3.2.2. Inline spiking viral clearance test for integrated flow-through two column and virus filter
To separately examine the virus removability of the virus filter, an inline spiking viral clearance test for the Planova BioEX filter implemented in the flow-through two column chromatography was carried out. Due to clogging from impurities in the virus spike, the flow rate was reduced to produce a flux of 40 LMH to the filter (compared to 76 LMH for the end-to-end continuous process) to avoid exceeding the operating pressure limit of Planova BioEX (0.35 MPa) before reaching the target throughput. It should be noted that viral clearance determined for the lower flux process (40 LMH) can be taken as confirming viral clearance at 76 LMH, as lower flux is a worse case condition for virus filtration.
As shown in Figure 5, a feed solution of 10 mg/mL mAb without virus spike was supplied to the two columns and the same mAb spiked at 10% MVM or X-MuLV was supplied through inline spiking. The pressure increases on the filter were observed until the peak after the process pause for the loading of mAb solution spiked with MVM or X-MuLV compared to the minimal pressure increase observed for loading of mAb solution without spike. The pressure profile of the virus-spiked mAb filtration shows fluctuations that are considered to be due to pulsation of the pump due to air bubbles. The pressure increases with increasing load volume are greater for the filtration of the X-MuLV spiked solution and approach the upper operating pressure limit of the filter at the target throughput. The more stable filtration pressure of the un-spiked mAb is similar to the pressure profile for the filter incorporated in the end-to-end continuous process shown in Figure 3.
Due to the concern that virus breakthrough to permeate may occur due to the pressure drop during a process pause as previously described (Ajayi et al., 2022; Fan et al., 2021; Johnson et al., 2021), a 60 min process pause was included after loading the virus-spiked mAb, and virus removability for inline spiking of MVM or X-MuLV was evaluated for both the flow-through fraction of the mAb and the flow-through fraction with the buffer wash. As shown in Table 2 for mAb spiked with MVM or X-MuLV, complete clearance was achieved for both the flow-through fraction and the flow-through plus wash with MVM LRV of ≥ 5 and X-MuLV LRV of ≥ 3. The relatively lower X-MuLV LRV for the filter than the two column process can be attributed to the lower X-MuLV spike titer. These results confirm that the Planova BioEX filter has high virus removability for both MVM and X-MuLV and that the process pause did not affect virus removal.