LAUSR

Monitoring of critical quality attributes (CQAs) is essential for the development of biotherapeutics. One example of a CQA is molecular fragmentation, which is often analyzed by capillary gel electrophoresis with sodium dodecyl sulfate (SDS). Sialylation is a post‐translational modification and form of glycosylation that can impact purity profiles of biotherapeutics, resulting in complex structure elucidation. Here, we studied the heterogeneity of Biotherapeutic 1 as a result of O‐linked glycosylation with sialylation. Biotherapeutic 1 displayed a second unidentified peak in SDS–capillary gel electrophoresis (SDS‐CGE) under reducing conditions that directly impacted peak integration practices and, therefore, method validation. The two peaks were highly reproducible in SDS‐CGE as well as complementary LabChip experiments. The apparent molecular weights were calculated using molecular weight ladders with known protein standards. A combination of ion exchange chromatography (IEX), hydrophilic interaction chromatography mass spectrometry (HILIC‐MS), and ultra‐high‐performance size exclusion chromatography (UP‐SEC) were used to identify O‐linked glycosylation as responsible for the production of reduced peak 1 and reduced peak 2 in SDS‐CGE. Specifically, reduced peak 2 contained sialylation that was not observed in reduced peak 1, resulting in two distinct migration times due to impacts in SDS binding efficacy. Enzymatic removal of the sialic acids simplified the heterogeneity into a single uniform peak (reduced peak 1). This work and methodologies highlight the impact a single O‐linked glycan can have on SDS‐CGE and is applicable to analyzing future biotherapeutics involving complex structure profiles resulting from sialylation.