Evaluating N-Glycosylation of a Therapeutic Monoclonal Antibody Using UHPLC-FLR-MS with RapiFluor-MS Labeling

Released N-glycan analysis using the fluorescent label 2-AB (2-aminobenzamide) has been the gold standard method for released glycan analysis for several years. The more recent RapiFluor-MS™ labeling technique, however, offers enhanced mass spectrometric detection of released N-glycans, improving the sensitivity and detection limits of the method. The optimized multidimensional detection offers increased confidence in glycan identification which can be further supported by an exoglycosidase digestion array (optional). Here we describe the PNGase F release of N-glycans from a typical IgG1 monoclonal antibody (mAb) with subsequent labeling with RapiFluor-MS™ for detection by HILIC-FLR-MS. The method output quantifies the relative proportion of each glycan species including core afucosylation, sialylation, and high-mannose content, and has a limit of detection (LOD) of 0.01% relative abundance.