Hydrophilic Interaction Chromatography HRMS with Acrylamide Monolithic Columns: A Novel Approach for Intact Antibody Glycoform Characterization.

Glycosylation significantly impacts the pharmacokinetics and efficacy of monoclonal antibody (mAb) biotherapeutics. Characterizing mAbs' glycoform profiles is crucial for optimizing therapeutic outcomes, and intact antibody analysis provides key information about the glycoform combinations present. While state-of-the-art RPLC-MS methods are commonly used for intact mAb analysis, they lack the selectivity to resolve glycoforms and, therefore, may not detect lower-abundance glycoforms. In contrast, HILIC methods have demonstrated good resolving power for middle-up mAb glycoform analysis. However, to date, no application of HILIC has been described to characterize mAb glycoforms at the intact level. This study describes the development of acrylamide monoliths for HILIC-MS intact mAb glycoprofiling. We studied how the porogen composition (octanol and DMSO ratio) in the polymerization mixture affects the column permeability and separation performance. Our findings indicated that increasing the DMSO content increased retention and decreased the peak widths. The optimized HILIC-MS method was applied to analyze five reference intact mAbs (IgG(1) and IgG(4)). The method demonstrated glycoform selectivity at the intact protein level, achieving baseline separations between single and double Fc glycosylation (e.g., for trastuzumab, resolution (Rs) of 3.62 for G0F vs G0F/G0F) and partial separations between glycoforms differing by one glycan unit (e.g., for trastuzumab, Rs of 1.06 between G0F/G0F and G0F/G1F). Compared to state-of-the-art RPLC-MS, acrylamide-monolith HILIC-MS enabled the measurement of low-abundance glycoforms (e.g., single G0F and M5/M5). The selectivity and sensitivity (ng of sample injection) of this method open opportunities for studies of IgG heterogeneity in bioanalytical applications.