1. Innocuity test
of a simple inactivation study could comprise evaluation of the titre of live
vaccine before and after inactivation and measuring the log10 fall
in titre during the inactivation process. This would give an indication of the
efficacy of the inactivation process. There is evidence that virus titration
tests may not have satisfactory sensitivity to ensure complete inactivation. In
these circumstances, a specific innocuity test would need to be developed and
validated to be fit for increased sensitivity. To increase sensitivity more
than one passage would be essential depending on the virus (OIE Terrestrial
manual tests of sterility, 2017).
sample representing at least 200 doses of vaccine (final formulated product)
should go through innocuity test to show absence of infectious virus by inoculation of sensitive cell culture monolayers.
following instructions should be considered for innocuity test:
The test method should be highly sensitive to the virus.
large enough number of vaccine doses to satisfy statistical requirements should
Under the selected conditions inactivated virus should not interfere with the
replication of non-inactivated virus (Barteling, 1983).
the final product, antigen must be extracted from adjuvant following an
appropriate validated method (OIE Terrestrial manual FMD, 2017). Antigen recovery
from oil-adjuvanted vaccines using n-butyl alcohol as nine volumes of an
oil-emulsion vaccine was mixed thoroughly with one volume of n-butyl alcohol
and centrifuged for 5 min at 4?, 5000g. Collection of the lower aqueous phases
to be tested (Feng et.al, 2016).
of antigen may be necessary in which case it must be shown that the
concentrated does not affect with the sensitivity or reading of the assay. Inspection
of The cell sheets is done daily for 2–3 days, then the consumed medium is
transferred to fresh monolayers and the original monolayers are replenished
with fresh medium. Using this method, traces of live virus can be propagated by
the passage technique and detected on the basis of CPE
observed. Three passages of the original virus preparation are usually used. A
variant on this method is to freeze–thaw the old monolayers to release
intracellular virus, which can be detected by additional passage (OIE Terrestrial
manual FMD, 2017).
long as the 146S antigen concentration was below 1 ?g per 106 cells,
suspension cultures of baby hamster kidney cells is proved to be the most
sensitive detection system for traces of infective virus. Above this level
interference may cover the presence of non-inactivated virus (Barteling, 1983).
is defined as the absence of viable microorganisms.
from contamination is defined as the absence of specified viable microorganisms
(OIE Terrestrial manual tests of sterility, 2017).
Ø Country regulations:
for sterility should be applied on every batch of vaccine. Based on their
animal health status, each country or regions will have specific requirements to
what microorganisms are necessary to eliminate and what appropriate testing
protocols are acceptable. As well as applying general testing procedures recognized
in national or regional standards, it may be necessary to apply rigorous
exclusion testing for specific agents that are exotic to the precise country or
region (OIE Terrestrial manual tests of sterility, 2017).
Ø Tests for sterility
for sterility shall be convenient to prove that the vaccine is free from
extraneous viruses, bacteria, fungi and protozoa.
Amplification of viable extraneous
agents arises by using of cell culture that is susceptible to particular
viruses of concern, bacterial, and fungal culturing techniques and, where
necessary and possible, tests including animal inoculation. Some techniques
have been accurately validated and found to be “fit for purpose”, whereas
others may have undertaken only partial validation studies. For instance, methods for bacterial and fungal sterility have not
been officially validated although they have been used for many years. In
particular, the in-vivo and cell culture methods have basically unknown
sensitivity and specificity (Sheets et al., 2012) though there is a believed
theoretical sensitivity of 1 colony-forming unit (CFU). It is therefore
important to interpret results in the light of specific circumstances of
cultures employed and bearing in mind sensitivity and specificity of detection
systems (OIE Terrestrial manual tests of sterility, 2017).
antibody test (FAT), presence of colonies or cytopathic effects (CPE) and
enzyme-linked immunosorbent assay (ELISA) will be used for detection purposes
after amplification using culturing techniques. Care must be taken when using ELISA
technique for detection as such tests do not distinguish viable from non-viable
agent detection (OIE Terrestrial manual tests of sterility, 2017).
sensitive techniques such as molecular assays may have the ability to detect agents,
which may not be fruitfully amplified in traditional culturing methods. The
detection range can be widened using family specific primers and probes when
designed properly. Yet, such new tests are mostly able to detect evidence for
non-infectious contaminants, such as remnants of nucleic acid from
inactivated contaminants. Additional tests would be required to determine the
nature of the contaminant such as non-infectious nucleic acid or infectious
virus. Trails at virus isolation or sequencing may remedy this. Molecular
assays if not designed as fit for purpose may miss detection of contaminating
agents or lack sensitivity to do so (Hodinka, 2013).
recent enhancements in protein and peptide separation proficiencies and highly precise
mass spectrometry have endorsed the identification and quantification of
proteins in a given sample. However, most of these new technologies cannot discriminate
between viable and non-viable organisms. Therefore, targeted assays, e.g.
amplification in cell culture followed by polymerase chain reaction (PCR) may
be superior (Wang et al., 2014).
Extraneous agents require specific methods
General procedures are useful as screening tests to detect extraneous
agents present in biological material as they will not necessarily detect all contaminants.
