Structural stability of symmetric bispecific antibodies: a case study showing potential compromise near linker regions.

Over recent decades, bispecific antibodies (bsAbs) have garnered significant attention for their superior therapeutic efficacy compared to progenitor monoclonal antibodies, enabling innovative treatment strategies. Despite their potential, the development of bsAbs presents significant challenges, with structural stability playing a pivotal role in manufacturability, therapeutic performance, and safety. Among the factors influencing stability, the design and incorporation of molecular linkers are particularly critical. In this study, we investigated the structural stability and fragmentation profiles of a symmetric bispecific antibody (Sym-bsAb), targeting HER2 and CD3, under forced degradation conditions. The Sym-bsAb exhibited pronounced fragmentation under prolonged thermal stress, particularly when combined with high pH and salt conditions. Intact mass analysis identified key degradation events, including sequential clipping along G4S and G4 linkers, fragmentations at interchain cystinyl residues and cleavage at the C-terminal of asparagine residues. The identification of G4S and G4 linkers as vulnerable regions prone to clipping in Sym-bsAb provided valuable insights into the stability and manufacturability of bsAbs incorporating linker sequences, underscoring critical considerations for their development.