Application of a Quantitative LC–MS Multiattribute Method for Monitoring Site-Specific Glycan Heterogeneity on a Monoclonal Antibody Containing Two N-Linked Glycosylation Sites
Autor: | Ken Lawson, Lily Chu, Tian Wang, Wenzhou Li, Tamer Eris, Izydor Apostol |
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Rok vydání: | 2017 |
Předmět: |
0301 basic medicine
Glycan Glycosylation medicine.drug_class Tandem mass spectrometry Monoclonal antibody 01 natural sciences Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound N-linked glycosylation Polysaccharides Tandem Mass Spectrometry Liquid chromatography–mass spectrometry medicine Deamidation biology Hydrophilic interaction chromatography 010401 analytical chemistry Antibodies Monoclonal 0104 chemical sciences carbohydrates (lipids) 030104 developmental biology chemistry Biochemistry biology.protein Protein Processing Post-Translational Chromatography Liquid |
Zdroj: | Analytical Chemistry. 89:3562-3567 |
ISSN: | 1520-6882 0003-2700 |
Popis: | A significant challenge of traditional glycan mapping techniques is that they do not provide site-specific glycosylation information. Therefore, for proteins containing multiple glycosylation sites, the individual glycan species present at a particular site cannot be differentiated from those species present at the other glycosylation sites on the molecule. Quantification of glycoform has previously been demonstrated using a multiattribute method (MAM), which can quantify multiple post-translational modifications including deamidation, oxidation, glycosylation variants, and fragmentation ( Rogers, R. S.; Nightlinger, N. S.; Livingston, B.; Campbell, P.; Bailey, R.; Balland, A. MAbs 2015 , 7 , 881 - 890 ; ref 1). In this paper we describe the application of an MAM based method for site specific quantification of N-linked glycan heterogeneity present on an IgG1 mAb molecule containing two distinct N-linked glycosylation sites: one present on the heavy chain (HC) variable region (Fab) and the other present on the conserved HC constant region (Fc). MAM is a peptide mapping method utilizing mass spectrometry to detect and quantify specific peptides of interest. The ionization properties of the glycopeptides with different classes of glycan structural variants, including high mannose, sialylated, and terminal galactosylated species were studied in detail. Our results demonstrate that MAM quantification of individual glycan species from both the Fab and Fc N-Linked glycosylation sites is consistent with quantification using the traditional hydrophilic interaction liquid chromatography (HILIC) analysis of enzymatically released and fluorescently labeled glycans. Furthermore, no significant impact from the glycoform on the ionization properties of the glycopeptide is observed. Our work demonstrates that the MAM method is a suitable approach for providing quantitative, site-specific glycan information for profiling of N-linked glycans on immunoglobulins. |
Databáze: | OpenAIRE |
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