SEA-Vaccines-131 Distribution: General
Hepatitis B Potency Testing Standardization Report of a Meeting WHO/SEARO, New Delhi, 9- 11 April 2001
WHO Project: ICP VAB 001
World Health Organization Regional Office for South-East Asia New Delhi August 2001
© World Health Organization 2001 This document is not a formal publication of the World Health Organization (WHO), and all rights are reserved by the Organization. The document may, however, by freely reviewed, abstracted, reproduced or translated, in part or in whole, but not for sale or for use in conjunction with commercial purposes. The views expressed in documents by named authors are solely the responsibility of those authors.
CONTENTS Page 1. INTRODUCTION ................................................................................................1 2. OBJECTIVES ........................................................................................................1 3. INAUGURATION ................................................................................................1 4. BACKGROUND ..................................................................................................2 5. IN VIVO VS IN VITRO METHODS OF POTENCY TESTING ..................................2 5.1 Background ................................................................................................2 5.2 Manufacturers’ Data on Characterization of the Vaccine.............................3 5.3 Conclusions ................................................................................................3 6. ISSUES FOR CONSIDERATION ON IN VITRO AND IN VIVO POTENCY TESTS....................................................................................3 6.1 Working Standards .....................................................................................4 6.2 International Reference Reagent .................................................................4 6.3 International Standard 80/549 ....................................................................4 6.4 Conclusions ................................................................................................4 7. STANDARDIZATION OF R HBV .........................................................................5 8. VACCINE VIAL MONITORS (VVMS)....................................................................5 9. RECOMMENDATIONS........................................................................................5 Annexes 1. 2. 3. 4. 5. Programme..........................................................................................................6 List of Participants................................................................................................8 Characterization of Vaccines Produced in SEAR.................................................10 Issues for Consideration .....................................................................................14 Points to Consider in the Standardization of Hepatitis B Vaccines .....................15
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1.
INTRODUCTION This is the first time that a regional WHO meeting on Standardization of Potency Testing of Hepatitis B Vaccines (HBV) was held in SEARO, New Delhi. A unique feature of the meeting was that representatives from the manufacturers of HBV from all the producing countries (having in place an established National Regulatory Authority (NRA) for vaccines) and their respective National Control Lab (NCL) staff participated. A total of 26 participants from four Member Countries of the Region attended the meeting. For programme of the meeting see Annex 1. The list of participants and faculty members is at Annex 2. Five manufacturers from India, one manufacturer each from Indonesia and Myanmar and the NCL staff from all the four vaccine-producing countries including Thailand participated in the meeting.
2.
OBJECTIVES The objectives of the meeting were: (1) (2) To discuss all issues related to the potency testing of Hepatitis B vaccines, and To standardize Hep B potency testing methods and standards used by the manufacturers and National Control Laboratories.
3.
INAUGURATION Welcoming the participants, Dr Palitha Abeykoon, Director, Department of Health Technology and Pharmaceuticals, WHO Regional Office for South-East Asia said that, presently four out of the ten Member Countries in the Region, namely Bhutan, Indonesia, Maldives and Thailand had already included HBV in the EPI schedule and under the initiative of GAVI, more countries in the Region were likely to introduce it shortly. India was likely to introduce it as an EPI vaccine gradually and in a phased manner.
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Hepatitis B Potency Testing Standardization
Dr (Ms) Nora Dellepiane, WHO-Headquarters was nominated the Chairperson and Dr (Ms) Jaspal Sokhey, WHO-SEARO the Rapporteur.
4.
BACKGROUND Dr Nora Dellepiane, WHO-HQ said that during the recent inspection of vaccine manufacturers for their prequalification to supply vaccines to UN Agencies, the problems faced by the manufacturers and the NCLs in the standardization of potency testing of HB vaccines by in vivo method and, in changing from in vivo method of testing to the in vitro method came to light. The WHO procedure as described in WHO/VSQ/97.06 for assessing the acceptability, in principle, of vaccines for purchase by UN Agencies or the prequalification of manufacturers to supply vaccines to UN Agencies was explained in detail by Dr Nora Dellepiane. It was stated that, WHO had recently introduced an additional requirement in the initial evaluation procedure for “Prequalification of manufacturers to supply vaccines to UN Agencies” of full evaluation of the NRA of the country against the indicators of the six NCA functions. However, for the reassessment evaluation (which is done every two years), the requirement for site visit to the manufacturer jointly with the NRA could be waived if a set of specified criteria are met.
