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,...

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Main Authors:	Higel, Fabian, Demelbauer, Uwe, Seidl, Andreas, Friess, Wolfgang, Sörgel, Fritz
Format:	Online
Language:	English
Published:	Springer-Verlag 2013
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581771/

id	pubmed-3581771
recordtype	oai_dc
spelling	pubmed-35817712013-02-26 Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals Higel, Fabian Demelbauer, Uwe Seidl, Andreas Friess, Wolfgang Sörgel, Fritz Original Paper 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. Springer-Verlag 2013-01-31 2013-03 /pmc/articles/PMC3581771/ /pubmed/23371526 http://dx.doi.org/10.1007/s00216-012-6690-3 Text en © The Author(s) 2013 Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Higel, Fabian Demelbauer, Uwe Seidl, Andreas Friess, Wolfgang Sörgel, Fritz
spellingShingle	Higel, Fabian Demelbauer, Uwe Seidl, Andreas Friess, Wolfgang Sörgel, Fritz Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals
author_facet	Higel, Fabian Demelbauer, Uwe Seidl, Andreas Friess, Wolfgang Sörgel, Fritz
author_sort	Higel, Fabian
title	Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals
title_short	Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals
title_full	Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals
title_fullStr	Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals
title_full_unstemmed	Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals
title_sort	reversed-phase liquid-chromatographic mass spectrometric n-glycan analysis of biopharmaceuticals
description	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.
publisher	Springer-Verlag
publishDate	2013
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581771/
_version_	1611957485171638272

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

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