A Comprehensive Approach for Monoclonal Antibody N-linked Glycan Analysis from Sample Preparation to Data Analysis

Significance of the Topic

Monoclonal antibody glycosylation is a critical quality attribute that directly impacts therapeutic efficacy, stability, pharmacokinetics and immunogenicity. Detailed characterization of N-linked glycans supports regulatory compliance and batch‐to‐batch consistency in biopharmaceutical production.

Objectives and Study Overview

This application note presents an end-to-end workflow for N-linked glycan analysis of monoclonal antibodies, combining high‐throughput sample preparation, optimized chromatographic separation, fluorescence and mass spectrometric detection, and automated data processing. The goals are to improve throughput, sensitivity, reproducibility and reporting for glycan profiling.

Methodology

• Enzymatic release of N-glycans from mAbs using PNGase F on an Agilent AssayMAP Bravo liquid handling platform with GlykoPrep-plus Rapid N-Glycan reagents and InstantPC fluorescent tag
• Cleanup of labeled glycans and injection of 1–2 µL into Agilent 1290 Infinity II LC with AdvanceBio Glycan Mapping column
• Dual detection by 1260 Infinity fluorescence detector (Ex 285 nm/Em 345 nm) and 6545XT AdvanceBio LC/Q-TOF with Agilent Jet Stream ESI
• Gradient elution with formic acid/ammonium hydroxide buffer and acetonitrile at 0.4–0.9 mL/min over 30 min
• Data acquisition in high-resolution mode (m/z 300–1,700 at 2 spectra/sec)

Used Instrumentation

• Agilent AssayMAP Bravo liquid handling system (G5542A)
• Agilent 1290 Infinity II LC system with 1260 Infinity fluorescence detector (G1321B)
• Agilent AdvanceBio Glycan Mapping column, 2.1 × 100 mm, 1.8 µm
• Agilent 6545XT AdvanceBio LC/Q-TOF system with Dual Agilent Jet Stream source
• Agilent MassHunter BioConfirm B.09.00 software with Personal Compound Database glycan library

Main Results and Discussion

• Fluorescence chromatograms resolved over 15 distinct glycan peaks from NISTmAb, including G0F, G1F, G1F′ and G2F
• MS extracted ion chromatograms confirmed glycan identities and adduct patterns with excellent isotopic fidelity
• Relative quantitation by MS closely matched FLD results across four mAb samples (NISTmAb, Herceptin, Sigma SiLu and CHO mAb1) using top five abundant glycans
• Automated batch processing in BioConfirm enabled rapid glycan profiling, peak annotation and reporting with customizable output formats

Benefits and Practical Applications

• Fully automated sample preparation improves throughput and reproducibility for high-volume workflows
• Dual FLD and high-resolution MS detection delivers robust quantitation and structural confirmation
• Scalable assay suitable for quality control, comparability studies and biosimilar development
• Customizable reporting accelerates data review and documentation for regulatory submissions

Future Trends and Opportunities

• Integration of machine learning for automated glycan structure prediction and anomaly detection
• Development of novel fluorescent and ionization tags to expand coverage of low-abundance glycans
• Miniaturized and parallelized platforms for ultra-high-throughput glycomics
• Real-time online glycan monitoring in bioreactor harvest streams

Conclusion

The combined Agilent AssayMAP Bravo, AdvanceBio LC/Q-TOF instrumentation and MassHunter BioConfirm software provide a streamlined, high-sensitivity workflow for N-glycan analysis of monoclonal antibodies. This solution offers high throughput, reliable quantitation by FLD and MS, and automated reporting, meeting the demands of modern biopharmaceutical R&D and QC laboratories.

References

1. Rademacher TW, Williams PM, Dwek RA. Agalactosyl glycoforms of IgG autoantibodies are pathogenic. Proc Natl Acad Sci U S A. 1994;91(13):6123-6127.
2. Anumula KR. Advances in fluorescence derivatization methods for HPLC analysis of glycoprotein carbohydrates. Anal Biochem. 2006;350(1):1-23.
3. Agilent Technologies. N-Glycan Analysis of mAbs and Other Glycoproteins with UHPLC and Fluorescence Detection. Pub. No. 5991-5253EN.
4. Agilent Technologies. Comparison of Relative Quantification of Monoclonal Antibody N-glycans Using Fluorescence and MS Detection. Pub. No. 5991-6958EN.
5. Agilent Technologies. Analysis of Monoclonal Antibody N-glycans by Fluorescence Detection and Robust Mass Selective Detection Using the Agilent LC/MSD XT. Pub. No. 5991-8071EN.