recommendations to assure the quality, safety and efficacy of … · who/bs/2014.2233 page 2 1 2...

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WHO/BS/2014.2233 3

ENGLISH ONLY 4

5

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION 6

Geneva, 13 to 17 October 2014 7

8

9

Recommendations to assure the quality, safety and efficacy of 10

poliomyelitis vaccine (inactivated) 11

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Proposed replacement of: TRS 910, Annex 2 13

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NOTE: 16

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This document has been prepared for the purpose of inviting comments and suggestions on the proposals 18

contained therein, which will then be considered by the Expert Committee on Biological Standardization 19

(ECBS). Publication of this draft is intended to provide information about the proposed WHO 20

Recommendations to assure the quality, safety and efficacy of poliomyelitis vaccine (inactivated) 21

to a broad audience and to improve transparency of the consultation process. 22

23

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

Committee. Written comments proposing modifications to this text MUST be received by 25 22 September 2014 in the Comment Form available separately and should be addressed to the World 26

Health Organization, 1211 Geneva 27, Switzerland, attention: Department of Essential Medicines and Health 27

Products (EMP). Comments may also be submitted electronically to the Responsible Officer: Dr TieQun Zhou 28

at email: [email protected]. 29

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The outcome of the deliberations of the Expert Committee will be published in the WHO Technical Report 31

Series. The final agreed formulation of the document will be edited to be in conformity with the "WHO style 32

guide" (WHO/IMD/PUB/04.1). 33

© World Health Organization 2014 34

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 35 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: [email protected]). Requests for 36 permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to 37 WHO Press, at the above address (fax: +41 22 791 4806; e-mail: [email protected]). 38

The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever 39 on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or 40 concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may 41 not yet be full agreement. 42 43 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the 44 World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names 45 of proprietary products are distinguished by initial capital letters.46

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WHO/BS/2014.2233

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All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. 2 However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for 3 the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages 4 arising from its use. The named authors alone are responsible for the views expressed in this publication. 5

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Recommendations and guidelines published by 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 Guidelines 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.

WHO/BS/2014.2233

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Contents 2

3

Introduction ..................................................................................................................................................... 5 4

Scope ............................................................................................................................................................... 6 5

General considerations .................................................................................................................................... 7 6

Part A. Manufacturing recommendations ..................................................................................................... 12 7

A.1 Definitions .......................................................................................................................................... 12 8

A.2 General manufacturing recommendations ......................................................................................... 17 9

A.3 Control of source materials ................................................................................................................ 17 10

A.4 Control of vaccine production ............................................................................................................ 25 11

A.5 Filling and containers ......................................................................................................................... 42 12

A.6 Control tests on the final lot ............................................................................................................... 42 13

A.7 Records............................................................................................................................................... 45 14

A.8 Retained samples ................................................................................................................................ 45 15

A.9 Labelling ............................................................................................................................................ 45 16

A.10 Distribution and shipping ................................................................................................................. 46 17

A.11 Stability, storage and expiry date ..................................................................................................... 46 18

Part B. Nonclinical evaluation of poliomyelitis vaccines (inactivated) .................................................... 47 19

B.1 Characterization of poliovirus seed lots derived from attenuated strains (Sabin strains and strains 20

derived by recombinant DNA technology) ............................................................................................... 48 21

B.2 Antigenic profile ................................................................................................................................ 48 22

B.3 D-antigen content of IPV derived from attenuated strains (Sabin strains and strains derived by 23

recombinant DNA technology) ................................................................................................................. 49 24

B.4 Evaluation of immunogenicity in animal models ............................................................................... 49 25

B.5 Nonclinical safety studies ................................................................................................................... 50 26

Part C. Clinical evaluation of poliomyelitis vaccine (inactivated) ............................................................ 50 27

C.1 General considerations ....................................................................................................................... 50 28

C.2 Immunogenicity studies ..................................................................................................................... 51 29

C.3 Concomitant administration with other vaccines ............................................................................... 54 30

C.4 Pre-licensure safety data ..................................................................................................................... 55 31

C.5 Post-marketing studies and surveillance ............................................................................................ 55 32

Part D. Recommendations for national regulatory authorities ...................................................................... 56 33

D.1 General ............................................................................................................................................... 56 34

D.2 Official release and certification ........................................................................................................ 56 35

Authors and acknowledgements ................................................................................................................... 58 36

References ..................................................................................................................................................... 65 37

Appendix 1 ................................................................................................................................................... 73 38

Overview of the virus seeds used in IPV production .................................................................................... 73 39

Appendix 2 ................................................................................................................................................... 81 40

WHO/BS/2014.2233

In vivo potency assay of IPV ....................................................................................................................... 81 1

Appendix 3 .................................................................................................................................................... 84 2

Model summary protocol for manufacture and control of poliomyelitis vaccine (inactivated) .................... 84 3

Appendix 4 .................................................................................................................................................. 106 4

Model certificate for the release of poliomyelitis vaccine (inactivated) by national regulatory authorities106 5

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WHO/BS/2014.2233

1

Introduction 2

The requirements for inactivated poliomyelitis vaccine (IPV) were first formulated in 1959 (1) 3

and revised in 1965 (2). Following several advances in vaccine production technology, the 4

requirements were further updated in 1981 (3) and amended in 1985 (4). At that time, the 5

introduction of continuous cells for the manufacture of IPV was a novel development. Therefore, 6

when the regulatory control of products manufactured in continuous cells had been standardized, 7

the requirements were again updated in 2000 (5). An addendum was developed in 2003 (6), 8

specifying the measures to be taken to minimize the accidental risk of reintroducing wild-type 9

poliovirus from a vaccine manufacturing facility into the community after global certification of 10

polio eradication. 11

12

Since the Recommendations for the production and control of poliomyelitis vaccine (inactivated) 13

were last revised in 2000 (5) and in 2003 (6), there have been several changes in vaccine 14

production, including the use of seed viruses derived from Sabin strains, which make a further 15

revision of the recommendations necessary. To facilitate this process, a meeting to discuss the 16

international specifications for IPV – attended by experts from academia, national regulatory 17

authorities (NRAs)/national control laboratories (NCLs) and industry involved in the research, 18

manufacture, authorization and testing/release of IPV around the world – was convened by 19

WHO on 29 March 2012. During the discussions, critical issues were considered both for the 20

quality control and evaluation of IPV (including Sabin-based IPV, or sIPV) and for the revision 21

of the recommendations described in Annex 2 of WHO Technical Report Series 910 (5). WHO 22

convened an international Technical Working Group meeting in Geneva on 1415 May 2013 – 23

attended by experts from academia, NRAs/NCLs and industry involved in the development, 24

manufacture, authorization and testing/release of IPV, including sIPV and other new 25

developments of novel IPV – to further discuss and reach consensus on critical issues relevant to 26

the revision of Annex 2 of WHO Technical Report Series 910 (7). WHO organized an informal 27

consultation at its headquarters in Geneva on 2526 March 2014 – attended by academics, 28

researchers, vaccine manufacturers and regulators involved in IPV development, production, 29

evaluation and regulatory licensure – to review the draft recommendations prepared by the 30

drafting group and to seek consensus on key technical and regulatory issues. 31

32

Major issues addressed in this revision include: 33

WHO/BS/2014.2233

- an update of “General considerations” and other sections to reflect the future 1

development of IPV in accordance with global programmatic need (e.g. use of Sabin 2

strains and strains derived by recombinant DNA technology); 3

- inclusion of a new Appendix 1 to update the history of the different virus seed strains 4

used by manufacturers for IPV production ; 5

- an update of the section on international standards and reference preparations; 6

- an update of the section on general manufacturing recommendations and control 7

tests; 8

- updated terminology; 9

- inclusion of specific tests for sIPV and IPV made from strains derived by 10

recombinant DNA technology; 11

- an update of the appendices; 12

- inclusion of new sections on nonclinical and clinical evaluation of IPV. 13

14

Additional changes have been made to bring the document into line with other WHO 15

recommendations published since the last revision. 16

Scope 17

This document provides guidance to NRAs and manufacturers on the quality and nonclinical and 18

clinical aspects of IPV in order to ensure the quality, safety and efficacy of the vaccines. 19

The scope of the present recommendations encompasses IPV derived from 1) the wild-type 20

strains that have been used in the manufacture of IPV for many years; 2) the attenuated Sabin 21

strains that have been used in the manufacture of oral poliomyelitis vaccine (OPV); and 3) new 22

alternative poliovirus strains currently under development, including those derived by 23

recombinant DNA technology. 24

25

This document does not cover vaccines which are based on virus-like particles (VLPs) and 26

replicons. However, some aspects described in the current document may be relevant to these 27

types of seeds and should be taken into consideration during vaccine development using such 28

seeds. 29

30

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

nonclinical (8) and clinical evaluation (9) of vaccines. 32

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WHO/BS/2014.2233

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

combination with other vaccines, such as diphtheria toxoid (D) and tetanus toxoid (T), which 2

raises considerations – such as interaction of the poliovirus antigens with other antigens and/or 3

adjuvants – that do not apply when IPV is used as a stand-alone product. These considerations 4

are dealt with in a separate WHO document (10) but not in the present Recommendations to 5

assure the quality, safety and efficacy of poliomyelitis vaccine (inactivated). However, to provide 6

further guidance on control of the vaccine, key tests that may be influenced by other antigens 7

and/or adjuvants in combined vaccines are identified. 8

9

General considerations 10

Poliomyelitis is an acute communicable disease of humans caused by three distinct poliovirus 11

serotypes – types 1, 2 and 3 – distinguished by neutralization test (11). Poliovirus is classified as 12

a species C human enterovirus of the Picornaviridae family and is composed of a single-13

stranded, positive-sense RNA genome and a protein capsid. 14

15

Where sanitation is poor, faecal-to-oral transmission predominates, whereas oral-to-oral 16

transmission may be more common where standards of sanitation are high. In most settings, 17

mixed patterns of transmission are likely to occur. In the pre-vaccine era, when poliovirus was 18

the leading cause of permanent disability in children, virtually all children became infected by 19

polioviruses. On average, in the absence of protection by humoral or maternal antibody, 1 in 200 20

susceptible individuals develops paralytic poliomyelitis (11). 21

22

Progress in polio control (and, since 1988, polio eradication) has been due mainly to the 23

widespread use of vaccines. An inactivated poliomyelitis vaccine (IPV Salk vaccine, wIPV1) was 24

first licensed in 1955; live, attenuated oral poliomyelitis vaccine (OPV, Sabin vaccine) was 25

licensed in the USA in 1961 as a monovalent (mOPV) vaccine, followed by a trivalent OPV 26

(tOPV) licensed for use in 1963 (11). In May 1988, the World Health Assembly resolved to 27

eradicate poliomyelitis globally by the year 2000 and the Global Polio Eradication Initiative 28

(GPEI) was established. Sustained use of polio vaccines worldwide since 1988 has led to a 29

1In 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 represents IPV derived from Sabin strains only.

WHO/BS/2014.2233

precipitous drop in the global incidence of poliomyelitis by more than 99% (11).Globally, the 1

last case of poliomyelitis caused by naturally circulating wild poliovirus (WPV) type 2 (WPV2) 2

occurred in India in 1999. No case due to WPV type 3 (WPV3) has been detected globally since 3

10 November 2012. Despite the overall success of the GPEI, in 2014 Afghanistan, Nigeria and 4

Pakistan remain endemic for transmission of WPV type 1 (WPV1). The Horn of Africa, 5

Cameroon, and parts of the Middle East (Egypt, Israel and Syria) also reported WPV1 6

circulation associated with imported WPV1 in 2013, resulting in clinical cases following a 7

period of elimination (11). 8

9

Given the progress towards polio eradication, countries have increasingly switched from using 10

OPV to wIPV in routine immunization programmes, primarily in order to eliminate the burden of 11

vaccine-associated paralytic poliomyelitis (VAPP), a rare adverse event associated with OPV. 12

The incidence of VAPP has been estimated at 2–4 cases per million birth cohort per year in 13

countries using OPV (11). The sole use of wIPV successfully eradicated polio in some countries, 14

notably the Netherlands and Scandinavia. In most of the countries that have introduced wIPV as 15

the only poliomyelitis vaccine over the past decade, there has been no evidence of continued 16

circulation of poliovirus strains, thus indicating that wIPV is able to inhibit community 17

transmission of poliovirus. However, Israel, which switched to an all-IPV routine immunization 18

schedule in 2004, reported detection of WPV1 in sewage samples from February 2013 onwards. 19

However, no clinical cases of paralytic poliomyelitis had been reported in Israel, the West Bank 20

or Gaza as of 31 December 2013 (11, 12). 21

22

In addition to VAPP, the live polio strains in OPV, currently predominantly Sabin type 2, can 23

occasionally revert to a transmissible form termed circulating vaccine-derived poliovirus 24

(cVDPV) (13, 14) which acts essentially the same as the wild type in causing poliomyelitis. This 25

is an obvious threat to polio eradication that is not posed by the use of IPV. 26

27

The GPEI of WHO, in conjunction with its partners, developed the comprehensive Polio 28

Eradication and Endgame Strategic Plan 20132018 with the goal of achieving a polio-free 29

world by 2018 (15). This plan involves detection of poliovirus, the interruption of spread, 30

immunization strengthening, OPV withdrawal, containment, certification and legacy planning, 31

and gives a timetable of events following the identification of the last wild-type poliovirus. Three 32

of the key features of the GPEI strategic plan are the withdrawal of the type 2 OPV strain from 33

WHO/BS/2014.2233

tOPV and the introduction of bivalent (types 1 and 3) OPV (bOPV), the introduction of routine 1

use of IPV for managing long-term poliovirus risks including type 2 cVDPV, and the cessation 2

of all OPV use following the global certification of total WPV serotype eradication (15). 3

4

Although the last type 2 WPV was detected in 1999, in 2013 eight countries reported cases of 5

paralytic poliomyelitis associated with cVDPVs, most of them derived from Sabin type 2 (11). 6

There are 250–500 VAPP cases per year and 40% are due to type 2 Sabin poliovirus. The need to 7

synchronize OPV cessation was identified in 2008, and withdrawal of the use of type 2 OPV and 8

the introduction of bOPV began in 2012 (15) in supplementary immunization activities or for 9

outbreak control. In 2012 the WHO Strategic Advisory Group of Experts on Immunization 10

(SAGE) recommended that all countries using OPV should introduce at least one dose of IPV in 11

their routine immunization programmes to mitigate the risks of withdrawal of OPV 2 (16). One 12

of the prerequisites for OPV 2 cessation is the availability of an affordable IPV option for all 13

OPV-using countries. This may include full dose, fractional dose and adjuvanted IPV, and the 14

intradermal use of IPV in addition to intramuscular/subcutaneous administration. 15

16

When poliomyelitis due to wild type and cVDPV polioviruses is eradicated (17), laboratories and 17

manufacturers that store or use wild type, vaccine-derived polioviruses, or any other related 18

viruses (materials) will become an important potential source of reintroduction of these viruses 19

into the community. To manage and control this risk, WHO is finalizing a Global Action Plan 20

that requires the implementation of appropriate primary safeguards of biorisk management with 21

poliovirus facility containment specifications, secondary safeguards of population immunity in 22

the country approving manufacturing operations, and tertiary safeguards of facility location in 23

countries with demonstrated good personal, domestic and environmental hygiene standards, 24

including closed sewage systems with effective effluent treatment (18). 25

26

To mitigate biosafety and biosecurity concerns associated with virulent wild-type viruses used in 27

the manufacture of wIPV, the use of attenuated strains for IPV production has been proposed 28

(19). Production of IPV from live-attenuated Sabin poliovirus seeds has been shown to be 29

technically feasible (2024), and the first Sabin IPVs have been licensed in Japan in the form of 30

two combination vaccines. Manufacturers in various countries are establishing the production of 31

IPV from live-attenuated Sabin strains or IPV using strains derived by recombinant DNA 32

technology and are at different stages in the development/licensing process. Additional new 33

WHO/BS/2014.2233

manufacturers and IPV manufacturers that currently use wild-type poliovirus strains may wish to 1

consider evaluating the potential offered by a Sabin-based IPV or IPV using strains derived by 2

these alternative means. 3

4

Wild-type polioviruses are both transmissible and virulent. They will have to be grown under 5

appropriate and strict containment if they are to be used to produce IPV after the elimination of 6

circulating WPV, according to defined timelines, beginning with type 2 (18). The Sabin vaccine 7

strains are attenuated, and transmission from vaccine recipients is limited. However, they are 8

unstable on passage in cell culture and the human gut and can revert to give cVDPVs. 9

10

Given these uncertainties, assurance is required of the characteristics of the live attenuated Sabin 11

virus before inactivation in order to justify the implementation of containment measures that may 12

be different from those required for wIPV production (18). Production conditions should be 13

validated by the full range of tests including in vivo and in vitro testing of the master seed and 14

working seed and successive monovalent bulks (with the number to be approved by the NRA), to 15

ensure that the attenuated phenotype of the Sabin strains in monovalent pools is maintained. 16

Subsequently, a limited range of tests, such as mutant analysis by polymerase chain reaction and 17

restriction enzyme cleavage (MAPREC), may be applied to a proportion of the monovalent pools 18

produced each year in order to ensure production consistency. The number of pools of each type 19

tested each year should be justified and agreed by the NRA. Furthermore, it is important that, at 20

intervals to be agreed with the NRA, pools should be tested with the full range of tests to ensure 21

that production conditions remain satisfactory. 22

23

In addition to the Sabin strains that are used in the manufacture of OPV, alternative attenuation 24

methods utilizing recombinant DNA technology are being investigated (2529). Strains derived 25

by such methodology may have properties specifically designed to be suitable for the safe 26

production of vaccine (e.g. inability to replicate in the human gut). They should be considered as 27

they become available and may require specific characterization. Biocontainment requirements 28

for such strains will need to be determined on a case-by-case basis. Only virus strains that are 29

approved by the NRA should be used. 30

31

An overview of the history of virus seeds that are currently used in IPV production is given in 32

Appendix 1. 33

WHO/BS/2014.2233

1

The in vivo potency assay in rats has been standardized and shown to have advantages over 2

those previously described in vivo tests for IPV (30).When the in vivo assay is required for 3

routine production batches, it should be performed at the level of the final bulk. The assay in rats 4

is described in detail in this document (Appendix 2). The in vivo assay should be used to 5

characterize the vaccine after substantial changes in the manufacturing process that may 6

influence the quality of the vaccine, unless otherwise justified and agreed by the NRA. The in 7

vivo assay should also be used for stability studies of the vaccine and for establishing 8

consistency of vaccine production. The in vivo potency test described in these recommendations 9

requires the assay of neutralizing antibodies to each of the three poliovirus types. This test 10

requires the use of live poliovirus and, for historical reasons, many laboratories use wild-type 11

strains of poliovirus. The attenuated Sabin strains of poliovirus have been shown to be suitable 12

for the assay of neutralizing antibodies in the in vivo test, in principle, by a collaborative study 13

and should be used (30), but validation of the use of the Sabin strains by each manufacturer 14

should be provided. 15

16

Immunization with OPV will cease at some point in the future when poliomyelitis has been 17

eradicated. After that time, the biocontainment levels for use of the Sabin strains for laboratory 18

work will be reviewed. Laboratories are therefore encouraged to investigate the use of 19

alternatives to live viruses for the assay of poliovirus neutralizing antibodies in order to comply 20

with future biocontainment requirements. 21

22

The development of transgenic mice that express the human poliovirus receptor (TgPVR mice) 23

