Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

Abstract

Recombinant human erythropoietin (rhEPO) has been extensively used as pharmaceutical product for treatment of 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 its 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 on an Orbitrap. Totally, 74 intact glycopeptides from four glycosylation sites at N24, N38, N83 and O126 were identified with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR/AM data allowed relative quantification of glycoforms. Our results could be extended to the quality control of rhEPO or help to establish detection approaches for glycosylation of other proteins.

MS Platform for Erythropoietin Glycome Characterization

“…[32][33][34] Recently, a few groups have utilized LC/MS-based platform to determine glycosylation sites with glycan microheterogeneity of the first generation EPO. 13,35,36 However, site-specific glycosylation analysis is still in its infancy due to the absence of proper technologies and methods. No doubt site-specific profiling will be a powerful platform to provide all of the information available with the localization of glycosylation with glycan heterogeneity.…”

“…It should come as no surprise, therefore, that MS has contributed significantly to recent progress towards understanding the role of the glycome in biotherapeutic glycoproteins including mAbs, recombinant proteins (such as EPOs), therapeutic enzymes, and fusion proteins. 4,[10][11][12][13][14][15][16] This review will cover the current state of the arts in MS-based analytical platforms for comprehensive characterization of EPO glycosylation in respect of N-and O-glycome, and site-specific glycoform profiling.…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…Mass spectrometric studies on rEPO and its next‐generation derivatives have been an ongoing endeavor for both in‐depth characterization of glycoforms in pharmaceutical products as well as alternative detection methods in sports drug testing. Okano et al .…”

“…Mass spectrometric studies on rEPO and its next-generation derivatives have been an ongoing endeavor for both in-depth characterization of glycoforms in pharmaceutical products [87] as well as alternative detection methods in sports drug testing. Okano et al utilized the fact that darbepoetin alpha comprises a modified protein backbone, enabling the generation of a peptide of unique amino acid composition, which serves as target structure for doping controls.…”

Comprehensive Characterization of N‐Glycosylation in Darbepoetin Alfa

“…3,[6][7][8][9] Glycan is a major structural moiety that has important effects on the pharmacokinetics and pharmacodynamics of EPO analogues such as darbepoetin alfa and epoetin alpha and beta. [10][11][12][13] The structure of glycan in EPO plays an important role in various biological activities. First, several studies have shown that sialic acid capping of the terminal galactose residue prevents hepatic clearance of EPO.…”