RESUMO
Bispecific antibodies containing single-chain variable fragment (scFv) appended to immunoglobulins G offer unique development challenges. Here, we describe the stability of a novel bispecific format, BiS5, where the scFv is tethered to the CH3 domain. BiS5 showed an improved conformational and chemical stability compared with that of BiS4 in which the scFv is appended in the hinge region between the Fab and Fc. By switching the location of the scFv from hinge region to the CH3, there was an improved stabilization of CH2 and scFv domains. Interestingly, no noticeable impact was observed on the conformational stability of CH3 and Fab domains. BiS4 and BiS5 showed different aggregation and fragmentation rates under accelerated temperature stress conditions. BiS4 showed higher fragmentation rates compared with BiS5 likely owing to fragmentation in the linker region on either side of the scFv while BiS5 is more resistant toward fragmentation owing to tethering of scFv to the CH3 domain at its N and C terminus. In conclusion, the location of scFv affects both aggregation and fragmentation kinetics. These insights into the molecular structure and correlations with their physical and chemical stability will help formulation development of these novel bispecific antibodies.