5. 5.1
IN VIVO VS IN VITRO METHODS OF POTENCY TESTING Background Following the two very exhaustive talks on “Hepatitis B Vaccine Standardization: A Historical Perspective” and “Establishment and Replacement of Standards for Use in the Control of Hepatitis B Vaccines“ delivered by Dr Morag Ferguson, all the participants actively participated in the discussions, and clarified their doubts. It was realized by all that, it was important that the clinical trials are conducted by the manufacturers with their respective Primary Working Standard (WS) for evaluating the safety and efficacy of the vaccine. The clinical trial data is to be submitted by the manufacturer to the NRA and WS is to be shared by the manufacturer with the NCL for the potency testing of the final vaccine. At the time of replacement of the Primary WS with the Secondary WS, it was agreed that, the in vivo method of potency test be retained.
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There are two methods available for the potency testing of HBV i.e. the in vivo and in vitro methods. To obtain comparable in vitro potency results between the manufacturer and the NCL, it is important that both adopt the same procedure and method of testing.
5.2
Manufacturers’ Data on Characterization of the Vaccine Presentations were made by all the seven manufacturers of HBV outlining the characterization of the vaccines produced by them in the Region. Annex 3 gives the comparison of the source of vaccine, yeast, adjuvant, shelf life, clinical trial data and other details as presented by the manufacturers. As can be seen, all manufacturers from the countries represented are producing the recombinant HBV except in Myanmar, where presently plasma-derived HBV is produced. But within the next 2-4 years, Myanmar is likely to start the manufacture of recombinant HBV with transfer of technology from the Republic of Korea.
5.3
Conclusions It was agreed that for switching over to the in vitro method of potency testing on the final vaccine, it (in vitro method) should be properly validated and parallel testing done by both the methods (in vivo and in vitro) initially to gain experience and confidence in obtaining comparable results. At least four dilutions of the test vaccine and WS be used in the performance of the in vitro test against the one or two dilutions as presently used by some manufacturers and the NCLs.
6.
ISSUES FOR CONSIDERATION ON IN VITRO AND IN VIVO POTENCY TESTS The participants were divided in two working groups (A & B) to discuss at length the issues for consideration on in vivo and in vitro potency tests, given in Annex 4. In group A were participants from M/s Shantha Biotech, M/s Bharat Biotech and Panacea Biotech and group B included participants from M/s Wockardt, Serum Institute of India, Biofarma and Myanmar. Both groups were assisted by the facilitators during the discussions on the procedure of potency testing of the vaccine by in vivo and in vitro methods, validation of the test methods, WS, replacement of the WS by the secondary WS and clinical trial material etc. Page 3
Hepatitis B Potency Testing Standardization
6.1
Working Standards It was unanimously agreed that the WS of the manufacturer is part of the same batch of HBV which has been used as clinical trial material demonstrating the safety and efficacy of the vaccine with known potency value. It is useful and economical to produce a large size batch of the vaccine for such activities and also to conduct the stability studies for assigning the shelf life of the vaccine. More information will be collected and provided by the manufacturers to WHO experts on assigning the shelf life (if any) to the WS. It is important that the Primary WS be replaced with the Secondary WS by calibrating the latter with the Primary WS and using the in vivo method of testing. It was emphasized that the in vivo test be retained and performed when the Primary WS is replaced with the Secondary WS.
6.2
International Reference Reagent The International Reference Reagent (IRR) should not be used as WS for batch release, as no specification is established for the individual recombinant vaccines. There is no single Reference Standard available for HB vaccines for the simple reason that each vaccine produced is a specific product of the manufacturer. Hence, the use of homologous WS is absolutely critical in the potency testing of the vaccine.
