Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals.
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.
Hexal AG, Sandoz Biopharmaceuticals, Oberhaching, Germany.
Fabian
F
Higel
Uwe
U
Demelbauer
Andreas
A
Seidl
Wolfgang
W
Friess
Fritz
F
Sörgel
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006031:Glycosylation; D007536:Isomerism; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011134:Polysaccharides