2.2.2. Virus filter in total flow-through integrated polishing process (inline spike test)
To confirm the virus removability of the virus filter in the total flow-through integrated polishing process shown in Figure 5, the inline spiking viral clearance study method designed by ViruSure as reported by Shirataki et. al. (2021a) was conducted. For this study, 10 mg/mL mAb spiked at 10% with one of two viruses, MVM or X-MuLV, was passed through a 0.1 μm PES prefilter or 0.2 μm PES prefilter, respectively before use. The flow-through two column chromatography process described in Section 2.2.1 was directly connected without pooling to a 0.0003 m2 Planova BioEX filter on an AKTA pure 25. Un-spiked 10 mg/mL mAb was supplied to the two column system by system pump at a flow rate of 0.18 mL/min while the 10% virus (MVM or X‐MuLV) spiked mAb was supplied by sample pump at a flow rate of 0.02 mL/min to the feed stream after the two chromatography columns and before the virus filter, resulting in mAb with 1% virus spike being applied to the filter at a constant flow rate of 0.2 mL/min, corresponding a flux of 40 LMH. After loading 300 mL of mAb solution, both pumps were stopped for a 60 min process pause. Then, only the system pump was turned on to supply 30 mL of buffer at a flow rate of 0.2 mL/min to wash out feed from the chromatography columns. Using this procedure, viral clearance was conducted at a constant flow rate and the effect of a process pause was evaluated (Table 2). The total mAb solution loaded to virus filter was 300 mL and loading of mAb on the filter by effective surface area was about 10 kg/m2 and 1000 L/m2.