Insights into the structural differences between homo- and heterodimers enriched from a cocktail of monoclonal antibodies against SARS-CoV-2

Heteromeric complexes observed within co-formulated products represent a unique class of impurities that are poorly understood in terms of their biochemical, biophysical, structural, and functional attributes. Recently, heterodimers from co-formulated REGEN-COV^® were enriched and purified to evaluate their stability and impact on potency. In this study, we extend our previous work and provide a side-by-side comparative study of these heterodimers to antibody homodimers enriched from the same combination product. More specifically, we evaluate shape and structural differences among the various dimer species using electron microscopy and analytical ultracentrifugation, as well as explore the distribution of covalent and non-covalent interactions among the dimer samples using denaturing capillary electrophoresis (CE-SDS) and post-column denaturation SEC-MS (PCD-nSEC-MS). Based on this study, the extent of covalent modification and distribution of the dimerization interface (Fab-Fab, Fab-Fc, and Fc-Fc) was observed to be highly correlated with potency for the REGEN-COV^® combination. Overall, we propose that a comprehensive and well-executed study which leverages deep biophysical, biochemical, and functional analyses to compare heteromeric aggregates, with unknown properties, to related homodimer aggregates, with known properties, may represent an effective non-clinical based strategy to investigate attribute-function relationships in antibody dimers and potentially support mitigating risks incurred by heterodimers in co-formulated mAb samples.