Leveraging digestion-assisted capillary electrophoresis sodium dodecyl sulfate (CE-SDS) to monitor galactosylation in monoclonal antibodies

CE-SDS and MCE-SDS have become industry-standard methods for characterizing product-related size variants and identifying potential product-related impurities of biologics. These methods are used as analytical tools to guide manufacturing process parameters, as well as to establish comparability among sample lots for release. An asymmetric heavy chain doublet was recently observed among multiple monospecific monoclonal antibody samples while performing routine lot-to-lot comparability assessments under reduced CE-SDS conditions. Notably, this splitting pattern was not observed under MCE-SDS. Using a combination of enzymatic digestions and oligosaccharide analysis, we confirm the specific identity of the slower-migrating heavy chain peak as galactosylation variants. Leveraging this method for extended product characterization, it was revealed that galactosylation variants are consistently elevated in enriched acidic charge variant fractions of several mAbs. This digestion-assisted CE-SDS method can also be applied successfully to Fc fusion proteins to enable domain-specific characterization of galactose content. Overall, we demonstrate that, in the absence of more complex mass-spectrometry based glycan profiling techniques, IdeS pre-treatment of mAbs and Fc fusion proteins prior to reduced CE-SDS analysis can readily detect differences in galactose content across various samples while simultaneously informing on N-glycan occupancy. Finally, we propose that the analytical strategy outlined in this study provides CE-SDS peak identification, specifically for galactosylation variants, and can support extended characterization.