Increased Resolving Power for Acidic Glycans with an MS-Compatible Anion Exchange Reversed Phase Separation

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Xiaoxiao Liu, Matthew A. Lauber
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Glycosylation are often assigned as critical quality attributes in certain biotherapeutics because they can affect stability, efficacy, and immunogenicity. To carefully control and monitor these moieties, N-glycans are frequently released and analyzed by hydrophilic interaction chromatography (HILIC) and detected by fluorescence or mass spectrometry. Several challenges have, however, been encountered upon attempting to implement this approach, most particularly the incomplete recovery of acidic glycans and the lack of charge-based separations for MS-enhancing glycan labels. Herein, a novel mixed-mode, anion-exchange RPLC stationary phase has been used to afford a class separation of different charge states and additional resolution of acidic glycans. Combined with MaxPeak High Performance Surface (HPS) Technology, this new stationary phase and the ACQUITY PREMIER Glycan BEH C18 AX Column addresses these shortcomings and brings with improved capabilities for the analysis of acidic N-glycans.

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