(31, 32) has led to the development of an in vivo immunization/challenge model (33, 34) that 24

may be useful for assessing the vaccine efficacy of new poliovirus strains. This test is not 25

proposed for lot release. Any work with transgenic mice should comply with WHO guidelines 26

for the maintenance, containment and transport of transgenic animals (35). 27

28

The manufacturer of the final lot must be responsible for ensuring conformity with all the 29

recommendations applicable to the final vaccine (Part A, sections A.5A.11) even where 30

manufacturing involves only formulating the final bulk from trivalent bulks supplied by another 31

manufacturing establishment and filling the final containers. The manufacturer of the final lot 32

WHO/BS/2014.2233

must also be responsible for any production and control tests performed by an external contract 1

laboratory, if applicable, with the approval of the NRA. 2

3

If an immunization schedule combining both IPV and OPV is to be claimed which could 4

potentially achieve both the high serum antibody levels and the intestinal protection (11), clinical 5

studies designed to establish such a combination (sequential) schedule should also examine 6

patterns of virus excretion following poliovirus challenge (with OPV), other than serum 7

neutralizing antibodies, in different sequential schedules. 8

9

Part A. Manufacturing recommendations 10

A.1 Definitions 11

A.1.1 International name and proper name 12

The international name should be poliomyelitis vaccine (inactivated). The proper name should be 13

equivalent to the international name in the language of the country of origin. 14

15

The use of the international name should be limited to vaccines that satisfy the recommendations 16

formulated below. 17

18

A.1.2 Descriptive definition 19

Poliomyelitis vaccine (inactivated) should consist of a sterile aqueous suspension of poliovirus 20

types 1, 2 and 3 grown in cell cultures, concentrated, purified and inactivated. The antigen may 21

be formulated with a suitable adjuvant. The preparation should satisfy all the recommendations 22

formulated below. 23

24

A.1.3 International reference materials 25

An International Standard of IPV is available for use in in vitro assays to measure the D-antigen 26

content of IPV containing classical wild-type strains. It is stored frozen in ampoules containing 1 27

mL of trivalent inactivated poliomyelitis vaccine. This material is for use in calibrating 28

secondary reference preparations of IPV which are included in each potency test so that 29

potencies in D-antigen units may be calculated. International standards and reference reagents 30

for the control of in vivo potency assays are under investigation. The need for an International 31

Standard for IPV based on the Sabin or other strains is also being investigated. 32

WHO/BS/2014.2233

1

An International Reference Preparation (IRP) of poliomyelitis vaccine 2

(inactivated) was established by the WHO Expert Committee on Biological 3

Standardization in 1963 (36). This preparation was a trivalent blend prepared in 4

1959 in primary monkey kidney cells from type 1 (Mahoney), type 2 (MEF) 5

and type 3 (Saukett) strains of poliovirus. After preparation of the IRP, 6

significant advances in production and control of IPV occurred and vaccines of 7

increased potency and purity were developed. An enhanced potency IPV 8

(PU78-02) from one manufacturer, the Rijksinstituut voor Volksgezondheid en 9

Milieuhygiene (RIVM), was widely used as a reference preparation for control 10

purposes. When stocks of this reagent were nearly exhausted, a new reference 11

material (91/574) was established by the Expert Committee as the second 12

WHO International Reference Reagent for in vivo and in vitro assays of IPV 13

(37). Potencies of 430, 95 and 285 D-antigen units per mL were assigned 14

respectively to poliovirus types 1, 2 and 3 of this preparation. A separate 15

aliquot of the preparation, established by the European Pharmacopeia 16

Commission as the Biological Reference Preparation (BRP) batch 1, has an 17

identical assigned titre (38). Material from a concentrated trivalent bulk from a 18

commercially available IPV vaccine was established as the BRP batch 2 in 19

2003, with an assigned potency of 320, 67 and 282 D-antigen units per mL for 20

types 1, 2 and 3 respectively (39). Following inconsistency in the performance 21

of some vials of 91/574, the use of this reference was discontinued in 2010. In 22

2013, the third International Standard for in vitro assays of IPV (12/104) was 23

established by the Expert Committee using BRP batch 2 as the reference in the 24

study. A potency of 277, 65 and 248 D-antigen units per mL was assigned to 25

poliovirus type 1, 2 and 3, respectively. 26

27

There are still gaps in the scientific knowledge of biological standardization of 28

IPV, and some inconsistency has been found in results obtained by different 29

laboratories and methods. Validation of international references suitable for 30

vaccines produced from different poliovirus strains will be required. 31

32

WHO/BS/2014.2233

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

the standardization of neutralizing antibody tests for poliovirus (40). 2

3

The International Standards listed above are available from the National 4

Institute for Biological Standards and Control, Potters Bar, United Kingdom. 5

6

A.1.4 Terminology 7

The definitions given below apply to the terms as used in these recommendations. They may 8

have different meanings in other contexts. 9

10

Adjuvant: A vaccine adjuvant is a substance, or a combination of substances, that is used in 11

conjunction with a vaccine antigen to enhance (e.g. increase, accelerate, prolong and/or possibly 12

target) the specific immune response to the vaccine antigen and the clinical effectiveness of the 13

vaccine. 14

15

Adventitious agents: Contaminating microorganisms of the cell culture, or source materials used 16

in its culture, that may include bacteria, fungi, mycoplasmas, and endogenous and exogenous 17

viruses that have been unintentionally introduced into the manufacturing process. 18

19

Cell-culture infective dose 50% (CCID50): The quantity of a virus suspension that will infect 20

50% of cell cultures. 21

22

Cell bank: A cell bank is a collection of appropriate containers whose contents are of uniform 23

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

pool of cells. 25

The individual containers (e.g. ampoules, vials) should be representative of the 26

pool of cells from which they are taken and should be frozen on the same day 27

by following the same procedure and by using the same equipment and 28

reagents. 29

30

Cell seed: A quantity of well-characterized cells derived from a single tissue or cell of human or 31

animal origin and stored frozen in liquid nitrogen in aliquots of uniform composition, one or 32

more of which may be used for the production of a master cell bank. 33

WHO/BS/2014.2233

1

D-antigen: The term refers to the antigen found in sucrose gradient fraction that contains native 2

virus particles, which are the target of neutralizing antibodies (41). D-antigen units were 3

originally defined on the basis of an agar precipitin test performed with D-antigen-specific 4

polyclonal sera. A vaccine preparation that produced precipitin line at the distance of 25 5

millimetres from the centre was arbitrarily assigned a value of 600 D-antigen units using a 6

particular antibody at a particular concentration. This test was used in the initial calibration of 7

reference materials. The D-antigen content of IPV is currently determined by an enzyme-linked 8

immunosorbent assay (ELISA) test. 9

10

Final bulk: The finished vaccine present in the container from which the final containers are 11

filled. The final bulk may be prepared from one or more trivalent bulks. 12

13

Final lot: A collection of sealed final containers of finished vaccine that is homogeneous with 14

respect to the risk of contamination during the filling process. All of the final containers must 15

therefore have been filled from a single vessel of final bulk in one working session. 16

17

Inactivated purified monovalent pool: A filtered purified monovalent pool which has been 18

inactivated through the use of a validated method. 19

20

Master cell bank (MCB): A quantity of well characterized cells of human or animal origin 21

derived from a cell seed at a specific population doubling level (PDL) or passage level, 22

dispensed into multiple containers, cryopreserved, and stored frozen under defined conditions, 23

such as the vapour or liquid phase of liquid nitrogen in aliquots of uniform composition. The 24

master cell bank is prepared from a single homogeneously mixed pool of cells and is used to 25

derive all working cell banks (WCBs). The testing performed on a replacement master cell bank 26

(derived from the same clone or from an existing master or working cell bank) is the same as for 27

the initial master cell bank, unless a justified exception is made. 28

29

Monovalent pool: A pool of a number of single harvests of the same virus type processed at the 30

same time. 31

32

WHO/BS/2014.2233

Production cell culture: A collection of cell cultures derived from one or more ampoules of the 1

WCB used for the production of IPV. 2

3

Purified monovalent pool: A concentrated and purified pool of a number of single harvests of the 4

same virus type processed at the same time. 5

6

Single harvest: A quantity of virus suspension of one virus type harvested from cell cultures 7

derived from the same WCB and prepared from a single production run. 8

9

sIPV: Inactivated poliomyelitis vaccine derived from Sabin strains only. 10

11

Trivalent bulk: A pool of a number of inactivated purified monovalent pools processed at the 12

same time and containing all three virus types, blended to achieve a defined D- antigen content 13

for each type. 14

15

Virus master seed lot: A quantity of virus suspension that has been processed at the same time to 16

assure a uniform composition and has been passaged for a specific number of times that does not 17

exceed the maximum approved by the NRA. It has been characterized to the extent that is 18

necessary to support development of the virus working seed lot. 19

20

Virus working seed lot: A quantity of virus of uniform composition derived from the virus master 21

seed lot made at the multiplicity of infection, ensuring that cytopathic effect develops within an 22

appropriate time frame and used at a passage level approved by the NRA for the manufacturing 23

of vaccine. 24

25

wIPV: Inactivated poliomyelitis vaccine derived from wild-type polio virus strains only. 26

27

Working cell bank (WCB): A quantity of cells of uniform composition derived from one or more 28

ampoules of the MCB at a finite passage level, stored frozen at –70 °C or below in aliquots, one 29

or more of which would be used for vaccine production. All containers are treated identically 30

and once removed from storage are not returned to the stock. 31

WHO/BS/2014.2233

A.2 General manufacturing recommendations 1

The general manufacturing requirements contained in Good manufacturing practices for 2

pharmaceutical products: main principles (42) and Good manufacturing practices for biological 3

products (43) should apply to establishments manufacturing IPV. 4

5

For vaccines prepared using wild-type poliovirus, guidance is provided in WHO’s Guidelines for 6

the safe production and quality control of IPV manufactured from wild polioviruses (6). This 7

document also gives some guidance on vaccines produced from attenuated poliovirus strains 8

(such as Sabin strains). In addition, facilities that manufacture IPV should comply with the 9

current global recommendations for poliovirus containment appropriate to the particular 10

poliovirus strains used for production in both the production and the quality control departments 11

(18). 12

13

Attenuated strains derived by recombinant DNA technology that are used in IPV production 14

should not be readily transmissible from person to person. Applicable containment conditions 15

will depend on the phenotype and production conditions, and should ensure an acceptable level 16

of phenotypic stability and should be assessed on a case-by-case basis. In any case, any 17

biocontainment arrangement should comply with the global recommendations for poliovirus 18

containment that are current at the time of production (18). 19

20

The staff involved in the production and quality control of IPV should be shown to be immune to 21

all three types of polioviruses. 22

23

The manufacturer should also be able to demonstrate the availability of appropriate means to 24

respond adequately to and manage an inadvertent release of unfinished product containing live 25

viruses. 26

A.3 Control of source materials 27

A.3.1 Virus strains and seed lot system 28

A.3.1.1 Virus strains 29

Strains of poliovirus used in the production of IPV should be identified by historical records, 30

which should include information on the strains’ origin and subsequent manipulation (e.g. wild, 31

attenuated or manipulated by recombinant DNA technology). The strain identity should be 32

WHO/BS/2014.2233

determined by infectivity tests and immunological methods. In addition, Sabin strains and strains 1

derived by recombinant DNA technology should be identified by nucleotide sequence analysis. 2

3

Only virus strains that are approved by the NRA and that yield a vaccine complying with the 4

recommendations set out in the present document should be used. 5

6

A.3.1.2 Virus seed lot system 7

Vaccine production should be based on the virus seed lot system. Unless otherwise justified and 8

authorized, the virus in the final vaccine should not have undergone more passages from the 9

virus master seed lot than were used to prepare the vaccine shown to be satisfactory with respect 10

to safety and efficacy and biocontainment requirements. 11

12

If Sabin virus master seeds are supplied by WHO, a virus sub-master seed lot 13

should be prepared by a single passage from the WHO master seed at a 14

multiplicity of infection that ensures the development of cytopathic effect 15

within an appropriate time frame. The virus sub-master seed lot should be 16

characterized to the extent that is necessary to support the development of the 17

virus working seed lot. The characterized virus sub-master seed lot is used for 18

the preparation of virus working seed lots (see section A.3.2.2 and Part B of the 19

Recommendations to assure the quality, safety and efficacy of live attenuated 20

poliomyelitis vaccine (oral)(44).The virus sub-master seed lot should be 21

subject to the same tests as a virus master seed lot. 22

23

Virus master and working seed lots should be stored in dedicated temperature-monitored freezers 24

at a temperature that ensures stability on storage (e.g. ≤ 60ºC). 25

26

A.3.1.3 Tests on virus master and working seed lots 27

Each virus master and working seed lot used for the production of vaccine batches should be 28

subjected to the tests listed in this section and certain tests applicable to single harvests listed in 29

sections A.4.3 (A.4.3.1 Sterility test for bacteria, fungi and mycoplasmas; A.4.3.2 Virus titration; 30

and A.4.3.3 Identity test). 31

32

WHO/BS/2014.2233

Each virus master and working seed lot should have been derived from materials that comply 1

with the Recommendations made in sections A.3.2 and A.3.3 and should be approved by the 2

NRA. 3

4

A.3.1.3.1 Tests in rabbit kidney cell cultures (only for virus master seeds derived from strains 5

which have previously been passaged on primary monkey kidney cells) 6

Virus master seeds that have previously been passaged on primary monkey kidney cells should 7

be tested in rabbit kidney cell cultures for the presence of herpes B virus and other viruses. A 8

sample of at least 10 mL of virus seeds should be tested. Serum used in the nutrient medium of 9

the cultures should have been shown to be free from B virus inhibitors using herpes simplex 10

virus as an indicator virus. The pooled fluid should be inoculated into bottles of these cell 11

cultures in such a way that the dilution of the pooled fluid in the nutrient medium does not 12

exceed 1 in 4. The area of the cell sheet should be at least 3 cm2 per mL of pooled fluid. At least 13

one bottle of each kind of cell culture should remain uninoculated and should serve as a control. 14

15

The inoculated and control cultures should be incubated at a temperature of 37 °C and should be 16

observed at appropriate intervals for a period of at least two weeks. 17

18

For the test to be valid, not more than 20% of the culture vessels should have been discarded for 19

nonspecific accidental reasons by the end of the test period. The sensitivity of each batch of 20

rabbit kidney cells should be demonstrated by challenge with a validated amount of herpes 21

simplex virus. The challenge test should be approved by the NRA. 22

23

A.3.1.3.2 Tests for adventitious viruses and freedom from detectable SV40 sequences 24

A.3.1.3.2.1 Tests for adventitious viruses in cell cultures 25

The virus master and working seed lot used for the production of vaccine batches should be free 26

from adventitious viruses in cell culture assays. 27

28

A sample of at least 40 mL of each virus master and working seed lot should be tested for the 29

presence of adventitious agents. The sample should be neutralized against the specific type of 30

poliovirus by a high-titred antiserum. 31

32

WHO/BS/2014.2233

The Sabin strains may be used as immunizing antigen for the preparation of the 1

antiserum. However, the immunizing antigen used for the preparation of the 2

antiserum should not be the same as the production seed. 3

4

The immunizing antigen should be shown to be free from adventitious agents 5

and should be grown in cell cultures free from adventitious microbial agents 6

that might elicit antibodies that could inhibit the growth of any adventitious 7

agents present in the single harvest. 8

9

The sample should be tested in primary Cercopithecus sp. kidney cell cultures, or cells that have 10

been demonstrated to be of equal susceptibility to SV40 virus, and in human diploid cells. The 11

tissue cultures should be incubated at 37 °C and observed for two weeks. At the end of this 12

observation period, at least one subculture of supernatant fluid should be made in the same tissue 13

culture system. The sample should be inoculated in such a way that the dilution of the 14

supernatant fluid in the nutrient medium does not exceed 1 in 4. The area of the cell sheet should 15

be at least 3 cm2 per mL of supernatant fluid. At least one bottle of the cell cultures should 16

remain uninoculated and should serve as a control. The cells inoculated with the supernatant 17

fluid and the uninoculated control cultures should be incubated at 37 °C and observed at 18

appropriate intervals for an additional two weeks. 19

20

If necessary, serum may be added to the primary cultures at this stage, provided 21

that the serum does not contain SV40 antibody or other inhibitors. 22

23

The virus master and working seed virus passes the test if there is no evidence of the presence of 24

adventitious agents. For the test to be valid, not more than 20% of the culture vessels should 25

have been discarded for nonspecific accidental reasons by the end of the observation period. 26

27

New molecular methods with broad detection capabilities are being developed 28

for the detection of adventitious agents. These methods include: degenerate 29

nucleic acid amplification technique (NAT) for whole virus families, with 30

analysis of the amplicons by hybridization, sequencing or mass spectrometry; 31

NAT with random primers followed by analysis of the amplicons on large 32

oligonucleotide micro-arrays of conserved viral sequencing or digital 33

WHO/BS/2014.2233

subtraction of expressed sequences; and high throughput sequencing. These 1

methods might be used in the future to supplement existing methods or as 2

alternative methods to both in vivo and in vitro tests after appropriate 3

validation and with the approval of the NRA (45). 4

5

The theoretical risk of the presence of potential human, simian, bovine or 6

porcine adventitious agents in the seed lots, which may be derived from the use 7

of bovine serum or porcine trypsin, should be assessed. If necessary, viruses 8

such as bovine polyomavirus, porcine parvovirus or porcine circovirus (PCV) 9

may be screened by using specific assays, such as molecular techniques like 10

NAT (45). 11

12

A.3.1.3.2.2 Tests for freedom from detectable SV40 sequences 13

The virus master seed lot should be shown to be free from detectable SV40 sequences by using 14

specific validated assays which are approved by the NRA, such as molecular techniques like 15

NAT (45). 16

17

DNA of SV40 is widely used as molecular biological reagent, and 18

contamination of polymerase chain reaction (PCR) assays is potentially a major 19

problem. One approach is to identify separate genomic regions of SV40 for 20

amplification, and to use one region for screening purposes and the other for 21

the confirmation of repeatedly positive samples. It is useful if the second 22

genomic region used for confirmation varies between isolates from different 23

sources, as it is then possible to show that it has a unique sequence and that 24

positive results are not due to contamination with laboratory strains of SV40. 25

The sensitivity of the PCR assays should be established for the genomic 26

regions used. 27

28

A.3.1.3.3 Additional tests on seeds from Sabin strains and other attenuated strains derived by 29

recombinant DNA technology 30

31

If live-attenuated Sabin strains are used for vaccine production, established master seeds should 32

be used and additional tests should be performed. The virus master seed lots used for the 33

WHO/BS/2014.2233

production of vaccine batches should be tested to monitor virus molecular characteristics – e.g. 1

by MAPREC – and should meet the specifications established in agreement with the NRA. 2

