Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals

• Published in: Analytical and bioanalytical chemistry 2013-03, Vol.405 (8), p.2481-2493
• Main Authors: Higel, Fabian, Demelbauer, Uwe, Seidl, Andreas, Friess, Wolfgang, Sörgel, Fritz
• Format: Article
• Language: English
• Subjects: Acids; Analytical Chemistry; anthranilic acid; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - metabolism; Biochemistry; biopharmaceuticals; Characterization and Evaluation of Materials; Chemical properties; Chemistry; Chemistry and Materials Science; Chromatography; Chromatography, High Pressure Liquid - methods; Chromatography, Reverse-Phase - methods; Drugs; Fluorescence; Food Science; Glycan; Glycosylation; Identification and classification; Isomerism; isomers; Labels; Laboratory Medicine; Liquid chromatography; Liquids; Mass spectrometry; Mass Spectrometry - methods; Mathematical analysis; Methods; Molecular Structure; Monitoring/Environmental Analysis; Monoclonal antibodies; Ochratoxin A; Original Paper; Peptides; Pharmaceutical chemistry; Pharmaceuticals; Polysaccharides - chemistry; Polysaccharides - metabolism; reversed-phase liquid chromatography; Scientific imaging; Spectroscopy
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-012-6690-3

N -Glycosylation is a common post-translational modification of monoclonal antibodies with a potential effect on the efficacy and safety of the drugs; detailed knowledge about this glycosylation is therefore crucial. We have developed a reversed-phase liquid chromatographic–mass spectrometric method, with different fluorescent labels, for analysis of N -glycosylation, and compared the sensitivity and selectivity of the methods. Our work demonstrates that anthranilic acid as fluorescent label in combination with reversed-phase liquid chromatography–mass spectrometry is an advantageous method for identification and quantification of neutral and acidic N -glycans. Our results show that mass spectrometry-based quantification correlates with quantification by fluorescence. Chromatographic discrimination between several structural glycan isomers was achieved. The sharp peaks of the eluting anthranilic acid-labeled N -glycans enabled on-line mass spectrometric analysis of even low-abundance glycan species. The method is broadly applicable to N -glycan analysis and is an orthogonal analytical method to the widely established hydrophilic-interaction liquid chromatography of 2-aminobenzamide-labeled N -glycans for characterization of N -glycans derived from biopharmaceuticals.