Profiling N-linked oligosaccharides from IgG by high-performance anion-exchange chromatography with pulsed amperometric detection

We report an improved HPAE-PAD method for IgG oligosaccharide separation. The neutral glycans are well resolved, including separation of high-mannose species from typical human IgG glycans. Oligosaccharide identification was performed by comparison to known standards in conjunction with selective exoglycosidase digestion of both standards and released glycans. Retention times (RTs) of known glycans were compared with the retention times of maltose, maltotriose and maltotetraose standards to define a retention index value for each glycan. These retention indices were used to aid identification of glycans from an example monoclonal antibody sample of unknown glycosylation. Method ruggedness was evaluated across duplicate systems, analysts and triplicate column lots. Comparing two systems with different analysts and columns, retention time precision relative standard deviations (RSDs) were between 0.63 and 4.0% while retention indices precision RSDs ranged from 0.27 to 0.56%. The separation is orthogonal to capillary electrophoresis-based separation of labeled IgG oligosaccharides.

Source: Glycobiology - April 26, 2016 Category: Biology Authors: Rohrer, J. S., Basumallick, L., Hurum, D. C. Tags: Analytical Glycobiology Source Type: research