition, there are two 11

common variants of MEF-1 differing by a few nucleotides in use in different laboratories 12

and production facilities. 13

14

The Saukett strains obtained from different laboratories and manufacturers differ 15

significantly (7, 8) and the degree of diversity (~10% nucleotide substitutions) 16

demonstrates that they are different strains. Some of the differences were observed in 17

antigenic sites and could affect immunogenicity, suggesting that better characterization of 18

vaccines in the future may need to include determination of the exact nucleotide sequences 19

of virus master seed lots used by manufacturers. 20

21

The flow diagrams in Figures 1, 2 and 3 show the history of the seed virus used to prepare 22

their respective master seed lots claimed to be used by the manufacturers of IPV from 23

Type 1, 2 and 3 strains respectively. The proper names of manufacturers shown on the 24

charts are given in Table 2. 25

26


The figures provide only an overview of the use of different seeds. They were compiled 1

based on a written survey conducted in August 2012 by the WHO among vaccine 2

manufacturers and information obtained from subsequent consultations. They do not 3

indicate any WHO "qualification" or "approval" of the strains or vaccines in the context of 4

this document. 5

6

7


1

Table 2 Proper names of manufacturers shown on the charts are: 2

3

2. IPV made from attenuated strains (Sabin) 4

Once circulation of wild type polio viruses is eliminated, IPV manufacturing 5

establishments will be the biggest potential source of virulent viruses which therefore must 6

be stringently contained to prevent reintroduction in the environment. The Sabin vaccine 7

strains used to manufacture Oral Polio Vaccine (OPV) are less virulent than the wild 8

strains and have been proposed as less hazardous seeds for IPV production to mitigate risks 9

of potential inadvertent release from production facilities. Sabin strains are known to be 10

genetically unstable in infected humans and to some extent in production. To retain the 11

attenuated phenotype they must be propagated under defined and well-controlled 12

conditions. In addition each viral harvest is tested for neurovirulence for use as OPV. As 13

the IPV product is inactivated neurovirulence testing may be unnecessary because the test 14

reflects safety for recipients of live vaccine and not necessarily biosafety during 15

manufacture of inactivated product. OPV strains are considered less transmissible that the 16

wild type so that should they escape from the production facility and start to circulate 17

within communities, they would pose a lesser risk. However they can revert to give rise to 18

circulating vaccine derived strains that are both transmissible and capable of causing 19

outbreaks. Therefore, use of Sabin strains in manufacture of IPV does not completely 20

eliminate biosecurity concerns, but reduces them compared to virulent wild strains. Some 21

testing for the attenuated phenotype may be required together with a level of containment 22

and production consistency. Two IPV containing combination products based on 23

attenuated Sabin strains have been licensed in Japan, and other sIPV vaccines are 24

RIVM National Institute of Public Health and the Environment

(RIVM), Bilthoven, The Netherlands

Bbio Bilthoven Biologicals B.V. (Bbio, former NVI),

Bilthoven, The Netherlands

SSI Staten Serum Institut (SSI), Copenhagen, Denmark

GSK GlaxoSmithKline Vaccines, Wavre, Belgium

Sanofi Pasteur

(France)

Sanofi Pasteur SP, Marcy L’Etoile, France

Sanofi Pasteur

(Canada)

Sanofi Pasteur Ltd. SP, Canada


undergoing clinical evaluation in other countries. The seed viruses used for sIPV are the 1

same as those used for manufacture of OPV. The derivation of Sabin strains was described 2

in the literature (1) and the detailed origin of seed viruses made from them can be found in 3

Appendix 1 of the Recommendations to Assure the Quality, Safety and Efficacy of Live 4

Attenuated Poliomyelitis Vaccine (oral) Revised 2012 (9). 5

6

3. Other strains in development 7

Alternative attenuated strains of poliovirus are being developed by genetic modifications. 8

They are intended to be both attenuated and genetically stable and also to possess low or no 9

infectivity for humans and therefore of negligible transmissibility. Such strains would pose a 10

lower risk of inadvertent release from production facilities or infecting a production worker. 11

Thus they could be used to produce IPV under lower levels of containment. They include 12

strains in which known attenuation determinants are stabilized by targeted genetic changes, 13

alterations in codon usage to introduce multiple mutations to reduce replication efficiency 14

and other strategies intended to greatly impair virus growth in a way that will be retained on 15

passage. The stability and the phenotypes of these strains will require confirmation. 16

17

18


1

2

Figure 1: Type 1 IPV 3

Francis and Mack

1941

Jonas Salk

Sanofi Pasteur

(Canada)

SSI

(Denmark)

RIVM

(The

Netherlands)

Sanofi Pasteur

(France)

GSK

(Belgium)

Mahoney

David Bodian

1939

Brunhilde

John Enders

Arne Svedmyr

Bbio

(The

Netherlands)


