High-performance anion-exchange chromatography with pulsed amperometric detection for carbohydrate analysis of glycoproteins

Abstract

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an established technique for the carbohydrate analysis of glycoproteins. HPAE-PAD is routinely used for determinations of monosaccharide, sialic acid, mannose-6-phosphate (M-6-P), and oligosaccharide contents of a glycoprotein. This is true for both the initial investigation of a glycoprotein and routine assays of recombinant therapeutic glycoproteins. This contribution reviews the fundamentals of HPAE-PAD, recent technological improvements, and advances in the last ten years in its application to carbohydrate analysis of glycoproteins. The application areas reviewed include monosaccharide determinations, sialic acid determinations, M-6-P determinations, sugar alcohol determinations, analysis of polysialic acids, neutral and charged oligosaccharide analysis, following glycosidase and glycosyltransferase reactions, and coupling HPAE-PAD to mass spectrometry (MS).