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. Additionally, higher molecular mobility at -20 °C (compared to -80 °C) propagates the aggregation. Molecular structure modeling of the single-chain variable fragment (scFv) revealed the steric clashes between amino acid residues at the core of the interface between the variable heavy (VH) and variable light (VL) domains, could be an intrinsic driver of the protein aggregation. The study presents an interesting case of sequence-specific product quality liability for bispecifics and provides insights into strategies for prevention and mitigation of the liability.