Discussion
Raw materials or cell culture media including specific components
thereof, like FBS, have been identified as source of viral contaminants
for biopharmaceutical products based on eucaryotic expression systems.
It has been reported that such contaminations have led to virus
contamination and the subsequent collapse of the cell population in
these fermenters. As a result, severe measures to clean up the upstream,
and should any intermediate have been forward-processed already also the
down-stream equipment, to minimize the recurrence of such an event had
to be taken. The downtime can lead to unmet patient needs, dramatic
economic consequences, and severe impact on the reputation of an
affected company (Bethencourt, (2009)).
The introduction of a contaminating virus might not necessarily become
obvious by degenerative effects presented by the production cells, as
“silent infections” have also occurred (Moody, (2011)). The specific
adventitious agent testing (AAT) program on fermenter harvest material
would be the point where a virus contaminant would be detected. However
not necessarily: although AAT is designed to detect as many potential
viral contaminants as possible, there might be viruses that would not be
picked up (Gombold, (2014)). Similarly, the testing of raw materials
does not necessarily reveal a viral contaminant, especially when the
concentration of the contaminant in the raw material is lower than the
detection limit of the testing procedure, or the contaminant is unevenly
distributed across the large volumes involved versus the small volumes
actually tested.
Therefore, to make sure that a potential contaminant would never reach
the patient, a series of effective virus clearance steps is implemented
in down-stream biomanufacturing operations, where feasible. Some
biopharmaceutical products can, however, not be subjected to dedicated
viral clearance steps in the down-stream processes as they would not
only inactivate or remove potentially present viral contaminations but
have the same clearance effect on the biopharmaceutical components. This
is true for live virus vaccines, ATMPs (e.g., cellular or gene
therapies) or particularly complex biotechnology products which are
simply too large to be subjected to filtration with virus filters with a
nominal pore size of 20 nm, e.g.: vWF (Parker, (2021)).
A potential solution is to introduce a method which ensures a
significant reduction of theoretically present viruses in the culture
media before it even enters a manufacturing process, i.e., an upstream
virus barrier.
Different methods capable of this feat have been investigated: high
temperature short time treatment (HTST), ultraviolet light irradiation
(UV-C) and finally virus filtration. The implementation of HTST and UV-C
have, however, been faced with complications as in both cases media
components are susceptible to degeneration (Cao, (2013)), (Meunier,
(2016)).
Virus filtration, however, is one of the most robust and effective virus
clearance procedures, as it conceptually removes all pathogens as long
as their size is larger than the stipulated pore-size of the filter,
while at the same time being neutral to the biological activity of the
biopharmaceutical compound(s) smaller than the pore size. It is,
therefore, best suited to reduce the risk of the introduction of
adventitious viruses through culture media. For this process to be
economically feasible the filter area must ideally be kept small while
at the same time large volumes of culture media are processed for a
potentially prolonged times of filtration, and the impact of low or no
flow incidents on the virus clearance capacity needs to be understood
also (Wieser, (2016)).
The studies presented here investigated the feasibility of implementing
culture media virus filtration with respect to their virus clearance
capacities under extreme conditions such as very high process feed
loading (up to ~ 19,000 L/m²), very long duration (up to
31 days), and multiple process interruptions (up to 21, with cumulative
interruptions of over 92 hours). MMV was used as a relevant target virus
as a contaminant for some cell cultures, and in general as a small
non-enveloped viruses which are the main challenge for the investigated
virus filters with a stipulated pore-size of about 20 nm.
It was found that a number of filters – especially of the newer
2nd generation – are capable of effective virus
clearance despite the harsh regimen they were subjected to. At the same
time the investigation of biochemical parameters for un-spiked control
runs showed the filters to have no measurable impact on the composition
of the culture media. Again, indicting this technology to be especially
suitable for large volume pre-manufacturing process culture media
preparation.