Versatile Glycan Labeling Tool for Therapeutic Glycoproteins

Glycosylation on therapeutic glycoproteins is a critical quality attribute (CQA) that impacts the quality, safety, and potency of drugs. Therefore, glycosylation should be characterized during the whole drug development by adequate analytical techniques. Since the multiple levels of glycan heterogeneity pose a daunting analytical challenge, Glycan analysis usually involves the use of complementary analytical techniques methods, which include HPLC and MS. Since it is very time-consuming to assessing glycans for each approach, an analytical method that can be flexibly applied to both LC and MS is highly demanded. Therefore, InstantPC that provides not only chromophore for analysis on LC-FLD but also improved MS ionization efficiency by attaching a tertiary amine is emerging as a novel glycan labeling. Thus, we optimized MS-based sample preparation and analytical method for InstantPC labeled N-glycans of therapeutic proteins. N-glycans labeled with InstantPC were profiled using hydrophilic interaction chromatography (HILIC) coupled with MS. InstantPC labeled N-glycans provide increased mass spectrometric sensitivity and tandem MS efficiency. In addition, we found that the results in LC-FLD and LC/MS show high similarity, suggesting our approach could be utilized for glycan quality assessment. This platform provides a flexibility for MS-based rapid and in-depth characterization of N-glycans as well as screening applications by robust fluorescence-based quantitation.