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Structural Elucidation of Fc- and Fab-Associated N-Glycans in Cetuximab Using Protein A-Assisted Domain-Resolved
Divya Chauhan1, Tarun Shivach1, Anurag S Rathore1
1Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, Delhi 110016, India.
Summary
This study introduces a novel Protein A-assisted method for analyzing N-glycans on specific antibody domains (Fc and Fab). This domain-resolved glycan profiling enhances detection of low-abundance glycoforms, crucial for biopharmaceutical development.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Immunology
Background:
- Monoclonal antibody glycosylation significantly impacts efficacy, stability, and safety.
- Accurate analysis of site-specific glycan heterogeneity is essential for biopharmaceutical quality control.
- Existing methods often struggle to detect low-abundance glycoforms.
Purpose of the Study:
- To develop a domain-resolved glycan profiling approach for mass spectrometry-based analysis of N-glycans in cetuximab.
- To enable sensitive and specific characterization of glycosylation on Fc and Fab domains.
- To improve the detection and quantification of low-abundance glycoforms.
Main Methods:
- Protein A affinity separation to isolate Fc and Fab antibody domains.
- Enzymatic release of N-glycans from isolated domains.
- High-resolution mass spectrometry for detailed glycan profiling and quantification.
- Comparative analysis of Fc- and Fab-associated glycan populations.
Main Results:
- The Protein A-assisted method effectively separates Fc and Fab domains, reducing spectral complexity.
- Domain-specific glycan profiling clearly distinguishes glycosylation patterns between Fc and Fab regions.
- The approach significantly improves the detection and measurement of low-abundance glycoforms (e.g., sialylated, high-mannose, afucosylated).
Conclusions:
- A reliable and repeatable platform for domain-level glycan characterization and quantification is established.
- This method facilitates thorough analysis of glycosylation patterns across antibody regions.
- The approach supports quality control, biosimilar comparability studies, and biopharmaceutical development.
