Analytical Platform for Glycomic Characterization of Recombinant Erythropoietin Biotherapeutics and Biosimilars by MS

Abstract

This platform may be applied to QC and batch analysis of not only recombinant erythropoietin, but also other complex, glycosylated biotherapeutics and biosimilars.

Mass spectrometry for glycosylation analysis of biopharmaceuticals

“…However, a more thorough evaluation of biopharmaceutical glycosylation at all stages of drug development and production becomes obligatory, since glyco-engineering is emerging. This is demonstrated by the targeted insertion of additional glycosylation sites in darbepoetin alpha or alpha 1-antitrypsin [9][10][11], concurrent with increasing availability of cost-effective glyco-analytical methods.…”

MS Platform for Erythropoietin Glycome Characterization

“…As shown in Figure 1, EPO is a heavily heterogeneous glycoprotein containing multiple N-and O-glycosylation sites with possible glycosyl modifications such as sialylation, phosphorylation, dehydration, O-acetylation on NeuAc, and polylactosaminylation. 4 Basic methods of glycan analysis often include chemical derivatization of the glycan (such as addition of fluorescent *Reprint requests to Hyun Joo An E-mail: hjan@cnu.ac.kr…”

“…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.…”

“…Rapid glycan profiling is possible with few seconds of acquisition times in a high-throughput manner. 4,5,20 Most of EPO N-glycans are multi-antennary glycans having high sialic acid residues, therefore, EPO Nglycans are analyzed in the negative ion detection mode to obtain overall profiling of N-glycan compositions during MALDI-MS. Figure 2a shows representative MALDI-TOF MS spectrum of native N-glycans released from the second generation EPO (NESP). High resolution TOF-MS provided accurate mass, simplifying identification of molecular ion, compositional assignment of each detected glycans.…”

Multi-Level Characterization of Protein Glycosylation

“…1−4 Glycosylation may be found on a variety of medically-and pharmaceutically-significant proteins, including biologic drugs (such as monoclonal antibodies or erythropoietin), 5,6 vaccines (e.g. viral proteins hemagluttinin and neuraminidase), 7,8 and clinical biomarkers (including cancer markers CA 15-3, CA-125, and PSA).…”