Specifications for OPV based on Sabin strains are described in the Recommendations to assure 3

the quality, safety and efficacy of live attenuated poliomyelitis vaccine (oral) (44). Tests to 4

monitor virus molecular characteristics are described in section A.3.2.4 of those 5

recommendations, while section A.3.2.4.1 deals with in vitro tests and section A.3.2.4.2 covers 6

neurovirulence tests. (See also section A.4.4.2.7 of this document). 7

8

Suitable in vitro tests should be performed on the master seed from attenuated strains derived by 9

recombinant DNA technology. The tests may include full genome characterization by nucleotide 10

sequencing or deep sequencing techniques and demonstration of genetic and phenotypic stability 11

on passage under production conditions. Such tests should be validated for this purpose by using 12

appropriate standards and materials, and should be approved by the NRA. 13

14

The need for testing virus master seed lots of attenuated strains derived by recombinant DNA 15

technology in in vivo neurovirulence tests should be considered and scientifically justified, in 16

agreement with the NRA. 17

18

Any new virus working seed derived from an established master seed, including Sabin strains 19

and other attenuated strains derived by recombinant DNA technology, and at least three 20

consecutive monovalent pools should be analysed in tests to monitor virus molecular 21

characteristics such as MAPREC, when relevant (see section A.4.4.2.7.1). 22

23

A.3.2 Cell lines 24

The general production precautions, as formulated in Good manufacturing practices for 25

biological products (43), should apply to the manufacture of IPV, with additional requirement 26

that, during production, only one type of cell should be introduced or handled in the production 27

area at any one time. Vaccines may be produced in a human diploid cell line or in a continuous 28

cell line. 29

30

A.3.2.1 Master cell bank (MCB) and working cell bank (WCB) 31

The use of a cell line for the manufacture of IPV should be based on the cell bank system. The 32

cell seed and cell banks should conform to the Recommendations for the evaluation of animal 33

WHO/BS/2014.2233

cell cultures as substrates for the manufacture of biological medicinal products and for the 1

characterization of cell banks (45).The MCB should be approved by the NRA. The maximum 2

number of passages (or population doublings) by which the WCB is derived from the MCB and 3

the maximum number of passages of the production cultures should be established by the 4

manufacturer and approved by the NRA. 5

6

Additional tests may include, but are not limited to: examination for the presence of retrovirus 7

and tumorigenicity in an animal test system (45) and propagation of the MCB or WCB cells to or 8

beyond the maximum in vitro passage for production. 9

10

The WHO Vero reference cell bank 10-87 is considered suitable for use as a 11

cell seed for generating an MCB (46) and is available to manufacturers on 12

application to the Coordinator, Technologies Standards and Norms, 13

Department of Essential Medicines and Health Products (EMP), Health 14

Systems and Innovation (HIS) Cluster, World Health Organization, Geneva, 15

Switzerland. 16

17

A.3.2.2 Identity test 18

Identity tests on the MCB and WCB are performed in accordance with the WHO 19

Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of 20

biological medicinal products and for the characterization of cell banks (45) and should be 21

approved by the NRA. 22

23

The WCB should be identified by means of tests such as biochemical tests (e.g. isoenzyme 24

analysis), immunological tests, cytogenetic marker tests and DNA fingerprinting or sequencing. 25

The tests should be approved by the NRA. 26

27

A.3.3 Cell culture medium 28

Serum used for the propagation of cells should be tested to demonstrate freedom from bacterial, 29

fungal and mycoplasmal contamination – as specified in Part A, sections 5.2 (47) and 5.3 (48) of 30

the General requirements for the sterility of biological substances – and freedom from infectious 31

viruses. Suitable tests for detecting viruses in bovine serum are given in Appendix 1 of the WHO 32

WHO/BS/2014.2233

Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of 1

biological medicinal products and for the characterization of cell banks (45). 2

3

Validated molecular tests for bovine viruses may replace the cell culture tests of bovine sera if 4

approved by the NRA. As an additional monitor of quality, sera may be examined for freedom 5

from bacteriophage and endotoxin. Gamma-irradiation may be used to inactivate potential 6

contaminant viruses, recognizing that some viruses are relatively resistant to gamma-irradiation. 7

8

The source(s) of animal components used in the culture medium should be approved by the 9

NRA. The components should comply with the current WHO guidelines on transmissible 10

spongiform encephalopathies in relation to biological and pharmaceutical products (49).The 11

serum protein concentration should be reduced by rinsing the cell cultures with serum-free 12

medium and/or purification of the virus harvests. 13

14

In some countries, control tests are carried out to detect the residual animal 15

serum content in the final vaccine (see section A.6.6). 16

17

Human serum should not be used. If human serum albumin is used at any stage of product 18

manufacture, the NRA should be consulted regarding the requirements, as these may differ from 19

country to country. As a minimum, it should meet the Requirements for the collection, 20

processing and quality control of blood, blood components and plasma derivatives (50). In 21

addition, human albumin and materials of animal origin should comply with current WHO 22

guidelines on transmissible spongiform encephalopathies in relation to biological and 23

pharmaceutical products (49). 24

25

Manufacturers are encouraged to explore the possibilities of using serum-free 26

media for the production of IPV. 27

28

Penicillin and other beta-lactams should not be used at any stage of manufacture because of their 29

nature as highly sensitizing substances in humans. 30

31

Other antibiotics may be used at any stage of manufacture, provided that the 32

quantity present in the final product is acceptable to the NRA. 33

WHO/BS/2014.2233

1

Nontoxic pH indicators may be added, such as phenol red at a concentration of 2

0.002%. 3

4

Only substances that have been approved by the NRA may be added. 5

6

Bovine or porcine trypsin used for preparing cell cultures should be tested and found free of 7

cultivable bacteria, fungi, mycoplasmas and infectious viruses, as appropriate (45). The methods 8

used to ensure this should be approved by the NRA. 9

10

In some countries, irradiation is used to inactivate potential contaminant 11

viruses. If irradiation is used, it is important to ensure that a reproducible dose 12

is delivered to all batches and to the component units of each batch. The 13

irradiation dose must be low enough for the biological properties of the 14

reagents to be retained but also high enough to reduce virological risk. 15

Therefore, irradiation cannot be considered a sterilizing process (45). 16

17

Recombinant trypsin is available and should be considered; however, it should 18

not be assumed to be free from risk of contamination and should be subject to 19

the usual considerations for any reagent of biological origin (45). 20

21

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

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

to biological and pharmaceutical products (49). 24

A.4 Control of vaccine production 25

A.4.1 Control cell cultures 26

When human diploid or continuous cell lines are used to prepare cultures for the production of 27

vaccine, a fraction equivalent to at least 5% of the total or 500 mL of cell suspension, or 100 28

million cells, at the concentration and cell passage level employed for seeding vaccine 29

production cultures, should be used to prepare control cultures. 30

31

If bioreactor technology is used, the NRA should determine the size and treatment of the cell 32

sample to be examined. 33

WHO/BS/2014.2233

1

A.4.1.1 Tests of control cell cultures 2

The treatment of the cells set aside as control material should be similar to that of the production 3

cell cultures, but they should remain uninoculated for use as control cultures for the detection of 4

any adventitious agents. 5

6

These control cell cultures should be incubated under conditions as similar as possible to the 7

inoculated cultures for at least two weeks, and should be tested for the presence of adventitious 8

agents as described below. For the test to be valid, not more than 20% of the control cell cultures 9

should have been discarded for nonspecific accidental reasons. 10

11

At the end of the observation period, the control cell cultures should be examined for evidence of 12

degeneration caused by an adventitious agent. If this examination, or any of the tests specified in 13

this section, shows evidence of the presence of any adventitious agent in the control culture, the 14

poliovirus grown in the corresponding inoculated cultures should not be used for vaccine 15

production. 16

17

If not tested immediately, samples should be stored at 60 °C or below. 18

19

A.4.1.2 Tests for haemadsorbing viruses 20

At the end of the observation period, at least 25% of the control cells should be tested for the 21

presence of haemadsorbing viruses using guinea pig red blood cells. If the latter cells have been 22

stored, the duration of storage should not have exceeded seven days and the storage temperature 23

should have been in the range of 2–8 °C. In tests for haemadsorbing viruses, calcium and 24

magnesium ions should be absent from the medium. 25

26

Some NRAs require, as an additional test for haemadsorbing viruses, that other 27

types of red cells, including cells from humans (blood group IV O), monkeys 28

and chickens (or other avian species), should be used in addition to guinea pig 29

cells. 30

31

A reading should be taken after incubation at 28 °C for 30 minutes, and again after a further 32

incubation for 30 minutes at 20–25 °C. 33

WHO/BS/2014.2233

1

If a test with monkey red cells is performed, readings should also be taken after 2

a final incubation for 30 minutes at 34–37 °C. 3

4

In some countries the sensitivity of each new batch of red blood cells is 5

demonstrated by titration against a haemagglutin antigen before use in the test 6

for haemadsorbing viruses. 7

8

A.4.1.3 Tests for other adventitious agents in cell supernatant fluid 9

At the end of the observation period, a sample of the pooled supernatant fluid from each group of 10

control cultures should be tested for other adventitious agents. For this purpose, 10 mL of each 11

pool should be tested in the same cells, but not the same batch of cells, as those used for the 12

production of vaccine. 13

14

A second indicator cell line should be used to test an additional 10 mL sample of each pool. 15

When a human diploid cell line is used for production, a simian kidney cell line should be used 16

as the second indicator cell line. When a simian kidney cell line is used for production, a human 17

diploid cell line should be used as the second indicator cell line (45). 18

19

The pooled fluid should be inoculated into bottles of these cell cultures in such a way that the 20

dilution of the pooled fluid in the nutrient medium does not exceed 1 part in 4. The area of the 21

cell sheet should be at least 3 cm2 per mL of pooled fluid. At least one bottle of each kind of cell 22

culture should remain uninoculated and should serve as a control. 23

24

The inoculated cultures should be incubated at a temperature of 35–37 °C and observed at 25

appropriate intervals for a period of at least 14 days. 26

27

Some NRAs require that, at the end of this observation period, a subculture is 28

made in the same culture system and observed for at least an additional 14 29

days. Furthermore, some NRAs require that these cells should be tested for the 30

presence of haemadsorbing viruses. 31

32

WHO/BS/2014.2233

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

nonspecific accidental reasons by the end of the test period. 2

3

If any cytopathic changes due to adventitious agents occur in any of the cultures, the virus 4

harvests produced from the batch of cells from which the control cells were taken should be 5

discarded. 6

7

Some selected viruses may be screened by using specific validated assays which are approved by 8

the NRA, such as molecular techniques (e.g. NAT) (45). 9

10

If these tests are not performed immediately, the samples should be kept at a temperature of 60 11

°C or below. 12

13

A.4.1.4 Identity tests 14

At the production level, the control cells should be identified by means of tests approved by the 15

NRA. 16

17

Suitable methods include, but are not limited to, biochemical tests (e.g. isoenzyme analyses), 18

immunological tests, cytogenetic tests (e.g. for chromosomal markers) and tests for genetic 19

markers (e.g. DNA fingerprinting or sequencing). 20

21

A.4.2 Control of vaccine production 22

A.4.2.1 Cell cultures for vaccine production 23

A.4.2.1.1 Observation of cultures for adventitious agents 24

On the day of inoculation with the virus working seed lot, each cell culture or a sample from 25

each culture vessel should be examined visually for degeneration caused by infective agents. If 26

this examination shows evidence of the presence in a cell culture of any adventitious agent, the 27

culture should not be used for vaccine production. 28

29

If animal serum is used for cell cultures before the inoculation of virus, the medium should be 30

removed and replaced with serum-free maintenance medium after the cells have been washed 31

with serum-free medium, if appropriate. 32

33

WHO/BS/2014.2233

A.4.3 Control of single harvests 1

After inoculation of the production cells with virus, the culture conditions of inoculated and 2

control cell cultures should be standardized and kept within limits agreed with the NRA. 3

4

Samples required for the testing of single harvests should be taken immediately on harvesting. 5

6

In some countries, samples are taken after storage and filtration with the 7

agreement of the NRA. 8

9

A.4.3.1 Sterility test for bacteria, fungi and mycoplasmas 10

A volume of at least 10 mL of each virus master and working seed lot (see A.3.1.3) and single 11

harvest should be tested for bacterial, fungal and mycoplasmal contamination by appropriate 12

tests, as specified in Part A, sections 5.2 (47) and 5.3 (48) of General requirements for the 13

sterility of biological substances, or by a method approved by the NRA. If the test is performed 14

outside the production facilities, adequate containment procedures (18) should be used according 15

to the virus strain used for production. 16

17

NAT alone or in combination with cell culture, with an appropriate detection 18

method, may be used as an alternative to one or both of the compendial 19

mycoplasma detection methods following suitable validation and the 20

agreement of the NRA (45). 21

22

In some countries this test is performed on the purified monovalent harvest 23

instead of on the single harvest. 24

25

A.4.3.2 Virus titration 26

The virus concentration of each virus master and working seed lot (see A.3.1.3.) and single 27

harvest should be determined by titration of infectious virus using tissue culture methods. This 28

titration should be carried out in not more than 10-fold dilution steps using 10 cultures per 29

dilution, or by any other arrangement yielding equal precision. 30

31

WHO/BS/2014.2233

The use of Hep-2C or Vero cells in microtitre plates is suitable for this purpose 1

(44).The same cells should be used for virus titrations on monovalent pools 2

throughout the production process. 3

Information on virus titre will help in selecting single harvests that can be 4

expected to meet potency requirements after inactivation. 5

6

In some countries the virus titration may be carried out on the purified, pooled 7

monovalent harvest after demonstration of consistency of production at the 8

stage of the single harvest. 9

10

A.4.3.3 Identity test 11

The poliovirus in each virus master and working seed lot (see A.3.1.3) and single harvest should 12

be tested for serotype and strain identity by neutralization with specific antiserum or molecular 13

methods approved by the NRA. 14

15

Care should be taken to ensure that the sera used are monospecific by titrating 16

them against homotypic and heterotypic viruses of known virus titre. 17

Monoclonal antibodies may be useful in this test. 18

19

The strain identity of each of the three serotypes may be determined by 20

standard or deep nucleotide sequence analysis or by a suitable molecular 21

technique. 22

23

In some countries this test is performed on the purified monovalent harvest 24

instead of on the single harvest. 25

26

A.4.4 Control of purified monovalent pools 27

A.4.4.1 Purification of monovalent pools 28

Each monovalent pool of virus, consisting of several single harvests of the same serotype, should 29

be purified before inactivation. Removal of host cell protein should be assessed during process 30

validation (45). 31

32

WHO/BS/2014.2233

An acceptable method is to clarify the virus suspension by filtration, to 1

concentrate the virus by ultrafiltration and, thereafter, to collect the virus peak 2

after passing it through a gel-filtration column. Further purification is achieved 3

by passing the virus through an ion-exchange column. Other purification 4

procedures, such as passing the preparation through an immobilized DNA-ase 5

column, may be used. 6

7

A.4.4.2 Tests on purified monovalent pools 8

A.4.4.2.1 Residual cellular DNA 9

For viruses grown in continuous cells, the purified monovalent pools should be tested for 10

residual cellular DNA (45). The purification process should be shown by calculation to reduce 11

consistently the level of cellular DNA to less than 10 ng per human dose. 12

13

In some countries, IPV produced in mammalian cells is required to contain less 14

than 100 pg DNA per human dose. 15

16

This test may be omitted from routine testing, with the agreement of the NRA, if the 17

manufacturing process is validated to achieve this specification (45). 18

19

If assessed, the size distribution of the DNA may be considered as a 20

characterization test, taking into account the amount of DNA detectable using 21

appropriate methods, as approved by the NRA (45). 22

23

In some countries this test is performed on the trivalent bulk following 24

validation and with the agreement of the NRA. 25

26

A.4.4.2.2 Virus titration 27

The virus concentration of each purified monovalent pool should be determined by titration of 28

infectious virus using tissue culture methods. This titration should be carried out in not more 29

than 10-fold dilution steps using 10 cultures per dilution, or by any other arrangement yielding 30

equal precision. 31

32

WHO/BS/2014.2233

The use of Hep-2C or Vero cells in microtitre plates is suitable for this purpose 1

(44). 2

3

Information on virus titre will help in selecting purified monovalent pools that 4

can be expected to meet potency requirements following inactivation. 5

6

A.4.4.2.3 Identity test 7

The poliovirus in each purified monovalent pool should be tested for serotype and strain identity 8

by neutralization, using specific antiserum or molecular methods approved by the NRA. 9

10

Care should be taken to ensure that the sera used are monospecific by titrating them against 11

homotypic and heterotypic viruses of known virus titre. Monoclonal antibodies may be useful in 12

this test. 13

14

The strain identity of each of the three serotypes may be determined by nucleotide sequence 15

analysis or a suitable molecular technique. 16

17

A.4.4.2.4 D-antigen content 18

The D-antigen content of each purified monovalent pool should be determined by use of a 19

validated immunochemical method and should be calculated by using a reference vaccine 20

calibrated against the WHO International Standard (see section A.1.3). 21

22

A.4.4.2.5 Protein content 23

The purified monovalent pool should be shown to contain no more than 0.1µg of protein per D-24

antigen unit of poliovirus or should be within the limits approved for that particular product by 25

the NRA. 26

27

A.4.4.2.6 Filtration before inactivation 28

Each purified monovalent pool should be filtered before inactivation. 29

30

Satisfactory results have been reported with several filter types but a final 31

filtration using a 0.22-µm filter should be used. 32

33

WHO/BS/2014.2233

Filters containing asbestos should not be used. 1

2

Inactivation should be initiated as soon as possible and in any case not later than 72 hours after 3

filtration. 4

5

It is preferable to start inactivation within 24 hours of filtration. Since the 6

purpose of the filtration step is to remove particulate matter and other 7

interfering substances that may diminish the effectiveness of the inactivation 8

process, and since aggregates tend to increase on standing after filtration, 9

efforts should be made to keep within this time limit. 10

11

A sample of the filtered purified monovalent pool should be retained and its virus titre 12

determined as described in A.4.4.2.2. 13

14

The main purpose of determining the titre of filtered virus pools destined for 15

inactivation is to provide the starting titre to monitor the kinetics of 16

inactivation. 17

18

A.4.4.2.7 Additional tests for purified monovalent pools produced from Sabin vaccine seeds or 19

from other attenuated seeds derived by recombinant DNA technology 20

21

Production conditions should be validated by the full range of tests, including in vivo and in vitro 22

testing of the master and working seed and successive monovalent bulks (the number to be 23

approved by the NRA), to ensure that the attenuated phenotype of the Sabin strains in monovalent 24

pools is maintained. Subsequently, a limited range of tests, such as MAPREC, may be applied to a 25

proportion of the monovalent pools produced each year in order to ensure production consistency. 26

The number of pools of each type tested each year should be justified and should be agreed with 27

the NRA. Furthermore, it is important that, at intervals to be agreed with the NRA, pools should 28

be tested with the full range of tests to ensure that production conditions remain satisfactory. 29

30

The use of the rct40 test is discouraged as it requires the use of WPV controls. 31

32

WHO/BS/2014.2233

In vitro tests to monitor virus molecular characteristics (consistency) and in vivo neurovirulence 1

tests which could be used for this purpose are described in A.4.4.2.7.1 and A.4.4.2.7.2 2

respectively. 3

4

Suitable in vitro tests should be performed on purified monovalent pools derived from attenuated 5

strains derived by recombinant DNA technology. Tests may include full genome characterization 6

by nucleotide sequencing or deep sequencing techniques. Such tests should be validated for this 7

purpose by the use of appropriate standards and materials, and should be approved by the NRA. 8

9

An in vitro test (described above) for the molecular consistency of production 10

may be performed on single harvests before preparing the purified monovalent 11

pool. If performed, the acceptance/rejection criteria should be updated 12

periodically and should be approved by the NRA. 13

14

A.4.4.2.7.1 Tests to monitor virus molecular characteristics (consistency) 15

In vitro tests such as MAPREC which are used to determine the molecular consistency of 16

production of monovalent pools should meet the specifications for the test used (44). 17

18

Results from MAPREC tests should be expressed as ratios relative to the relevant type-specific 19