6.3
International Standard 80/549 The International Standard (IS) 80/549 which is non-adjuvanted and is a dilution of partially purified plasma-derived HBsAg in PBS/BSA is not suitable for use in in vitro potency testing of adjuvanted vaccines.
6.4
Conclusions (1) Each manufacturer’s product is a specific product and the NCL should receive the WS from the manufacturer for testing the product of the respective manufacturer. Primary WS could be replaced with the Secondary WS provided the latter’s method of preparation, validation and testing is the same as that of the Primary WS. However, the conduct of additional clinical trials is not required for the Secondary WS if it is calibrated against the Primary
(2)
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WS (clinical trials conducted) using the same method of testing i.e. the in vivo method. It is imperative that the WSs are always tested by in vivo test. (3) In vitro method of potency testing of the vaccines could be adopted only after it has been proper validated with the in vivo method.
7.
STANDARDIZATION OF r HBV Points to consider for the standardization of recombinant HBV included in the monograph on recombinant Hep B were discussed in great detail (see Annex 5). Some changes in the text of this Annex and addition of more details are likely to be included by Dr Morag Ferguson and Dr Nora Dellepiane after having discussions with their colleagues both at NIBSC, London and WHOHQ. A revised version of the Annex will be circulated later on to all the participants for information and review.
8.
VACCINE VIAL MONITORS (VVMS) So far, the manufacturers of HBV in the Region have no experience with VVMs. However, the specifications for VVMs available for various vaccines were discussed. It is important that the appropriate VVM is selected and used for the vaccine by the manufacturer.
9.
RECOMMENDATIONS (1) (2) The NCL should obtain the WS from the manufacturer for testing the potency of the final vaccine. The manufacturer should replace the Primary WS with the Secondary WS only after the latter has been calibrated against the Primary WS that has been earlier used (as clinical trial material) in the clinical trials. In vivo test to be retained by the manufacturer for the potency testing of the Secondary WS.WHO-HQ will propose revision of Hepatitis B vaccine monograph to the Expert Committee on Biological Standardization. NIBSC, UK will review the use of IRR.
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(4)
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Hepatitis B Potency Testing Standardization
Annex 1 PROGRAMME Monday, 9 April 2001 9:00-9:30 hrs 9:30- 10:15 hrs Opening of workshop HTP, WHO/SEARO- Dr Palitha Abeykoon Aims of the workshop Overview of the pre-qualification process - Dr Nora Dellepiane- WHO/HQ Discussion Hepatitis B vaccine standardization: A historical perspective - Dr Morag Ferguson- Consultant SEARO Discussion Establishment and replacement of standards for use in the control of hepatitis B vaccines - Dr Morag Ferguson- Consultant SEARO Presentations by manufactures on in vivo potency tests and the choice, standardization and use of reference vaccine, correlation with clinical efficacy, selection of the antigen concentration in an adult dose - Shantha Biotech - Bharat Biotech - Panecea Biotech - Wockhardt - Serum Institute of India 15:30-16:00 hrs 16:00-17:00 hrs Tuesday, 10 April 2001 9:00-9:30 hrs Recap: Dr Jaspal Sokhey SEARO Continuation of presentations by manufacturers Serum Institute of India Manufacturers Manufacturers
10:15-10:30 hrs 11:00-11:45 hrs 11:45-12:00 hrs 13:00-14:00 hrs
14:00-15:00 hrs
General discussion, input of National Regulatory Authorities from the Region
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9:30-10:30 hrs
