Profiling and Characterization of N- and O-linked Glycans Released from Glycoproteins using RP-HPLC with Charged Aerosol Detection and Mass Spectrometry

摘要

Characterization of therapeutic glycoproteins by mass spectrometry (MS) includes a thorough accounting of the resident N- and O-linked oligosaccharides at each site. The most abundant and common oligosaccharide structures are confidently assigned through accurate mass determinations using combined top-down and bottom-up approaches. Here, because the glycans remain attached to the polypeptide, site and occupancy levels can be readily determined. However, elucidation of structural isomers, rare motifs, and low-level species clearly benefits from releasing the oligosaccharides from the polypeptide, fractionating the glycan pool, and ultimately performing detailed characterization with glycosidases and off-line tandem MS. We developed a unified LC/MS-based method for release, cleanup, profiling, and characterization of N- and O-glycans from glycoproteins containing O-sialomucin regions. A universal detector is employed for routine HPLC in biopharmaceutical development laboratories.