Production, quality control and characterization of an inactivated hepatitis A vaccine Julien Peetermans The isolation and adaptation of hepatitis A virus to cell culture opened the way to the development of vaccines. Based on experience with inactivated poliovaccines, a similar approach was chosen for the development of an inactivated hepatitis A vaccine. Strain HM175, adapted to MRC-5 human diploid cells, was used as the virus strain. Vaccine production starts with growth and multiplication of the seed virus in MRC-5 cells. The harvests are clarified, purified and concentrated. Inactivation by formaldehyde is carried out on a pool of purified harvests. Close control of all process parameters results in consistent production of completely inactivated and highly immunogenic vaccine lots. Quality control testing is based on the general requirements for biologicals of WHO and National Control Authorities. Tests have been developed and validated to show the purity of the cell substrate used for each production cycle, the quality of the virus harvest, the adequacy of the purification and inactivation processes, and the conformity to stringent specifications for purity, safety and potency of the final bulk vaccine filled in final containers. The vaccine is characterized by adequate identity tests, by its reaction with polyclonal and monoclonal antibodies, by its immunogenicity in laboratory animals and by the detailed study of the immune response in primates and human volunteers. The final result of the development of adequate production and testing methods, confirmed by extensive characterization studies, is the availability of a consistent, safe and potent hepatitis A vaccine. Keywords: Virus strain; substrate; assessment; characterization
INTRODUCTION The isolation and adaptation of hepatitis A virus (HAV) to cell culture opened the way to the development of vaccines. Successful development depends on a number of factors. First, the isolated virus strain should grow well and give high antigen yields. One of the best candidates is the HM 175 strain isolated in African green monkey primary kidney cell cultures and adapted to MRC-5 cells 1,2. The substrate chosen for vaccine production was the MRC-5 human diploid cell line which was developed at the National Institute of Biological Standards and Control in the UK 3 and appeared to be the best available cell line. Other important factors are the possibility of developing reliable and standardized production methods controlled by validated and reproducible quality control tests and an adequate quality assurance organization 4.
SmithKline Beecham Biologicals, 89, Rue de I'lnstitut, 13--1330 Rixensart, Belgium 0264-410X/92/100S99-03 © 1992 Butterworth-HeinemannLtd
PRODUCTION
AND QUALITY CONTROL
Virus strain
The HMI75 strain of HAV has been adapted to human diploid cells. This adaptation has resulted in an increase in antigen yield and a speeding up of the growth cycle. The MRC-5 cells-adapted strain was developed and studied as a live vaccine candidate. It is attenuated for chimpanzees, marmosets and man. Production is based on the working seed principle. A large quantity of working seed material is stored frozen. Each production lot is infected with a constant quantity of this material under strictly standardized conditions. Cell substrate
The MRC-5 cell line is a normal, human diploid cell line. It meets the requirements of WHO and National Control Authorities for continuous cell lines suitable for the production of vaccines. It is free of extraneous agents, shows normal karyology and lacks tumorigenicity. It has been widely used for > 20 years in the production of live and inactivated vaccines such as live
Vaccine, Vol. 10, Suppl. 1, 1992 $99
Inactivated hepatitis A vaccine." J. Peetermans
polio, rubella, measles and mumps, and inactivated rabies vaccines. It has a good record of safety. There are no reports of the induction of hypersensitivity related to the presence of residual MRC-5 proteins. The clinical trials carried out with the inactivated hepatitis A vaccine have confirmed the safety of this substrate.
Table 2
Purification and inactivation
Production steps
Quality control
6.