Development and validation of in vitro potency tests for hepatitis B vaccines Dr Morag Ferguson - Consultant SEAROS General discussions on the development of in vitro potency tests Presentation by manufacturers on their experience in establishment of an in vitro potency assay Working groups The participants in three working groups to review the data presented by manufacturers (both for in vivo and in vitro) and make comments on aspects correctly addressed and gaps in: • Correlation between the in vivo test and clinical efficacy. • Potency testing in vivo and in vitro. • Establishment and replacement of standards Facilitators: Dr. Ferguson, Sokhey and Dellepiane
11:00-12:00 hrs 13:00-14:00 hrs 14:00-16:30 hrs
16:30-17:00 hrs 17:00-17:15 hrs
VVMs on all vaccines: Aspects for consideration. Dr Nora Dellepiane - WHO/HQ Discussion
Wednesday, 11 April 2001 9:00- 10:00 hrs Presentations from manufacturers on how the meeting has helped them address issues. What they have learned and how to approach the standardization of hepatitis B vaccines Review of Draft Points to consider - All participants Finalization of Points to consider- All participants General discussion on issues to consider for the production of DTPHep B combination vaccines in the region. Closure
10:30-12:00 hrs 13:00-15:00 hrs 15:30-16:00 hrs 16:00 hrs
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Annex 2 LIST OF PARTICIPANTS WHO/SEARO Dr Jaspal Sokhey STP VSQ Ms Vidhya Ganesh STP VSQ
Manufacturers from India Shantha Biotechnics Pvt Ltd III Floor Serene Chambers Road No. 7 Banjara Hills Hyderabad Andhra Pradesh Dr Varaprasad Reddy Managing Director Mr K S N Prasad Head QA & RD Dr Sudarshan Jagannathan Head Quality Control Bharat Biotech Intl. Ltd #726 Road #3 Banjara Hills Hyderabad Andhra Pradesh Dr M Kuppusamy Vice President Mr K Gopinathan Manager, Quality Control Mr G Ravi Manager Production Wockhardt Research Centre D-4 MIDC Chikalthana Aurangabad -431210 Maharashtra Dr H Nellaiah Research Scientist - Biotechnology Dr A A Walvekar Q.A. Manager
WHO/HQ WHO/HQ (Facilitator) Dr Nora Dellepiane Consultant NIBSC UK (Facilitator) Dr Morag Ferguson Consultant Division of Virology NIBSC Blanche Lane South Mimms Hertfordshire EN6 3QG
India Government Dr A K Tahlan Joint Director Central Drugs Laboratory Central Research Institute Kasauli Himachal Pradesh Mr S D Vijayaraghavan Assistant Drugs Controller (I) Dte. General of Health Services Nirman Bhawan New Delhi-110011
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Report of a Meeting Dr M K Mishra Production In-Charge Mr Syed Abrar Ali Research Scientist (Observer) Serum Institute of India Ltd 212/2 Hadapsar Pune 400 028 Maharashtra Dr Suresh Jadhav Executive Director (QA) Dr K. Suresh Senior Manager Quality Control Dr S P Dhorje Manager Panacea Biotec Limited B-1 Ext A-27 Mohan Cooperative Industrial Estate Mathura Road New Delhi Mr A.K. Chawla Dy. Chief Production Dr Mrs Suniti B. Sharma Dy.Chief Quality Control Mr Ganesh R Kumraj Chief Production Mr Jaspreet Singh Sr. Scientist Vaccine Division (Observer) Dr Mrs Deepika R. Sharma Manager – Clinical Research(Observer) Mr Paramjit Singh Asst. Manager Medical Services(Observer)
Indonesia Dr Benny Kaligis Bio Farma 28 Pasteur Bandung Dr Sri Kusmartini Harsodjo National Quality Control Laboratory of Drug and Food National Board of Drug and Food Control Jl Percetakan Negara 23 Jakarta
Thailand Dr Amnat Thapchula Biological Products Division Department of Medical Sciences National Control Laboratory Nonthaburi
Myanmar Dr Khin Pyone Kyi Deputy Director and Head Vaccine Division Department of Medical Research Ministry of Health Yangon Dr Khin Yi Oo Consultant Microbiologist National Health Laboratory 35 Hmawkun Daik Rd Dagon P O Yangon
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Page 10 Shanta Shanvac B Source of vaccine construct Shanta Bharat Bharat Yeast species Pichia pastoris Sacc.cerevisiae Formulation Based on protien HBsAg internal standard, target 20µg HBsAg Aluminium hydroxide 0.05 mg/ml 20 ug Adjuvant Thiomersal Aluminium hydroxide 0.05 mg/ml Aluminium hydroxide
