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Comparison of ZIC-HILIC and graphitized carbon-based analytical approaches combined with exoglycosidase digestions for analysis of glycans from monoclonal antibodies

Citation

Mauko, L and Lacher, NA and Pelzing, M and Nordborg, A and Haddad, PR and Hilder, EF, Comparison of ZIC-HILIC and graphitized carbon-based analytical approaches combined with exoglycosidase digestions for analysis of glycans from monoclonal antibodies, Journal of Chromatography. B, Analytical Technologies in The Biomedical and Life Sciences, 911 pp. 93-104. ISSN 1570-0232 (2012) [Refereed Article]

Copyright Statement

Copyright 2012 Elsevier B.V.

DOI: doi:10.1016/j.jchromb.2012.10.043

Abstract

Two LC approaches for analysis of therapeutic monoclonal antibodies (MAbs) are presented and compared. In the first approach, zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC) of 2-aminobenzamide-labelled glycans was coupled with fluorescence or electrospray ionisation mass spectrometric (ESI-MS) detection. The ZIC-HILIC method enabled relative quantification and identification of major glycan species. The sensitivity of fluorescence detection was higher compared to ESI-MS; however, MS detection enabled identification of co-eluted peaks. The new ZIC-HILIC approach was compared with porous graphitized carbon (PGC) separation of reduced glycans coupled with ESI-MS. Using PGC higher sensitivity was achieved compared to ZIC-HILIC due to the lower chemical background originating from the mobile phase and the derivatisation step, providing detailed information on minor glycan species. Furthermore, PGC exhibited excellent capability for separation of isobaric glycans with various degrees of mannosylation and galactosylation. The structures of glycans from MAbs used in this study were confirmed by exoglycosidase digestions. The two methods were applied to two monoclonal antibodies expressed in Chinese Hamster ovary cell lines and a monoclonal antibody expressed in a murine NS0 cell line. While the fluorescence-based approach is more suitable for routine glycan profiling due to the simplicity of data analysis, MS-based approaches were shown to provide detailed glycosylation analysis of complex glycoprotein samples.

Item Details

Item Type:Refereed Article
Keywords:monoclonal antibodies, glycans, 2-Aminobenzamide, porous graphitized carbon, ZIC-HILIC; ESI-MS, fluorescence
Research Division:Chemical Sciences
Research Group:Analytical Chemistry
Research Field:Separation Science
Objective Division:Expanding Knowledge
Objective Group:Expanding Knowledge
Objective Field:Expanding Knowledge in the Chemical Sciences
Author:Mauko, L (Ms Lea Mauko)
Author:Nordborg, A (Dr Anna Nordborg)
Author:Haddad, PR (Professor Paul Haddad)
Author:Hilder, EF (Professor Emily Hilder)
ID Code:82766
Year Published:2012
Deposited By:Austn Centre for Research in Separation Science
Deposited On:2013-02-18
Last Modified:2013-11-28
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