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Analysis of three epoetin alpha products by LC and LC-MS indicates differences in glycosylation critical quality attributes, including sialic acid content

Thomson, R. I., Gardner, R. A., Strohfeldt, K., Fernandes, D. L., Stafford, G. P., Spencer, D. I. R. and Osborn, H. M. I. (2017) Analysis of three epoetin alpha products by LC and LC-MS indicates differences in glycosylation critical quality attributes, including sialic acid content. Analytical Chemistry, 89 (12). pp. 6455-6462. ISSN 0003-2700

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To link to this item DOI: 10.1021/acs.analchem.7b00353

Abstract/Summary

Erythropoietin (EPO) is one of the main therapeutics used to treat anaemic patients, greatly improving their quality of life. In this study, biosimilars Binocrit and a development product, called here CIGB-EPO, were compared to the originator product, Eprex. All three are epoetin alpha products, reputed to have similar glycosylation profiles. The quality, safety and efficacy of this biotherapeutic depend on the following glycosylation critical quality attributes (GCQAs): sialylation, N-glycolyl-neuraminic acid (Neu5Gc) content, branching, N-acetyl-lactosamine (LacNAc) extensions and O-acetylation pattern. Reverse-phase ultra high pressure liquid chromatography (RP-UHPLC) analysis of acid-released, 1,2-diamino-4,5-methylenedioxybenzene (DMB) labelled sialic acid derivatives and hydrophilic interaction liquid chromatography (HILIC) in combination with mass spectrometry (HILIC-UHPLC-MS) of procainamide (PROC) labelled N-glycans were the analytical tools used. An automated method for enzymatic release and PROC labelling was applied for the first time to the erythropoiesis stimulating agent (ESA) products, which facilitated novel, in-depth characterisation, and allowed identification of precise structural features including the location of O-acetyl groups on sialic acid (SA) moie-ties. Samples were digested by a sialate-O-acetylesterase (NanS) to confirm the presence of O-acetyl groups. It was found that Eprex contained the greatest relative abundance of O-acetylated derivatives, Binocrit expressed the least Neu5Gc, and CIGB-EPO showed the greatest variety of high-mannose-phosphate structures. The sialylation and LacNAc extension patterns of the three ESAs were similar, with a maximum of four N-acetyl-neuraminic acid (Neu5Ac) moieties detected per glycan. Such differences in SA derivatisation, particularly O-acetylation, could have consequences for the quality and safety of a biotherapeutic, as well as its efficacy.

Item Type:Article
Refereed:Yes
Divisions:Faculty of Life Sciences > School of Chemistry, Food and Pharmacy > School of Pharmacy > Medicinal Chemistry Research Group
ID Code:70676
Publisher:American Chemical Society

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