Target precipitation is a bulk separation technique that takes advantage of ever-increasing monoclonal antibody (mAb) titers, providing an alternative to capture via protein A (ProA) affinity chromatography. The determination of solubility behaviors is fundamental to the development of precipitation-based processes. Two precipitants with orthogonal 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 and harvested cell culture fluid (HCCF) forms. High throughput methodologies were used to quickly generate a large set of mAb solubility data that enabled a deeper understanding of the mAb solubility behaviors, and the biophysical and solution physicochemical properties that impact these behaviors. The solubility behaviors of purified mAbs at neutral pH could be classified as Zn²⁺-dependent or synergistically Zn²⁺ and PEG co-dependent. The mAb isoelectric point and the presence of weak zinc ligands in solution determined the classification. Solubility behaviors in HCCF form were often significantly different than those in purified form but pre-processing via dialysis restored mAb solubility behavior to that for the purified material or even reduced the solubility below that of the purified material. HCCF pre-processing using combined DNA-depletion, diafiltration, and mAb pre-concentration permitted the standardization or platformization of the precipitation conditions for mAbs to a common low precipitant concentration range (1~3 mM ZnCl₂ and 0~3 w/v% PEG) that achieved precipitation yields >95% for titers in the 3 to 10 g/L range. Product quality analyses of redissolved mAb precipitates using SEC and ELISA indicated that increasing ZnCl₂ and PEG concentrations resulted in increases in both mAb yield and HCP impurity content for a given titer. We precipitated three different pre-processed HCCFs using similar precipitant concentrations with yields ranging from 95% - 98.5%, HCP levels ranging from 8,300 ppm - 26,000 ppm (sufficient for subsequent polishing step), and no additional HMW species generated. This work provides the mAb solubility behavior information that is basic to the design of precipitation-based capture processes in the Zn²⁺ and PEG3350 precipitant system and demonstrates that precipitation can provide satisfactory mAb yield and product quality.

Yuncan (Olivia) Zhu

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