International Standard for MAPREC analysis of poliovirus (Sabin). The acceptable variation of 20

mutant content from batch to batch should be agreed with the NRA in the light of production and 21

testing experience. 22

23

For type 3 (472-C), a purified monovalent pool should be rejected if the level of mutations 24

ismore than 1.0% when normalized against the International Standard. The limits for types 1 and 25

2 should be approved by the NRA. 26

27

Levels of mutations obtained by manufacturers who have implemented the test 28

for types 1 and 2 virus have been less than 2.0% for type 1 Sabin (for the sum 29

of both mutations 480-A, 525-C) and 1.5% for type 2 Sabin (481-G) (44, 51). 30

31

The test(s) used should be approved by the NRA. The MAPREC assay provides a sensitive and 32

quantitative measure for consistency for monovalent pools derived from Sabin viruses. 33

WHO/BS/2014.2233

1

A.4.4.2.7.2 Neurovirulence tests 2

Appropriate in vivo tests which may be used to evaluate the phenotype of virus in monovalent 3

pools produced from the Sabin vaccine strains are described in section A.4.4.7.2 of 4

Recommendations to assure the quality, safety and efficacy of live attenuated poliomyelitis 5

vaccine (oral) (44). 6

7

For other attenuated strains derived by recombinant DNA technology, the need for testing 8

purified monovalent virus pools by in vivo neurovirulence tests should be considered and should 9

be scientifically justified with the agreement of the NRA. 10

11

A.4.5 Control of inactivated purified monovalent pools 12

A.4.5.1 Inactivation procedure 13

The virus in the filtered purified monovalent pools should be inactivated by a method approved 14

by the NRA. Prior to inactivation, the concentration of the filtered monovalent pool, based on 15

viral titre or D-antigen content, should be adjusted to the acceptable range established during the 16

process validation. The acceptable range should be established during validation studies. 17

18

Most manufacturers during the past 4050 years have used formaldehyde as 19

the method for inactivation. 20

21

The method of inactivation should be shown to give consistent inactivation for the production of 22

acceptable vaccine. The progress of inactivation should be monitored by suitably spaced 23

determinations of virus titres. The inactivation period should exceed the time taken to reduce the 24

titre of live virus to undetectable amounts by a factor of at least 2. 25

A second filtration should be made during the process of inactivation. 26

27

This step is made after the virus titre has fallen below detectable levels but 28

before the first sample for the safety test is taken. 29

30

The kinetics of viral inactivation should be established by each manufacturer and should be 31

approved by the NRA. Adequate data on viral inactivation kinetics should be obtained and 32

consistency of the inactivation process should be monitored. For this purpose, the virus titre and 33

WHO/BS/2014.2233

D-antigen content of each filtered purified monovalent pool before, during and at the end of 1

inactivation should also be determined, as specified in A.4.4.2.2 and A.4.4.2.4 respectively. 2

3

A record of consistency (effective inactivation and kinetic of inactivation) should be established 4

by the production of at least five consecutive lots and, if broken, a root cause analysis should be 5

performed and a further five consecutive filtered purified monovalent pools should be prepared 6

and shown to be satisfactory for establishing this record. 7

8

A.4.5.2 Test for effective inactivation 9

Two samples should be taken of a volume equivalent to at least 1500 human doses of each 10

inactivated purified monovalent pool. One sample should be taken at the end of the inactivation 11

period and the other not later than three-quarters of the way through this period. After removal or 12

neutralization of the inactivating agent, the samples should be tested for the absence of infective 13

poliovirus by inoculation into tissue cultures. Kidney cells from some monkey species, for 14

instance those of the genera Macaca, Cercopithecus and Papio sps, appear to be more sensitive 15

than others. If other tissue culture systems, including continuous cell lines (e.g. L20B), are used, 16

they should have been shown to possess at least the same sensitivity to poliovirus as those 17

specified above by inoculating with partially formalin-inactivated virus (as opposed to 18

infectious, untreated virus) as formalin treatment changes biological properties of poliovirus (see 19

below). When primary monkey kidney cells are used for this test, the two samples should be 20

inoculated into bottles of tissue cultures derived from different batches of cells. 21

22

The dilution of the sample in the nutrient fluid should not exceed 1 in 4 and the area of the cell 23

sheet should be at least 3 cm2

per mL of sample. One or more bottles of each batch of cultures 24

should be set aside to serve as uninoculated control bottles with the same medium. 25

26

The formaldehyde in samples of vaccine for tissue culture tests is generally 27

neutralized at the time of sampling by the addition of bisulfite. Usually, the 28

samples are subsequently dialysed. 29

30

It is possible to conduct tissue culture tests on non-dialysed material. However, 31

this is often found to be toxic to cells, even with a dilution of 1 in 4. If in such 32

tests nonspecific degeneration of cells occurs, or if the sensitivity of the tissue 33

WHO/BS/2014.2233

culture system is reduced, the test should be repeated on dialysed material. The 1

virus D-antigen content after dialysis should be determined to ascertain 2

whether any D-antigen was lost during the dialysis process. 3

4

The tissue culture bottles should be observed for at least three weeks. Not less than two 5

subcultures should be made from each original bottle. The first subculture from each bottle 6

should be made prior to the first medium change, and the second subculture should be made at 7

the end of the observation period. The subcultures should be observed for at least two weeks. 8

9

If infectious poliovirus is detected in samples taken at the end of inactivation, or in the samples 10

taken no later than three-quarters through the inactivation process, the inactivated purified 11

monovalent pool should not be used for further processing. The isolation of live poliovirus from 12

an inactivated purified monovalent pool must be regarded as a break in the manufacturing 13

consistency record and a review of production process and revalidation should be undertaken. 14

15

If primary monkey kidney cells are used in this test, they may contain adventitious agents that 16

could interfere with the test result. It is important to demonstrate that each test retains sensitivity 17

to detect partially inactivated polioviruses. At the end of the observation period, the cell culture 18

used for the detection of residual live virus should be challenged with a validated amount of live 19

Sabin virus of the same type as that of the inactivated purified monovalent pool. The details of 20

the challenge procedure should be approved by the NRA. 21

22

If other cells, such as continuous cell lines, are used in this test, they must be shown to possess at 23

least the same sensitivity as primary monkey kidney cells or must be challenged as described 24

above. The problem of detecting residual active poliovirus in an inactivated vaccine is not the 25

same as that of measuring infective virus in untreated suspensions. Poliovirus that has been 26

exposed to the action of formaldehyde without becoming inactivated has been shown to require a 27

much longer period to produce cytopathic changes than does untreated virus. For this reason it is 28

desirable that tissue cultures in tests for the presence of residual active virus are observed for as 29

long a time as is technically possible. A satisfactory tissue culture system for this purpose 30

depends, therefore, not only on the sensitivity of the cells used for the preparation of the cultures 31

but also on the nutrient fluid. 32

33

WHO/BS/2014.2233

The serum added to the nutrient fluid should be tested for inhibitors to 1

poliovirus at serum concentrations up to 50%. Only serum free from inhibitors 2

to all three types of poliovirus should be used. 3

4

Maintenance of the cultures in good condition may require frequent changes of 5

culture medium. However, it should be borne in mind that early changes of 6

fluid may result in unadsorbed virus being removed and the validity of the test 7

would thus be impaired. Therefore, the fluid should be changed no earlier than 8

5–7 days after inoculation. 9

10

A.4.5.4 Sterility test for bacteria and fungi 11

Each inactivated purified monovalent pool should be tested for bacterial and fungal sterility, as 12

specified in Part A, section 5.2 of the General requirements for the sterility of biological 13

substances (47), or by methods approved by the NRA. 14

15

A.4.5.5 D-antigen content 16

The D-antigen content of each inactivated purified monovalent pool should be determined by use 17

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

calibrated against the WHO International Standard (see section A.1.3). The results obtained 19

should be within the required limits established by the NRA. 20

21

A.4.6 Control of trivalent bulk 22

Only those inactivated purified monovalent pools that have been shown to be satisfactory should 23

be blended to form a trivalent bulk. 24

25

A.4.6.1 Test for absence of infective poliovirus 26

A sample of at least 1500 mL or, if purified and concentrated vaccine is prepared, the equivalent 27

of at least 1500 doses of each trivalent bulk should be tested in cell cultures for the absence of 28

infective poliovirus by the procedure described in section A.4.5.2. If infective poliovirus is 29

isolated, this trivalent bulk, or product derived from it, should not be used. 30

31

In some countries this test may be omitted on the trivalent bulk if, following a 32

review of manufacturing records, the test for inactivation has been performed 33

WHO/BS/2014.2233

with satisfactory results on the inactivated purified monovalent pool, subject to 1

approval by the NRA. 2

3

When a trivalent bulk is supplied by one manufacturer to another, the 4

validation of inactivation may rely on the inactivation tests performed by the 5

bulk supplier. 6

7


The trivalent bulk should be tested for bacterial and fungal sterility, as specified in Part A, 9

section 5.2 of the General requirements for the sterility of biological substances (47), or by 10

methods approved by the NRA. 11

12

A.4.6.3 Residual formaldehyde 13

The content of free residual formaldehyde in the trivalent bulk should be determined by a 14

method approved by the NRA. The limits should be approved by the NRA. 15

16

A.4.6.4 D-antigen content 17

The D-antigen content of each trivalent bulk should be determined by use of a validated 18

immunochemical method and should be calculated by use of a reference vaccine calibrated 19

against the WHO International Standard (see section A.1.3). The results obtained should be 20

within the required limits established by the NRA. 21

22

A.4.7 Control of final bulk 23

Preservatives, excipients or other substances that might be added to or combined with the 24

trivalent bulk to form the final bulk should have been shown, to the satisfaction of the NRA, to 25

have no deleterious effect on the immunizing potency and the safety profile of the poliovirus 26

antigens. 27

28

The operations necessary for preparing the final bulk from trivalent bulk should be conducted in 29

such a manner as to avoid contamination of the product. In preparing the final vaccine bulk, any 30

substances such as diluents, stabilizers or adjuvants that are added to the product should have 31

been shown, to the satisfaction of the NRA, not to impair the safety and efficacy of the vaccine 32

in the concentration used. Until the final bulk is filled into containers, the final vaccine bulk 33

WHO/BS/2014.2233

suspension should be stored under conditions shown by the manufacturer to retain the desired 1

biological activity. 2

3


The final bulk should be tested for bacterial and fungal sterility, as specified in Part A, section 5

5.2 of the General requirements for the sterility of biological substances (47), or by methods 6


8

A.4.7.2 Potency tests 9

Each final bulk should be tested in an in vivo assay for immunogenicity by tests approved by the 10

NRA. An in vivo potency assay in rats has been standardized and shown to be a suitable test for 11

IPV (see Appendix 2). Product-specific reference preparations may be used in these tests (see 12

Appendix 2). 13

14

The D-antigen content of each final bulk should be determined using a validated 15

immunochemical method and calculated using a reference vaccine calibrated against the WHO 16

International Standard (see section A.1.3). The results obtained should be within the required 17

limits established by the NRA. This test may be omitted on the final bulk if conducted on the 18

final lot. 19

20

When consistency of production has been established on a suitable number of consecutive final 21

bulks, the in vivo assay may be omitted with the agreement of the NRA. This can occur once it 22

has been demonstrated that the acceptance criteria for the D-antigen determination are such that 23

the in vitro test yields a comparable result to the in vivo assay in terms of acceptance or rejection 24

of a batch. This demonstration must include testing of subpotent batches, produced 25

experimentally if necessary by heat treatment or other means of diminishing the immunogenic 26

activity. 27

28

Where there is a significant change in the manufacturing process of the antigens or in their 29

formulation, the in vivo test should be performed to demonstrate the comparability of the new 30

manufacturing process to the established process. If the process change affects the in vivo test, 31

the need for revalidation should be considered and clinical data may be required for the approval 32

by the NRA. 33

WHO/BS/2014.2233

1

The in vitro assay that has been found most suitable for measuring the antigen content is the D-2

antigen ELISA. Although this assay is widely used, particular attention is required for its 3

standardization. Some NRAs accept the use of polyclonal antisera whereas others accept the use 4

of monoclonal antibodies in the test. The use of different antibodies may give different results. 5

The D-antigen specificity of the antibodies should be demonstrated. Whichever types of antisera 6

are used, the validation studies should show that the assay can determine consistency of 7

production. For a D-antigen ELISA to be valid, it should comply with specified criteria of 8

linearity and parallelism. The effect of a change in the method of calculation of the D-antigen 9

content on registered specifications should also be taken into account. 10

11

Other validated tests such as multiplex antibody test or plasmon resonance 12

technology (52, 53) may be used subject to the approval of the NRA. 13

14

If the use of an adjuvant in the final bulk interferes with the assay, a desorption or treatment step 15

may be necessary before performing the D-antigen ELISA. 16

17

If the final bulk is formulated with poliovirus trivalent bulk and with other antigens into a 18

combination vaccine, the suitability of performing the D-antigen ELISA on the final bulk will 19

have to be determined. If the D-antigen ELISA is not suitable for a particular combination, an in 20

vivo assay should be used. 21

22

The potency of the final bulk for each virus type should be approved by the NRA. 23

24

A.4.7.3 Preservative content 25

If preservative is added, the content in the final bulk should be determined by a method approved 26

by the NRA. The preservative used and content permitted should be approved by the NRA. 27

28

2-Phenoxyethanol has been the only preservative used by IPV manufacturers 29

during the past 50 years as the use of thiomersal can result in loss of D-antigen 30

content. 31

32

A.4.7.4 Adjuvant (if applicable) 33

WHO/BS/2014.2233

Each final vaccine bulk should be assayed for the content of adjuvant. This test may be omitted 1

if it is performed on the final lot. Where aluminium compounds are used, the content of 2

aluminium should not be greater than 1.25 mg per single human dose. 3

A.5 Filling and containers 4

The requirements concerning filling and containers given in Good manufacturing practices for 5

biological products (43) should apply to vaccine filled in the final form. 6

7

Single- and multiple-dose containers may be used. 8

A.6 Control tests on the final lot 9

Samples should be taken from each final lot for the tests described in the following sections. The 10

following tests should be performed on each final lot of vaccine (i.e. in the final containers). 11

Unless otherwise justified and authorized, the tests should be performed on labeled containers 12

from each final lot by means of validated methods approved by the NRA. The permissible limits 13

for the different parameters listed under this section, unless otherwise specified, should be 14


16

A.6.1 Inspection of final containers 17

Every container in each final lot should be inspected visually or mechanically, and those 18

showing abnormalities should be discarded and recorded for each relevant abnormality. A limit 19

should be established for the percentage of rejection. 20

21

A.6.1.1 Appearance 22

The appearance of the vaccine should be described with respect to its form and colour. 23

24

A.6.2 Identity test 25

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

appropriate method. 27

The potency test described in section A.6.5 may serve as the identity test. 28

29

A.6.3 Sterility test for bacteria and fungi 30

WHO/BS/2014.2233

Each final lot should be tested for bacterial and fungal sterility, as specified in Part A, section 5.2 1

of the General requirements for the sterility of biological substances (47), or by methods 2


4

A.6.4 General safety test 5

Each final lot should be tested for the absence of abnormal toxicity in mice or guinea pigs using 6

a test approved by the NRA. This test may be omitted for routine release once consistency of 7

production has been established to the satisfaction of the NRA. 8

9

A.6.5 Potency test 10

Each final lot should be tested by a validated immunochemical method for D-antigen content 11

(see sections A.4.5.5 and A.4.7.2) and calculated by use of a reference vaccine calibrated against 12

the WHO International Standard (see A.1.3). 13

14

In some countries, this test is omitted provided that the determination of the D-15

antigen content has been carried out with satisfactory results on the final bulk 16

product and provided that a validation has been performed to demonstrate that 17

there is no loss of potency between the final bulk product and the final lot, 18

subject to approval by the NRA. 19

20

If the use of an adjuvant in the final bulk interferes with the assay, a desorption or treatment step 21

may be necessary before performing the D-antigen ELISA. If treatment/desorption is not 22

possible, the interference of the adjuvant should be documented and an in vivo assay should be 23

performed (see A.4.7.2 and Appendix 2). 24

25

In general, wIPV that have been formulated to contain 40, 8 and 32 D-antigen 26

units or more per dose for types 1, 2 and 3, respectively, are effective (54). 27

Vaccines with lower D-antigen contents may be acceptable if supported by 28

clinical data. Vaccines in which adjuvants are used, or vaccines produced from 29

other seed viruses (e.g. Sabin viruses), may also be licensed with a different 30

antigenic composition if supported by clinical data. 31

32

WHO/BS/2014.2233

If the final bulk is formulated from a trivalent bulk and other antigens into a 1

combination vaccine, the suitability of performing the D-antigen ELISA on the 2

final lot will have to be determined. If the D-antigen ELISA is not suitable for 3

a particular combination, an in vivo assay such as that described in Appendix 2 4

should be used. 5

6

The potency of the vaccines for each virus type should be approved by the NRA. 7

8

A.6.6 Protein content 9

Poliomyelitis vaccine (inactivated) should not contain more than 10 µg of protein per human 10

dose. This test may be omitted for routine lot release once consistency of production has been 11

established to the satisfaction of the NRA. 12

13

If animal serum is used for the growth of cell cultures, the serum protein concentration (bovine 14

serum albumin) in the final lot should be no more than 50 ng per human dose. The test for 15

bovine serum albumin may be omitted if performed on the trivalent or final bulk, subject to 16

approval by the NRA. 17

18

A.6.7 Preservative content 19

Where appropriate, the preservative content of each final lot should be determined by a method 20

approved by the NRA. The method used and content permitted should be approved by the NRA. 21

This test may be omitted if conducted on the final bulk. 22

23

A.6.8 Endotoxin content 24

The endotoxin content of each final lot should be determined by a method approved by the NRA. 25

Levels should be consistent with levels found to be acceptable in vaccine lots used in pre-26

licensure clinical trials and approved by the NRA. 27

28

A.6.9 Test for residual formaldehyde 29

The content of free residual formaldehyde in each final lot should be determined by a method 30

approved by the NRA, The limit should be approved by the NRA. This test may be omitted if 31

performed on the trivalent bulk or on the final bulk. 32

33

WHO/BS/2014.2233

A.6.10 Test for pH 1

The pH of each final lot should be determined and should be within limits approved by the NRA. 2

3

A.6.11 Adjuvant and degree of adsorption (if applicable) 4

If an adjuvant is used in the formulation, each final lot should be assayed for the content of 5

adjuvant. Where aluminium compounds are used, the content of aluminium should not be greater 6

than 1.25 mg per single human dose. This test may be omitted on the final lot if performed on the 7

final bulk. 8

9

The degree of adsorption of the antigen to the aluminium compounds (aluminium hydroxide or 10

hydrated aluminium phosphate) in each final lot should be assessed. 11

12

A.6.12 Residual antibiotics (if applicable) 13

If any antibiotics are added in the vaccine production, the content of the residual antibiotics 14

should be determined and should be within limits approved by the NRA. This test may be 15

omitted for routine lot release once consistency of production has been established to the 16

satisfaction of the NRA. 17

A.7 Records 18

The requirements given in Good manufacturing practices for biological products (43) should 19

apply. 20

A.8 Retained samples 21


apply. 23

A.9 Labelling 24


apply, and additionally the label on the container or package should include the following 26

information: 27

− the designation(s) of the strain(s) of poliovirus contained in the vaccine; 28

− the cell substrate used for the preparation of vaccine; 29

− the D-antigen content of each poliovirus type; 30

− the method and inactivating agent used to inactivate the virus; 31

− the nature and amount of any stabilizer and preservative present in the vaccine; 32