Purification
7
Inactivation
On purified virus Protein content Bovine albumin Antigen content During inactivation Inactivation kinetics: days 0, 1,2,3 Effective inactivation at day 10 End of inactivation Effective inactivation Antigen content
Production Procedure
Preparation of crude bulk vaccine. Production is carried out following the classical steps used for viral vaccines (Table 1): preparation of cell cultures starting from the Manufacturer's Working Cell Bank (MWCB), followed by inoculation with working seed. Virus replication takes place at constant temperature and the incubation time is identical for all lots. The virus is extracted from the cells by freezing and thawing and the harvests are stored frozen. Quality control tests are carried out in parallel. They cover the control cell cultures which are part of the same cell batch as the production cells but not inoculated. They are tested for absence of extraneous agents, microbiological sterility (bacteria, fungi and mycoplasma) and identity. Tests for identity, virus titre, antigen content and sterility are carried out on the crude harvest. Purification and inactivation (Table 2) The purification process comprises several steps : sterile filtration, ultrafiltration and concentration by column chromatography. The purified virus is assayed for antigen concentration, total protein and bovine albumin content. Bovine serum is used in the medium for cell growth. As it is a potential agent for the induction of hypersensitivity in vaccinees its elimination is necessary. Extensive washing of cell cultures before harvest and the purification method applied reduce the presence of serum to the trace level. Its quantification is based on a very sensitive test for bovine albumin in the concentrated vaccine. W H O requirements state a maximum content of 50 ng/ml. The hepatitis A vaccine contains < 1 ng/ml. The inactivation process is monitored daily during the first three days and the test for effective inactivation is carried out at day 10. The total inactivation time is 15 days. At that time, a final sterile filtration is done and tests for complete inactivation and antigen content are carried out. Inactivation is the most important production step. The procedure used was developed in analogy with the well-known process for inactivated polio vaccine (IPV) for which W H O requirements exist and of which we have great experience. Sterile filtration is carried out immediately before adding formaldehyde. As aggregates were thought to be the cause of incomplete inactivation of early IPV batches, a second sterile filtration is carried out at day 6. The formaldehyde concentTable 1
Preparation of the crude butk vaccine
Production steps
Quality control
1
Preparation of the cell culture (PDL 35)
Test on control cell cultures, microbiological sterility, adventitious agents, identity (karyotype)
2 3 4 5
Inoculation with working seed Virus replication Harvest Frozen storage
$100
Test on crude harvest
Vaccine, Vol. 10, Suppl. 1, 1992
Final sterile filtration: day 15
ration is monitored during the first part of the process and the temperature is kept at 37°C during the entire inactivation period. The inactivation curve is determined from results on samples tested at days 0, I, 2 and 3. The observed infectivity titres are plotted on a semi-logarithmic scale against time and the intersection with the x-axis determines the time needed to inactivate the virus present in I ml. The constant slope of the straight line obtained on different vaccine lots provides proof of consistent, reproducible inactivation. The total inactivation time should be at least three times greater than the time to reach the x-axis intersection. In the case of hepatitis A vaccine, this period lasts three to four days. The total inactivation time has been set at 15 days. Proof of complete inactivation is carried out on samples corresponding to 1500 doses taken at days 10 and 15 of the process. Two passages in MRC-5 cells do not show the presence of non-inactivated virus in the test samples. Formulation
The vaccine contains per dose of 1 ml : 720 antigen units, determined by a standardized enzyme-linked immunosorbent assay (ELISA), 0.5 mg aluminium as aluminium hydroxide AI(OH)3 and 5.0 mg 2-phenoxyethanol. The antigen content is determined and standardized by immunological methods : ELISA and/or radioimmunoassay (RIA). The specific antigen activity, responsible for the immune response is quantified by comparison to a reference preparation. A similar method is used for the determination of the antigen content of IPV. AI(OH)3 is generally used as an adjuvant in inactivated vaccines, while 2-phenoxy-ethanol has been widely used as a preservative in IPV for ~ 30 years. Quality control on final vaccine for lot release
The final vaccine is tested following the general requirements for biologicals : description of the physical aspects of the vaccine, identity, volume, pH, sterility and general safety. The aluminium and 2-phenoxyethanol contents are quantified and have to comply with the requirements of the formula. The endotoxin content is determined by the Limulus amoebocyte lysote (LAL) test LAL and has been found to be consistently less than one endotoxin unit per dose. The antigen content is determined on the bulk vaccine before adsorption. The immunogenicity of the final adsorbed vaccine is evaluated in a mouse potency test.