Some agents may involve specific methods for detection (Table 1). Remarkably, some subtypes of an
agent type may be detectable by general methods, and some may require specified
testing for detection. Such as, bovine adenovirus subgroup 1 (serotypes 1, 2, 3
and 9) can be readily isolated using general methods (Vero cells) however
bovine adenovirus subgroup 2 (serotypes 4, 5, 6, 7, 8 and 10) are not readily
isolated and required specialized methods for isolation (OIE sterility test).
Ø Precautions against microbial contamination
The test for sterility is performed under aseptic conditions. In
order to achieve such circumstances, the test environment has to be adapted to avoid
contamination in order not to affect any microorganisms which are to be
revealed in the test.
The working conditions in which the tests are performed are
monitored regularly by appropriate sampling of the working area and by carrying
out appropriate controls (WHO, 2012).
Representative samples of ?nal lot to be tested shall be taken. For
?nal lots containing 500 or more containers, at least 20 samples shall be
taken. For ?nal lots containing fewer than 500 containers, not less than 10
containers from the lot should be taken for a sample, except that for lots of
less than 100 containers, only 10% of the lot need be tested.
A suggested rule for calculating the number of samples is to take
samples, Where N is the number of ?nal
containers in the lot (WHO, 1973).
Ø Culture media and incubation
Media for the test may be prepared as mentioned below, or corresponding
commercial media may be used provided that they fulfill the growth promotion
The following culture media have been found to be suitable for the
test for sterility. Fluid thioglycollate medium is primarily intended for the
culture of anaerobic bacteria; however, it will also detect aerobic bacteria.
Soya-bean casein digest medium is suitable for the culture of both fungi and
Fluid thioglycollate medium
L-Cystine 0.5 g
Agar 0.75 g
Sodium chloride 2.5 g
Glucose monohydrate/anhydrous 5.5/5.0 g
Yeast extract (water-soluble) 5.0 g
Pancreatic digest of casein 15.0 g
Sodium thioglycollate or 0.5 g
Thioglycollic acid 0.3 ml
Resazurin sodium solution (1 g/l of resazurin sodium), freshly
prepared 1.0 ml
Water R 1000 ml
pH after sterilization 6.9 to 7.3.
Mix the L-cystine, agar, sodium chloride, glucose, water-soluble
yeast extract and pancreatic digest of casein with the water R and heat until
solution is effected. Liquefy the sodium thioglycollate or thioglycollic acid
in the solution and, if needed, add sodium hydroxide (1 mol/l) so that, after
sterilization, the solution will have a pH of 9 to 7.3. If filtration is required
heat the solution again without boiling and filter while hot through moisturized
filter paper. Add the resazurin sodium solution, mix and place the medium in
suitable vessels which provide a ratio of surface to depth of medium to permit
not more than the upper half of the medium has undergone a color change revealing
oxygen uptake at the end of the incubation period. Sterilize using a validated
process. Medium is stored at a temperature between 2 °C and 25 °C in a sterile, airtight
container. If more than the upper one third of the medium has acquired a pink color,
the medium may be reestablished once by warming the vessels in a water-bath or
in free-flowing steam until the pink color disappears and cooling quickly, care
must be taken to prevent the introduction of non-sterile air into the
container. Longer storage period of medium requires validation. Fluid
thioglycollate medium is to be incubated at 30–35 °C.
Soya-bean casein digest medium
Pancreatic digest of casein 17.0 g
Papaic digest of soya-bean meal 3.0 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate 2.5 g
Glucose monohydrate/anhydrous 2.5/2.3 g
Water R 1 000 ml
pH after sterilization 7.1 to 7.5.
Preparation, Usage and storage:
Dissolve the solids in water R, warming slightly to effect
solution. Cool the solution to room temperature. Add sodium hydroxide (1 mol/l),
if necessary, so that after sterilization the solution will have a pH of 7.1 to
7.5. Filter, if necessary, to clarify, dispense into suitable vessels and sterilize
using a validated process. Store is at a temperature between 2 °C and 25 °C in
a sterile well-closed container, unless it is planned for direct use. Do not
use the medium for a longer storage period than has been validated. Soya-bean
casein digest medium is to be incubated at 20–25 °C (WHO, 2012).
Ø Sterility test of Medium and growth promotion of aerobes, anaerobes
Test of sterility
The sterility of the media should be tested by incubating
representative containers at the suitable temperature for the length of time definite
for each test.
The ability of the culture media to support growth in the presence
and absence of tested material, for each new batch or lot of culture media.
To test for capability to support growth in the absence of the tested
product, 10–100 viable control organisms of
the suggested ATCC strains listed in Table 2 and incubated in media according
to the conditions specified.
To test for ability of the culture media to support growth in the
presence of the test material, both
the test material and 10–100 viable control organisms should be inoculated in
media. The number of containers used should be at least one-half the number
used to test the product.
The test media are acceptable if within 7 days, clear sign of
growth of the control organisms appears in all inoculated media containers. In
the event that growth is evident, identification of the organism id performed to
confirm that it is the organism formerly added to the medium. If any of the
media show insufficient growth response, or if the organism growed, is not the
organism used to inoculate the material, the sterility test is considered
The presence or absence of microbial growth cannot be readily
determined by visual examination if the material being tested renders the medium turbid, therefore
14 days after the beginning of incubation transfer portions (each not less than
1 ml) of the medium to fresh containers of the same medium and then incubate
the original and transfer containers for not less than 4 days (OIE Terrestrial manual Tests of