WHO/BS/2014.2233

− the nature and amount of adjuvant, if applicable. 1

2

It is desirable for the label to carry the names both of the producer and of the 3

source of the bulk material if the producer of the final vaccine did not prepare 4

it. The nature and amount of the antibiotics present in the vaccine, if any, may 5

be included. 6

A.10 Distribution and shipping 7


apply. Further guidance is provided in WHO’s Model guidance for the storage and transport of 9

time- and temperature-sensitive pharmaceutical products (55). 10

A.11 Stability, storage and expiry date 11

A.11.1 Stability testing 12

Adequate stability studies form an essential part of vaccine development. Current guidance on 13

the evaluation of vaccine stability is provided in the WHO Guidelines on stability evaluation of 14

vaccines (56). Stability testing should be performed at different stages of production when 15

intermediate product is stored, namely on single harvests, inactivated purified monovalent pool, 16

trivalent bulk, final bulk and final lot. Stability-indicating parameters should be defined or 17

selected appropriately according to the stage of production. During vaccine production a shelf-18

life should be assigned to all in-process materials – particularly intermediates such as single 19

harvests, inactivated purified monovalent pool, trivalent bulk and final bulk. 20

21

The stability of the vaccine in its final containers, maintained at the recommended storage 22

temperature up to the expiry date, should be demonstrated to the satisfaction of the NRA. As a 23

guide, containers from at least three consecutive final lots, and derived from different 24

monovalent pools and different trivalent bulks, may be tested. 25

26

Where manufacturing involves only formulation of the final bulk from trivalent bulks supplied 27

by another manufacturing establishment and the filling of final containers, stability data on the 28

trivalent bulks in their storage conditions and the shelf-life established until use should be 29

generated by the manufacturer performing the final fill. 30

31

WHO/BS/2014.2233

The formulation of the vaccine should be stable throughout its shelf-life. Acceptable limits for 1

stability should be agreed with the NRA. Following licensure, ongoing monitoring of vaccine 2

stability is recommended to support shelf-life specifications and to refine the stability profile 3

(56). Data should be provided to the NRA in accordance with local regulatory requirements. 4

5

The efficacy of the preservative should be confirmed at the end of shelf-life. In the case of multi-6

dose presentations, efficacy of the preservative should be confirmed for the period during which 7

the vial can be open, in compliance with the WHO policy on the use of opened multi-dose vials 8

of vaccine in subsequent immunization sessions (57). 9

10

The final stability testing programme should be approved by the NRA and should include an 11

agreed set of stability-indicating parameters, procedures for the ongoing collection and sharing 12

of stability data, and criteria for rejecting vaccine(s). 13

14

A.11.2 Storage conditions 15

Poliomyelitis vaccine (inactivated) should be stored at all times at a temperature between 2 °C 16

and 8 °C. For novel vaccines, appropriate storage conditions should be validated and approved 17

by the NRA. 18

19

A.11.3 Expiry date 20

The expiry date should be based on the shelf-life, and should be supported by stability studies 21

and approved by the NRA. The expiry date should relate to the date of blending of the final bulk, 22

the date of filling or the date of the first valid potency test on the final lot, which should be 23

performed in an assay as described in Appendix 2. 24

25

Where an in vivo potency test is used, the date of the potency test is the date on 26

which the test animals were inoculated with the final bulk. 27

28

Part B. Nonclinical evaluation of poliomyelitis vaccines 29

(inactivated) 30

31

The nonclinical evaluation of candidate inactivated poliomyelitis vaccines should be based on 32

the WHO guidelines on nonclinical evaluation of vaccines (8). The following specific issues are 33

WHO/BS/2014.2233

intended for new IPV candidates and should also be referred to when a significant change is 1

made in the manufacturing process or vaccine formulation of a licensed IPV. 2

3

B.1 Characterization of poliovirus seed lots derived from attenuated 4

strains (Sabin strains and strains derived by recombinant DNA 5

technology) 6

The virus master and working seed lots derived from attenuated strains (Sabin strains and strains 7

derived by recombinant DNA technology) that are used to manufacture a candidate IPV should 8

be extensively characterized, as described in section A.3.1. Ideally, the characterization studies 9

should be performed on seed lots used to prepare the vaccine batches tested in preclinical and 10

clinical studies. 11

12

When attenuated poliovirus strains derived by recombinant DNA technology are used to prepare 13

a candidate IPV, the mutations responsible for attenuation should be identified along with the 14

mutations that can revert to partial or full virulence phenotype. The need for testing virus master 15

seed lots of attenuated strains derived by recombinant DNA technology with in vivo 16

neurovirulence tests should be considered and the decision whether to test or not should be 17

scientifically justified. In addition, the genetic stability of the strains derived by recombinant 18

DNA technology should be confirmed at the passage level (or beyond) used to prepare the 19

vaccine. Efforts should also be made to develop an in vitro test to detect reversion to partial and 20

full virulent virus. 21

B.2 Antigenic profile 22

The available evidence suggests that there may be significant differences in the antigenic 23

composition of various IPV products developed independently (58, 59), particularly when 24

comparing sIPV to wIPV. It is likely that antigenic profiles of IPV are influenced by virus 25

strains, cell substrates and process parameters used in manufacture. The antigenic structure of a 26

candidate IPV should ideally be established using monoclonal antibodies (59, 60) of known 27

specificity at the early stage of product development and should be used as a characterization 28

tool for investigating vaccine stability and demonstrating manufacturing consistency during 29

product development. 30

WHO/BS/2014.2233

B.3 D-antigen content of IPV derived from attenuated strains (Sabin 1

strains and strains derived by recombinant DNA technology) 2

The type-specific antigen content of current licensed wIPV is measured using various ELISA 3

procedures (60) and is reported as D-antigen units relative to a reference preparation traceable to 4

the International Standard. When attenuated strains (e.g. Sabin strains and strains derived by 5

recombinant DNA technology) are used to prepare the candidate IPV, an in-house ELISA should 6

be developed and implemented to determine type-specific D-antigen content. An in-house 7

reference standard should be established at the early stage of product development and should be 8

calibrated against the International Standard. A monitoring programme should be put in place to 9

ensure the stability of the in-house reference standard and the comparability of its subsequent 10

replacement. In addition, the ratio between virus titre (per mL) and D-antigen content (per mL) 11

of purified monovalent pools prior to inactivation should also be established for each polio type 12

during product development and should be monitored during commercial production. This 13

provides further assurance that the D-antigen content of commercial lots, throughout the product 14

life cycle, is comparable to lots shown to be safe and immunogenic in clinical studies. 15

16

Most licensed wIPV products have been formulated to contain 40, 8 and 32 D-antigen units per 17

human dose. However, the D-antigen unit is not well defined with respect to various poliovirus 18

strains used in manufacture and known to be influenced by the specificity of the antibodies used 19

as ELISA reagents. Therefore, it is not possible to compare directly the D-antigen content of 20

various IPV (e.g. sIPV versus wIPV) measured using different monoclonal or polyclonal 21

antibody-based ELISA procedures (61). It is recognized that IPV derived from attenuated strains 22

or adjuvanted IPV may require different D-antigen content to induce adequate immune responses 23

in humans. 24

B.4 Evaluation of immunogenicity in animal models 25

Prior to initiating clinical trials, the immunogenic properties of a candidate IPV should be 26

studied in suitable animal models (e.g. rats). Proof-of-concept nonclinical studies should include 27

the comparison of immunogenicity between a candidate IPV and a currently licensed wIPV 28

based on type-specific serum neutralizing antibody titres against both Sabin and wild-type 29

strains. Those studies may also assist in the selection of D-antigen content to be tested in the 30

dose-finding studies in humans. However, it is important to note that immunogenicity data in 31

animals do not reliably predict the antigen content that might be appropriate for inclusion as a 32

single human dose in the final vaccine formulation. The assay using transgenic mice may be 33

WHO/BS/2014.2233

performed to compare the immune response and protection against virulent challenge induced by 1

a candidate IPV to that induced by a licensed wIPV (31, 32). In vivo tests are also important 2

tools to be used as characterization tests to demonstrate comparable manufacturing processes 3

when major changes are introduced. 4

5

When an adjuvant is included in the formulation, manufacturers should provide a rationale and 6

immunogenicity data to support the use of an adjuvant in the vaccine (62). 7

B.5 Nonclinical safety studies 8

If a candidate IPV is formulated with a novel adjuvant or excipient (e.g. stabilizer), nonclinical 9

safety studies should be conducted based on WHO’s Guidelines on the nonclinical evaluation of 10

vaccine adjuvants and adjuvanted vaccines (62). The use of a delivery device as well as 11

alternative administration routes (e.g. intradermal) may affect vaccine potency/immunogenicity, 12

tolerability, toxicity and long-term safety. The design of nonclinical safety studies should follow 13

special considerations outlined in section 5 of the WHO guidelines on nonclinical evaluation of 14

vaccines (8). 15

16

Part C. Clinical evaluation of poliomyelitis vaccine 17

(inactivated) 18

19

Clinical trials should adhere to the principles described in WHO’s Guidelines for good clinical 20

practice (GCP) for trials on pharmaceutical products (63) and Guidelines on clinical evaluation 21

of vaccines: regulatory expectations (9). 22

23

Some of the issues that are specific to the clinical evaluation of IPV are discussed in the 24

following sections, which are applicable to IPV derived from wild-type strains as well as 25

attenuated strains (e.g. Sabin strains and strains derived by recombinant DNA technology). 26

C.1 General considerations 27

The global poliomyelitis eradication initiative following the 1998 World Health Assembly 28

resolution led to a dramatic decrease in poliomyelitis cases globally (11). Consequently, clinical 29

efficacy studies to support the licensure of a candidate IPV are no longer feasible, and the 30

clinical evaluation should be based on the comparative assessment of safety and immunogenicity 31

of a candidate vaccine with a licensed vaccine (comparator vaccine). The assessment of 32

WHO/BS/2014.2233

seroconversion should be based on the elicitation of serum neutralizing antibodies, which have 1

been established as the basis of protection (64). The licensure of a candidate IPV should be based 2

on a clear demonstration of non-inferiority in terms of immunogenicity when compared to a 3

comparator vaccine. 4

C.2 Immunogenicity studies 5

C.2.1 Assessment of the immune response 6

A serum neutralizing antibody titre of ≥8 is considered to be a marker of clinical protection 7

against poliomyelitis (65). The demonstration of an immune response to IPV vaccination should 8

be based on the measurement of neutralizing antibody titres at pre- and post-vaccination time 9

points. Seroconversion for polio antigen is defined as: 10

− for subjects seronegative at the pre-vaccination time point, post-vaccination antibody 11

titres of ≥ 8; 12

− for subjects seropositive at the pre-vaccination time point, a ≥4-fold rise in antibody titres 13

post-vaccination. In the event that the pre-vaccination titre is due to maternal antibodies, 14

a 4-fold rise above the expected titre of maternal antibodies based on the pre-vaccination 15

titre declining with a half-life of 28 days indicates seroconversion. 16

17

It is recommended that the assay used to assess serum neutralizing antibodies should be 18

standardized, as described in WHO’s Manual for the virological investigation of polio (66), 19

particularly with respect to the use of appropriate cell lines, International Standards of anti-20

poliovirus sera and other important reagents. The level of neutralizing antibody present in a 21

serum sample is expressed as a titre, which is the reciprocal of the highest serum dilution that 22

inhibits the viral cytopathic effect in 50% of cell culture. 23

24

For the evaluation of IPV derived from attenuated strains (Sabin strains and strains derived by 25

recombinant DNA technology), serum neutralizing antibody titres against both Sabin and wild-26

type poliovirus should be determined in order to ensure that the conclusions of clinical studies are 27

applicable to both types of strains. In view of the antigenic differences between the wild-type 28

poliovirus strains, it may be useful to assess the neutralizing antibody titres using both recent 29

wild-type isolates and the conventional strains in a subset of study subjects, if relevant. 30

31

WHO/BS/2014.2233

The presence of neutralizing antibody against polioviruses is considered a reliable correlate of 1

protection against poliomyelitis. However, immunity induced by one serotype does not provide 2

protection against the other two serotypes. 3

4

C.2.2 Immunogenicity studies 5

A candidate IPV should be directly compared with at least one well established and licensed IPV 6

in prospective controlled studies. In the event that none of the IPV products can be used in the 7

country where the clinical studies are conducted, the use of OPV as a comparator may be 8

acceptable to the NRA, bearing in mind the differences in the route of administration and the 9

types of immune responses generated. However, the use of OPV in clinical studies should be in 10

compliance with the Polio Eradication and Endgame Strategic Plan 20132018 (15), and it must 11

be noted that the use of tOPV is time-limited as bOPV will be introduced and will be followed 12

by the complete cessation of all OPV vaccination. 13

14

Non-inferiority studies to evaluate immunogenicity after completion of the primary vaccination 15

series in the target population (e.g. naive infants) are required for regulatory approval of a 16

candidate IPV. Persistence of the serum neutralizing antibodies after the primary series should 17

also be investigated to recommend whether and when a booster dose is required. However, data 18

concerning long-term antibody persistence might not be available prior to regulatory approval. 19

Waning of antibodies over time is inevitable and should not be interpreted to indicate the need 20

for a booster dose per se, as available data suggest that persistent immune memory may be 21

sufficient to protect against poliomyelitis (67, 68). 22

23

C.2.3 Study population and region 24

In general, the first clinical study (Phase I) of a candidate IPV should be performed in healthy 25

adults to assess vaccine safety. Due to wide use of IPV and OPV, the immunogenicity of a 26

candidate IPV can be reliably evaluated only in a naive target population, such as infants. 27

28

Exposure of study subjects to circulating wild-type or OPV-derived poliovirus may enhance the 29

immune response induced by IPV and, in turn, may affect study conclusions. Therefore, clinical 30

trials to evaluate the immunogenicity of a candidate IPV should ideally be performed in regions 31

where IPV is used exclusively. In the event that a clinical trial is conducted in areas where OPV 32

WHO/BS/2014.2233

is used routinely, special measures should be taken to minimize the potential exposure of study 1

participants to live poliovirus. 2

3

C.2.4 Endpoints and analyses 4

The primary study analysis should be based on the rate of seroconversion (as described in section 5

C.2.1) against both Sabin and wild-type strains measured at approximately 4 weeks following 6

completion of the primary infant immunization. The primary study objectives should be based on 7

the demonstration of the non-inferiority of the seroconversion rates achieved with the candidate 8

IPV versus the comparator vaccine. 9

10

The predefined clinical margins of non-inferiority should be justified, and the calculations of the 11

proposed sample size required should be clearly explained in the study protocol. Further 12

guidance on demonstrating non-inferiority trials is described in WHO’s Guidelines on clinical 13

evaluation of vaccines: regulatory expectations (9). 14

15

Comparison of geometric mean titres (GMTs) and reverse cumulative distributions of individual 16

titres against both Sabin and wild-type poliovirus at 4 weeks post-primary should also be 17

performed as a secondary analysis. While it may be that the GMT(s) for one or more poliovirus 18

types induced by the candidate IPV derived from attenuated strains is lower than that induced by 19

the comparator, it is not clear if a lower GMT at 4 weeks post-primary affects long-term 20

antibody persistence. Consequently, any significant differences in GMT observed (e.g. not 21

meeting pre-specified criteria) should be carefully considered by the NRA and a decision should 22

be supported by additional studies of antibody persistence (as described in section C.2.2) and 23

commitment to post-marketing studies (described in section C.5). 24

25

The minimal D-antigen content required for the candidate vaccine at the end of its shelf-life 26

should be based on the D-antigen content of the clinical lots shown to induce acceptable immune 27

responses in clinical studies (e.g. lots used in the dose-finding study). 28

29

C.2.5 Immunization schedule 30

Different immunization schedules are used for licensed wIPV in various regions and countries. It 31

is common for IPV to be administered according to the same schedule as DTP-containing 32

vaccines in order to achieve a high compliance rate. Clinical trial data have shown that the 33

WHO/BS/2014.2233

immune response induced by licensed wIPV varies according to the immunization schedule 1

used. In general, longer intervals in the primary immunization series (e.g. 2, 4 and 6 months) 2

induce higher neutralizing antibody titres and a better seroconversion rate (69, 70). An 3

immunization schedule should be defined for the targeted country or region wherever possible, 4

and dose-finding and non-inferiority studies for a candidate IPV should be conducted according 5

to this immunization schedule. However, it is not feasible to study a candidate vaccine using 6

every possible schedule in all target regions. Manufacturers should justify the relevance of the 7

clinical data provided to each country in which approval is sought and should discuss the basis 8

for extrapolation of the findings. For example, satisfactory immune responses using a schedule 9

with a short interval between immunizations (e.g. 2, 3 and 4 months) supports an expectation 10

that satisfactory immune responses would also be observed using a schedule with a longer 11

interval (e.g. 2, 4 and 6 months). However, the local and systemic reactogenicity associated with 12

a candidate IPV may also differ between schedules within a specific population so there is still a 13

need to collect some safety data, prior to regulatory approval, for the proposed schedules (e.g. 2, 14

4 and 6 months). 15

16

The use of an IPV that is prepared from attenuated poliovirus strains or that contains a fractional 17

antigen dose in a sequential IPV-OPV immunization schedule should be supported by clinical 18

studies to ensure adequate serum neutralizing antibodies against both wild-type and Sabin 19

poliovirus. 20

C.3 Concomitant administration with other vaccines 21

IPV is commonly co-administered with other infant and toddler vaccines. Consequently, it is 22

essential to evaluate the immune responses to a candidate IPV as well as to all other antigens co-23

administered in all the co-administration situations claimed. Due to the large number of licensed 24

vaccines that may need to be co-administered with IPV in infants and toddlers using a variety of 25

schedules, it is not feasible for manufacturers to study every possible combination. The data on 26

the effects of co-administration that are available at the time of initial licensure may be limited 27

and should be expanded in post-approval studies. If study results indicate that immune responses 28

are lower on co-administration with other vaccine(s), the NRA will need to consider the potential 29

clinical consequences on a case-by-case basis. 30

WHO/BS/2014.2233

C.4 Pre-licensure safety data 1

The general approach to safety assessment of a candidate IPV during pre-licensure clinical 2

studies should be in accordance with WHO’s Guidelines on clinical evaluation of vaccines: 3

regulatory expectations (9). The safety profile of a candidate IPV derived from attenuated strains 4

is expected to be very similar to that of current licensed wIPV, which is very well tolerated. The 5

NRA may decide that large safety studies are not required. However, if a candidate IPV includes 6

novel adjuvants and/or excipients, or is administered using an alternative route and/or delivery 7

device, a safety database similar in size to that requested for any new vaccine entity might be 8

needed. This should be discussed and approved by the NRA on a case-by-case basis. In addition, 9

it is likely that adverse events at the injection site are more frequent if a candidate IPV contains 10

an adjuvant. This may be acceptable if the incidence of adverse events is comparable to that 11

observed for other licensed adjuvanted vaccines and the benefit clearly outweighs the risks. 12

13

An appropriate pharmacovigilance plan should be developed and approved by the NRA prior to 14

licensure. 15

C.5 Post-marketing studies and surveillance 16

Post-marketing surveillance should be undertaken during the initial post-approval years in 17

collaboration with the NRA. Manufacturers and health authorities should work in collaboration 18

with the global polio surveillance laboratory network to monitor new vaccines once they are 19

introduced in immunization programmes. The enhanced safety surveillance is particularly 20

important for vaccines which include novel adjuvants and/or excipients. Due to the possibility 21

that the sIPV may induce a lower GMT for one or more poliovirus types, the persistence of 22

antibody and the need for a booster dose should be studied in the post-marketing period. 23