1 2


4

Van Rooyen et al

1941

Schlessinger, Morgan,

and Olitsky

Jonas Salk

Sanofi Pasteur

(Canada) SSI

(Denmark)

RIVM

(The

Netherlands)

Sanofi Pasteur

(France)

GSK

(Belgium)

MEF-1

Bbio

(The

Netherlands)


1 2


4

5

6

References 7

8

1 Sabin, A.B. and L.R. Boulger, History of Sabin attenuated poliovirus oral live 9

vaccine strains. Journal of Biological Standardization, 1973. 1: 115-118. 10

2. Salk, J.E., Studies in human subjects on active immunization against poliomyelitis. I. 11

A preliminary report of experiments in progress. Journal of the American Medical 12

Association, 1953. 151(13): 1081-98. 13

3. van Rooyen, C.E. and A.D. Morgan, Poliomyelitis. Experimental work in Egypt. 14

Edinburgh Medical Journal, 1943. 50: 705-720. 15

4. Schlesinger, R.W., I.M. Morgan, and P.K. Olitsky, Transmission to Rodents of 16

Lansing Type Poliomyelitis Virus Originating in the Middle East. Science, 1943. 17

98:452-4. 18

5. Howe, H.A. and D. Bodian, Poliomyelitis in the chimpanzee; a clinical pathological 19

study. Bulletin of the Johns Hopkins Hospital, 1941. 69: 149-181. 20

Jonas Salk

1950

Sanofi Pasteur

(Canada)

SSI

(Denmark)

RIVM

(The

Netherlands)

Sanofi Pasteur

(France)

GSK

(Belgium)

Sauke

Bbio

(The

Netherlands)


6. Dragunsky, E.M., A.P. Ivanov, V.R. Wells, A.V. Ivshina, G.V. Rezapkin, S. Abe, 1

S.G. Potapova, J.C. Enterline, S. Hashizume, and K.M. Chumakov, Evaluation of 2

immunogenicity and protective properties of inactivated poliovirus vaccines: a new 3

surrogate method for predicting vaccine efficacy. Journal of Infectious Diseases, 4

2004. 190: 1404-1412. 5

7. Minor, P.D., G.C. Schild, M. Ferguson, A. MacKay, D.I. Magrath, A. John, P.J. 6

Yates, and M. Spitz, Genetic and antigenic variation in type 3 polioviruses: 7

characterization of strains by monoclonal antibodies and T1 oligonucleotide 8

mapping. Journal of General Virology, 1982. 61: 167-176. 9

8. Huovilainen, A., L. Kinnunen, T. Poyry, L. Laaksonen, M. Roivainen, and T. Hovi, 10

Poliovirus type 3/Saukett: antigenic and structural correlates of sequence variation 11

in the capsid proteins. Virology, 1994. 199: 228-32. 12

9 Recommendations to Assure the Quality, Safety and Efficacy of Live Attenuated 13

Poliomyelitis Vaccine (oral) Revised 2012. Annex x in WHO Expert Committee on 14

Biological Standardization. Sixty third report. Geneva, World Health Organization, 15

xxxx (WHO Technical Report Series, No. xxx). 16

http://www.who.int/entity/biologicals/vaccines/BS2185_OPV_Post_ECBS_DB_TZ17

_DBFinal12Feb2013.pdf 18

19

20


Appendix 2. In vivo potency assay of IPV 1

2

Tests for evaluating the potency of inactivated polio vaccines include an in vivo assay for 3

immune response. The WHO International Standard has not been validated for use in in 4

vivo tests. Because of the diversity in the reactivity of vaccines, it is unlikely that an 5

International Standard will be suitable for the standardization of in vivo assays of vaccines 6

from all manufacturers. If this is shown to be the case, manufacturers should establish a 7

product specific reference preparation which is traceable to a lot of vaccine shown to be 8

efficacious in clinical trials. The NRA should approve the reference preparation used and 9

agree with the potency limits applied. The performance of this reference vaccine should be 10

monitored by trend analysis using relevant test parameters and it should be replaced when 11

necessary. 12

13

In recent investigations the in vivo potency assay in rats has been standardized (1) and 14

shown to have advantages over previously described in vivo tests for IPV (2). 15

16

A suitable in vivo assay method consists of intramuscular injection into the hind limb(s) of 17

rats of four dilutions of the vaccine to be examined and a reference vaccine, using for each 18

dilution a group of not fewer than 10 rats of a suitable strain, and which are specific 19

pathogen-free. The number of animals used should enable the calculation of potency with 20

95% confidence limits within the 25-400% range. The number of dilutions used and the 21

number of animals used per dilution may be different from that specified here, provided 22

that any alternative scheme gives at least the same sensitivity in the test. For each dilution, 23

the weight of the individual animals should not vary by more than 20% from the group 24

mean. An inoculum of 0.5 ml is used per rat. The dose range is chosen such that a dose 25

response to all three poliovirus types is obtained. The animals are bled after 20–22 days. 26

