Cell Culture / Recovery & Purification
Acoustic Wave Separation as a Novel Clarification Technique for the Removal of Aggregate–Enriched Calcium Phosphate Precipitates During Bispecific Antibody Purification
High aggregate levels (20-30%) are commonly observed during bivalent bispecific antibody (BisAb) process development and often exceed the capabilities of the monoclonal antibody (mAb) purification platform process in removing the impurity to acceptable levels. Calcium phosphate precipitation has recently been demonstrated to be an effective method for removing approximately ~10-20%of the aggregates present. One disadvantage is the high turbidity (1,000-3,000 NTU) generated as a result of precipitate formation. Although depth filtration has been used to remove these precipitates, the large filter areas required are not practical for large scale production. Acoustic wave separation (AWS) devices have been marketed as a companion technology to cell culture harvest as an alternative to centrifugation. In this study, a novel application for the use of the AWS system (Pall Cadence Acoustic Separator) is described, and improvements to depth filtration capacity in the purification process are showcased. Varying concentrations of calcium phosphate were used to identify the optimal operating conditions. Studies were performed with clarified cell culture fluid and with neutralized low pH viral inactivated product. Turbidity reduction of greater than 90% was achievable for most product pools. Depth filtration capacity evaluation of the AWS-treated feed streams showed > 2x improvement in filter capacity compared to feed streams under similar conditions. Interestingly, at lower calcium phosphate additions to feed streams, AWS treatment was able to reduce turbidity from 1100 NTU to 50 NTU, allowing for direct sterile filtration without the need for depth filtration. The ability of this technology to significantly reduce turbidity improves the scalability of calcium phosphate precipitation processes and has great promise for its use in removing aggregates present in bispecific antibody purification processes.