24

The total duration of enhanced surveillance should be regularly reviewed by the NRA. If 25

particular issues arise during pre-licensure studies or during post-licensure safety surveillance, it 26

may be necessary to conduct specific post-licensure safety studies. 27

28

WHO/BS/2014.2233

Part D. Recommendations for national regulatory authorities 1

D.1 General 2

The general recommendations for NRAs and NCLs given in the Guidelines for national 3

authorities on quality assurance for biological products (71) and Guidelines for independent lot 4

release of vaccines by regulatory authorities (72) should apply. 5

6

The detailed production and control procedures, as well as any significant changes in them that 7

may affect the quality, safety and efficacy of poliomyelitis vaccine (inactivated), should be 8

discussed with and approved by the NRA. 9

10

For control purposes, the International Standards that are currently in force should be obtained 11

for the purpose of calibration of the national/regional/working standards (73). The NRA may 12

obtain from the manufacturer the product-specific or working reference to be used for lot release 13

until international/national standard preparation is established. 14

15

Consistency of production has been recognized as an essential component in the quality 16

assurance of poliomyelitis vaccine (inactivated). In particular, the NRA should carefully monitor 17

production records and quality control test results for clinical lots as well as for a series of 18

consecutive lots of the vaccine. 19

D.2 Official release and certification 20

A vaccine lot should be released only if it fulfils the national requirements and/or satisfies Part A 21

of the present recommendations (72). 22

23

A protocol based on the model given in Appendix 3, signed by the responsible official of the 24

manufacturing establishment, should be prepared and submitted to the NRA in support of a 25

request for the release of vaccine for use. 26

27

A statement signed by the appropriate official of the NRA should be provided to the 28

manufacturing establishment and should certify that the lot of vaccine in question meets all 29

national requirements, as well as Part A of these recommendations. The certificate should 30

provide sufficient information on the vaccine lot. A model certificate is given in Appendix 4. 31

WHO/BS/2014.2233

The official national release certificate should be provided to importers of the vaccines. The 1

purpose of the certificate is to facilitate the exchange of vaccines between countries. 2

WHO/BS/2014.2233

Authors and acknowledgements 1

2

A pre-draft of this document was prepared by Dr M. Lennon, Horning, England; Dr C. Li, 3

National Institutes for Food and Drug Control, Beijing, China; Dr J. Martin, National Institute 4

for Biological Standards and Control, Potters Bar, England; Dr P. Minor, National Institute for 5

Biological Standards and Control, Potters Bar, England; Dr K. Chumakov, Center for Biologics 6

Evaluation and Research, Rockville, MD, USA; Dr T. Wu, Health Canada, Ottawa, Canada; with 7

support from the WHO Secretariat: Dr T.Q. Zhou, Dr J. Fournier-Caruana, Dr I. Knezevic and 8

Dr D.J. Wood, Department of Essential Medicines and Health Products, World Health 9

Organization, Geneva, Switzerland; Dr H. Okayasu and Dr R. Sutter, Research, Policy and 10

Product Development, World Health Organization, Geneva, Switzerland, taking into 11

considerations the discussions at a Working Group meeting on Technical Specifications for 12

Manufacturing and Evaluating the WHO Recommendations for IPV: TRS No. 910 in Geneva, 13

Switzerland, 2729 March 2012 attended by: Dr M. Baca-Estrada, Health Canada, Ottawa, 14

Canada; Dr W.A.M. Bakker, National Institute for Public Health and the Environment, 15

Bilthoven, Netherlands; Dr J. Fournier-Caruana, Department of Essential Medicines and Health 16

Products, World Health Organization, Geneva, Switzerland; Mr B.S. Chauhan, Bharat Biotech 17

International Limited, Hyderabad, India; Dr K. Chumakov, Center for Biologics Evaluation and 18

Research, Bethesda, MD, USA; Dr E. Coppens, Sanofi Pasteur, Marcy l’Etoile, France; Dr M. 19

Duchêne, GSK Biologicals, Wavre, Belgium; Ms G. Dunn, National Institute for Biological 20

Standards and Control, Potters Bar, England ; Dr D. Felnerova, Crucell, Berne, Switzerland; Dr 21

M. Lennon, Horning, England; Dr L. Fiore, Istituto Superiore di Sanita, Rome, Italy; Mr J.B. 22

González, Laboratorios de Biológicos y Reactivos de México S.A. de C.V., Mexico City, 23

Mexico; Dr M.A. Gonzalez, Federal Commission for the Protection from Sanitary Risks, 24

Ministry of Health, Mexico; Dr V. Grachev, Russian Academy of Medical Sciences, Moscow, 25

Russian Federation; Ms H. Wang, Tiantan Biological Products Co. Ltd., Beijing, China; Mrs T. 26

Jivapaisarnpong, Ministry of Public Health, Bangkok, Thailand; Dr I. Knezevic, Department of 27

Essential Medicines and Health Products, World Health Organization, Geneva, Switzerland; Dr 28

H. Okayasu, Research, Policy and Product Development, World Health Organization, Geneva, 29

Switzerland; Dr D. Kusmiaty, National Quality Control Laboratory of Drug and Food, Ministry 30

of Health, Jakarta, Indonesia; Dr K. Katayama, National Institute of Infectious Diseases, Tokyo, 31

Japan; Dr C. Li, National Institutes for Food and Drug Control, Beijing, China; Dr J. Martin, 32

National Institute for Biological Standards and Control, Potters Bar, England; Dr. C. Milne, 33

WHO/BS/2014.2233

European Directorate for the Quality of Medicines & HealthCare, Council of Europe, 1

Strasbourg, France; Dr R. Modi, Cadila Pharmaceuticals Limited, Ahmedabad, India; Ms E. 2

Niogret, Sanofi Pasteur, Marcy L'Etoile, France; Dr L.V. Phung, National Institute for Control of 3

Vaccine and Biologicals, Hanoi, Viet Nam; Dr V. Pithon, Agence nationale de sécurité du 4

médicament et des produits de santé, Lyon, France; Dr A. Sinyugina, Russian Academy of 5

Medical Sciences, Moscow, Russian Federation; Dr R. Sutter, Research, Policy and Product 6

Development, World Health Organization, Geneva, Switzerland; Mr D. Ugiyadi, BioFarma, 7

Bandung, Indonesia; Dr G. Waeterloos, Scientific Institute of Public Health, Brussels, Belgium; 8

Dr S. Yamazaki, Japan Poliomyelitis Research Institute, Tokyo, Japan; Dr D.J. Wood, 9

Department of Essential Medicines and Health Products, World Health Organization, Geneva, 10

Switzerland; Mr L. Yi, Institute of Medical Biology, Kunming, China; Dr T.Q. Zhou, 11


Switzerland. 13

Draft 1 was prepared by Dr M. Lennon, Horning, England; Dr C. Li, National Institutes for 14

Food and Drug Control, Beijing, China; Dr J Martin, National Institute for Biological Standards 15

and Control, Potters Bar, England; Dr P. Minor, National Institute for Biological Standards and 16

Control, Potters Bar, England; Dr K. Chumakov, Center for Biologics Evaluation and Research, 17

Rockville, MD, USA; Dr T. Wu, Health Canada, Ottawa, Canada; with support from the WHO 18

Secretariat: Dr T.Q. Zhou, Dr J. Fournier-Caruana, Dr I. Knezevic and Dr D.J. Wood , 19


Switzerland; Dr H. Okayasu and Dr H.S. Jafari, Policy and Product Development, World Health 21

Organization, Geneva, Switzerland;taking into considerations the discussions at a Working 22

Group meeting on Technical Specifications for Manufacturing and Evaluating the WHO 23

Recommendations for IPV: TRS No. 910 in Geneva, Switzerland, 1415 May 2013, attended by 24

Ms P. Agsiri, Ministry of Public Health, Nonthaburi, Thailand; Dr W A.M. Bakker, Institute for 25

Translational Vaccinology, Bilthoven, Netherlands; Dr K. Chumakov, Center for Biologics 26

Evaluation and Research, Rockville, MD, USA; Dr J. Fournier-Caruana, Department of Essential 27

Medicines and Health Products, World Health Organization, Geneva, Switzerland; Dr E. 28

Griffiths, Consultant, Kingston upon Thames, England; Dr I. Hansenne, Scientific Institute of 29

Public Health, Brussels, Belgium; Dr K. Katayama, National Institute of Infectious Diseases, 30

Tokyo, Japan; Dr J. Korimbocus, Agence nationale de sécurité du médicament et des produits de 31

santé, Lyon, France; Dr M. Lennon, Horning, Norfolk, England; Dr C. Li, National Institutes for 32

Food and Drug Control, Beijing, China; Dr J Martin, National Institute for Biological Standards 33

WHO/BS/2014.2233

and Control, Potters Bar, England; Dr P. Minor, National Institute for Biological Standards and 1

Control, Potters Bar, England; Mr M. Mitsuki, Pharmaceuticals and Medical Devices Agency, 2

Tokyo, Japan; Dr P. Neels, Federal Agency for Medicinal and Health Products, Brussels, 3

Belgium; Dr M. van Oijen, Institute for Translational Vaccinology, Bilthoven, Netherlands; Dr 4

V.G. Somani, Central Drugs Standard Control Organization, New Delhi, India; Dr T. Wu, Health 5

Canada, Ottawa, Canada; Professor H. Yin, State Food and Drug Administration, Beijing, China; 6

Dr N. Benno Chukilizo, Tanzania Food and Drugs Authority, Dar-es-Salaam, United Republic of 7

Tanzania; Mrs D. Darko, Food and Drugs Board, Accra, Ghana; Dr K. Mahmood, PATH, 8

Seattle, WA, USA; Dr C. Milne, European Directorate for the Quality of Medicines & 9

HealthCare, Council of Europe, Strasbourg, France; Dr D-Y. Choi, Lucky Goldstar Life 10

Sciences, Seoul, Korea; Dr R. Dhere, Serum Institute of India Ltd., Pune, India; Dr S. Konda, 11

Panacea Biotec Ltd., New Delhi, India; Ms X. Li, National Vaccine and Serum Institute, Beijing, 12

China; Mr D. Ugiyadi, PT Biofarma, Bandung, Indonesia; Ms H. Wang, Tiantan Biological 13

Products, Beijing, China; Dr S. de Walque, GlaxoSmithKline Vaccines, Wavre, Belgium; Dr T. 14

de Rosa, Crucell Switzerland Ltd., Berne, Switzerland; Dr E. Vidor, Sanofi Pasteur, Lyon, 15

France; Dr Y. Kino, The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan; Dr Q. 16

Li, Kunming Institute of Medical Biology, Kunming, China; Dr G. Stawski, Statens Serum 17

Institut, Copenhagen, Denmark;Dr K. Wakabayashi, Japan Poliomyelitis Research 18

Institute,Tokyo, Japan; Dr D.J. Wood, Department of Essential Medicines and Health Products, 19

World Health Organization, Geneva, Switzerland. 20

Draft 2 was prepared by Dr M. Lennon, Horning, England and Dr T.Q. Zhou, Department of 21

Essential Medicines and Health Products, World Health Organization, Geneva, Switzerland 22

taking into consideration comments from Ms P. Agsiri, Ministry of Public Health, Nonthaburi, 23

Thailand; Dr W.A.M. Bakker, Institute for Translational Vaccinology, Bilthoven, Netherlands; 24

Dr J. Fournier-Caruana, Department of Essential Medicines and Health Products, World Health 25

Organization, Geneva, Switzerland; Dr J. Korimbocus, Agence nationale de sécurité du 26

médicament et des produits de santé, Lyon, France; Dr C. Li, National Institutes for Food and 27

Drug Control, Beijing, China; Dr J. Martin, National Institute for Biological Standards and 28

Control, Potters Bar, England; Dr P. Minor, National Institute for Biological Standards and 29

Control, Potters Bar, England; Dr M. van Oijen, Institute for Translational Vaccinology, 30

Bilthoven, Netherlands; Dr T. Wu, Health Canada, Ottawa, Canada; Dr K. Mahmood, PATH, 31

Seattle, WA, USA; Dr C. Milne, European Directorate for the Quality of Medicines & 32

HealthCare, Council of Europe, Strasbourg, France; Dr S. Konda, Panacea Biotec Ltd., New 33

WHO/BS/2014.2233

Delhi, India; Dr E. Vidor, Sanofi Pasteur, Lyon, France; Dr Y. Kino, The Chemo-Sero-1

Therapeutic Research Institute, Kumamoto, Japan; Dr Q. Li, Kunming Institute of Medical 2

Biology, Kunming, China; Dr K. Wakabayashi, Japan Poliomyelitis Research Institute, Tokyo, 3

Japan. 4

Draft 3 was prepared Dr M. Lennon, Horning, Norfolk, England and Dr T.Q. Zhou, Department 5

of Essential Medicines and Health Products, World Health Organization, Geneva, Switzerland 6

and the drafting group: Dr K. Chumakov, Center for Biologics Evaluation and Research, 7

Rockville, MD, USA; Dr C. Li, National Institutes for Food and Drug Control, Beijing, China; 8

Dr J. Martin, National Institute for Biological Standards and Control, Potters Bar, England; Dr P. 9

Minor, National Institute for Biological Standards and Control, Potters Bar, England; Dr T. Wu, 10

Health Canada, Ottawa, Canada; with support from the WHO Secretariat: Dr J. Fournier-11

Caruana, Dr I. Knezevic and Dr D.J. Wood, Department of Essential Medicines and Health 12

Products, World Health Organization, Geneva, Switzerland; Dr H. Okayasu and Dr R. Sutter, 13

Research, Policy and Product Development, World Health Organization, Geneva, Switzerland; 14

and Dr N. Previsani, Global Polio Eradication Initiative, World Health Organization, Geneva, 15

Switzerland, following a first round public consultation of the second draft document on the 16

WHO Biologicals website during December 2013January 2014, taking into consideration 17

comments provided by Dr S. Abe and Dr S. Yamazaki, Japan Poliomyelitis Research Institute, 18

Tokyo, Japan; Professor Y Che, and Professor S Jiang, and Professor Q Li, Chinese Academy of 19

Medical Sciences, Kunming, China; Dr Y-K. Lee, Ministry of Food & Drug Safety, Cheongwon-20

gun, Republic of Korea; Dr R. Lundquist, Center for Biologics Evaluation and Research, 21

Bethesda, MD, USA; Dr C. Milne, European Directorate for the Quality of Medicines & 22

HealthCare, Council of Europe, Strasbourg, France; Dr M. Li, China Food and Drug 23

Administration, Beijing, China; Dr S. Morgeaux, Agence Nationale de Sécurité du Médicament 24

et des Produits de Santé, Lyon, France; Dr M. van Oijen, Institute for Translational Vaccinology, 25

Bilthoven, Netherlands; Dr E. Vidor, Sanofi Pasteur, Lyon, France; Dr K. Wakabayashi, Japan 26

Poliomyelitis Research Institute, Tokyo, Japan; Dr S. de Walque, GlaxoSmithKline Vaccines, 27

Wavre, Belgium; Dr K. Mahmood, PATH, Seattle, WA, USA; Ms P. Agsiri, Ministry of Public 28

Health, Bangkok, Thailand; Dr J. Prakash, Indian Pharmacopoeia Commission, Ghaziabad, 29

India. 30

Draft 4 was prepared Dr M. Lennon, Horning, England and Dr T.Q. Zhou, Department of 31

Essential Medicines and Health Products, World Health Organization, Geneva, Switzerland and 32

the drafting group: Dr K. Chumakov, Center for Biologics Evaluation and Research, Food & Drug 33

WHO/BS/2014.2233

Administration, Rockville, MD, USA; Dr C. Li, National Institutes for Food and Drug Control, 1

Beijing, China; Dr J. Martin, National Institute for Biological Standards and Control, Potters Bar, 2

England; Dr P. Minor, National Institute for Biological Standards and Control, Potters Bar, 3

England; Dr T. Wu, Health Canada, Ottawa, Canada; with support from the WHO Secretariat: Dr 4

J. Fournier-Caruana, Dr I. Knezevic and Dr D.J. Wood, Department of Essential Medicines and 5

Health Products, World Health Organization, Geneva, Switzerland; Dr H. Okayasu, Research, 6

Policy and Product Development, World Health Organization, Geneva, Switzerland; Dr N. 7

Previsani, Surveillance, Monitoring and Information, World Health Organization, Geneva, 8

Switzerland; and Dr M.R. Balakrishan, Department of Essential Medicines and Health Products, 9

World Health Organization, Geneva, Switzerland, taking into consideration the discussions at an 10

Informal Consultation on the WHO Recommendations to assure the quality, safety and efficacy of 11

poliomyelitis (inactivated) in Geneva, Switzerland on 1415 May 2013 attended by Dr R. Arora, 12

Indian Council of Medical Research, New Delhi, India; Ms P. Agsiri, Ministry of Public Health, 13

Nonthaburi, Thailand; Dr W.A.M. Bakker, Institute for Translational Vaccinology, Bilthoven, 14

Netherlands; Dr J.J. Bergers, Medicines Evaluation Board, Utrecht, Netherlands; Ms Juliati 15

Dahlan, National Agency of Drug and Food Control, Jakarta, Indonesia; Dr L. Fiore, Istituto 16

Superiore di Sanita, Rome, Italy; Dr M. Ayala Gonzalez, Federal Commission for the Protection 17

from Sanitary Risks, Mexico City, Mexico; Dr E. Griffiths, Consultant, Kingston upon Thames, 18

England; Dr K. Katayama, National Institute of Infectious Diseases, Tokyo, Japan; Ms Y-K. Lee, 19

Ministry of Food and Drug Safety, Seoul, Republic of Korea; Dr M. Mitsuki, Pharmaceuticals and 20

Medical Devices Agency, Tokyo, Japan; Dr M. van Oijen, Institute for Translational Vaccinology, 21

Bilthoven, Netherlands; Dr S. Pakzad, Food and Drug Control Laboratory, Tehran, Iran; Dr V. 22

Pithon, Agence nationale de sécurité du médicament et des produits de santé, Lyon, France; Dr A. 23

Utami, National Agency of Drug and Food Control, Jakarta, Indonesia; Dr G. Waeterloos, 24

Scientific Institute of Public Health, Brussels, Belgium; Dr R. Wagner, Paul-Ehrlich-Institut, 25

Langen, Germany; Dr B. Voordouw, Medicines Evaluation Board, Utrecht, Netherlands; 26

Professor H. Yang, China Food and Drug Administration, Beijing, China; Professor H. Yin, China 27

Food and Drug Administration, Beijing, China; Dr A.F. Nkayamba, Tanzania Food and Drugs 28

Authority, United Republic of Tanzania; Dr K. Mahmood, PATH, Seattle, WA, USA; Dr C. 29

Milne, European Directorate for the Quality of Medicines & HealthCare, Council of Europe, 30

Strasbourg, France; Representatives of Developing Countries Vaccine Manufacturers Network: 31

Mr B. Alirezaie, Razi Vaccine and Serum Research Institute, Alborz, Iran; Mr B. Ballabh Baluni, 32

Panacea Biotec Ltd., New Delhi, India ; Dr C. Breda, BE Vaccines SAS, Nantes, France; Dr S. 33

WHO/BS/2014.2233

Gairola, Serum Institute of India Ltd., Pune, India; Professor X. Li, National Vaccine and Serum 1

Institute, Beijing, China; Dr W. Meng, Sinovac, Beijing, China; Mr D. Ugiyadi, PT Biofarma, 2

Bandung, Indonesia; Representatives of International Federation of Pharmaceutical 3