Inactivated hepatitis A vaccine: J. Peetermans
Mice are injected with graded doses of the vaccine and bleeding is carried out after four weeks. The proportion of seroconverters among inoculated mice at each vaccine dilution is used to calculate the median effective dose (EDs0) by probit analysis. All lots have to pass the specifications which were developed on the basis of the results of consistency lots. These consistency lots were also found to be satisfactory in all respects in clinical trials. TJae reference vaccine is one of these consistency lots. The stability of the vaccine is evaluated by using the same mouse potency test. Vaccine lots stored for > 24 months still meet the specifications for vaccine at release. Accelerated stability tests on vaccine lots exposed at 37°C for 1, 2 and 3 weeks show no loss of immunogenicity. The accelerated stability tests were carried out both on vaccine lots at release and on vaccine lots stored for 15 months at + 2°C to + 8°C. Characterization The particulate character of the virus is preserved during the production and inactivation steps. Purified virus particles and formaldehyde-inactivated vaccine sediment homogeneously with a maximum peak at 1.321.34 g/ml. Electron micrographs of vaccine preparations show particles with a mean diameter of ~ 27 nm. The viral protein of the vaccine is identified by immunoblots using antibodies against the whole viral capsid, the VP0, VPI and Px proteins. Antigenicity is identified by routine immunological tests using RIA and ELISA. By using well characterized neutralizing monoclonal antibodies, it was possible to show that the inactivated vaccine carries the immunodominant epitopes of the infectious virus and that formaldehyde treatment does not impair the reactivity of the epitopes. A most important characteristic of a vaccine is the immune response it stimulates. This vaccine induces antibodies in animals and man which can be quantified by several immunological methods such as ELISA, RIA (modified Abbott HAVAB test) and radioimmunofocus
inhibition test (RIFIT). The correlation between the different methods is satisfactory. Most important is the fact that the induced antibodies neutralize HAV. Further, competition studies with the panel of monoclonal antibodies used in the characterization of the viral proteins show that the antobodies elicited by vaccination are directed to the immunodominant epitopes. The vaccine induces protection against challenge in chimpanzees and marmosets, lmmunoglobulins prepared from plasma of vaccinees induce passive protection against challenge in chimps. CONCLUSION The inactivated hepatitis A vaccine developed by SmithKline Beecham Biologicals (Havrix) is produced in a safe, well characterized cell substrate using an extensively studied attenuated virus strain. The purification and inactivation methods are reproducible. The quality control tests are adequate for the assessment of all production steps and for the analysis of the final vaccine. The vaccine has an excellent stability profile and routine production lots show consistency in laboratory tests and clinical trials. REFERENCES 1 Gust, I.D., Lehmann, N.I., Crowe, S., McCrorie, M., Locarnini, S.A. and Lucas, C.R. The origin of the HM 175 strain of hepatitis A virus. J. Infect. Dis., 1985, 151,365-367 2 Daemer, R.J., Feinstone, S.M., Gust, I.D. and Purcell, R.H. Propagation of human hepatitis A virus in African green monkey cell culture, primary isolation and serial passage. Infect. Immun. 1981, 32, 388393 3 Jacobs, J.P. The status of human diploid cell strain MRC-5 as an approved substrate for the production of viral vaccines. J. BioL Stand. 1976, 4, 97-99 4 Andre, F.E., Hepburn, A, and D'Hondt, E. Inactivated candidate vaccines for hepatitis A. Prog. Med. ViroL 1990, 37, 72-95
Vaccine, Vol. 10, Suppl. 1, 1992
$101
INTRODUCTION The isolation and adaptation of hepatitis A virus (HAV) to cell culture opened the way to the development of vaccines. Successful development depends on a number of factors. First, the isolated virus strain should grow well and give high antigen yields. One of the best candidates is the HM 175 strain isolated in African green monkey primary kidney cell cultures and adapted to MRC-5 cells 1,2. The substrate chosen for vaccine production was the MRC-5 human diploid cell line which was developed at the National Institute of Biological Standards and Control in the UK 3 and appeared to be the best available cell line. Other important factors are the possibility of developing reliable and standardized production methods controlled by validated and reproducible quality control tests and an adequate quality assurance organization 4.
SmithKline Beecham Biologicals, 89, Rue de I'lnstitut, 13--1330 Rixensart, Belgium 0264-410X/92/100S99-03 © 1992 Butterworth-HeinemannLtd
PRODUCTION
AND QUALITY CONTROL
Virus strain
The HMI75 strain of HAV has been adapted to human diploid cells. This adaptation has resulted in an increase in antigen yield and a speeding up of the growth cycle. The MRC-5 cells-adapted strain was developed and studied as a live vaccine candidate. It is attenuated for chimpanzees, marmosets and man. Production is based on the working seed principle. A large quantity of working seed material is stored frozen. Each production lot is infected with a constant quantity of this material under strictly standardized conditions. Cell substrate
The MRC-5 cell line is a normal, human diploid cell line. It meets the requirements of WHO and National Control Authorities for continuous cell lines suitable for the production of vaccines. It is free of extraneous agents, shows normal karyology and lacks tumorigenicity. It has been widely used for > 20 years in the production of live and inactivated vaccines such as live
Vaccine, Vol. 10, Suppl. 1, 1992 $99
Inactivated hepatitis A vaccine." J. Peetermans
polio, rubella, measles and mumps, and inactivated rabies vaccines. It has a good record of safety. There are no reports of the induction of hypersensitivity related to the presence of residual MRC-5 proteins. The clinical trials carried out with the inactivated hepatitis A vaccine have confirmed the safety of this substrate.