Laboratory Diagnosis of HIV Opportunistic Infections
Annex 3 CHARACTERIZATION OF VACCINES PRODUCED IN SEAR Panacea Enivac Filling of adjuvanted bulks from CIGB Cuba Wockardt Biovac B Rhein Biotech, Germany Serum Institute of India Genevac B Rhein Biotech, Germany
Biopharma Formulation and Filling of bulks produced by KGC (Rhein Biotech, Germany) Final bulk formulated at Biopharma Hansenula
Myanmar NY Blood Centre (plasma derived). Transfer of technology for rHBV from Republic of Korea
Pichia pastoris
Hansenula
Hansenula
Plasma (Proposed Hansenula from Republic of Korea 10 ug
Protien
20 ug
20 ug
Aluminium hydroxide 0.05 mg/ml
Aluminium hydroxide 0.05 mg/ml
Aluminium hydroxide 0.05 mg/ml
Aluminium hydroxide 0.05 mg/ml
0.05 mg/ml
Shanta Shanvac B Dose (adult/ paediatric) 20µg/10µg Pediatric up to 10 yrs
Bharat 20µg/10µg Pediatric up to 10 yrs
Panacea Enivac Pediatric up to 10 yrs (CIGB data support up to 19 yrs, submitted to DCGI) 20µg/10µg 0,1 2 and 12 months, 0,1,6 3 years
Wockardt Biovac B 20µg/10µg up to 10 yrs
Serum Institute of India Genevac B 20µg/10µg up to 10 yrs
Biopharma 20µg/10µg up to 10 yrs
Myanmar 10/5 ug up to 10 yrs
Immunisation schedule Shelf life
0,1,2 and 12 months or 0,1 6 months 3 years
0,1,2 months or 0,1 6 months 3 years
0,1,6
0,1,6 and 0,1,2 and 12 months (non-adjuvanted bulks 2 years) vaccine 2 years 95.8% to 98.4 % seroprotection in different studies (i.e. >10 mIU)
2, 3, 4 months 0, 2, 3 months 2 years
0, 1, 2
2 years
3 years
Clinical trials
Highly immunogenic, 100 % seroconversion
Tested in CT. At least 98% seroconversion
95% seroconversion in infants, 100% in healthy young adults. GMT>cf Engerix. potency upper confidence limit >1.0 (set by CIGB)
98.3 % seroconversion
Infants newborn or 2 months. 0,2,3 96% seroconversion, both in vials and in uniject Upper confidence interval>1.0
500 volunteers, 90-94 % seroconversion
Report of an Intercountry Training Workshop
In vivo potency test specification Page 11
Upper confidence interval>1.0
Mean relative potency>1.0
Upper confidence interval>1.0
Upper confidence interval>1.0
Mean relative potency of 1
Page 12 Mouse strain/sex Number per dilution/ dilutions Diluent Cut-off Antibody assay kit used Reference used
Laboratory Diagnosis of HIV Opportunistic Infections
Shanta Shanvac B Balb C 5week old females At beginning 20; now 10 1:32,1:64, 1:128, 1:256 Containing Al(OH)3 Based on mean response of control mice + 2sd AUSAB IRR but now product specific
Bharat Balb C
Panacea Enivac Balb C, 5 weeks, females 10 mice, 1:16,1:64, 1:256, 1:1024 Containing Al(OH)3 Based on mean response of control mice + 2 sd AUSAB CIGB standards New Ref prep.received. Tested in clinical trials? Specifications?
Wockardt Biovac B Balb C, 1416g, 4-5wk, both sexes 12, 1251000ng
Serum Institute of India Genevac B Balb C, 4-5 weeks, 14-16 g, both sexes 10 male, 10 females; 1:16,1:32, 1:64,1:128, Containing Al(OH)3 Based on mean response of control mice+2sd AUSAB Tested in clinical trials
Biopharma Balb C, both sexes, 4-5 weeks 20 mice, 1:4,1:16,1:64, 1:256, Containing Al(OH)3 Based on mean response of control mice AUSAB KGC references
Myanmar ICR, 5 weeks both sexes 15 mice, four fold dilutions
5 male, 5 females doubling 1/16 to 1/128 Containing Al(OH)3 10mIU, (Abbott quantitation panel) AUSAB IRR but now product specific
Containing Al(OH)3 Based on mean response of control mice+ 2sd AUSAB Initial clinical trial batch, then new batch compared with previous standard in 10 in vitro and 7 in vivo potency tests
Containing Al(OH)3 Based on mean response of control mice + 2 sd AUSAB IRR
Shanta Shanvac B Characterisation Tested in clinical trials Potency of clinical lots to be checked Clinical trials Highly immunogenic, 100% seroconversion
Bharat Tested in clinical trials Working std included in CT. Data to be circulated
Panacea Enivac Tested in clinical trials Lots produced in new facility? 95% seroconversion in infants, 100% in healthy young adults. GMT> cf Engerix.
