High-selectivity profiling of released and labeled N-glycans via polar-embedded reversed-phase chromatography

N-Glycosylation is the most complex post-translational modification of proteins and involved in many physiological processes and is therefore of major interest in academic research and in the biopharmaceutical industry. Reliable, robust, reproducible, and selective analysis of N-glycans is essential to understand the multitude of biological roles of N-glycosylation. So far, hydrophilic interaction liquid chromatography analysis of 2-AB or 2-AA derivatized N-glycans has been the standard method. In this work, the superiority of reversed-phase chromatography for complex N-glycosylation analysis is demonstrated. Separation of N-glycans derivatized with anthranilic acid on polar-embedded stationary alkyl phases with sub-2-μm particles results in outstanding selectivity and resolution. In combination with the highly mass spectrometry-compatible mobile phase, even very complex glycan mixtures can be separated, identified, and quantified precisely and accurately. The presented methodology can be applied broadly from basic research to analytical control and release testing of biological drug products and can be implemented in analytical laboratories with minimal effort. Graphical abstract ᅟ.