Summary
Oligosaccharides in glycoproteins by their very nature infl uence 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.
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Acknowledgments
We thank Ms. Ping Du and Ms. Mary Beth Ebert for their contributions to validation of methods.
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Saddic, G.N., Dhume, S.T., Anumula, K.R. (2008). Carbohydrate Composition Analysis of Glycoproteins by HPLC Using Highly Fluorescent Anthranilic Acid (AA) Tag. In: Kannicht, C. (eds) Post-translational Modifications of Proteins. Methods in Molecular Biology™, vol 446. Humana Press. https://doi.org/10.1007/978-1-60327-084-7_15
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DOI: https://doi.org/10.1007/978-1-60327-084-7_15
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