Wockardt Biovac B
Serum Institute of India Genevac B
Biopharma Tested in clinical trials, both in vials and in uniject
Myanmar
95% seroprotection (ie >10mIU), GMT9200
Infants newborn or 2months. 0,1,2 96% seroconversion
500 volunteers, 90-94% seroconversion
In vitro potency test assay kit Pre-treatment step Specification
Report of an Intercountry Training Workshop
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Annex 4 ISSUES FOR CONSIDERATION
In vivo test • • Demonstrate consistency of products against the product-specific working standard Show that the existing specification is meaningful either by using existing or new data (as example upper confidence limit). This is basically a retrospective validation. Develop a working standard checked in clinical trials, or a secondary standard calibrated against a standard checked in clinical trials. Need to go through immunogenicity studies as characterization of the vaccine and the standard and use it always when a new standard is being set. Consider review of cut-off if criterion used is different from negative control mice OD
• •
•
In vitro test • • Is it validated? Is it suitable for your specific product? Changes in Abbott kit Correlation against immunogenicity test.
•
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Annex 5 POINTS TO CONSIDER IN THE STANDARDIZATION OF HEPATITIS B VACCINES Recombinant hepatitis B vaccines were licensed in the mid-1980s. WHO held two meetings to review progress in their development and formulate requirements. WHO requirements for hepatitis B vaccines made by recombinant techniques in yeast were published in 1987 and revised in 1989 to include all vaccines produced by recombinant DNA techniques. Although these requirements provide guidance on the characterization and tests to be undertaken, it is the responsibility of the NRA to approve many aspects of production and characterization of the antigen and also several quality control test methods and specifications. Recombinant hepatitis B vaccines produced by different manufacturers must be considered as different products. Both manufacturers and NRAs have queried many aspects of the characterization and quality control tests to be undertaken on individual vaccines. This document has been produced to highlight points that must be considered in evaluating the immunogenicity and efficacy of a recombinant hepatitis B vaccine, testing vaccines for potency and the establishment of suitable reference preparations.
Evaluation of vaccines in clinical trials According to the WHO requirements for recombinant hepatitis B vaccines, a vaccine will ‘reliably induce antibody responses to HBsAg in human recipients and the frequency and titre should be at least equivalent to those induced by plasma derived vaccines which meet WHO requirement’. The NRA is required to approve the data showing that the vaccine produces and adequate antibody response (titre, duration and quality) in human beings. There is no minimum requirement for antigen content of a hepatitis B vaccine and based on the results of clinical trials, manufacturers should establish the protein /antigen content of their vaccine.
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Points to consider: • • • • Assay of antibody responses to HBsAg in human recipients Cut-off value for sero-conversion Standard included in assays Titre and sero-conversion rates
Formulation According to the WHO requirements for recombinant hepatitis B vaccines, final adjuvanted vaccine bulks should be produced from aqueous bulks, which meet the specifications of all QC tests undertaken. Antigen purified by different methods and produced on different substrates may react differently when assayed using different HBsAg detection kits. The International Standard for HBsAg (80/549) was established for use in the determination of the sensitivity of assay kits for the detection of HBsAg. It is assigned a unitage in International Units, and although 1 IU can be considered as approximately equal to 1ng, this standard is not ideal for evaluating the HBsAg content of a vaccine in ng. Manufacturers should use a highly purified preparation of known protein content for this purpose and establish an in-house standard against which the protein content of future batches could be determined. The HBsAg content of the aqueous bulk should be determined by appropriate methods and by the lower limit of the ratio of HBsAg to total protein approved by the NRA.
