Comprehensive Characterization of the N and O-Linked Glycosylation of a Recombinant Human EPO

Significance of the Topic

Erythropoietin (EPO) glycosylation critically influences the therapeutic efficacy and serum half life of recombinant human EPO products.
Detailed characterization of both N- and O-linked glycans is essential to ensure biosimilar comparability and regulatory compliance in biopharmaceutical development.

Objectives and Study Overview

This study presents two complementary liquid chromatography strategies to profile N- and O-linked glycosylation of recombinant human EPO (rhEPO).
The first approach focuses on rapid release and RapiFluor-MS labeling of N-glycans followed by HILIC-fluorescence–mass spectrometry profiling.
The second strategy employs rapid enzymatic deglycosylation of intact rhEPO and HILIC profiling of O-glycoforms via intact protein analysis.

Methodology and Instrumentation

• Released N-glycan analysis:
  • N-glycans released with PNGase F, labeled with RapiFluor-MS, purified by SPE.
  • Separation on ACQUITY UPLC Glycoprotein BEH Amide 300Å column using HILIC gradient coupled to fluorescence detection (Ex 265/Em 425 nm) and Xevo G2-XS QTof MS.
• Intact protein O-glycoform profiling:
  • Rapid N-deglycosylation using GlycoWorks Rapid PNGase F and RapiGest SF surfactant.
  • Intact rhEPO separated on the same widepore amide column by HILIC at 45 °C with intrinsic fluorescence detection (Ex 280/Em 320 nm) and SYNAPT G2-S HDMS MS.

Main Results and Discussion

• N-glycan profiling revealed a dominant tetra-antennary, tetrasialylated glycan species with N-acetyl lactosamine extensions alongside a significant biantennary disialylated glycan (FA2G2S2).
• High chromatographic resolution enabled clear assignment of 15 major N-glycan structures with mass errors under 3 ppm.
• Intact protein HILIC analysis of N-deglycosylated rhEPO resolved ~10 proteoforms, identifying O-glycan occupancy and heterogeneity including mono- and disialylated core structures.

Benefits and Practical Applications of the Method

• Rapid sample preparation and RapiFluor-MS labeling provide high sensitivity and throughput for N-glycan analysis.
• Widepore HILIC columns enhance resolution of highly branched glycans and intact glycoprotein proteoforms.
• Combined N- and O-glycosylation profiling supports biosimilar comparability, quality control, and regulatory submissions.

Future Trends and Potential Applications

Advances in labeling chemistries and ultra-high-resolution MS will further improve glycan isomer separation and structural elucidation.
Integration of automated sample preparation and microfluidic LC-MS platforms is expected to streamline glycoanalytics for biopharmaceutical development.
Application of these workflows to other glycoprotein therapeutics will enhance understanding of structure–function relationships.

Conclusion

The dual LC-MS strategies described enable comprehensive, high-throughput characterization of rhEPO glycosylation, capturing critical N- and O-linked features with high sensitivity and chromatographic resolution.
This approach offers a robust platform for supporting biotherapeutic development, comparability assessments, and quality control of glycoprotein drugs.

References

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