The quality control of vaccines is intended to provide assurances of both the probable efficacy and the safety of every batch of every product. It is achieved in three ways:
The quality
control of vaccines is intended to provide
assurances of both the probable efficacy
and the safety
of every batch
of every product. It is achieved
in three ways:
(1) in-process control; (2) final product control;
and (3) requirements
that for each
product the starting materials, intermediates, final product
and processing methods
are consistent.
The results
of all quality control tests must be recorded
in detail
and authorized by a qualified person as, in those
countries in which the manufacture of vaccines is regulated by law, they are part of the evidence on which
control authorities judge the acceptability or otherwise of each batch of each preparation.
A) In-Process Control
In-process quality
control is the control exercised over starting materials and intermediates. Its importance stems from the opportunities that
it provides for the
examination of a product
at the stages in its manufacture
at which
testing is most likely to provide the most meaningful information. The WHO
recommendations and national
authorities stipulate many in-process controls but manufacturers often perform
tests in excess
of those stipulated, especially sterility tests as, by so doing, they obtain assurance
that production is proceeding normally and
that the final
product is likely
to be satisfactory.
Numerous examples of in-process control exist for various types
of vaccine but
three demonstrate the principle.
The quality control of
both diphtheria and tetanus vaccines requires that
the products are
tested for the
presence of free toxin,
i.e. for specific
toxicity due to inadequate detoxification with formaldehyde, at the final product stage. By this stage,
however, the toxoid concentrates used in the preparation of the vaccines have been much diluted
and, as the volume of vaccine that
can be inoculated into the test animals (guinea-pigs) is limited, the tests are relatively
insensitive. In-process control, however, provides for tests on the undiluted concentrates and thus increases the sensitivity of the method
at least 100-fold.
An example from virus
vaccine manufacture is the titration, prior to inactivation, of the infectivity of the pools of live poliovirus used to make inactivated poliomyelitis vaccine. Adequate infectivity of the virus from
the tissue cultures is an indicator of the adequate virus content of the starting material
and, as infectivity is destroyed in the inactivation process, there is no possibility of performing such an assay after formaldehyde treatment.
A more general example
from virus vaccine
production is the rigorous
examination of tissue
cultures to exclude contamination with
infectious agents from
the source animal or, in the cases
of human diploid
cells or cells from continuous cell lines, to detect cells with
abnormal characteristics. Monkey kidney cell
cultures are tested for simian
herpes B virus,
simian virus 40,
mycoplasma and tubercle bacilli. Cultures of human
diploid cells and continuous line cells are subjected to detailed
karyological
examination (examination of chromosomes by microscopy) to ensure that
the cells have not undergone any
changes likely to impair the quality of a vaccine or lead to adverse effects.
B) Final
Product Control
i) Assays
Vaccines containing killed microorganisms or their products are generally tested for potency
in assays in which
the amount
of the vaccine that is required to protect
animals from a defined
challenge dose of the appropriate pathogen, or its product, is compared with the amount of a standard
vaccine that is required to provide the same protection. The usual format
of the test
is the 3 + 3 dose
quantal assay that is used to estimate
the potency of whole-cell pertussis vaccine (British
Pharmacopoeia, 2010). Three logarithmic serial doses of the test
vaccine and 3 of the standard
vaccine are made
and each is used
to inoculate a group of 16 mice.
In the case of both the
test
vaccine and the standard, the middle dose
is chosen on the basis of experience, so that it is sufficient to induce a protective response
in about 50% of the animals to which it is given. Each
lower dose may then be expected
to protect less than 50% of the mice to which it is given and each higher dose
to protect more
than 50% of the
animals. Fourteen days later
all of the mice are
inoculated (‘challenged’) with a suitable virulent
Bordetella pertussis strain and, after a further 14 days, the number of mice
surviving in each of the 6 groups
is counted. The number
of survivors in each group
is used to calculate the potency
of
the test vaccine
relative to the potency of the standard vaccine by the statistical method of probit
analysis (Finney, 1971).
