General Procedure
The laboratory scale experiments were designed to simulate the feeding
of a perfusion fermenter with a fermenter feed media solution containing
MMV as a contaminant. The intention of each laboratory scale experiment
was to investigate the maximum process feed load capacity of an
investigated filter when loading it with fermenter feed media spiked
with MMV at a ratio of 1:250 (i.e., 0.4% spike load). The MMV-spiked,
0.2 µm filtered fermenter feed media solution (“spiked starting
material”) was passed over a filter train consisting of one 0.1 µm
prefilter followed by a virus removal filter (each with a stipulated
pore size of approximately 20 nm, see Materials and Methods). During
each investigation the impact of multiple interruptions during the
loading process on virus clearance of a given filter was also
investigated. This was done when the filtrate collecting flask was
replaced by a fresh one (samples for virus titration were drawn from the
filtrate and stored at ≤-60°C until the end of the experiment when all
filtrate samples were titrated together) – during this activity
filtration was interrupted intentionally for at least one hour. One of
these intentional interruptions lasted at least 72 hours to represent
the pause between the first filling of the fermenter and the build-up of
the inoculum before the actual perfusion fermentation started. Filtrate
collection flask replacement – and thus process interruption – took
place approximately once a day. The overall number of interruptions per
experiment ranged from 1 to 21 depending on filter performance.
Filtration was continued until a flow decay of around 90% was reached
or the duration of the experiment had exceeded 30 days (duration ranged
from one to 34 days). Virus removal filtration was performed at constant
transmembrane pressures (TMP, i.e., the pressure difference between feed
and filtrate – which was at ambient pressure and, therefore, taken as
0.0 bar for calculations) over the virus removal filter. The TMP was set
to 1.0 to 2.6 bar for each experiment (the maximum TMP set-point was
determined either by the maximum value suggested by the filter
manufacturer or by the maximum pressure allowed by the tubing used) and
to 0.0 bar during the intentional interruptions. Filtration was
performed under ambient temperature for all experiments. pH was measured
for the spiked starting material and for each filtrate fraction.
Controls for aggregates (EMEA, (2010)) were performed for (a) the virus
stock and (b) the spiked starting material matrix. For (a) the titers of
the thawed virus stock and of the 0.1 µm filtered virus stock (which
then was used for spiking) were compared – a titer reduction of less
than 1 log10 is not considered to be significant
according to regulatory guidance (EMEA, (1996)). For (b) the titer of
the spiked starting material was compared to the titer of the
pre-filtrate drawn during the first interruption.
In order to evaluate the influence of the freeze/thaw cycle, to which
the filtrate fractions were subjected, on the virus titer of these
samples, freeze controls were performed (EMEA, (1996)). Two freeze
control levels were investigated: (c) “high titer” and (d) “low
titer”. For (c) the titer of the spiked starting material (titrated
immediately) was compared to the titer of the same matrix which had been
stored at ≤-60°C throughout the experiment and titrated together with
all other samples which had been stored frozen. For (d) the titer of the
spiked starting material diluted with starting material to a theoretical
MMV titer of 2.0 log10 [TCID50/mL]
(titrated immediately) was compared to the titer of the same matrix
which had been stored at ≤-60°C throughout the experiment and titrated
together with all other samples which had been stored frozen.
In order to evaluate the influence of ambient conditions during an
experiment on the virus titer, process controls were performed. To this
purpose an aliquot of the spiked starting material was kept at ambient
temperature in the biosafety cabinet where the virus filtration was
performed, until the end of the NF procedure. At evenly spaced intervals
samples were drawn and stored at ≤-60°C throughout the experiment and
titrated together with all other samples which had been stored frozen.
The virus titers of these samples were compared to that of the spiked
starting material (titrated immediately).
Un-spiked control runs were performed for the virus clearance validation
studies. All settings were identical to those for the virus spiked runs
with the exception that no intentional pressure interruptions were
performed. Samples were drawn for the analysis of glucose concentration
and osmolality. Furthermore, a cell proliferation use-test was performed
with the pooled filtrate in order to evaluate if the composition of the
fermenter feed media was significantly altered during filtration with
respect to supporting cell growth. The use test consisted of the
analysis of the cell density, cell viability and cell productivity after
a 3-day incubation period with the filtered media.