Carbohydrate composition analysis of glycoproteins by HPLC using highly fluorescent anthranilic acid (AA) tag.
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.
Saddic
George N
GN
Charles River Laboratories, Malvern, PA, USA.
Dhume
Shirish T
ST
Anumula
Kalyan R
KR
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D012015:Reference Standards; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D005456:Fluorescent Dyes; D006023:Glycoproteins; D062367:ortho-Aminobenzoates; C031385:anthranilic acid