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Comprehensive Glycosylation Characterization of Recombinant Human Erythropoietin by Electron-Activated Dissociation Mass Spectrometry

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Abstract

Recombinant human erythropoietin (rhEPO) is a glycoprotein that acts as the main hormone involved in regulating red blood cell production to treat anemia caused by chronic kidney disease or chemotherapy, which has three N-glycosylation sites and one O-glycosylation site. It contains a variety of different glycosylation modifications, such as sialyation, O-acetylation on sialic acids, etc., which causes a big challenge for the glycosylation analysis of rhEPO. In this study, a liquid chromatography-mass spectrometry (LC–MS) method combined with electron-activated dissociation (EAD) technology was used in qualitative and quantitative characterization of rhEPO N-glycosylation and O-glycosylation in just one injection. The usage of EAD not only generated abundant MS/MS fragment ions of glycopeptides and improved the MS/MS sequence coverage but also preserved the glycan structures in the MS/MS fragment ions and the integrity of the glycosidic bond between the glycans and peptides. Three N-glycosylation sites (N24, N38, and N83) and one O-glycosylation site (S126) of rhEPO samples were successfully identified. Among them, the glycosylation ratios of N24, N38, and N83 sites were 82.7%, 100%, and 100% respectively, and 15, 10, and 12 different N-glycans could be identified at the glycopeptide level. The total average number of sialic acids, N-hydroxyacetylneuraminoic acid, and O-acetylation on sialic acid were 7.28, 4.21, and 0.66 at the intact protein level, respectively. For O-glycosylation site S126, O-glycosylation ratios analyzed at the intact protein level and the glycopeptide level were 80.2% and 80.3%, respectively, and two O-glycans were identified, including Core1_S1 and Core1_S2. This study also compared the difference of the glycans and their relative contents in batch-to-batch rhEPO samples. The results proved that the workflow using EAD fragmentation in LC–MS method could be effectively applied for characterizing the glycosylation analysis of rhEPO samples and batch-to-batch consistency analysis, which would help to reasonably guide the optimization of rhEPO production process.

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Data Availability

Data are available from the corresponding authors on reasonable request.

Abbreviations

rhEPO:

Recombinant human erythropoietin

MS:

Mass spectrometry

PTMs:

Post-translational modifications

LC–MS/MS:

Liquid chromatography-tandem mass spectrometry

LC-MS:

Liquid chromatography-mass spectrometry

CE:

Capillary electrophoresis

CE-MS:

Capillary electrophoresis-mass spectrometry

RP:

Reversed phase

CID:

Collision induced dissociation

EAD:

Electron-activated dissociation

KE:

Kinetic energy

IAM:

Iodoacetamide

DTT:

Dithiothreitol

FA:

Formic acid

IDA:

Information-dependent acquisition

TIC:

Total ion chromatogram

MS/MS:

Tandem mass spectrometry

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Funding

This study was supported by Beijing Natural Science Foundation (No. L232013).

Author information

Authors and Affiliations

  1. Division of Recombinant Biological Products, National Institutes for Food and Drug Control (NIFDC), Beijing, 100501, People’s Republic of China

    Xiang Li & Yong Zhou

  2. SiChuan Institute for Drug Control, NMPA Key Laboratory for Quality Control and Evaluation of Vaccines and Biological Products, Chengdu, 611731, People’s Republic of China

    Yan Li

  3. SCIEX, Beijing, 100015, People’s Republic of China

    Wentao Wang, Ji Luo, Lihai Guo & Hong-Xu Chen

Authors
  1. Xiang Li
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  2. Wentao Wang
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  3. Ji Luo
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  5. Yong Zhou
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  6. Yan Li
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  7. Hong-Xu Chen
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Contributions

YZ, YL, and HXC conceived and designed the research; XL and WTW collected and analyzed the data and wrote the manuscript; JL and LHG revised the manuscript and gave meaningful discussion and suggestions. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Yong Zhou, Yan Li or Hong-Xu Chen.

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Li, X., Wang, W., Luo, J. et al. Comprehensive Glycosylation Characterization of Recombinant Human Erythropoietin by Electron-Activated Dissociation Mass Spectrometry. Appl Biochem Biotechnol (2024). https://doi.org/10.1007/s12010-024-04954-y

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