Points to consider: • • • Assay of the antigen content of non-adjuvanted bulks – standard used. Formulation of their vaccine - is this based on the protein content of the vaccine or on the results from assays of HBsAg Monitoring of consistency of production of non-adjuvanted bulks through the protein/antigen ratio
Potency testing When WHO requirements for recombinant hepatitis B vaccines were published, it was considered that assays of antigenicity of adjuvanted vaccines
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would be difficult to standardize. It was proposed that the content of pure HBsAg in the product should be used as the basis for comparing immunogenicity in mice and responses in human subjects. The relationship between antigenicity of the product in in vitro tests, in mouse immunogenicity tests and in human beings should therefore be established. The potency requirement stated ‘that an appropriate quantitative test for antigen content and an immunogenicity assay be performed on samples representative of the final filling lot. The vaccine potency should be compared with that of a reference preparation and the NRA shall determine the lower limit of potency’. Several manufacturers have since developed and validated in vitro potency tests which are suitable for use with their individual vaccines. Mouse potency tests are no longer being performed by these manufacturers on every final lot. Since the vaccines in question were well established and had been used in millions of individuals, the WHO requirements for recombinant vaccines were therefore modified by ECBS in October 1997 to permit the use of an in vitro test that has been validated in correlation with immune response in humans or with the results obtained in mouse immunogenicity tests for which an appropriate specification has been set by a manufacturer. The requirement for potency testing now reads: ‘The vaccine shall be identified as envelope antigens of hepatitis B virus by appropriate methods. An appropriate quantitative test for potency by an in vitro or in vivo method shall be performed on samples representative of the final filling lots. Any in vitro method shall be appropriately validated and the test method approved by the NRA. The vaccine shall be compared with a reference vaccine and the NRA shall establish limits of potency’. Because of the diversity in the reactivity of vaccines containing HBsAg produced by different manufacturing processes and to which adjuvants have been added by different methods, it was considered unlikely that a single international standard would be suitable for the standardization of vaccines in both in vivo and in vitro assays. Manufacturers were encouraged to consider establishing a product-specific reference which could be related to the immunogenicity of the vaccine in humans. This vaccine will serve as a working standard and be included in all potency tests. The International Reference Reagent for plasma-derived vaccine was shown in a collaborative study to be suitable for use as reference preparation in mouse immunogenicity tests on recombinant vaccines. This reference reagent has no assigned unitage and although the potency of successive production batches of a given product should give consistent potencies Page 17
Hepatitis B Potency Testing Standardization
relative to this material, it has always been emphasized that hepatitis B vaccines which are suitable for use in man need not be qualitatively equivalent to this reference reagent. In addition, there is not necessarily a constant relationship between the immunogenicity in animal tests of hepatitis B vaccines prepared by different procedures and their immunological potential in man.
In vivo potency tests A suggested test in mice is described in the WHO requirements. This test involves the immunization of groups of mice with dilutions of test and reference vaccines and bleeding them out four weeks later. Individual sera are assayed for anti-HBs and the mice scored positive or negative for anti-HBs. The potency of the test vaccine relative to the reference is calculated by statistical analysis.
Points to consider • • • • • • • • • • Strain and sex of mice used must give a suitable dose response to the reference and test antigen Number of mice per dilution Diluent Number of dilutions The concentrations of vaccine tested should be selected to permit the calculation of the dilution giving 50% seroconversion ie ED50 Assay of sera Calculation of the cut-off value Statistical analysis Interpretation of results - ED50/relative potency Specification
In vitro potency tests In vitro potency tests should be able to distinguish vaccines of low potency, which may affect the immune response in man. Several factors must be considered when validating an assay for an individual vaccine, as vaccines
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contain antigen manufactured by different processes and different forms of adjuvant. These include the need for a pre-treatment step to disaggregate the antigen, eg with detergent, to ensure consistent responses in vaccines of all ages, the HBsAg test kit used, the diluent in which dilutions of the vaccine are prepared and the reference preparation. Test and reference vaccines should be tested concurrently in an assay designed to produce dose-response curves suitable for quantitative analysis by an appropriate statistical method (eg parallel line analysis). An assessment of statistical validity of the assay, an estimate of the potency of the test vaccine relative to the reference and a measure of the assay precision (confidence interval) should be produced.
