No abstract

Oligosaccharides in glycoproteins by their very nature influence many aspects of protein function, e.g., half-life and activity/potency. Recombinant IgGs constitute a major portion of therapeutic proteins. Though the glycans in IgGs account for about 2% of the total weight, they influence biologic activity apart from antigen binding. Characterization of the carbohydrates is not only a regulatory requirement but it may allow understanding of structure-function of proteins. Current advances in analytical techniques permit structural elucidation of small quantities of glycoproteins. At a fi rst glance monosaccharide analysis may provide insight into the types of glycosylation similar to information afforded by amino acid composition. It is the only stand-alone technique by which individual sugar residues can be identifi ed and quantitated (mol/mol). Fluorescent anthranilic acid (AA) has been extensively used as a high sensitivity detection tag for carbohydrates. HPLC methods with fl uorescence detection described in this chapter are suitable for the analysis of monosaccharides (including sialic acids) on a routine basis. AA is used for the determination of hexoses and hexosamines, and o-phenylenediamine for sialic acids. These methods were validated and found to be highly reproducible compared to HPAEC-PAD and CE methods.

No abstract

Majority of protein drugs in development today are glycoproteins e.g. recombinant antibodies expressed in various cell lines. Oligosaccharides through conformational changes can modulate therapeutic value (potency) of glycoproteins e.g. complement dependent cell cytotoxicity (CDCC) and antibody-dependent cell cytotoxicity (ADCC) activities of MAbs. Carbohydrate structure analysis in detail is an integral part of protein drug characterization. This not only allows understanding of carbohydrates, but may allow deeper insight into the structure-function of the whole protein molecule. Oligosaccharide mapping by HPLC with fluorescence detection is a powerful technique that sheds considerable light into understanding of glycan structures with minimal effort. Oligosaccharide analysis using pulsed amperometric and/or chromophore detection methods lack resolution, sensitivity and ease of operations. In addition, these older methods are not highly reproducible. Simple labeling chemistry of anthranilic acid (AA) described here provide robust methods with the highest sensitivity and resolution for oligosaccharide analysis. Further, post-chromatography techniques such as mass spectrometry and NMR are amenable to this AA technology for detailed structure analysis.