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A chemoenzymatic method for simultaneous profiling N- and O-glycans on glycoproteins using one-pot format
Uriel Ortega-Rodriguez1, John Q Bettinger1, Guozhang Zou1
1Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, United States Food and Drug Administration, Silver Spring, MD 20993, USA.
Cell Reports Methods
|August 8, 2024
Summary
This study introduces a simplified one-pot chemoenzymatic method for simultaneously profiling N- and O-glycans in therapeutic glycoproteins using mass spectrometry. This approach enhances quality assessment by streamlining glycan characterization.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Glycobiology
Background:
- Glycosylation is crucial for therapeutic glycoprotein quality, affecting efficacy, stability, and safety.
- Characterizing N-glycans is established, but O-glycan analysis is complex and lacks tools.
- Accurate glycan profiling is essential for biopharmaceutical development and quality control.
Purpose of the Study:
- To develop a simplified, simultaneous method for profiling both N- and O-glycans from therapeutic glycoproteins.
- To overcome the challenges associated with traditional O-glycan analysis.
- To facilitate routine quality assessment of therapeutic glycoproteins.
Main Methods:
- A streamlined, one-pot chemoenzymatic strategy was employed.
- N-glycans were released using PNGase F.
- O-glycopeptides were generated, followed by selective N-glycan reduction and O-glycan release via β-elimination during permethylation.
Main Results:
- Simultaneous profiling of N- and O-glycans was achieved in a single mass spectrometry analysis.
- Glycan structural assignments and the N- to O-glycan ratio were determined.
- The method demonstrated reliability for comprehensive glycan characterization.
Conclusions:
- The developed one-pot method simplifies and enhances the simultaneous characterization of N- and O-glycans.
- This approach provides a reliable tool for advanced quality assessment of therapeutic glycoproteins.
- The streamlined workflow facilitates routine analysis and improves biopharmaceutical quality control.