Manufacturers & Associations: Dr S. de Walque, GlaxoSmithKline Vaccines, Wavre, Belgium; 4

Dr L. Mallet, Sanofi Pasteur, Lyon, France; Dr E. Vidor, Sanofi Pasteur, Lyon, France. 5

Representatives of other industries: Dr M. de Bruijn, Bilthoven Biologicals B.V., Netherlands; Dr 6

Y. Kino, The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan; Dr H. Ogawa, 7

Research Foundation for Microbial Diseases of Osaka University, Kagawa, Japan; Mr K. 8

Wakabayashi, Japan Poliomyelitis Research Institute, Tokyo, Japan. WHO secretariat: Dr M.R. 9

Balakrishan, Dr J. Fournier-Caruana, Dr I. Knezevic, Dr D.J. Wood and Dr T.Q. Zhou, 10


Switzerland; Dr H. Okayasu, Dr N. Previsani, Polio Eradication Initiative, World Health 12

Organization, Geneva, Switzerland. 13

14

Draft 5 was prepared by Dr M. Lennon, Horning, England and Dr T.Q. Zhou, Department of 15

Essential Medicines and Health Products, World Health Organization, Geneva, Switzerland and 16

the drafting group: Dr K. Chumakov, Center for Biologics Evaluation and Research, Rockville, 17

MD, USA; Dr C. Li, National Institutes for Food and Drug Control, Beijing, China; Dr J. Martin, 18

National Institute for Biological Standards and Control, Potters Bar, England; Dr P. Minor, 19

National Institute for Biological Standards and Control, Potters Bar, England; Dr T. Wu, Health 20

Canada, Ottawa, Canada; with support from the WHO Secretariat: Dr J. Fournier-Caruana and Dr 21

I. Knezevic, Department of Essential Medicines and Health Products, World Health Organization, 22

Geneva, Switzerland and Dr N. Previsani, Global Polio Eradication Initiative, World Health 23

Organization, Geneva, Switzerland; following comments received on the fourth draft from Ms P. 24

Agsiri, Ministry of Public Health, Nonthaburi, Thailand; Dr W.A.M. Bakker, Institute for 25

Translational Vaccinology, Bilthoven, Netherlands; Mr B. Ballabh Baluni, Panacea Biotec Ltd., 26

New Delhi, India; Dr Y. Li, Chinese Academy of Medical Sciences, Kunming, China; Dr K. 27

Mahmood, PATH, Seattle, WA, USA; Dr L. Mallet, Sanofi Pasteur, Lyon, France; Dr W. Meng, 28

Sinovac, Beijing, China; Dr C. Milne, European Directorate for the Quality of Medicines & 29

HealthCare, Council of Europe, Strasbourg, France; Dr V. Pithon, Agence nationale de sécurité du 30

médicament et des produits de santé, Lyon, France; Dr E. Vidor, Sanofi Pasteur, Lyon, France; Dr 31

G. Waeterloos, Scientific Institute of Public Health, Brussels, Belgium; Dr R. Wagner, Paul-32

WHO/BS/2014.2233

Ehrlich-Institut, Langen, Germany; Professor H. Yang, Center for Drug Evaluation, China Food 1

and Drug Administration, Beijing, China. 2

3

The following experts provided responses to a WHO survey on IPV seeds and quality 4

control information conducted during 20122013: 5

S. Yamazaki and S. Abe, Japan Poliomyelitis Research Institute, Tokyo, Japan; R.M. Dhere, 6

L.R. Yeolekar and S. Gairola, Serum Institute of India Ltd., India; M.B. Sun, G.Y. Liao and Y. 7

Li, Chinese Academy of Medical Sciences, Kunming, China; H. Wang, Beijing TianTan 8

Biological Products Co. Ltd., Beijing, China; G. Stawski, Statens Serum Institut, Copenhagen, 9

Denmark; M. van Oijen and W. Bakker, Institute for Translational Vaccinology, Bilthoven, 10

Netherlands; D. Ugiyadi, PT Bio Farma (Persero), Bandung, Indonesia; R.K. Suri, H.S. Mali and 11

D. Rastogi, Panacea Biotec Ltd., New Delhi, India; E. Niogret, Sanofi Pasteur, Marcy L'Etoile, 12

France; X. Bouwstra, Bilthoven Biologicals B.V., Netherlands; M. Duchêne and C. Saillez, 13

GlaxoSmithKline Vaccines, Wavre, Belgium; D. Rudert-Dolby, Sanofi Pasteur, Toronto, 14

Canada. 15

16

WHO/BS/2014.2233

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WHO/BS/2014.2233



64. Brown GC, Rabson AS, Schieble JH. The effect of gamma globulin on subclinical 3

infection in familial associates of poliomyelitis cases: II. Serological studies and virus 4

isolations from pharyngeal secretions. J. Immunol. 1955;74:7180. 5

65. Nathanson N. David Bodian’s contribution to the development of poliovirus vaccine. 6

Am. J. Epidemiol. 2005;161: 20712. 7

66. Manual for the virological investigation of polio. Geneva: World Health Organization; 8

1997 (http://whqlibdoc.who.int/hq/1997/WHO_EPI_GEN_97.01.pdf, accessed 13 May 9

2014). 10

67. Salk J, Salk D. Control of influenza and poliomyelitis with killed virus vaccines. 11

Science. 1977;195:834–47. 12

68. Salk J. Immune response and minimum requirement for immunity to disease. Scand. J. 13

Infect. Dis. Suppl. 1982;36:65–7. 14

69. Taranger J, Trollfors B, Knutsson N, Sundh V, Lagergard T, Ostergaard E. Vaccination 15

of infants with a four-dose and a three-dose vaccination schedule. Vaccine. 16

2000;18:884–91. 17

70. Salk J. One-dose immunization against paralytic poliomyelitis using a non-infectious 18

vaccine. Rev. Infect. Dis. 1984;6(Suppl. 2):S444–50. 19

71. Guidelines for national authorities on quality assurance for biological products. In: 20

WHO Expert Committee on Biological Standardization: forty-second report. Geneva: 21

World Health Organization; 1992: Annex 2 (WHO Technical Report Series, No. 822; 22

whqlibdoc.who.int/trs/WHO_TRS_822.pdf, accessed 13 May 2014). 23

72. Guidelines for independent lot release of vaccines by regulatory authorities. In: WHO 24

Expert Committee on Biological Standardization: sixty-first report. Geneva: World 25


http://www.who.int/biologicals/TRS_978_Annex_2.pdf , accessed 13 May 2014). 27

73. WHO manual for the establishment of national and other secondary standards for 28

vaccines. Geneva: World Health Organization; 2011 29

(http://whqlibdoc.who.int/hq/2011/WHO_IVB_11.03_eng.pdf, accessed 13 May 2014). 30

31

http://whqlibdoc.who.int/trs/WHO_TRS_850.pdf?ua=1

http://whqlibdoc.who.int/hq/1997/WHO_EPI_GEN_97.01.pdf

http://www.who.int/biologicals/TRS_978_Annex_2.pdf

http://whqlibdoc.who.int/hq/2011/WHO_IVB_11.03_eng.pdf

WHO/BS/2014.2233

Appendix 1 1

Overview of the virus seeds used in IPV production 2

3

This appendix gives an overview of the history of virus seeds that are currently used in 4

production or may be used in the future. They include the wild strains used in current production 5

of inactivated poliomyelitis vaccine (IPV) and the attenuated Sabin strains which are considered 6

to pose a lower risk and are being developed as alternative seeds. Novel strains intended to be 7

safer for use in production are also in development. 8

9

1. IPV made from virulent strains 10

Both classic IPV, which was developed by Jonas Salk and others and was licensed in 1955, and 11

the enhanced-potency IPV which was introduced in the late 1980s, are prepared from wild 12

(virulent) polioviruses of three serotypes. The strains selected by Salk were Mahoney, MEF-1, 13

and Saukett, representing types 1, 2 and 3, respectively. The Mahoney strain was isolated in 14

1941 by Drs Thomas Francis and Walter Mack from the pooled faeces of three healthy children 15

in Cleveland, Ohio, USA (1). It was subsequently passaged by Salk, including 14 times in living 16

monkeys and twice in monkey testicular cultures (2). The MEF-1 strain was isolated by 17

inoculation of monkeys in Egypt in 1940 (3) during a polio outbreak among allied troops of the 18

Mediterranean Expeditionary Force (hence the name MEF). It was adapted by Schlesinger and 19

Olitsky to growth in mice (4), and then transferred by Salk from the spinal cord of a paralysed 20

mouse to tissue culture (2). The original Saukett strain was isolated by Salk in 1950 by direct 21

inoculation of tissue culture with a faecal specimen from a paralysed patient (2). Seed stocks of 22

the viruses were provided by Salk to most manufacturers and were used to establish their virus 23

master seeds. An alternative strain of type 1 poliovirus (Brunhilde) is used by the Statens Serum 24

Institute (SSI) in Denmark. The strain was isolated in 1939 by David Bodian from a pool of stool 25

specimens from seven patients in Maryland (5). The strain was provided to the laboratory of Dr. 26

John Enders at Harvard Medical School in Boston, MA, USA, and from there to Dr. Arne 27

Svedmyr’s laboratory in Stockholm, Sweden. Dr Svedmyr supplied SSI with the virus. Table 1 28

summarizes the history of isolation and early passaging of these viruses. 29

30

WHO/BS/2014.2233

1

Table 1. History of isolation and early passaging of wild polioviruses used in the 2

production of IPV 3

4

Strain name Source of

isolation

Location Year Reference

Mahoney

Stool of 3

healthy children Cleveland,

Ohio,

USA

1941 Francis & Mack. See Sabin AB,

Boulger LR, 1973 (1)

MEF-1

CNS of a

paralysed

patient

Egypt 1941 van Rooyen CE, Morgan AD,

1943 (3)

Saukett

Stool of a

paralysed

patient

USA 1950 Salk JE, 1953 (2)

Brunhilde

Stool of 7

patients

Maryland 1939 Howe HA, Bodian D, 1941 (5)

5

Subsequent studies raised questions regarding these strains. The nucleotide sequence of MEF-1 6

was found to be very close to the sequence of another type-2 strain (Lansing, isolated in 1937 in 7

Michigan, USA), with only 17 nucleotide and 2 amino acid differences (6). Since the strains 8

were isolated four years apart in the Middle East and USA, it is unlikely that the similarity 9

represents a natural relatedness. MEF-1 from the spinal cords of monkeys was adapted to growth 10

in mice (4) and was found in this early study to be indistinguishable in pathogenicity and 11

immunological properties from Lansing, which was also adapted to growth in mice. A plausible 12

explanation is that the Lansing strain used as a reference strain in Schlesinger and Olitsky’s 13

laboratory was inadvertently substituted for MEF-1, and all subsequent stocks of MEF-1 are 14

derivatives of the Lansing strain. In addition, two common variants of MEF-1, differing by a few 15

nucleotides, are in use in different laboratories and production facilities. 16

17

The Saukett strains obtained from different laboratories and manufacturers differ significantly (7, 18

8) and the degree of diversity (~10% nucleotide substitutions) demonstrates that they are 19

different strains. Some of the differences were observed in antigenic sites and could affect 20

immunogenicity, suggesting that better characterization of vaccines in the future may need to 21

include determination of the exact nucleotide sequences of virus master seed lots used by 22

manufacturers. 23

24

WHO/BS/2014.2233

The flow diagrams in Figures 1, 2 and 3 show the history of the seed virus used to prepare the 1

respective master seed lots claimed to be used by the manufacturers of IPV from types 1, 2 and 3 2

strains respectively. The full names of manufacturers shown on the charts are given in Table 2. 3

4

The figures provide only an overview of the use of different seeds. They were based on a written 5

survey conducted in 2012 by WHO among vaccine manufacturers and on information obtained 6

from subsequent consultations. They do not indicate any WHO qualification or approval of the 7

strains or the vaccines in the context of this document. 8

9

Table 2. Proper names of manufacturers shown on the charts are: 10

11

2. IPV made from attenuated strains (Sabin) 12

Once circulation of wild-type polio viruses is eliminated, IPV manufacturing establishments will 13

be the biggest potential source of virulent viruses which must therefore be stringently contained 14

to prevent their reintroduction into the environment. The Sabin vaccine strains used to 15

manufacture oral polio vaccine (OPV) are less virulent than the wild strains and have been 16

proposed as less hazardous seeds for IPV production in order to mitigate the risks of potential 17

inadvertent release from production facilities. Sabin strains are known to be genetically unstable 18

in infected humans and, to some extent, in production. To retain the attenuated phenotype the 19

Sabin strains must be propagated under defined and well-controlled conditions. In addition, in 20

the manufacture of OPV each harvest is tested to monitor the molecular consistency – e.g. by 21

mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) – and 22

each monovalent bulk is tested for neurovirulence to be sure that the attenuated phenotype is 23

retained. As the IPV product is inactivated, full characterization on every batch is not necessary 24

RIVM National Institute of Public Health and the Environment

(RIVM), Bilthoven, Netherlands

BBio Bilthoven Biologicals B.V. (BBio, former NVI), Bilthoven,

Netherlands

SSI Statens Serum Institute (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

WHO/BS/2014.2233

but some assurance of consistent production is required. Sabin strains are considered less 1

transmissible than the wild type so that, should they escape from the production facility and start 2

to circulate within communities, they would pose a lesser risk. However, they can revert and 3

may give rise to circulating vaccine-derived strains that are both transmissible and capable of 4

causing outbreaks. Therefore, the use of Sabin strains in the manufacture of IPV may reduce 5

biosecurity concerns compared to manufacture from virulent wild strains but does not eliminate 6

the concerns entirely. Some testing or process validation will be required to show that the 7

product is consistent and that the attenuated phenotype is retained in order to justify the level of 8

containment used (see also General considerations and Part A of this document). Two sIPV-9

containing combination products based on attenuated Sabin strains have been licensed in Japan, 10

and other sIPV vaccines are undergoing clinical evaluation in some countries. The derivation of 11

Sabin strains was described in the literature (1) and the detailed origin of seed viruses made from 12

them can be found in Appendix 1 of the Recommendations to assure the quality, safety and 13

efficacy of live attenuated poliomyelitis vaccine (oral), revised 2012 (9). 14

15

3. Other strains in development 16

Alternative attenuated strains of poliovirus, such as strains derived from recombinant DNA 17

technology, are under development. They are intended to be both attenuated and genetically 18

stable and also to possess low or no infectivity for humans, thus being of negligible 19

transmissibility. Such strains should pose a lower risk of inadvertent release from production 20

facilities or of infecting production workers. They may include strains in which known 21

attenuation determinants are stabilized by targeted genetic changes, strains with alterations in 22

codon usage in order to introduce multiple mutations to reduce virus replication efficiency, or 23

viruses produced by other strategies. The phenotypes and the stability of these strains will 24

require confirmation. 25

26

27

WHO/BS/2014.2233

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

Figure 1. History of seed virus used to produce type 1 IPV 36

Francis & Mack 1941

Jonas Salk

Sanofi Pasteur (Canada)

SSI (Denmark)

RIVM (Netherlands)

Sanofi Pasteur (France)

GSK (Belgium)

Mahoney

David Bodian 1939

Brunhilde

John Enders

Arne Svedmyr

BBio (Netherlands)

WHO/BS/2014.2233

1 2


4

VanRooyenetal1941

Schlessinger,Morgan,andOlitsky

JonasSalk

SanofiPasteur(Canada) SSI

(Denmark)

RIVM(Netherlands)

SanofiPasteur(France)

GSK(Belgium)

MEF-1

Bbio(Netherlands)

WHO/BS/2014.2233

1 2


4

5

6

References 7

8

1. Sabin AB,Boulger LR. History of Sabin attenuated poliovirus oral live vaccine strains. J 9

Biol Stand. 1973;1:1158. 10

2. Salk JE. Studies in human subjects on active immunization against poliomyelitis. I. A 11

preliminary report of experiments in progress. JAMA. 1953;151:108198. 12

3. van Rooyen CE, Morgan AD. Poliomyelitis. Experimental work in Egypt. Edinburgh 13

Medical Journal. 1943;50:70520. 14

4. Schlesinger RW, Morgan IM, Olitsky PK. Transmission to rodents of Lansing type 15

poliomyelitis virus originating in the Middle East. Science. 1943;98:4524. 16

5. Howe HA, Bodian D. Poliomyelitis in the chimpanzee: a clinical pathological study. 17

Bulletin of the Johns Hopkins Hospital. 1941;69:14981. 18

6. Dragunsky EM, Ivanov AP, Wells VR, Ivshina AV, RezapkinGV, Abe S et al. 19

Evaluation of immunogenicity and protective properties of inactivated poliovirus 20

JonasSalk1950

SanofiPasteur(Canada)

SSI(Denmark)

RIVM(Netherlands)

SanofiPasteur(France)

GSK(Belgium)

Sauke

Bbio(Netherlands)

WHO/BS/2014.2233

vaccines: a new surrogate method for predicting vaccine efficacy. J Infect Dis. 1

2004;190:140412. 2

7. Minor PD, Schild GC, Ferguson M, MacKay A, Magrath DI, John A et al. Genetic and 3

antigenic variation in type 3 polioviruses: characterization of strains by monoclonal 4

antibodies and T1 oligonucleotide mapping. J Gen Virol. 1982;61:16776. 5

8. Huovilainen A, Kinnunen L, Poyry T, Laaksonen L, Roivainen M, Hovi T. Poliovirus 6

type 3/Saukett: antigenic and structural correlates of sequence variation in the capsid 7

proteins. Virology. 1994;199:22832. 8

9. Recommendations to assure the quality, safety and efficacy of poliomyelitis vaccines 9

(oral, live, attenuated). Replacement of Annex 1 of WHO Technical Report Series, No. 10

904, and Addendum to Annex 1 of WHO Technical Report Series, No. 910. In: WHO 11



http://www.who.int/biologicals/vaccines/OPV_Recommendations_TRS_980_Annex_2.p14

df, accessed 13 May 2014). 15

16

17



WHO/BS/2014.2233

Appendix 2 1

In vivo potency assay of IPV 2

3

Tests for evaluating the potency of inactivated polio vaccines include an in vivo assay for 4

immune response. An appropriate WHO International Standard should be used to validate the 5

assay. Because of the diversity in the reactivity of vaccines, it is unlikely that an International 6

Standard will be suitable for the standardization of in vivo assays of vaccines from all 7

manufacturers. If this is shown to be the case, manufacturers should establish a product-specific 8

reference preparation which is traceable to a lot of vaccine shown to be efficacious in clinical 9

trials. The NRA should approve the reference preparation used and should agree with the 10

potency limits applied. The performance of this reference vaccine should be monitored by trend 11

analysis using relevant test parameters and it should be replaced when necessary. 12

13

In recent investigations the in vivo potency assay in rats has been standardized (1) and has been 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 rats 17

of four dilutions of the vaccine to be examined and a reference vaccine, using for each dilution a 18

group of not fewer than 10 rats of a suitable strain and which are specific pathogen-free. The 19

number of animals used should enable the calculation of potency with 95% confidence limits 20

within the 25400% range. The number of dilutions and the number of animals used per dilution 21

may differ from that specified here, provided that any alternative scheme gives at least the same 22

sensitivity in the test. For each dilution, the weight of the individual animals should not vary by 23

more than 20% from the group mean. An inoculum of 0.5 mL is used per rat. The dose range is 24

chosen so that a dose response to all three poliovirus types is obtained. The animals are bled after 25

20–22 days. Neutralizing titres against all three poliovirus types are measured separately using 26

