The resolution of the neutral N-linked oligosaccharides of IgG by high pH anion-exchange chromatography

Abstract

The introduction of high pH anion-exchange chromatography (HPAEC) has represented a major development in the qualitative analysis of glycoprotein derived oligosaccharides. When coupled with pulsed amperometric detection, the technique permits the detection of picomole quantities of heterogeneous mixtures of oligosaccharide without the need for derivatisation. The applications of HPAEC have generally been limited to the analysis of sialylated oligosaccharides, however, it is now possible to analyse heterogenous mixtures of neutral oligosaccharides with the latest systems. We have used such a system to separate completely a panel of seven commercially available neutral N-linked oligosaccharides and found the influence of monosaccharide substitution on elution position to be identical to that for sialylated structures. A standard monosialylated N-linked oligosaccharide was modified by sequential digestion with specific exoglycosidases to produce a monogalactosylated, diantennary oligosaccharide which is commercially unavailable. This standard's elution position was confirmed by HPAEC. The technique was applied to the identification of neutral N-linked oligosaccharides released from human immunoglobulin G using the enzyme peptide- N-glycosidase F.