The potency of the test vaccine may be
expressed as a percentage of the potency
of the standard vaccine. However,
as the standard vaccine will have an assigned potency in international units (IU), it is more usual to express the potency of the test
vaccine in similar units. Tests similar
to that used
to estimate the
potency of pertussis vaccine are prescribed for the potency
determinations of diphtheria vaccine and tetanus
vaccines. In these cases the respective bacterial toxins are used as the
challenge material (British Pharmacopoeia,
2010). Tests that do not involve
challenge but involve titration of the antitoxin response
in vitro, e.g. by ELISA (enzyme-linked immunosorbent assay), are now being
adopted.
Vaccines
containing live
microorganisms are generally tested for potency by determining their
content of viable particles. In the case of the most widely used live bacterial vaccine, BCG vaccine, dilutions of vaccine
are prepared in a medium which
inhibits clumping of cells, and fixed
volumes are dropped on to solid media
capable of supporting mycobacterial growth. After
a fortnight the colonies generated by the drops
are counted and the live count of the undiluted vaccine
is calculated. The potency
of live viral
vaccines is estimated in much the same way except that
a substrate of living cells
is used. Dilutions of vaccine are inoculated on to tissue culture monolayers in Petri dishes
or in plastic trays, and
the infective particle count of the vaccine
is calculated from
the infectivity of the dilutions as indicated by plaque
formation, cytopathic effect, haemadsorption or other effect and the dilution factor involved.
ii) Safety tests
Because many vaccines are derived from basic materials of intense pathogenicity—the lethal
dose of tetanus toxin for a mouse
is estimated to be 3 × 10−2 ng—safety testing is of paramount importance. Effective testing provides a guarantee of the safety
of each batch
of every product
and most vaccines in the final
container must pass one or more safety tests as prescribed in a pharmacopoeial monograph. This generality does not absolve
a manufacturer from the need to perform in-process tests as required, but it is relaxed for those preparations that have a final
formulation that makes safety tests on the final product either
impractical or meaningless.
Bacterial vaccines
are regulated by relatively simple safety tests. Those vaccines composed of killed
bacteria or bacterial products
must be shown
to be completely free from the living microorganisms used in the production process. Inoculation of appropriate bacteriological media with the
final product provides an assurance that all organisms have been killed. Those vaccines
prepared from toxins, for example, diphtheria and tetanus toxoids,
require in addition, a test system
capable of revealing inadequately
detoxified toxins; this can be done by inoculation of guinea-pigs, which are exquisitely sensitive to both diphtheria and tetanus toxins.
A test for sensitization of mice
to the lethal
effects of histamine is used to detect active pertussis toxin in pertussis
vaccines. An improved non-lethal method is also
available. The trend is to replace in vivo
assays by cell
culture methods where possible but these do not always
emulate in vivo effects. Inoculation of guinea-pigs
is also used to exclude the presence of abnormally virulent organisms in BCG
vaccine. Molecular
genetic methods, such as nucleic
acid amplification to probe for genes specific
to virulent strains, are now available but not yet in routine
use for vaccine testing.
Viral vaccines can present problems of safety testing far more complex than
those experienced with
most bacterial vaccines. With killed viral vaccines the potential hazards are those due to incomplete virus inactivation
and the consequent presence of residual live
virus in the preparation. The tests
used to detect
such live virus consist of the inoculation of susceptible tissue
cultures and of susceptible animals.
The cultures are examined for cytopathic effects, and the animals
for symptoms of disease and histological evidence of infection at autopsy. This test
is of particular importance in inactivated poliomyelitis vaccines, the vaccine being injected intraspinally into monkeys or mice transgenic for the poliovirus receptor. At autopsy, sections of brain
and spinal cord are
examined microscopically for the
histological lesions indicative of
proliferating poliovirus.