Neutralizing titres against all three poliovirus types are measured separately using 100 27

CCID50 of the Sabin strains as challenge viruses, Vero or Hep-2C as indicator cells, and 28

neutralization conditions of 3h at 35–37 °C followed by 18h at 2–8 °C. Results should be 29

read after fixation and staining after 7 days of incubation at 35 °C. For the antibody assay 30

to be valid, the titre of each challenge virus must be shown to be within the range of 10 - 31

1000 CCID50 and the neutralizing antibody titre of a control serum must be within two 2-32

fold dilutions of its geometric mean titre. The potency is calculated by comparison of the 33

proportion of animals defined as responders to the test vaccine and the reference vaccine 34


by the probit method. To define an animal as a responder, it is necessary to establish a cut-1

off neutralizing antibody titre for each poliovirus type. Owing to between-laboratory 2

variation, it is not possible to define cut-off values that could be applied by all laboratories. 3

Rather, the cut-off values should be determined by each laboratory, based on a minimum 4

series of three tests with the reference vaccine. The mid-point on a log2 scale of the 5

minimum and maximum geometric mean titres of the series of three or more tests is used 6

as the cut off value. For each of the three poliovirus types, the potency of the vaccine 7

should not be statistically significantly less than that of the reference preparation. The test 8

is not valid unless: 9

• the median effective dose (ED50) for both the test and reference vaccines lies 10

between the smallest and the largest doses given to the animals; 11

• the statistical analysis shows no significant deviation from linearity or parallelism; 12

• the confidence limits of the estimated relative potency fall between 25% and 400% 13

of the estimated potency. 14

15

Laboratories that have established the parallel line method of analysis of antibody titres for 16

the rat test may use it instead of converting titres to proportions of responders as in the 17

probit method of analysis. 18

19

Laboratories are encouraged to validate alternative methods for the assay of neutralizing 20

antibody to reduce the use of live polioviruses in laboratories. If IPV is formulated with 21

other antigens into a combination vaccine, then the suitability of performing the rat 22

immunogenicity test will have to be determined. If the immunogenicity test is performed, 23

the potency of the final bulk for each virus type should be approved by the national control 24

authority. 25

26

The development of transgenic mice that express the human poliovirus receptor (TgPVR 27

mice) (3, 4, 5) has led to the development of an immunization/challenge model that may be 28

useful for assessment of vaccine efficacy. This test is not proposed for lot release. Any 29

work with transgenic mice should comply with WHO guidelines (6). 30

31

References 32


1. Wood DJ, Heath AB. Collaborative study for the establishment of a rat bioassay for 1

inactivated poliovaccine. Pharmeuropa special issue BIO 2000–1:25–49. 2

2. Van Steenis A van Wezel AL, Sekhuis VM. Potency testing of killed polio vaccine 3

in rats. Developments in Biological Standardization, 1981, 47:119–128. 4

3. Ren R, Costantini F, Gorgacz EI, Lee II, and Racaniello VR. Transgenic mice 5

expressing a human poliovirus receptor: a new model for poliomyelitis. Cell, 1990, 6

63: 353−362. 7

4. Koike S, Taya C, Kurata T, Abe W, Ise I, Yonekawa H, Nomoto A. Transgenic 8

mice susceptible to poliovirus. Proceedings of the National Academy of Sciences of 9

the United States of America, 1991, 88:951−955. 10

5. Dragunsky E, Nomura T, Karpinski K, Furesz J, Wood DJ, Pervikov Y, Abe S, 11

Kurata T, Vanloocke O, Karganova G, Taffs R, Heath A, Ivshina A, Levenbook I. 12

Transgenic mice as an alternative to monkeys for neurovirulence testing of live oral 13

poliovirus vaccine: validation by a WHO collaborative study. Bulletin of the World 14

Health Organization, 2003, 81:251−260. 15

6. Maintenance and distribution of transgenic mice susceptible to human viruses: 16

memorandum from a WHO meeting. Bulletin of the World Health Organization, 17

1993, 71:493−502. 18

19

20


Appendix 3. Model summary protocol for manufacturing and control of poliomyelitis 1

vaccine (inactivated) 2

3

The following protocol is intended for guidance, and indicates the information that should be 4

provided as a minimum by the manufacturer to the NRA. 5

6

Information and tests may be added or deleted as required by the NRA, if applicable. 7

It is thus possible that a protocol for a specific product may differ in detail from the model 8

provided. The essential point is that all relevant details demonstrating compliance with the 9

license and with the relevant WHO recommendations of a particular product should be given 10

in the protocol submitted. 11

The section concerning the final lot must be accompanied by a sample of the label and a 12

copy of the leaflet that accompanies the vaccine container. If the protocol is being submitted 13

in support of a request to permit importation, it should also be accompanied by a lot release 14

certificate from the NRA of the country in which the vaccine was produced/released stating 15

that the product meets the national requirements as well as Part A recommendations of this 16

document published by WHO. 17

18

Summary information on the finished product (final vaccine lot)