Table 2
Purification and inactivation
Production steps
Quality control
6.
Purification
7
Inactivation
On purified virus Protein content Bovine albumin Antigen content During inactivation Inactivation kinetics: days 0, 1,2,3 Effective inactivation at day 10 End of inactivation Effective inactivation Antigen content
Production Procedure
Preparation of crude bulk vaccine. Production is carried out following the classical steps used for viral vaccines (Table 1): preparation of cell cultures starting from the Manufacturer's Working Cell Bank (MWCB), followed by inoculation with working seed. Virus replication takes place at constant temperature and the incubation time is identical for all lots. The virus is extracted from the cells by freezing and thawing and the harvests are stored frozen. Quality control tests are carried out in parallel. They cover the control cell cultures which are part of the same cell batch as the production cells but not inoculated. They are tested for absence of extraneous agents, microbiological sterility (bacteria, fungi and mycoplasma) and identity. Tests for identity, virus titre, antigen content and sterility are carried out on the crude harvest. Purification and inactivation (Table 2) The purification process comprises several steps : sterile filtration, ultrafiltration and concentration by column chromatography. The purified virus is assayed for antigen concentration, total protein and bovine albumin content. Bovine serum is used in the medium for cell growth. As it is a potential agent for the induction of hypersensitivity in vaccinees its elimination is necessary. Extensive washing of cell cultures before harvest and the purification method applied reduce the presence of serum to the trace level. Its quantification is based on a very sensitive test for bovine albumin in the concentrated vaccine. W H O requirements state a maximum content of 50 ng/ml. The hepatitis A vaccine contains < 1 ng/ml. The inactivation process is monitored daily during the first three days and the test for effective inactivation is carried out at day 10. The total inactivation time is 15 days. At that time, a final sterile filtration is done and tests for complete inactivation and antigen content are carried out. Inactivation is the most important production step. The procedure used was developed in analogy with the well-known process for inactivated polio vaccine (IPV) for which W H O requirements exist and of which we have great experience. Sterile filtration is carried out immediately before adding formaldehyde. As aggregates were thought to be the cause of incomplete inactivation of early IPV batches, a second sterile filtration is carried out at day 6. The formaldehyde concentTable 1
Preparation of the crude butk vaccine
Production steps
Quality control
1
Preparation of the cell culture (PDL 35)
Test on control cell cultures, microbiological sterility, adventitious agents, identity (karyotype)
2 3 4 5
Inoculation with working seed Virus replication Harvest Frozen storage
$100
Test on crude harvest
Vaccine, Vol. 10, Suppl. 1, 1992
Final sterile filtration: day 15
ration is monitored during the first part of the process and the temperature is kept at 37°C during the entire inactivation period. The inactivation curve is determined from results on samples tested at days 0, I, 2 and 3. The observed infectivity titres are plotted on a semi-logarithmic scale against time and the intersection with the x-axis determines the time needed to inactivate the virus present in I ml. The constant slope of the straight line obtained on different vaccine lots provides proof of consistent, reproducible inactivation. The total inactivation time should be at least three times greater than the time to reach the x-axis intersection. In the case of hepatitis A vaccine, this period lasts three to four days. The total inactivation time has been set at 15 days. Proof of complete inactivation is carried out on samples corresponding to 1500 doses taken at days 10 and 15 of the process. Two passages in MRC-5 cells do not show the presence of non-inactivated virus in the test samples. Formulation
The vaccine contains per dose of 1 ml : 720 antigen units, determined by a standardized enzyme-linked immunosorbent assay (ELISA), 0.5 mg aluminium as aluminium hydroxide AI(OH)3 and 5.0 mg 2-phenoxyethanol. The antigen content is determined and standardized by immunological methods : ELISA and/or radioimmunoassay (RIA). The specific antigen activity, responsible for the immune response is quantified by comparison to a reference preparation. A similar method is used for the determination of the antigen content of IPV. AI(OH)3 is generally used as an adjuvant in inactivated vaccines, while 2-phenoxy-ethanol has been widely used as a preservative in IPV for ~ 30 years. Quality control on final vaccine for lot release
The final vaccine is tested following the general requirements for biologicals : description of the physical aspects of the vaccine, identity, volume, pH, sterility and general safety. The aluminium and 2-phenoxyethanol contents are quantified and have to comply with the requirements of the formula. The endotoxin content is determined by the Limulus amoebocyte lysote (LAL) test LAL and has been found to be consistently less than one endotoxin unit per dose. The antigen content is determined on the bulk vaccine before adsorption. The immunogenicity of the final adsorbed vaccine is evaluated in a mouse potency test.