Points to consider Test method • • • • • • • Documentation of test method Selection of test kits and how to use for quantitation Diluent Need for pre-treatment to disaggregate adjuvanted antigen Statistical analysis Assessment of statistical validity of the assay Precision (confidence intervals) of assay
Establishment of test specification based on correlation with mouse potency data or response in man or on assay results from a series of batches, which pass the mouse immunogenicity test Demonstration that a batch which fails in vivo test would also fail the in vitro test Validation of in vitro potency tests • • • • Linearity /Parallelism/Specificity/Robustness Linearity - 3 lots /6 independent runs Parallelism - acceptable parallelism to standard Reproducibility- 3 lots /6 independent runs/Multiple operators
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Expect to be within ?+/- 15% of mean potency and for each dilution, expect errors in the preparation of dilutions to be ?+/- 10% response for each dilution
Standard As per WHO requirements, vaccine potency assayed in in vivo and in vitro assays should be compared with a reference preparation. As indicated above, an International Reference Preparation has been established, but no unitage is assigned to this material and it cannot be used for the establishment and calibration of secondary standards. There is no requirement that vaccines, which are suitable for use in man, need be qualitatively equivalent to this reference reagent. Although the potency of successive production batches of a given product should give consistent potencies relative to the International Reference Reagent, manufacturers should consider establishing a productspecific reference which could be related to the immunogenicity of the vaccine in humans. This vaccine will serve as a working standard and be included in all potency tests.
Points to consider • • • • Source Characterization Immunogenicity in man Stability studies
Replacement of standard The standard should be replaced before it starts showing loss of activity. The shelf-life of standard could be longer than the shelf life of vaccine, if data to demonstrate stability of an individual vaccine for this period are available. The shelf life should be established under defined storage conditions and maintenance of sterility. Real time studies on vaccines should be supported by accelerated degradation studies A replacement working standard should be a typical batch of vaccine preferably of similar potency to the previous standard. If not, an adjustment factor may have to be incorporated into potency calculations if the specification is based on relative potency; or the specification may have to be amended. For example, if current reference has an assigned potency of 1.0
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and the replacement a potency of 1.2 relative to these standard, vaccines will be of lower potency relative to the replacement
Points to consider • • • • • • Documentation of the procedure for replacing standards Evaluation of stability of standards and establishment of shelf-life Identification of loss of potency e.g. lower responses in potency tests changes in dose response curves. Definition of acceptable potency loss (e.g. mean initial potency minus 3 standard deviations) Full characterization available in summary batch protocol Calibration Vs previous standard in in vivo and in vitro tests
References (1) Ferguson M, Seagroatt V and Schild GC. A collaborative study to establish the International Reference Reagent for hepatitis B vaccine containing plasma-derived hepatitis B surface antigen. Journal of Biological standardisation (1989) 17 151-160 Ferguson, M, Heath, A. and Minor, P.D. (1990) Report of a collaborative study for assessing the potency of recombinant hepatitis B vaccine. Biologicals 18, 345-350. EDQM; Project leader Roland Dobbelaer.( 1997) Collaborative study for the establishment of biological reference preparations for rDNA hepatitis B vaccine 1997 Pharmeuropa Special edition 97-2, 3-18. Seagroatt, V., Ferguson, M., Magrath, D.I., Schild, G.C. and Cameron, C.H. (1982). The British Reference Preparation of Hepatitis B Surface Antigen. Lancet (ii) 391-392. World Health Organization. International Reference Reagent for hepatitis B vaccine (plasma derived) WHO Technical Report Series 1987 760 29 World Health Organization (1989) Requirements for hepatitis B vaccines made by recombinant DNA techniques. WHO Technical Report Series 786, 38-71 World Health Organization (1999) Requirements for hepatitis B vaccines made by recombinant DNA techniques. WHO Technical Report Series 889, 94-95 Page 21
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(3)
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(5) (6)
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