To tackle the process bottlenecks in throughput, mass index, and cost in traditional protein A (ProA) packed-bed affinity chromatography due to the advancement in upstream production of monoclonal antibodies (mAbs), precipitation via the addition of inexpensive reagents is an attractive alternative to serve as the primary capture step. Precipitants with orthogonal and potentially synergistic mechanisms, polyethylene glycol (PEG) 3350, as a volume excluder, and zinc chloride, a reversible crosslinker, were examined in a high throughput screening format with several commercially relevant mAbs in both ProA-purified form – to explore the fundamental interaction mechanisms between precipitants, buffer species, and target mAb molecules; and harvested cell culture fluid (HCCF) form – to support the process development of continuous mAb precipitation operation. Platformized mAb precipitation conditions in HCCFs have been identified (1~3 mM ZnCl2 and 0~3 w/v% PEG) to achieve desirable > 95% yield for titers in the 3 to 10 g/L range, with the aid of HCCF pre-processing. Product purity analyses of redissolved mAb precipitates using SEC and CHO ELISA indicated that increasing precipitant concentrations leads to mAb yield and purity trade-off for a given titer. A limited study of the impact of additives on mAb precipitate yield and purity has been conducted. A case study of high-titer mAb precipitation process development and analysis will be demonstrated. This work provides the fundamental understanding of mAb precipitation behavior that is critical to the design of precipitation-based capture processes in the PEG-Zinc precipitant system and demonstrates that precipitation can provide satisfactory mAb yield and product quality as the primary capture step.