Inactivated hepatitis A vaccine: J. Peetermans
Mice are injected with graded doses of the vaccine and bleeding is carried out after four weeks. The proportion of seroconverters among inoculated mice at each vaccine dilution is used to calculate the median effective dose (EDs0) by probit analysis. All lots have to pass the specifications which were developed on the basis of the results of consistency lots. These consistency lots were also found to be satisfactory in all respects in clinical trials. TJae reference vaccine is one of these consistency lots. The stability of the vaccine is evaluated by using the same mouse potency test. Vaccine lots stored for > 24 months still meet the specifications for vaccine at release. Accelerated stability tests on vaccine lots exposed at 37°C for 1, 2 and 3 weeks show no loss of immunogenicity. The accelerated stability tests were carried out both on vaccine lots at release and on vaccine lots stored for 15 months at + 2°C to + 8°C. Characterization The particulate character of the virus is preserved during the production and inactivation steps. Purified virus particles and formaldehyde-inactivated vaccine sediment homogeneously with a maximum peak at 1.321.34 g/ml. Electron micrographs of vaccine preparations show particles with a mean diameter of ~ 27 nm. The viral protein of the vaccine is identified by immunoblots using antibodies against the whole viral capsid, the VP0, VPI and Px proteins. Antigenicity is identified by routine immunological tests using RIA and ELISA. By using well characterized neutralizing monoclonal antibodies, it was possible to show that the inactivated vaccine carries the immunodominant epitopes of the infectious virus and that formaldehyde treatment does not impair the reactivity of the epitopes. A most important characteristic of a vaccine is the immune response it stimulates. This vaccine induces antibodies in animals and man which can be quantified by several immunological methods such as ELISA, RIA (modified Abbott HAVAB test) and radioimmunofocus
inhibition test (RIFIT). The correlation between the different methods is satisfactory. Most important is the fact that the induced antibodies neutralize HAV. Further, competition studies with the panel of monoclonal antibodies used in the characterization of the viral proteins show that the antobodies elicited by vaccination are directed to the immunodominant epitopes. The vaccine induces protection against challenge in chimpanzees and marmosets, lmmunoglobulins prepared from plasma of vaccinees induce passive protection against challenge in chimps. CONCLUSION The inactivated hepatitis A vaccine developed by SmithKline Beecham Biologicals (Havrix) is produced in a safe, well characterized cell substrate using an extensively studied attenuated virus strain. The purification and inactivation methods are reproducible. The quality control tests are adequate for the assessment of all production steps and for the analysis of the final vaccine. The vaccine has an excellent stability profile and routine production lots show consistency in laboratory tests and clinical trials. REFERENCES 1 Gust, I.D., Lehmann, N.I., Crowe, S., McCrorie, M., Locarnini, S.A. and Lucas, C.R. The origin of the HM 175 strain of hepatitis A virus. J. Infect. Dis., 1985, 151,365-367 2 Daemer, R.J., Feinstone, S.M., Gust, I.D. and Purcell, R.H. Propagation of human hepatitis A virus in African green monkey cell culture, primary isolation and serial passage. Infect. Immun. 1981, 32, 388393 3 Jacobs, J.P. The status of human diploid cell strain MRC-5 as an approved substrate for the production of viral vaccines. J. BioL Stand. 1976, 4, 97-99 4 Andre, F.E., Hepburn, A, and D'Hondt, E. Inactivated candidate vaccines for hepatitis A. Prog. Med. ViroL 1990, 37, 72-95
Vaccine, Vol. 10, Suppl. 1, 1992
$101