International name: _______________________________________

Trade name: _______________________________________

Product licence (marketing

authorization) number

_______________________________________

Country: _______________________________________

Name and address of

manufacturer:

_______________________________________

Name and address of licence

holder if different:

_______________________________________

Virus strain _______________________________________

Origin and short history _______________________________________

Finished product (Final lot) _______________________________________

Batch number _______________________________________


Final bulk: _______________________________________

Type of container: _______________________________________

Number of doses per

container:

_______________________________________

Number of filled containers

in this final lot:

_______________________________________

Volume of single human

dose

Composition (D-antigen

unit) of a single human dose

Type 1 Type 2 Type 3

Bulk numbers of monovalent bulk Type 1 Type 2 Type 3

suspensions:

Site of manufacture of each

monovalent bulk:

_______________________________________

Date of manufacture of each

monovalent bulk:

_______________________________________

Date of manufacture of

trivalent bulk (blending):

_______________________________________

Date of manufacture of final

bulk

_______________________________________

Date of manufacture

(filling) of final lot:

_______________________________________

Date on which last

determination of potency

was started or date of start

of period of validity:

_______________________________________

Shelf-life approved

(months):

_______________________________________

Expiry date: ___________________________________

Storage conditions: _______________________________________

Nature and concentration of

Stabilizer

_______________________________________

Nature of any antibiotics

present in

vaccine and amount per

human dose

_______________________________________

Release date: _______________________________________

1

2


Starting materials 1

The information requested below is to be presented on each submission. Full details on 2

master and working seed-lots should be provided upon first submission only and whenever a 3

change has been introduced. 4

5

The following sections are intended for the recording of the results of the tests performed 6

during the production of the vaccine, so that the complete document will provide evidence 7

of consistency of production; thus if any test has to be repeated, this must be indicated. Any 8

abnormal result must be recorded on a separate sheet. 9

10

If any cell lot or virus harvest intended for production was rejected during the control 11

testing, this should also be recorded either in the following sections or on a separate sheet. 12

13

Control of source materials A.3

Virus seed A.3.1 (Every submission) Vaccine virus strain(s) and serotype(s):

____________________________________

___ Substrates used for preparing seed lots:

____________________________________

___ Origin and short history:

____________________________________

___ Authority who approved virus strains

____________________________________

___

Date of approval ____________________________________

___

Information and seed lot preparation A.3.1.3 (Every submission) Virus Master seed (VMS) and virus working seed (VWS) (to be provided upon first

submission only and whenever a change has been introduced)

Strain used

__________________________________

____ Source of VMS __________________________________

____ VMS and VWS lot number __________________________________

____ Name and address of manufacturer __________________________________

____

VWS passage level from VMS __________________________________

____

Dates of inoculation __________________________________

____


Dates of harvest __________________________________

____ Numbers of containers __________________________________

____ Conditions of storage __________________________________

____ Dates of preparation __________________________________

____ Maximum passages levels authorized __________________________________

____

Tests on Virus Master seed (VMS) and virus working seed (VWS) (First submission

only)

Tests for bacteria, fungi and

mycoplasma

Tests for bacteria and fungi Method used

__________________________________

_____ Number of vials tested

__________________________________

_____ Volume of inoculum per vial

__________________________________

_____ Volume of medium per vial

__________________________________

_____ Observation period (specification)

__________________________________

_____ Incu

bati

on

Med

ia

used

Inocul

um

Date of start of test Date of

end of

test

Results

20–

25 °

___

___

__

_____

____

___________ _______

____

______

____

30–

36 °

___

___

__

_____

____

___________ _______

____

______

____

Neg

ativ

e

cont

rol

___

___

__

_____

____

___________ _______

____

______

____

Test for mycoplasma

Method used

_______________________________________

Volume tested

_______________________________________

Media used

_______________________________________

Temperature of incubation

_______________________________________

Observation period

(specification) _______________________________________


Positive controls (list of

species used and results) _______________________________________

Date of start of

test

Date of

end of test

Results

Sub cultures at 3rd

day

____________

___

_________

______

___________

____ Sub cultures at 7

th day

____________

___

_________

______

___________

____

Sub cultures at 14th day

____________

___

_________

______

___________

____ Sub cultures at 21th day

____________

___

_________

______

___________

____

Indicator cell-culture method (if

applicable)

Cell substrate used

______________________________________

_ Inoculum

______________________________________

_

Date of test

______________________________________

_ Passage number

______________________________________

_ Negative control

______________________________________

_ Positive controls

______________________________________

_ Date of staining

______________________________________

_

Results

______________________________________

_

Virus titration Date of test:

______________________________________

Reference batch number:

