Results
The aim of this investigation was to evaluate which virus filters were
suitable for culture media virus filtration. This was done by
investigating the MMV removal capacity of different virus filters under
the conditions which can be expected when filtering culture media. The
intention of each laboratory scale experiment was to investigate the
maximum process feed load capacity of an investigated filter when
loading it with MMV-spiked fermenter feed media whilst demonstrating an
effective virus clearance capacity with no to minimal virus
breakthrough.
The culture media used for all experiments contained a base media with
an anti-foaming agent and a wide variety of nutrients typical for high
performance process. Soy-peptone (as animal-derived component free
nutrient) was added in different concentrations and from different
manufacturers – these variations were, however, not seen to have any
impact on the virus clearance capacity of the investigated filters and
are, therefore, not discussed in further detail here.
MMV was chosen as – on the one side – it is a target virus as a
contaminant for some cell cultures – such as CHO (Barone, (2020)), and
– on the other hand – it represents small non-enveloped viruses which
are the main challenge for the investigated virus filters with a
stipulated pore-size of about 20 nm.
The worst-case conditions investigated in this study were transmembrane
pressure (TMP) – including pressure interruptions, duration of
filtration, and process feed load (PDA, (2022)).
The TMP set-point (which is reflected in the mean TMP measured during
the filtration phases) was determined either by the maximum value
suggested by the filter manufacturer or by the maximum pressure allowed
by the tubing used (i.e., 2.6 bar). The complete TMP range was bracketed
by intentionally introducing multiple process interruptions. These were
typically one ≥ 72 hour pause (representing e.g., the duration of
inoculum build-up in the fermenter), followed by daily ≥ 1 hour pauses.
During some experiments a lower mean TMP (i.e., 1 bar) was also
investigated in order to broaden the data base.
Duration was a decision point, as the goal was to continue filtration
for at least 30 days or until flow decay reached or exceeded 90%
(sometimes earlier if a decision had to be made whether the feed tank
should be refilled). As a result of these decision points the maximum
load and duration possible whilst maintaining effective virus clearance
could be determined.
The filters investigated are grouped into first- and second-generation
filters (Miesegaes, (2010)). First generation filters are designed for
down-stream manufacturing processes. Typically, these processes need
virus filters designed to handle up to 1,000 L/m² of an aqueous protein
solution as rapidly as possible. Furthermore, first-generation filters
were not typically designed to tolerate the impact of pressure
interruptions or low flow phases on their virus retention capacities.
Second generation filters do generally have improved virus retention
capabilities even if pressure interruptions occur.
Table 1. Filtration conditions and results for MMV retention using first
generation (FG) filters.