Freezing-induced protein aggregation in a bispecific antibody: Characterization and mechanistic insights.

Aggregation or formation of high molecular weight species (HMWS) was observed with a pharmaceutical bispecific antibody (BsAb) during freeze-thaw and storage at -20 °C, but not at -80 °C. Several freezing stresses that may drive the protein aggregation were evaluated, including temperature, freeze concentration, ice formation, cooling rate, and cold denaturation. Experiments designed to identify the main aggregation mechanism and the drivers revealed that protein dimerization is the main mechanism and protein interaction with ice is the main driver of the protein aggregation.