100 CCID50 of the Sabin strains as challenge viruses, Vero or Hep-2C as indicator cells, and 27

neutralization conditions of 3 hours at 35–37 °C followed by 18 hours at 2–8 °C. Results should 28

be read after fixation and staining after 7 days of incubation at 35 °C. For the antibody assay to 29

be valid, the titre of each challenge virus must be shown to be within the range of 30300 30

CCID50 and the neutralizing antibody titre of a control serum must be within two 2-fold dilutions 31

of its geometric mean titre. The potency is calculated by comparing the proportions of animals 32

defined as responders to the test vaccine and to the reference vaccine by the probit method. To 33

WHO/BS/2014.2233

define an animal as a responder, it is necessary to establish a cut-off neutralizing antibody titre 1

for each poliovirus type. Owing to inter-laboratory variation, it is not possible to define cut-off 2

values that could be applied by all laboratories. Instead, the cut-off values should be determined 3

by each laboratory on the basis of a minimum series of three tests with the reference vaccine. 4

The mid-point on a log2 scale of the minimum and maximum geometric mean titres of the series 5

of three or more tests is used as the cut-off value. For each of the three poliovirus types, the 6

potency of the vaccine should not be statistically significantly less than that of the reference 7

preparation. The test is not valid unless: 8

− the median effective dose (ED50) for both the test and reference vaccines lies between the 9

smallest and the largest doses given to the animals; 10

− the statistical analysis shows no significant deviation from linearity or parallelism; 11

− the confidence limits of the estimated relative potency fall between 25% and 400% of the 12

estimated potency. 13

14

Laboratories that have established the parallel line method of analysis of antibody titres for the 15

rat test may use it instead of converting titres to proportions of responders as in the probit 16

method of analysis. 17

18

Laboratories are encouraged to validate alternative methods for the assay of neutralizing 19

antibody to reduce the use of live polioviruses in laboratories. If IPV is formulated with other 20

antigens into a combination vaccine, then the suitability of performing the rat immunogenicity 21

test will have to be determined. If the immunogenicity test is performed, the potency of the final 22

bulk for each virus type should be approved by the NCL. 23

24

The development of transgenic mice that express the human poliovirus receptor (TgPVR mice) 25

(3, 4, 5) has led to the development of an immunization/challenge model that may be useful for 26

assessment of vaccine efficacy. This test is not proposed for lot release. Any work with 27

transgenic mice should comply with WHO guidelines (6). 28

29

References 30

1. Wood DJ, Heath AB. Collaborative study for the establishment of a rat bioassay for 31

inactivated polio vaccine. Pharmeuropa special issue Bio. 2000;1:25–49. 32

WHO/BS/2014.2233

2. van Steenis A, van Wezel AL, Sekhuis VM. Potency testing of killed polio vaccine in 1

rats. Dev Biol Stand. 1981;47:119–28. 2

3. Ren R, Costantini F, Gorgacz EI, Lee II, Racaniello VR. Transgenic mice expressing a 3

human poliovirus receptor: a new model for poliomyelitis. Cell. 1990;63:35362. 4

4. Koike S, Taya C, Kurata T, Abe W, Ise I, Yonekawa H et al. Transgenic mice susceptible 5

to poliovirus. Proc Natl Acad Sci U S A. 1991;88:9515. 6

5. Dragunsky E, Nomura T, Karpinski K, Furesz J, Wood DJ, Pervikov Y et al. Transgenic 7

mice as an alternative to monkeys for neurovirulence testing of live oral poliovirus 8

vaccine: validation by a WHO collaborative study. Bull World Health Organ. 9

2003;81:25160. 10

6. Maintenance and distribution of transgenic mice susceptible to human viruses: 11

memorandum from a WHO meeting. Bull World Health Organ. 1993;71:493502. 12

13

14

http://www.sciencedirect.com/science/article/pii/009286749090168E?_alid=1768461607&_rdoc=11&_fmt=high&_origin=search&_docanchor=&_ct=23&_zone=rslt_list_item&md5=7853c992578bf395a191b4c56c966001

http://www.sciencedirect.com/science/article/pii/009286749090168E?_alid=1768461607&_rdoc=11&_fmt=high&_origin=search&_docanchor=&_ct=23&_zone=rslt_list_item&md5=7853c992578bf395a191b4c56c966001

WHO/BS/2014.2233

Appendix 3 1

Model summary protocol for manufacture and control of 2

poliomyelitis vaccine (inactivated) 3

4

The following protocol is intended for guidance. It indicates the type of information that should 5

be provided as a minimum by the manufacturer to the NRA. Information and tests may be added 6

or omitted as necessary, with the authorization of the NRA. 7

8

It is possible that a protocol for a specific product may differ in detail from the model provided. 9

The essential point is that all relevant details demonstrating compliance with the licence and with 10

the relevant WHO recommendations for a particular product should be given in the protocol 11

submitted. 12

13

The section concerning the final product must be accompanied by a sample of the label and a 14

copy of the leaflet that will accompany the vaccine container. If the protocol is being submitted 15

in support of a request to permit importation, it should also be accompanied by a lot release 16

certificate from the NRA or from the NCL of the country where the vaccine was produced or 17

released stating that the product meets the national requirements as well as the recommendations 18

in Part A of this document. 19

20

Summary information on finished product (final lot)

International name: _______________________________________

Trade name/commercial 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: _______________________________________

WHO/BS/2014.2233

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 pool Type 1 Type 2 Type 3

suspensions:

Site of manufacture of each

monovalent pool:

_______________________________________

Date of manufacture of each

monovalent pool:

_______________________________________

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: _______________________________________

WHO/BS/2014.2233

1

Starting materials 2

The information requested below is to be presented on each submission. Full details on master 3

and working seed lots should be provided upon first submission only and whenever a change has 4

been introduced. 5

6

The following sections are intended for recording the results of the tests performed during the 7

production of the vaccine, so that the complete document will provide evidence of consistency of 8

production. If any test has to be repeated, this must be indicated. Any abnormal result must be 9

recorded on a separate sheet. 10

11

If any cell lot or virus harvest intended for production was rejected during the control testing, 12

this should also be recorded either in the following sections or on a separate sheet. 13

14

Control of source materials (Section A.3)

Virus seed (every submission) (Section A.3.1)

Vaccine virus strain(s) and serotype(s):

____________________________________

Substrates used for preparing seed lots:

____________________________________

Origin and short history:

____________________________________

Authority that approved the virus strains:

______________________________________

Date of approval: ______________________________________

Information on seed lot preparation (every submission) (Section A.3.1.3)

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: ____________________________________

WHO/BS/2014.2233

Dates of harvest: ____________________________________

Number of containers: ____________________________________

Conditions of storage: ____________________________________

Dates of preparation: ____________________________________

Maximum passage levels authorized: ____________________________________

Tests on virus master seed (VMS) and virus working seed (VWS) (first submission only)

Tests for 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

Results

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

____________ ________ ___________

WHO/BS/2014.2233


____________ ________ ___________


____________ ________ ___________


____________ ________ ____________

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 of start of test:

_____________________________________

Date of end of test:

_____________________________________

Result:

_____________________________________

Test in rabbit kidney cell cultures:

Number of cell cultures:

_____________________________________

Total volume inoculated:

_____________________________________

Period of observation: _____________________________________

WHO/BS/2014.2233

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:

_____________________________________


_____________________________________


_____________________________________

Result:

_____________________________________

Absence of SV40

Method used:

_____________________________________

Date of start of test :

_____________________________________


_____________________________________

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 and 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

WHO/BS/2014.2233

Ratio of % of 481-G of bulk sample to the

International Standard, or level of

mutations:

_____________________________________


_____________________________________



_____________________________________

Type 3

Ratio of % of 472-C of bulk sample to the

International Standard, or

level of mutations:

_____________________________________


_____________________________________



_____________________________________

In vivo tests for neurovirulence (if

applicable)

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

WHO/BS/2014.2233

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 (Section A.3.2) (every

submission)

Information on cell banking system

WHO/BS/2014.2233

Name and identification of substrate:

Origin and short history:

_____________________________________

Authority that approved the cell bank:

_____________________________________

Master cell bank (MCB) and working cell

bank (WCB) lot numbers and date of

preparation:

_____________________________________

Date MCB and WCB were established:

_____________________________________

Date of approval by NRA:

_____________________________________

Total number of ampoules stored:

_____________________________________

Passage level (or number of population

doublings) of cell bank:

_____________________________________

Maximum passage approved:

_____________________________________

Storage conditions:

_____________________________________

Method of preparation of cell bank in

terms of number of freezes and efforts

made to ensure that a homogeneous

population is dispersed into the ampoules:

_____________________________________

Tests on MCB and WCB (Section A.3.2) (first submission only)

Percentage of total cell-bank ampoules

tested:

_____________________________________

Identity test:

_____________________________________

Method:

_____________________________________

Specification:

_____________________________________

Date of test:

_____________________________________

Result:

_____________________________________

Growth characteristics:

_____________________________________

Morphological characteristics:

_____________________________________

Immunological marker:

_____________________________________

WHO/BS/2014.2233

Cytogenetic data:

_____________________________________

Biochemical data:

_____________________________________

Results of other identity tests:

_____________________________________

Tests for adventitious agents:

Method used:

_____________________________________


_____________________________________


_____________________________________

Date of start of test

_____________________________________

Date of end of test

_____________________________________

Freedom from bacteria, fungi and

mycoplasmas


Method used:

______________________________________


______________________________________


______________________________________


______________________________________


______________________________________

Incubation Media used Inoculum Date of start

of test

Date of end

of test

Results

20–25 °C

______ _______ _______ _______ _________

30–36 °C

______ _______ _______ _______ _________

Negative control

______ _______ _______ _______ ____

Test for mycoplasma

Method used:

_______________________________________

Volume tested:

_______________________________________

Media used:

_______________________________________


_______________________________________

Observation period (specification): _______________________________________

WHO/BS/2014.2233

Positive controls (list of species used

and results):

_______________________________________

Date of start of test Date of end of test Results


_______________ ______________ _____________


_______________ ______________ _____________


_______________ ______________ _____________


_______________ ______________ _____________

Indicator cell culture method (if

applicable)


_______________________________________

Inoculum:

_______________________________________

Date of test:

_______________________________________

Passage number:

_______________________________________

Negative control:

_______________________________________

Positive controls:

_______________________________________

Date of staining:

_______________________________________

Results:

_______________________________________

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

(Provide information on control cells

corresponding to each single harvest.)

WHO/BS/2014.2233

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:

_______________________________________


_______________________________________


_______________________________________

Results:

_______________________________________

Tests for adventitious agents on

supernatant culture fluids

Method used:

_______________________________________


_______________________________________


_______________________________________

Result:

_______________________________________

Identity test

Method used:

_______________________________________


_______________________________________


_______________________________________

Result:

_______________________________________

WHO/BS/2014.2233

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)

Batch number(s) and virus type:

_______________________________________

Date of inoculation:

_______________________________________

Date(s) of harvest:

_______________________________________

Volume(s), storage temperature,

storage time and approved storage

period:

_______________________________________

Freedom from bacteria, fungi and

mycoplasmas


Method used:

_______________________________________


_______________________________________


_______________________________________


_______________________________________


_______________________________________


of test

Date of

end of test

Result

20–25 °C

_________ _________ _________ ________ ________

30–36 °C

_________ _________ _________ ________ ________

Negative control

_________ _________ _________ ________ ________

Test for mycoplasmas

Method used:

_______________________________________

Volume tested:

_______________________________________

Media used: _______________________________________

WHO/BS/2014.2233


_______________________________________


_______________________________________

Positive controls (list of species used and

results):

_______________________________________

Date of start of test Date of end of test Result


_______________ _______________ ______________


_______________ _______________ ______________


_______________ _______________ ______________


_______________ _______________ ______________

Indicator cell culture method (if applicable)


_______________________________________

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:

_______________________________________


______________________________________

WHO/BS/2014.2233


_______________________________________

Result:

______________________________________

Purified monovalent pools before

inactivation (Section A4.4)

Batch number(s) and virus type:

_______________________________________

Date of inoculation:

______________________________________

Date(s) of harvest:

_______________________________________

Volume(s), storage temperature, storage

time and approved storage period:

______________________________________

Test for residual cellular DNA _______________________________________

Method used:

_______________________________________


______________________________________


_______________________________________

Virus titration

Date of test:

______________________________________


_______________________________________

Date of test:

______________________________________

Result:

_______________________________________

Identity test

Method used:

_______________________________________


______________________________________


_______________________________________

Result:

______________________________________

D-antigen content

Reference used:

Method used:

_______________________________________

Date of test:

______________________________________

WHO/BS/2014.2233

Result:

______________________________________

Protein content

Method used:

_______________________________________


_______________________________________


_______________________________________

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 seeds derived by

recombinant DNA technology: e.g. in vitro tests (such as 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

(Section A.4.5):

Agent(s) and concentration at the

beginning and end of inactivation:

_______________________________________

Temperature of inactivation:

_______________________________________

Date of start of inactivation:

_______________________________________

D-antigen units at start of inactivation:

_______________________________________

Date of taking first sample:

_______________________________________

Date of completion of inactivation:

_______________________________________

D-antigen units at end of inactivation:

_______________________________________

Test for effective inactivation(after

removal/neutralization of inactivating

agent)

_______________________________________

Sample size tested: _______________________________________

WHO/BS/2014.2233

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 mycoplasmas


Method used:

_______________________________________


_______________________________________


_______________________________________


_______________________________________


_______________________________________


of test

Date of end

of test

Result

20–25 °C

_________ _________ __________ __________ __________

30–36 °C

_________ _________ __________ __________ __________

Negative

control

_________ _________ __________ __________ __________

D-antigen content

Method used: _______________________________________

Reference used:

Date of test:

______________________________________

Result:

______________________________________

Trivalent bulk product

(monovalent pools incorporated)

Date of preparation: _______________________________________

WHO/BS/2014.2233

Preservative (if added, type and

concentration):

_______________________________________

Tests on trivalent bulk (Section 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:

_______________________________________


_______________________________________


_______________________________________


_______________________________________


_______________________________________


of test

Result

20–25 °C

__________ _________ __________ __________

30–36 °C

__________ _________ __________ __________

Negative

control

__________ _________ __________ __________

Residual formaldehyde

Method used:

_______________________________________

Result:

_______________________________________

D-antigen content

Method used and acceptance limits for test

results:

_______________________________________

Reference used:

Date of test: ______________________________________

WHO/BS/2014.2233

Result:

______________________________________

Control of final bulk (Section A.4.7)

Tests for bacteria and fungi:

Method used:

_______________________________________


_______________________________________


_______________________________________


_______________________________________


_______________________________________


of test

Date of end of

test

Result

20–25 °C

_________ _________ __________ __________ __________

30–36 °C

_________ _________ __________ __________ __________

Negative

control

_________ _________ __________ __________ __________

Potency tests

D- antigen test

Method used and acceptance limits for test

results:

_______________________________________

Reference used:

_______________________________________

Date of test:

_______________________________________

Result:

_______________________________________

Results (and date) of in vivo tests (in rats), if

performed:

_______________________________________

Preservative content (if applicable):

Date of test:

_______________________________________

Method used:

_______________________________________

Result:

_______________________________________

Adjuvant (if applicable):

Date of test:

_______________________________________

WHO/BS/2014.2233

Method used:

_______________________________________

Result:

_______________________________________

Filling and containers (Section A.5)

Final lot number:

_______________________________________

Total volume for final filling:

_______________________________________

Date of filling:

______________________________________

Number of vials after inspection:

_______________________________________

Number of vials filled:

_____________________________________

Control tests on final lot (Section A.6)

Inspection of final containers

Date of test:

______________________________________

Results:

_______________________________________

Appearance:

_______________________________________

Date of test:

______________________________________

Results:

_______________________________________

Identity test

Method used:

_______________________________________


______________________________________


_______________________________________

Result:

______________________________________


Method used:

_______________________________________


_______________________________________


_______________________________________


_______________________________________

Observation period (specification): _______________________________________

WHO/BS/2014.2233

Incubation Media

used

Inoculum Date of start of

test

Date of end

of test

Result

20–25 °C

_______ _________ __________ __________ _________

30–36 °C

_______ _________ __________ __________ _________

Negative

control

_______ _________ __________ __________ _________

General safety test (if applicable)


_____________________________________


_____________________________________

Result:

_____________________________________

Potency test(s):

D- antigen test

Method used and acceptance limits for

test results:

_______________________________________

Reference used:

_______________________________________

Date of test:

_______________________________________

Result:

_______________________________________

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: _______________________________________

WHO/BS/2014.2233

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

Certification by the manufacturer 3

Name of the manufacturer____________________________________ 4

Name of head of production (typed) ___________________________ 5

6

Certification by the person from the control laboratory of the manufacturing company taking 7

responsibility for the production and control of the vaccine 8

9

I certify that lot no. ______________ of trivalent poliomyelitis vaccine (inactivated), whose 10

number appears on the label of the final container, meets all national requirements 11

and/or satisfies Part A of WHO’s Recommendations to assure the quality, safety and efficacy of 12

poliomyelitis vaccine (inactivated) (WHO Technical Report Series, No. XXXX). 13

14

Signature: ________________________________________________ 15

Name (typed): ______________________________________________ 16

Date: ________________ 17

18

WHO/BS/2014.2233

Appendix 4 1

Model certificate for the release of poliomyelitis vaccine 2

(inactivated) by national regulatory authorities 3

4

Lot release certificate 5

6

Certificate no. ________________ 7

8

The following lot(s) of poliomyelitis vaccine (inactivated) produced by 9

____________________________1 in _______________

2 whose numbers appear on the 10

labels of the final containers, comply with the relevant specification in the marketing 11

authorization3 and provisions for the release of biological products and Part A

4 of WHO’s 12

Recommendations to assure the quality, safety and efficacy of poliomyelitis vaccines 13

(inactivated) (_____)5 and comply with Good manufacturing practices for pharmaceutical 14

products6, Good manufacturing practices for biological products

7 and Guidelines for 15

independent lot release of vaccines by regulatory authorities8. 16

The release decision is based on _____________________________________________9. 17

18

The certificate may include the following information: 19

Name and address of manufacturer 20

Site(s) of manufacturing 21

Trade name and/common name of product 22

Marketing authorization number 23

Lot number(s) (including sub-lot numbers, packaging lot numbers if necessary) 24

Type of container 25

Number of doses per container 26

Number of containers/lot size 27

Date of start of period of validity (e.g. manufacturing date) and/or expiry date 28

Storage condition 29

Signature and function of the authorized person and authorized agent to issue the 30

certificate 31

Date of issue of certificate 32

WHO/BS/2014.2233

Certificate number. 1

2

The director of the National Regulatory Authority (or control authority, as appropriate): 3

Name (typed) _______________________________________________ 4

Signature __________________________________________________ 5

Date ______________________________________________________ 6

7

Footnote 8 1 Name of manufacturer. 9

2 Country of origin. 10

3 If any national requirements are not met, specify which one(s) and indicate why release 11

of the lot(s) has nevertheless been authorized by the NRA. 12 4 With the exception of provisions on distribution and shipping, which the NRA may not be 13

in a position to assess. 14 5 WHO Technical Report Series, No. (xx, xxxx). 15

6 WHO Technical Report Series, No. 961, Annex 3, 2011. 16



9 Evaluation of summary protocol, independent laboratory testing, and/or specific 19

procedures laid down in defined document etc., as appropriate. 20

21

22

= = = 23

recommendations to assure the quality, safety and efficacy of … · who/bs/2014.2233 page 2 1 2...

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