Comparison of Relative Quantification of Monoclonal Antibody N-glycans Using Fluorescence and MS Detection

Importance of the Topic

Monoclonal antibodies carry complex N-glycans that influence their stability, activity and immunogenicity. Monitoring glycosylation profiles is essential for quality control in biotherapeutic development and manufacturing

Study Objectives and Overview

This work compares relative quantification of InstantPC-labeled monoclonal antibody N-glycans by fluorescence detection versus mass spectrometry. Two mAb samples were processed in quadruplicate, separated by HILIC chromatography, and analyzed using both detection modes to evaluate reproducibility and agreement

Methodology

Released N-glycans were derivatized with the InstantPC amine-reactive tag, which enhances both fluorescence emission and MS ionization efficiency. A HILIC UHPLC gradient was optimized to resolve common glycoforms. Fluorescence peak areas provided relative abundances of 21 glycan compositions. MS-based quantification used extracted ion chromatograms and a custom Personal Compound Database for 35 compositions, leveraging accurate mass and tandem MS for structural assignment

Instrumentation

• Agilent 1290 Infinity UHPLC with binary pump, autosampler, thermostat and fluorescence detector
• Agilent 6550 iFunnel Q-TOF LC/MS with dual-nebulizer AJS source
• AdvanceBio Glycan Mapping columns (2.1 × 150 mm and 2.1 × 100 mm, 1.8 µm)
• Agilent MassHunter Workstation, PCDL Manager and Mass Profiler software

Main Results and Discussion

Fluorescence analysis quantified 21 major glycoforms with excellent reproducibility (RSD <4% for components ≥0.1% abundance). MS detection extended quantification to 35 species with an average RSD of 3.2%. Chromatograms from both methods aligned closely, demonstrating that InstantPC imparts sufficient ionization efficiency for reliable MS quantification. Tandem MS resolved isobaric compositions and confirmed structural assignments

Benefits and Practical Applications

• Simultaneous fluorescence and MS detection streamlines glycan profiling workflows
• High sensitivity of InstantPC labeling enables detection of low-abundance species
• Accurate mass and MS/MS data support confident glycan identification
• Reproducible relative quantification supports robust QC and comparability studies

Future Trends and Opportunities

Advances may include automated sample preparation, integration with multi-attribute methods, expansion of glycan databases and AI-driven spectral interpretation. Emerging labels and microfluidic platforms could further increase throughput and sensitivity in biopharmaceutical glycoanalysis

Conclusion

The combination of InstantPC labeling and Agilent LC/MS provides a reliable dual detection strategy for relative quantification of mAb N-glycans. MS-based quantification closely matches fluorescence results while offering extended coverage and structural insight, making it a powerful tool for biotherapeutic characterization

References

1. Potter O., Staples G. Comparison of Relative Quantification of Monoclonal Antibody N-glycans Using Fluorescence and MS Detection. Agilent Technologies Application Note 5991-6958EN, 2016
2. Consortium for Functional Glycomics. Nomenclature Guidelines for Glycan Representation. 2016