Carbohydrate characterization of recombinant glycoproteins of pharmaceutical interest

Carbohydrate characterization of recombinant glycoproteins entails determination of the primary structures and points of attachment of the oligosaccharide moieties. This article reviews several methods for oligosaccharide- and glycosylation-site characterization. A major recent advance in carbohydrate analysis has been the use of high-pH anion exchange (HPAE) chromatography for separation of glycoprotein-derived oligosaccharides. These separations are sensitive to molecular size, carbohydrate composition, linkage positions, and anomeric configurations. As a result, HPAE chromatography is a powerful technique for glycoprotein characterization and can serve as the basis of an "oligosaccharide map." Characterization of potential N-glycosylation sites involves determining whether each potential site is glycosylated, the extent of oligosaccharide processing at each site, and ideally, a detailed description of the distribution of oligosaccharides at each site. Several approaches to characterizing glycosylation sites are described, including peptide mapping and mass spectrometry. Treatment of a glycoprotein with endo-beta-N-acetylglucosaminidase H (endo H) followed by peptide:N-glycosidase F (PNGase F) can be used to distinguish sites that are not glycosylated from those carrying high-mannose structures and from those containing attached complex oligosaccharides. After individual glycosylation sites have been labeled by this series of reaction, the resulting peptides are characterized by automated Edman degradation. This technique is particularly valuable for characterizing peptides that contain more than one potential N-glycosylation site. An example is also given in which HPAE chromatography is used in conjunction with reversed-phase high-performance liquid chromatography tryptic mapping to obtain detailed information on the distribution of oligosaccharides at individual glycosylation sites.