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- Title
Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin.
- Authors
Jiang, Jing; Tian, Fang; Cai, Yun; Qian, Xiaohong; Costello, Catherine; Ying, Wantao
- Abstract
Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR-AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N, N, N, and O were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR-AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins. [Figure not available: see fulltext.]
- Subjects
ERYTHROPOIETIN; RECOMBINANT erythropoietin; ANEMIA treatment; GLYCOSYLATION; TANDEM mass spectrometry
- Publication
Analytical & Bioanalytical Chemistry, 2014, Vol 406, Issue 25, p6265
- ISSN
1618-2642
- Publication type
Article
- DOI
10.1007/s00216-014-8037-8