______________________________________

_ Date of test:

______________________________________

Result:

______________________________________

_

Identity test

Method used

______________________________________

Date test on

______________________________________

Date test off

______________________________________

Result

______________________________________


Test in rabbit kidney cell cultures:

No. of cell cultures

______________________________________

Total volume inoculated

______________________________________

Period of observation

______________________________________

Result

______________________________________

Test for adventitious agents

Date(s) of satisfactory test(s) for

freedom from adventitious agent

______________________________________

Volume of virus seed samples for

neutralization and testing ______________________________________

Batch number of antisera used for

neutralization virus seed ______________________________________

Method used

______________________________________

Date test on

______________________________________

Date test off

______________________________________

Result

______________________________________

Absence of SV40

Method used

______________________________________

Date test on

______________________________________

Date test off

______________________________________

Results

______________________________________

_

Tests for neurovirulence (if

applicable)

In vitro tests: MAPREC test for

attenuated strains (if applicable)

MAPREC

Date of test:

______________________________________

_ Type 1

Ratio of % of the sum of both

mutations 480-A, 525-C of bulk

sample to the International Standard

or

level of mutations:

______________________________________

_

Result of test of consistency of

production: ______________________________________

_


Result of test of comparison with

the International Standard: ______________________________________

_ Type 2

Ratio of % 481-G of bulk sample to

the International Standard or

level of mutations:

______________________________________

_


production:

______________________________________

_ Result of test of comparison with

the International Standard: ______________________________________

_ Type 3

Ratio of %472C of bulk sample to

the International Standard or

level of mutations:

______________________________________

_


production

______________________________________

_

Result of test of comparison with

the International Standard ______________________________________

_

In vivo tests for neurovirulence

Neurovirulence test in monkeys:

______________________________________

_ Result of blood serum test in

monkeys prior to inoculation:

______________________________________

_

Number and species of monkeys

inoculated:

______________________________________

_

Quantity (CCID50) inoculated in

each test monkey:

______________________________________

_

Number of “valid” monkeys

inoculated with test sample: ______________________________________

_ Number of positive monkeys

observed inoculated with test

sample or with reference:

______________________________________

_

Reference preparation:

______________________________________

_ Number of "valid" monkeys

inoculated with reference:

______________________________________

_

Number of positive monkeys

observed:

______________________________________

_

Mean Lesion Score of test sample:

______________________________________

_ Mean Lesion Score of reference:


(see also attached forms giving

details of histological observations

and assessment)

______________________________________

_

C1 constant value: ______________________________________

_

Neurovirulence test in transgenic

mice for attenuated strains (if

applicable)

Strain of mice inoculated:

______________________________________

_ For each dose of the seed sample:

______________________________________

_ Number of mice inoculated:

______________________________________

_ Number of mice excluded from

evaluation:

______________________________________

_

Number of mice paralysed:

______________________________________

_ Results of validity tests for each

dose of the reference virus:

______________________________________

_

Number of mice inoculated:

______________________________________

_ Number of mice excluded from

evaluation:

______________________________________

_

Number of mice paralysed:

______________________________________

_ Virus assay results for each dose

inoculated (residual inoculums): ______________________________________

_ Paralysis rates for test vaccine at

each dose:

Paralysis rates for reference virus at

each dose: ______________________________________

_ Results:

______________________________________

_ Log odds ratio:

______________________________________

_ L1 and L2 values:

______________________________________

_ Pass/fail decision:

______________________________________

_

Cell banks (Every submission) Information on cell banking system

Name and identification of substrate


Origin and short history

______________________________________

_ Authority that approved cell bank ______________________________________

_ Master cell bank (MCB) and

working cell bank (WCB) lots

number and date of preparation

______________________________________

_

Date MCB and WCB were

established

______________________________________

_

Date of approval by NRA

______________________________________

_ Total number of ampoules stored

______________________________________

_ Passage level (or no. of population

doublings) of cell bank ______________________________________

_ Maximum passage approved

______________________________________

_ Storage conditions

______________________________________

_

Method of preparation of cell bank

in terms of no. of freezes and efforts

made to ensure that a homogeneous

population is dispersed into the

ampoules

______________________________________

_

Tests on MCB and WCB A. 3.2 (First submission only) Percentage of total cell-bank

ampoules tested

______________________________________

_

Identification of cell substrate

______________________________________

_ Method

______________________________________

_ Specification

_____________________________________

__ Date of test

_____________________________________

__ Result

_____________________________________

__ Growth characteristics

_____________________________________

__ Morphological characteristics

_____________________________________

__ Immunological marker

_____________________________________

__ Cytogenetic data

_____________________________________

__ Biochemical data

_____________________________________

__


Results of other identity tests

_____________________________________

__

Tests for adventitious agents

Method used

_____________________________________

__ Number of vials tested

_____________________________________

__ Volume of inoculum per vial

_____________________________________

__ Date test on

_____________________________________

__ Date test off

_____________________________________

__

Freedom from bacteria, fungi and

mycoplasmas

Tests for bacteria and fungi

Method used:

