Equipment
The virus removal filtration equipment consisted of an electronic recorder, two pairs of redundant pressure sensors (placed up- and down-stream of the pre-filter) and temperature sensors as generally described elsewhere (Wieser, (2015)).
A pressure vessel made of electropolished 316L steel with a capacity of 5 Liters that could be pressurized to 7 bar (Millipore Cat. No. XX6700P05) was used as a feed tank.
Pressure was applied to the tank via a manual controller for nitrogen gas pressure. The nitrogen gas was passed over a 0.2µm air filter in order to avoid introduction of bacterial contaminants.
The filtrate was collected in vessels placed on an electronic balance. The data collected by electronic recorder was used to calculate process feed loads, feed flow rates, process duration and duration of pressure interruptions.
The virus removal filters used were:
Each set-up included a Millipore Durapore pre-filter (rating: 0.1 µm (nominal), 13 cm²) set-up in-line in a dead-end filtration configuration. The 0.1 µm pre-filter was flushed and conditioned with water for injection before it was autoclaved at 100°C for 30 minutes. Then it was re-introduced into the filtration equipment and conditioned with WFI again (together with the virus removal filter).
0.1µm Pall Acrodisc syringe filters were used to remove virus aggregates from the thawed virus stocks.
The virus-spiked fermenter feed media was passed over a 0.2 µm Pall Vacucap 90PF filter as needed in order to avoid the introduction of bacterial contaminants.
The pre-use suitability of each viral filter used was verified according to the vendors instructions:
Asahi filters were flushed with WFI, subjected to a pre-use air leakage test and then filled with WFI again. Only filters passing this pre-use test were used for individual experiments – none of the filters tested failed.
All other filters were subjected to normalized water flow tests during which water was passed through the filter at specified pressures and the flow rate measured. Only filters with water flow rates within the ranges specified by the manufacturer were used for individual experiments – none of the filters failed.
After each experiment the integrity of each viral filter used was verified according to the vendors instructions. For the Asahi filters this was identical to the pre-use leakage test. For all other filters the test was a pressure hold test. Only filters for which no air was seen exiting the filter at the specified testing pressure within a given time period were seen to be integral.