Monitoring glycosylation of therapeutic glycoproteins for consistency by HPLC using highly fluorescent anthranilic acid (AA) tag.
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.
Dhume
Shirish T
ST
Alexion Pharmaceuticals, Cheshire, CT, USA.
Saddic
George N
GN
Anumula
Kalyan R
KR
D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D005456:Fluorescent Dyes; D006023:Glycoproteins; D062367:ortho-Aminobenzoates; C031385:anthranilic acid