Preferential precipitation of acidic variants from monoclonal antibody pools

• Published in: Biotechnology and bioengineering 2023-01, Vol.120 (1), p.114-124
• Main Authors: Rumanek, Tomasz, Kołodziej, Michał, Piątkowski, Wojciech, Antos, Dorota
• Format: Article
• Language: English
• Subjects: Acids; Antibodies, Monoclonal - chemistry; charge variants; Chemical Precipitation; Ionic strength; Microheterogeneity; Monoclonal antibodies; Polyethylene glycol; Polyethylene Glycols - chemistry; precipitation; Production costs; Proteins; Regulatory approval
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.28257

Microheterogeneity of monoclonal antibodies (mAbs) can impact their activity and stability. Formation of charge variants is considered as the most important source of the microheterogeneity. In particular, controlling the content of the acidic species is often of major importance for the production process and regulatory approval of therapeutic proteins. In this study, the preferential precipitation process was developed for reducing the content of acidic variants in mAb downstream pools. The process design was preceded by the determination of phase behavior of mAb variants in the presence of different precipitants. It was shown that the presence of polyethylene glycol (PEG) in protein solutions favored precipitation of acidic variants of mAbs. Precipitation yield was influenced by the variant composition in the mAb feed solutions, the concentration of the precipitant and the protein, and the ionic strength of the solutions. To improve yield, multistage precipitation was employed, where the precipitate was recycled to the precipitation process. The final product was a mixture of supernatants pooled together from the recycling steps. Such an approach can be potentially used either instead or in a combination with chromatography for adjusting the acidic variant content of mAbs, which can benefit in improvement in throughput and reduction in manufacturing costs. PEG‐aided preferential precipitation was exploited to reduce the content of acidic variants (av) in mAb downstream pools. To improve the operation yield, three‐stage precipitation was employed, in which the precipitate after re‐dissolution was subjected to precipitation two times more. Finally, precipitate highly enriched with av was discarded, while the supernatants were pooled together as the product with the av content reduced to an acceptance level.