______________________________________

_ Number of vials tested:

______________________________________

_ Volume of inoculum per vial:

______________________________________

_ Volume of medium per vial:

______________________________________

_ Observation period (specification)

______________________________________

_ Incubation Media

used

Inoculum Date of

start of

test

Date of

end of

test

Res

ults

20–25 °C

______

____

________

__

________

___

_______

____

___

___

___ 30–36 °C

______

____

________

__

________

___

_______

____

___

___

___ Negative

control

______

____

________

__

________

___

_______

____

___

___

___

Test for mycoplasma

Method used:

___________________________________

____

Volume tested:

___________________________________

____

Media used:

___________________________________

____


Temperature of incubation:

___________________________________

____

Observation period (specification)

:

___________________________________

____

Positive controls (list of species

used and results) : ___________________________________

____

Date of start of test Date of end of

test

Results

Subcultures at day 3

_______________ _____________

__

_________

______ Subcultures at day 7

_______________ _____________

__

_________

______

Subcultures at day

14

_______________ _____________

__

_________

______

Subcultures at day

21

_______________ _____________

__

_________

______


applicable)

Cell substrate used:

_______________________________________

Inoculum:

_______________________________________

Date of test:

_______________________________________

Passage number:

_______________________________________

Negative control:

_______________________________________

Positive controls:

_______________________________________

Date of staining:

_______________________________________

Results:

_______________________________________

1

2

Control of vaccine production

(section A.4.1)

Virus type (1, 2 or 3) (A separate

protocol should be completed for

each type.)

Control of production cell

cultures

_____________________________________


__

Lot number of MCB: _____________________________________

__

Lot number of WCB: _____________________________________

__

Date of thawing of ampoule of

WCB: _____________________________________

__

Passage number of production cells: _____________________________________

__

Date of preparation of control cell

cultures: _____________________________________

__

Results of microscopic observation: _____________________________________

__

Tests on control cell cultures

Ratio of control to production cell

cultures:

_____________________________________

__

Incubation conditions:

_____________________________________

__

Period of observation of cultures:

Dates observation started/ended:

_____________________________________

__

Ratio or proportion of cultures

discarded for nonspecific reasons:

_____________________________________

__

Results of observation:

_____________________________________

__

Tests for haemadsorbing viruses

Quantity of cell tested:

_____________________________________

__

Method used:

_____________________________________

__


Date of start of test:

_____________________________________

__

Date of end of test:

_____________________________________

__

Results:

_____________________________________

__

Tests for adventitious agents on

supernatant culture fluids

Method used:

_____________________________________

__


_____________________________________

__


_____________________________________

__

Result:

_____________________________________

__

Identity test

Method used:

_____________________________________

__


_____________________________________

__


_____________________________________

__

Result:

_____________________________________

__

Control of vaccine production

Control of production cell cultures Observation of cultures for

adventitious agents on day of

inoculation

Results of microscopic observation: _____________________________________

__


Control of single harvests (section

A.4.3)

Freedom from bacteria, fungi and

mycoplasmas


Method used:

______________________________________

_ Number of vials tested:

______________________________________

_ Volume of inoculum per vial:

______________________________________

_ Volume of medium per vial:

______________________________________

_


______________________________________

_ Incubation Media

used

Inoculu

m

Date of

start of

test

Date

of end

of test

Result

20–25 °C

______

____

______

____

______

_____

_____

_____

_____

_____

30–36 °C

______

____

______

____

______

_____

_____

_____

_

_____

_____

Negative control

______

____

______

____

______

_____

_____

____

_____

_____

Test for mycoplasma

Method used:

______________________________________

_ Volume tested:

______________________________________

_ Media used:

______________________________________

_

Temperature of incubation:

______________________________________

_ Observation period (specification) :

______________________________________

_ Positive controls (list of species used

and results) : ______________________________________

_

Date of start of

test

Date of end of test Result


_____________

__

_______________ ___________

____


_____________

__

_______________ ___________

____


_____________

__

_______________ ___________

____


_____________

__

_______________ ___________

____



applicable)

Cell substrate used:

______________________________________

_ Inoculum:

______________________________________

_

Date of test:

______________________________________

_ Passage number:

______________________________________

_ Negative control:

______________________________________

_ Positive controls:

______________________________________

_ Date of staining:

______________________________________

_ Results:

______________________________________

_

Virus titration

Date of test:

______________________________________


______________________________________

_ Date of test:

______________________________________

Result:

______________________________________

_

Identity test

Method used:

______________________________________

_ Date of start of test:

______________________________________


______________________________________

_ Result:

______________________________________

Monovalent pools before

inactivation (A4.4)

Test for residual cellular DNA ______________________________________

_ Method used:

