Comparison of ZIC-HILIC and graphitized carbon-based analytical approaches combined with exoglycosidase digestions for analysis of glycans from monoclonal antibodies
| Autor: | Nathan A. Lacher, Anna Nordborg, Paul R. Haddad, Matthias Pelzing, Lea Mauko, Emily F. Hilder |
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| Přispěvatelé: | Mauko, Lea, Lacher, Nathan A, Pelzing, Matthias, Nordborg, Anna, Haddad, Paul R, Hilder, Emily F. |
| Rok vydání: | 2012 |
| Předmět: |
porous graphitized carbon
Glycan Electrospray Spectrometry Mass Electrospray Ionization Glycosylation Glycoside Hydrolases medicine.drug_class Electrospray ionization Clinical Biochemistry CHO Cells Monoclonal antibody Biochemistry Analytical Chemistry chemistry.chemical_compound Mice Cricetulus Exoglycosidase Polysaccharides Cricetinae medicine Animals Humans 2-Aminobenzamide ZIC-HILIC Chromatography biology Hydrophilic interaction chromatography ESI-MS Antibodies Monoclonal Cell Biology General Medicine carbohydrates (lipids) Spectrometry Fluorescence chemistry Mannosylation biology.protein glycans Graphite monoclonal antibodies fluorescence Hydrophobic and Hydrophilic Interactions Chromatography Liquid |
| Zdroj: | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. 911 |
| ISSN: | 1873-376X |
| Popis: | 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. Refereed/Peer-reviewed |
| Databáze: | OpenAIRE |
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