Comparison of enriched charge variants from different anti-CD3 bispecific antibodies reveals differential susceptibility of each bispecific arm to post-translational modification.

Charge heterogeneity is an important quality attribute of therapeutic antibodies, and a detailed understanding of charge heterogeneity arising from post-translational modifications (PTMs) is required by regulatory agencies during drug development. Among antibody therapeutics, the bispecific antibody with two distinct Fab domains targeting distinct antigens provides additional complexity to the charge profile. In this study, charge variant species were enriched from three bispecific antibodies (bsAbs) each containing one anti-CD3 binding arm designed with differential affinity to CD3. The charge heterogeneity corresponding to each anti-CD3 arm within each enriched fraction was evaluated using a domain-specific, digestion-assisted imaged capillary isoelectric focusing (icIEF) method known as DiCE. Through fractionation, we observed that the anti-CD3 arm of each bispecific antibody exhibited different distributions of acidic variants, even when the anti-CD3 arms were identical based on primary sequence. Reduced peptide mapping was performed on specific fractions to identify unique site-specific PTMs that were uncovered or enriched through fractionation. In each case, the bispecific arm that was most susceptible to PTMs exhibited a more basic isoelectric point. Conformational stability analysis of each bispecific antibody using differential scanning calorimetry suggested that the more basic Fab arm tended to be correlated with a lower melting temperature, although it is unclear the extent to which PTMs on the basic arm may contribute to reduced conformational stability. Overall, these results provide additional evidence that each of the two arms of a bispecific antibody may exhibit differential susceptibility to post-translational modification and that this susceptibility is likely correlated with subtle differences in overall bispecific antibody structure, which is influenced by electrostatic properties inherent to the primary sequence. Future studies to obtain high-resolution structures of full-length bispecific antibodies by crystallography or cryo-electron microscopy may help to elucidate the driving force for susceptibility to PTMs in bispecific antibodies.