______________________________________


______________________________________


______________________________________

_

Virus titration

Date of test: ______________________________________



______________________________________

_

Date of test:

______________________________________

Result:

______________________________________

_

Identity test

Method used:

______________________________________

_


______________________________________


______________________________________

_ Result:

______________________________________

D-antigen content

Method used:

______________________________________


______________________________________


______________________________________

_ Result:

______________________________________

Protein content

Method used:

______________________________________

_


______________________________________

_ Date of end of test:

______________________________________

_

Result:

______________________________________

Details of filtration and/or

clarification and/or purification (if

applied)

Date

______________________________________

_

Additional tests on monovalent pools produced from Sabin vaccine seeds or from novel

seeds derived by recombinant DNA technology eg In vitro tests: MAPREC test for

attenuated strains or in vivo Neurovirulence test in transgenic mice for attenuated strains

(if applicable) (see above in tests on virus seeds) ______________________________________

Inactivation of monovalent

product:


Agent(s) and concentration

______________________________________

_ Date of start of inactivation

______________________________________

_ Date of taking first sample

______________________________________

_ Date of completion of inactivation

______________________________________

_ Test for effective inactivation (after

removal/neutralization of inactivating

agent):

______________________________________

_

Sample size tested

______________________________________

_ Date of first sample

______________________________________

_ Date of second sample

______________________________________

_ Details of testing procedure

______________________________________

_ Period of observation of cell cultures

______________________________________

_ Period of observation of subcultures

______________________________________

_ Result

______________________________________

_

Result of challenge of used culture with

live virus

______________________________________

_

Tests for bacteria, fungi and

mycoplasma


Method used

______________________________________

_ Number of vials tested

______________________________________

_ Volume of inoculum per vial

______________________________________

_ Volume of medium per vial

______________________________________

_ Observation period (specification)

______________________________________

_

Incubatio

n Media

used

Inoculu

m

Date of

start of

test

Date of

end of

test

Result

20–25 °

______

____

______

____

_______

___

_______

____

_________

_ 30–36 °

______

____

______

____

_______

___

_______

____

_________

_


Negative

control

______

____

______

____

_______

___

_______

____

_________

_

D-antigen content

Method used:

_____________________________________

__ Date of start of test:

_____________________________________


_____________________________________

__

Result:

_____________________________________

_

Trivalent bulk product

(monovalent pools incorporated)

Date of preparation

_____________________________________

__ Preservative (if added, type and

concentration)

_____________________________________

__

Tests on trivalent bulk (A.4.6)

Test for absence of infective poliovirus:

_____________________________________

__ Sample size tested

_____________________________________

__

Details of testing procedure

_____________________________________

__

Period of observation of cell cultures

_____________________________________

__

Period of observation of subcultures

_____________________________________

__

Result

_____________________________________

__


Method used

_____________________________________


_____________________________________

__ Volume of inoculum per vial

_____________________________________

__ Volume of medium per vial

_____________________________________

__


_____________________________________

__ Incubatio

n Media used Inocul

um

Date test

began

Result

20–25 °

__________ _____

_____

_______

___

__________


30–36 °

__________ _____

_____

_______

___

__________

Negative

control __________ _____

_____

_______

___

__________

Residual formaldehyde

Method used

_____________________________________

__

Result

_____________________________________

__

D-antigen content

Method used:

_____________________________________

__


_____________________________________


_____________________________________

__

Result:

_____________________________________

_

Control of final bulk (A.4.7)


Method used

_____________________________________


_____________________________________

__

Volume of inoculum per vial

_____________________________________

__

Volume of medium per vial

_____________________________________

__ Observation period (specification)

_____________________________________

__

Incubati

on Media

used

Inoculu

m

Date of

start of

test

Date of

end of

test

Result

20–25 °

______

____

______

____

_______

___

________

__

__________

30–36 °

______

____

______

____

_______

___

________

__

__________

Negativ

e

control

______

____

______

____

_______

___

________

__

__________

Potency test:

Results (and date) of in vitro tests

(D-antigen) _____________________________________

__ Results (and date) of in vivo tests, (in

rats) if performed _____________________________________

__ Result

_____________________________________

__


Preservative content (if applicable)

Date of test

_____________________________________

__

Method used

_____________________________________

__ Result

_____________________________________

__

Endotoxin content

Date of test

_____________________________________

__ Method used

_____________________________________

__ Result

_____________________________________

__

Adjuvant (if applicable)

Date of test

_____________________________________

__

Method used

_____________________________________

__

Result

_____________________________________

__

Tests on final lot (A.6)

Filling and containers (section A.5)

Total volume for final filling:

_____________________________________

__

Date of filling:

_____________________________________

_ Number of vials after inspection:

_____________________________________

__ Number of vials filled:

_____________________________________

Control tests on final lot (A.6)

Inspection of final containers

Appearance:

