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.