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.