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 con-
tainers. 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 PRODUCTION AND QUALITY CONTROL

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 candi-
dates is the HM 175 strain isolated in African green mon-
key 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 meth-
ods controlled by validated and reproducible quality
control tests and an adequate quality assurance organi-
zation 4.
SmithKline Beecham Biologicals, 89, Rue de I'lnstitut, 13--1330
Rixensart, Belgium
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 tumorigeni-
city. It has been widely used for > 20 years in the
production of live and inactivated vaccines such as live

0264-410X/92/100S99-03
© 1992 Butterworth-HeinemannLtd 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.
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, micro-
biological 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, ultra-
filtration 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 cul-
tures before harvest and the purification method applied
reduce the presence of serum to the trace level. Its quanti-
fication is based on a very sensitive test for bovine albu-
min 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 produc-
tion step. The procedure used was developed in analogy
with the well-known process for inactivated polio vac-
cine (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 filt-
ration is carried out at day 6. The formaldehyde concent-
Table 1 Preparation of the crude butk vaccine
Production steps Quality control
1 Preparation of the cell culture Test on control cell cultures,
(PDL 35) microbiological sterility, ad-
ventitious agents, identity
(karyotype)
2 Inoculation with working seed
3 Virus replication
4 Harvest Test on crude harvest
5 Frozen storage
$100 Vaccine, Vol. 10, Suppl. 1, 1992
Table 2 Purification and inactivation
Production steps Quality control
6. Purification On purified virus
Protein content
Bovine albumin
Antigen content
7 Inactivation During inactivation
Inactivation kinetics: days 0,
1,2,3
Effective inactivation at day
10
Final sterile filtration: day 15 End of inactivation
Effective inactivation
Antigen content
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 infecti-
vity 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 intersec-
tion. 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-phenox-
yethanol. The antigen content is determined and stan-
dardized 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 inacti-
vated 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.

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 speci-
fications 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 immunogeni-
city. 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 1.32-
1.34 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 anti-
bodies in animals and man which can be quantified by
several immunological methods such as ELISA, RIA
(modified Abbott HAVAB test) and radioimmunofocus
Inactivated hepatitis A vaccine: J. Peetermans
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 monoclo-
nal antibodies used in the characterization of the viral
proteins show that the antobodies elicited by vaccination
are directed to the immunodominant epitopes. The vac-
cine 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 extensi-
vely studied attenuated virus strain. The purification and
inactivation methods are reproducible. The quality
control tests are adequate for the assessment of all pro-
duction 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. Propaga-
tion of human hepatitis A virus in African green monkey cell culture,
primary isolation and serial passage. Infect. Immun. 1981, 32, 388-
393
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 vac-
cines for hepatitis A. Prog. Med. ViroL 1990, 37, 72-95
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