Resolving charge and glycosylation variants of cetuximab combining offline ion-exchange and hydrophilic interaction chromatography–HRMS

Biopharmaceuticals, such as monoclonal antibodies (mAb), are complex molecules that require thorough analysis due to post-translational modifications, including charge variants and glycoform patterns. When analyzing intact mAbs, single liquid chromatography (LC) mass spectrometry (MS) methods often lack the resolution to distinguish this heterogeneity because they offer limited separation in both chromatographic and mass dimensions. Consequently, these methods struggle to detect low-abundance species, highlighting the importance of 2DLC-MS workflows. Here, we demonstrate the potential of the offline ion exchange chromatography (IEC)-hydrophilic interaction chromatography (HILIC)-MS method to effectively separate charge and glycoform variants of complex mAbs. Cetuximab served as the model antibody due to its multiple charge variants (arising from sialylation and C-terminal lysine clipping) and four glycosylation sites (in both Fab and Fc regions). Charge variants were first separated using non-volatile IEC and collected as individual fractions. These fractions were then analyzed using a low-flow HILIC-MS setup to increase sensitivity for low-abundance species. A RPLC trap column was used to desalt and reduce the volume transferred to the HILIC separation. The IEC-HILIC results showed that both Fc- and Fab-associated glycoforms of cetuximab were resolved to a level not achievable with HILIC-MS alone, allowing deeper insights into charge heterogeneity and glycosylation. In addition to resolving combined Fab/Fc glycoforms, the method also distinguished Fc-only glycoforms from the main isoform. This orthogonal IEC-HILIC method provides additional insights into charge and glycosylation heterogeneity, enabling a more comprehensive analysis of cetuximab. It provides an effective approach for detailed characterization of charge variations and glycoform diversity in next-generation protein therapeutics.