ASSESSMENT OF PROTEIN GLYCOSYLATION USING HIGH-pH ANION-EXCHANGE CHROMATOGRAPHY WITH PULSED ELECTROCHEMICAL DETECTION

Abstract

As more recombinant glycoproteins are being developed for therapeutic Purposes, there is an increasing need for sensitive, simplified methods for analysis of their oligosaccharide chains. High-pH anion-exchange liquid chromatography (HPAE) with pulsed amperometric detection (PAD) has been shownto be useful in a numberof areas of structural elucidation of glycoprotein glycans. Higher resolution of oligosaccharide mixtures, containing both neutral and anionic components, has been reported using HPAE chromatography with micropellicular resins. Sensitive composition analysis of both glycoproteins and oligosaccharides, without derivatization steps, was accomplished using HPAE/PAD. Oligosaccharides were purified in high yield using HPAE andin-line desalting through flat micro-membranes. These purified oligosaccharides were found to be suitable for mass spectrometric and ‘H-NMR analysis. Measurement of both released monosaccharides and changesinretention times of purified oligosaccharides in exoglycosidase digests have been performedin a single step. The developmentof additional AE/PAD-based methods should further simplify carbohydrate analysis of glycoproteins.