Reversed-phase liquid-chromatographic mass spectrometric N -glycan analysis of biopharmaceuticals
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ORIGINAL PAPER
Reversed-phase liquid-chromatographic mass spectrometric N-glycan analysis of biopharmaceuticals Fabian Higel & Uwe Demelbauer & Andreas Seidl & Wolfgang Friess & Fritz Sörgel
Received: 31 October 2012 / Revised: 5 December 2012 / Accepted: 20 December 2012 / Published online: 31 January 2013 # The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract 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 acidlabeled 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 hydrophilicinteraction liquid chromatography of 2-aminobenzamidelabeled N-glycans for characterization of N-glycans derived from biopharmaceuticals. F. Higel : U. Demelbauer : A. Seidl (*) Hexal AG, Sandoz Biopharmaceuticals, Keltenring 1+3, 82041 Oberhaching, Germany e-mail: [email protected] W. Friess Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwigs-Maximilians-Universität, Butenandtstrasse 5-13, Building B, 81377 Munich, Germany F. Sörgel IBMP—Institute for Biomedical and Pharmaceutical Research, Paul-Ehrlich-Straße 19, 90562 Nürnberg-Heroldsberg, Germany
Keywords Anthranilic acid . 2-aminobenzamide . Mass spectrometry . N-glycosylation . Reversed-phase chromatography . Monoclonal antibody Abbreviations 2-AA 2-Aminobenzoic acid 2-AB 2-Aminobenzamide ACN Acetonitrile ANTS 8-Aminonaphthalene-1,3,6-trisulfonic acid DMSO Dimethyl sulfoxide EIC Extracted ion chromatogram ESI Electrospray ionization FLD Fluorescence detector GlcNAc N-Acetyl-D-glucosamine HILIC Hydrophilic interaction liquid chromatography HPLC High-performance liquid chromatography LC Liquid chromatography mAb Monoclonal antibody MALDI Matrix-assisted laser desorption ionization MS Mass spectrometry PNGaseF Peptide N-glycosidase F PGC Porous graphitized carbon RP Reversed phase
Introduction Recombinant protein drugs belong to the most complex active pharmaceutical ingredients. Monoclonal antibodies (mAbs), glycoproteins with a molecular mass of approximately 150 kDa are one important class. Glycosylation has
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