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