Introduction
The production processes of biopharmaceutical products based on
eucaryotic expression systems carry an intrinsic risk for viral
contaminations. An industry wide data collection of such contamination
events revealed that contamination sources were traced primarily to raw
materials or cell culture media including specific components thereof,
like FBS (Barone, 2020).
Traditionally, to mitigate this risk, the safety tripod concept (Kreil,
2018) is applied; (a) raw materials used in manufacturing of
biopharmaceuticals are selected for low risk, under consideration of
viral safety aspects, (b) raw materials are tested for potential viral
contaminants, and most importantly the main proportion of safety margins
is achieved through (c) virus inactivation or removal steps integrated
in the “down-stream” manufacturing process. These steps are either
dedicated virus clearance steps, or steps with an intrinsic capacity to
clear viruses. As required by current regulations (EMEA 1996; ICH, 1999,
FDA, 1997), dedicated virus clearance steps are integrated into
manufacturing processes to increase the safety margins of
biopharmaceuticals, in a well-controlled manner.
However, for active (“live”) biopharmaceuticals, such as live virus
vaccines, or Advanced Therapy Medicinal Products (ATMPs) like cellular
therapies and gene therapies, dedicated viral clearance procedures
cannot always be applied to the down-stream processes as they would not
only inactivate or remove potentially present viral contaminations but
equally compromise activity of the biopharmaceutical ingredient.
This is especially true for virus filtration, one of the most robust and
effective viral clearance procedures that can conceptually remove all
pathogens larger than the stipulated pore-size of the filter.
Unfortunately, for most live virus vaccines or ATMPs the therapeutic
biomolecule is also larger, and thus effective virus clearance is not
possible.
Furthermore, some large biomolecules, such as von Willebrand Factor
(VWF) complexes cannot pass through the smallest type of virus filters
which are suitable for the removal of e.g. Parvovirus without
significant losses or severe clogging events due to the nature of the
molecules (Parker, 2021). In these cases, the introduction of
down-stream virus filtration as dedicated virus clearance step is
technically impossible.
However, taking specifically the main source of virus risk for cell
culture processes into account, the virus filtration procedure can be
directly applied to mitigating that risk by filtration of culture media
prior to its use in the manufacturing process.
The studies presented here investigated the feasibility of implementing
culture media virus filtration with respect to its virus clearance
capacity under extreme conditions, which have been shown to be
worst-case for virus removal, such as high process feed loading, long
duration of filtration, and multiple process interruptions.
For the many experiments reported here, Minute virus of mice (MMV) was
chosen as small challenge virus. This is based on the fact that although
culture media increasingly do not contain animal derived components
(Grillberger (2009)) viral contaminations of cell derived bulk harvests
have still occurred, e.g., with MMV (Garnick, (1998); Nims, (2006)).
Therefore, MMV is a target virus. Furthermore, MMV represents small
non-enveloped viruses which are the main challenge for virus filters
with a stipulated pore-size of 20 nm and is thus also a suitable model
virus (EMEA 1996).
Materials
and Methods