Carbohydrate Composition Analysis of Glycoproteins by HPLC Using Highly Fluorescent Anthranilic Acid (AA) Tag

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.

Polysaccharide Capsule Composition of Pneumococcal Serotype 19A Subtypes Is Unaltered among Subtypes and Independent of the Nutritional Environment

“…The extracted polysaccharides were then completely hydrolyzed by trifluoroacetic acid (TFA) (31). The monosaccharide composition of capsular polysaccharides of strains grown in CDM was determined by highperformance liquid chromatography (HPLC) analysis of fluorescently labeled monosaccharides as previously described (25,(32)(33)(34)(35), whereas the PIM extracts were analyzed on a system consisting of an ASI-100 autosampler and P680 HPLC pump (Dionex, Sunnyvale, CA) with an injection volume of 20 l per sample. Separation of the monosaccharide was done at a flow rate of 0.85 ml/min as follows: 6% solvent B isocratic for 35 min followed by a linear gradient from 6 to 12% solvent B over 20 min.…”

Lysosomal Targeting Enhancement by Conjugation of Glycopeptides Containing Mannose-6-phosphate Glycans Derived from Glyco-engineered Yeast

“…The M6PgPs were eluted from the TiO 2 beads by using elution buffer (28% ammonium hydroxide solution). The concentrations of the glycopeptides were measured by using a fluorescamine fluorescence assay 35 and monosaccharide composition analysis 36 . For the fluorescamine assay, the glycopeptides eluted from the TiO 2 beads were dried in a speedvac and then were redissolved in 160 μl of 0.5 M sodium borate buffer (pH 8.0).…”

“…The fluorescence was analyzed on a SpectraMax i3 plate reader (Molecular Devices, San Jose, CA) with 390 nm excitation and 470 nm emission (10 nm bandwidth). For the monosaccharide composition analysis, the carbohydrates released from glycopeptides by acid hydrolysis were labeled with 2-AA, and then analyzed by using reverse phase HPLC with fluorometric detection as described previously 36 . Monosaccharide standard solutions (Prozyme, Hayward, CA) were used for peak identification and quantification.…”