_____________________________________

__ Date of test:

_____________________________________

_ Results:

_____________________________________

__

Identity test

Method used:

_____________________________________

__ Date of start of test:

_____________________________________


_____________________________________

__


Result:

_____________________________________

_


Method used

_____________________________________


_____________________________________

__

Volume of inoculum per vial

_____________________________________

__

Volume of medium per vial

_____________________________________

__ Observation period (specification)

_____________________________________

__

Incubati

on Medi

a

used

Inocul

um

Date of

start of test

Date of

end of

test

Result

20–25 °

____

____

_____

_____

_________

_

_______

___

_________

30–36 °

____

____

_____

_____

_________

_

_______

___

_________

Negativ

e

control

____

____

_____

_____

_________

_

_______

___

_________

General safety test (if applicable)


_____________________________________


_____________________________________

Result:

_____________________________________

Potency test:

Results (and date) of in vitro tests

(D-antigen)

_______________________________________

Results (and date) of in vivo tests,

(in rats) if performed

_______________________________________

Protein content:

Content of protein in mg per human

dose

_______________________________________

Serum protein tests (if applicable):

Result

_______________________________________


Preservative content (if

applicable)

Date of test _______________________________________

Method used _______________________________________

Result _______________________________________

Endotoxin content

Date of test _______________________________________

Method used _______________________________________

Result _______________________________________

Test for residual formaldehyde

Date of test _______________________________________

Method used _______________________________________

Result _______________________________________

pH

Date of test _______________________________________

Result _______________________________________

Adjuvant (if applicable)

Date of test _______________________________________

Method used _______________________________________

Result _______________________________________

Residual antibiotics (if applicable)

Date of test

_______________________________________

Method used

_______________________________________

Result

_______________________________________

1

2

Submission addressed to NRA 3

4

Name of Head of Production (typed) 5

6

Certification by the person from the control laboratory of the manufacturing company 7

taking over responsibility for the production and control of the vaccine: 8

9

I certify that lot no. ______________ of trivalent poliomyelitis vaccine (inactivated) 10

satisfied Part A of the W H O Recommendations to Assure the Quality, Safety and Efficacy 11

of Poliomyelitis Vaccine (inactivated), revised xxxx. 12

13


Signature: __ 1

2

Name (typed): ______________________________________________ 3

Date: ____ 4

5

6


Appendix 4. Model certificate for the release of poliomyelitis vaccine 1

(inactivated) by national regulatory authorities 2

3

Lot release certificate 4

5

Certificate no. ________________ 6

7

The following lot(s) of poliomyelitis vaccine (inactivated) produced by 8

____________________________1 in _______________

2 whose numbers appear on 9

the labels of the final containers, complies with the relevant specification in the 10

marketing authorization3 and provisions for the release of biological products and 11

Parts A4, of WHO recommendations to assure the quality, safety and efficacy of 12

poliomyelitis vaccines (inactivated) (_____)5 and comply with Good Manufacturing 13

Practices for Pharmaceutical Products6,Good Manufacturing Practices for Biological 14

Products7 and Guidelines for Independent Lot Release of Vaccines by Regulatory 15

Authorities 8. 16

The release decision is based on 17

_____________________________________________9. 18

19

The certificate may include the following information: 20

• Name and address of manufacturer; 21

• Site(s) of manufacturing; 22

• Trade name and/common name of product; 23

• Marketing authorization number; 24

• Lot number(s) (including sub-lot numbers, packaging lot numbers if 25

necessary); 26

• Type of container; 27

• Number of doses per container; 28

• Number of containers/lot size; 29

• Date of start of period of validity (e.g. manufacturing date) and/or expiry 30

date; 31

• Storage condition; 32


• Signature and function of the authorized person and authorized agent to issue 1

the certificate; 2

• Date of issue of certificate; and 3

• Certificate number. 4

5

The director of the NRA (or authority as appropriate): 6

Name (typed) _______________________________________________ 7

Signature __________________________________________________ 8

Date ______________________________________________________ 9

10

Footnote 11 1 Name of manufacturer. 12

2 Country of origin. 13

3 If any national requirements are not met, specify which one(s) and indicate why 14

release of the lot(s) has nevertheless been authorized by the NRA. 15 4

With the exception of provisions on distribution and shipping, which the NRA may 16

not be in a position to assess. 17 5 WHO Technical Report Series, No. (xx, xxxx). 18

6 WHO Technical Report Series, No. 961, 2011, Annex 3. 19

7 WHO Technical Report Series, No. 822, 1992, Annex 1. 20

8 WHO Technical Report Series, No. 978, 2013, Annex 2 21

9 Evaluation of summary protocol, independent laboratory testing, and/or specific 22

procedures laid down in defined document etc., as appropriate 23

24

recommendations to assure the quality, safety and efficacy ......who/ ipv_draft/ 2 december 2013...

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