In-depth characterization analysis of a bispecific antibody acidic charge variant

Developing the chemical manufacturing and control (CMC) process for bispecific antibodies (BsAbs) remains challenging due to their complex structures and poor conformational and chemical stability. During molecular design, introducing additional disulfide bonds can improve a BsAb's structural stability; however, it may also cause unintended disulfide bridging and increase product heterogeneity. Here, we present a case study in which a single-chain variable fragment (scFv) is converted into a single-chain disulfide-stabilized Fv fragment (scdsFv) by introducing a pair of disulfide bonds between the heavy-chain variable region (VH) and the light-chain variable region (VL). This engineering strategy considerably enhances the stability of the tetravalent "2 + 2" format of a BsAb and effectively mitigates reversible aggregation. However, it also results in a distinct acidic peak in the ion exchange chromatography (IEC) analysis, attributed to an unexpected interchain disulfide bond linking two scdsFv segments. Despite this modification, the acidic and main peaks exhibit comparable antigen-binding affinity and potency, indicating that the unexpected disulfide linkage does not compromise antigen-binding functionality.