Parallel Sequencing of the Two Arms on a Bispecific Antibody by Electron Capture Dissociation on a timsOmni Platform Reveals Several By-products in the Controlled Fab-arm Exchange Process

Bispecific antibodies (bsAbs) can simultaneously target two distinct antigens, offering enhanced therapeutic efficacy compared to conventional antibodies. However, their complex manufacturing process, involving pairing of four distinct polypeptide chains (two heavy and two light chains), enhances the analytical challenges in quality control. Here, we applied top-down mass spectrometry using electron capture dissociation (ECD) on a novel timsOmni platform to characterize the bsAb LaC49 (AR4A/AR3C) and its monospecific precursors, IgG1-AR4A and IgG1-AR3C. This bsAb was developed to target different epitopes of the E1E2 envelope glycoprotein on the surface of hepatitis C virus (HCV). The bsAb was prepared via controlled Fab-arm exchange. Native mass spectrometry revealed that the sample contained the intended bsAb but also products that displayed substantial structural heterogeneity, including residual monospecific IgG1-AR4A and multiple other mass variants. ECD MS/MS analysis of the F(ab') 2 of these products provided comprehensive sequence coverage of the CDR3 regions from all four polypeptide chains, generating clear sequence ladders potentially suitable for de novo sequencing. Important impurities could be identified that could not be characterized by MS1 (intact mass) analysis alone, including multiple (alternative) signal peptide cleavages in the AR4A antibody and partial reduction of a functionally important disulfide bridge in the CDR3 region of the AR3C heavy chain, likely induced during bsAb preparation. Our findings demonstrate that top-down MS/MS analysis is key for the molecular-level characterization of bsAb modifications, as standalone intact mass analysis proved insufficient to distinguish between closely related molecular variants (i.e., separated by only 0.04 Da). This study also emphasizes the need for comprehensive batch-to-batch quality control during bsAb production, as structural modifications can impact binding specificity, therapeutic efficacy, and safety. The timsOmni platform with ECD fragmentation provides a powerful novel analytical tool for ensuring consistent product quality in bispecific antibody manufacturing and for antibody sequencing applications.