Comparative analysis of manufacturability and binding of various bispecific antibody architectures.

Bispecific monoclonal antibodies (BsAbs) enable novel therapeutic mechanisms with multiple binding specificities, but their architecture can influence manufacturability and function. We compared the production of five formats of a BsAb recognizing a viral epitope with an added function through binding to the transferrin receptor (TfR) for blood-brain barrier passage. They had symmetric and asymmetric architectures, with additional binding domains fused to either the heavy or light chains, and a dual-variable domain immunoglobulin (DVD). We characterized growth kinetics, productivity, downstream recovery, product related variants and in vitro binding to Zika virus and TfR. BsAb architecture had a pronounced impact on culture performance. The formats involving light-chain modification or asymmetric assembly produced here resulted in reduced cell culture performance, including lower growth and viability, and up to 70% lower productivity, compared to the parental antibody. In contrast, symmetric BsAbs with C-terminal single chain variable fragment (scFv) fusion to the heavy chain maintained growth, productivity, and purification performance comparable to the parental IgG and achieved high purity after protein A purification (> 95%). Asymmetric BsAb production resulted in imbalanced chain expression and the formation of homodimeric and half-antibody byproducts, reducing purity after protein A purification to around 68%. Binding analysis using ELISA demonstrated that modifications affecting the Fab region modified apparent binding to Zika virus, whereas fusion of scFv domains to the heavy chain distal to the Fab preserved viral recognition. Apparent binding to TfR depended on BsAb valency and configuration, with bivalent heavy-chain scFv formats showing stronger apparent binding than monovalent formats. These results show that, within the architectures evaluated here, bispecific antibody architecture determines manufacturability, product quality, and binding. The results provide guidance for selecting BsAb formats that balance functional requirements with scalable production.