With attenuated viral vaccines the potential hazards are those associated with reversion of the virus during
production to a degree of virulence capable
of causing disease in recipients. To a large extent
this possibility is controlled by very careful selection of a
stable seed but, especially with live attenuated poliomyelitis vaccine,
it is usual to compare the neurovirulence of the vaccine with that of a vaccine known to be safe in field use. The
technique involves the
intraspinal inoculation of monkeys
with both the reference vaccine and the test vaccine
followed by comparison of the neurological lesions and
symptoms, if any, that are caused.
If the vaccine causes abnormalities in excess of those caused
by the reference it fails the
test. A modification of this test
which uses transgenic mice instead of monkeys is now available. An in
vitro method (MAPREC test) which relies on detecting RNA sequences
specific to virulent
virus has also been
developed. A widespread problem with safety testing of live viral
vaccines is that
the host specificity of many viruses limits
the availability of suitable animal models.
iii) Tests of general application
In addition
to the tests
designed to estimate
the potency and to exclude
the hazards peculiar
to each vaccine
there are a number of tests of more general
application. These relatively simple tests are as follows.
Sterility.
In general, vaccines are required to be sterile. The exceptions to this requirement are smallpox vaccine made from the dermis of animals and bacterial vaccines such as BCG, Ty21A and tularaemia vaccine, which consist of living but attenuated strains. These have a bioburden limit which defines the number of permissible microorganisms but excludes pathogens. WHO recommendations and pharmacopoeial monographs stipulate, for vaccine batches of different size, the numbers of containers that must be tested and found to be sterile. The preferred method of sterility testing is membrane filtration, as this technique permits the testing of large volumes without dilution of the test media. The test system must be capable of detecting aerobic and anaerobic bacteria and fungi .
Freedom from abnormal or general toxicity.
The purpose
of this simple
test is to exclude the presence in a final container of a highly
toxic contaminant. Five
mice of 17–22 g and two guinea-pigs of 250–350 g are inoculated with one human dose
or 1.0 ml, whichever is less, of the
test preparation. All must survive
for 7 days without signs of illness. Current pharmacopoeial
monographs usually do not require this
test if another
in vivo test has been
performed on the product.
Pyrogenicity or endotoxin content.
The pyrogenicity of a specified dose of product
when administered to rabbits
can be assayed by a standard pharmacopoeial method but the trend is to replace
this with an in vitro assay
for endotoxin . The capacity
of the product to induce gelation of Limulus polyphemus amoebocyte lysate is determined against a reference endotoxin preparation
and the result is expressed as IU of endotoxin. For pyrogens other than endotoxin, a monocyte stimulation test is available.
Presence
of aluminium and calcium.
The quantity
of aluminium in vaccines containing aluminium hydroxide or aluminium phosphate as an adjuvant is
limited to 1.25 mg per dose and it is usually estimated compleximetrically. The
quantity of calcium
is limited to 1.3 mg per dose and is usually
estimated by atomic
absorption spectrometry.
Free formaldehyde.
Inactivation of bacterial
toxins with formaldehyde may lead to the presence
of small amounts of free formaldehyde in the final product. The concentration, as estimated by colour
development with acetylacetone, must
not exceed 0.02%.
Phenol
concentration.
When phenol is used to preserve a vaccine
its concentration must
not exceed 0.25%
w/v or, in the case
of some vaccines, 0.5% w/v. Phenol
is usually estimated by
the colour reaction with aminophenazone
and hexacyanoferrate.
pH.
The potentiometric determination of pH is made by measuring the potential difference between two appropriate electrodes immersed in the solution to be
examined: one of these electrodes is sensitive to hydrogen
ions and the other is the reference electrode. The pH apparatus is calibrated with the
buffer solution of potassium
hydrogen phthalate and one other buffer solution of different pH. The pH in the test sample should comply
with the limits
approved for the particular
products.
Osmolality.
Osmolality is a practical means of giving an overall measure
of the contribution of the
various solutes present
in a solution to the osmotic pressure
of the
solution. Osmolality is determined by measurement of the depression of freezing point
of the test sample using appropriate apparatus. The osmolality of the test sample should comply
with the limits
approved for the particular products, for example minimum 240 mOsmol/kg.
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