3.1.2. mAb purification with impurity removal
The impurity levels and mAb concentration are shown in Figure 2. For all three batches, the concentrations of mAb and impurities showed a similar trend for all steps. The mAb concentration increased in the capture step and decreased in the subsequent low pH virus inactivation and polishing steps due to volume increases accompanying pH and conductivity adjustment and the addition of wash buffer, resulting in 3.4 - 5.2 mg/mL mAb after the polishing step including virus filter. The mAb recovery for the total flow-through integrated polishing step was 77%, 93% and 95% for Batches 1, 2 and 3, respectively. The lower recovery rate for Batch 1 was likely due to using less buffer volume for wash and increasing the volume from 15 to 75 mL produced a recovery rate of more than 90%.
Impurities were overall reduced by the process. The HCP concentration exponentially decreased at each process step, especially in the capture and polishing steps, decreasing to 12.0 ng/mg-mAb or less. The DNA concentration also decreased exponentially, particularly in the capture and low pH virus inactivation steps, reaching 1 pg/mg-mAb or less after the polishing step. The aggregate concentration was around 1% in the harvest step, increased to around 3% in the capture and low pH virus inactivation steps, and then decreasing to less than 1% after the polishing step. High aggregate content was produced by the long-term (20-22 h) exposure of mAb to low pH in the capture elution pool, although the KANEKA KanCapATM 3G Protein A columns can efficiently reduce aggregates (Pabst et al., 2018). SEC analysis of the process solution confirmed that the aggregates were primarily dimers, and trimers or higher aggregates were below the detection limit. Since the virus filter removes virus particles by size exclusion, it is thought to not remove small-sized HCP and dimers. The HCP and aggregate contents were reduced by the polishing step as shown in Figure 2, corroborating that the flow-through two column chromatography shows excellent HCP and aggregate removability as reported previously (Shirataki et. al., 2021a; 2021b).