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).