3.2.1. Viral clearance for flow-through two column chromatography
Previously, the virus clearance test for individual column (AEX and CEX) and the inline virus spiking test for virus filter directly connected to a column, showed excellent virus removability as reported (Shirataki et al., 2021a). However, in that report, the residence time for the column was as short as 2 min compared to 13 to 35 min in this study, and each column was evaluated individually with throughput of 300 mg mAb /mL-resin. In this study, virus removability was examined for the flow-through two column chromatography with a residence time of 20 min and a mAb throughput of 600 mg/mL-resin for each column.
For the flow-through two column chromatography system with AEX and CEX, the UV absorbance profile of the MVM-spiked mAb solution run shows that buffer in the columns is rapidly replaced by mAb solution as indicated by the UV absorbance sharply rising and remaining at the maximum level throughout the run (Figure 4). Following application of the specified volume of solution to the two columns and a 60 min process pause, the buffer wash cleared the mAb from the columns as shown by the gradually decreasing UV absorbance. As shown in Table 1 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 LRV of ≥ 4 for MVM and X-MuLV. These results confirm that the two column chromatography has high virus removability for both X-MuLV and MVM for a process with residence time of 20 min in the columns and that process pause did not affect virus removal.