Profiling Glycosylation of Monoclonal Antibodies at Three Levels Using the Agilent 6545XT AdvanceBio LC/Q‑TOF

Significance of the Topic

• Monoclonal antibodies (mAbs) are a cornerstone of modern biopharmaceuticals, and their glycan decorations critically influence therapeutic efficacy, immunogenicity and pharmacokinetics.
• Regulatory agencies and quality control laboratories require robust analytical strategies to ensure consistency of glycosylation profiles throughout development and manufacturing.

Objectives and Overview of the Study

This study presents a comprehensive evaluation of four complementary liquid chromatography–mass spectrometry (LC/MS) workflows for quantitative profiling of N-glycosylation on the NISTmAb standard. The goals were to compare intact mAb analysis, subunit (heavy and light chain) profiling, glycopeptide mapping, and released glycan characterization, all using the Agilent 6545XT AdvanceBio LC/Q-TOF platform coupled with Agilent AssayMAP Bravo automation and MassHunter BioConfirm software.

Methodology and Instrumentation

• Sample Preparation: Minimal handling for intact mAb; DTT/TCEP reduction for subunit workflows; tryptic digestion for glycopeptide mapping; PNGase F release and InstantPC fluorescent labeling for released glycans.
• Chromatography: Agilent 1290 Infinity II with PLRP-S and AdvanceBio Glycan Mapping columns for separation; optimized gradients and temperatures for each level.
• Detection: Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Jet Stream electrospray source; fluorescence detection (λEx 285 nm, λEm 345 nm) for labeled glycans.
• Data Analysis: MassHunter BioConfirm B.09.00 workflows for intact mass deconvolution, peptide mapping, glycopeptide assignment and glycan identification using the Personal Compound Database and Library (PCDL).

Main Results and Discussion

• Intact mAb analysis resolved five major glycoforms (G0F-G2F variants) with relative standard deviations (RSD) <1% by peak height quantitation.
• Subunit profiling achieved baseline separation of heavy and light chains and quantified G0F, G1F and G2F on the heavy chain with RSD <0.3%.
• Glycopeptide mapping identified N-glycosylation site occupancy and relative abundances of glycoforms, demonstrating enhanced resolution of isobaric structures.
• Released glycan workflow detected over fifteen labeled species with excellent chromatographic resolution and fluorescence/MS quantitation, yielding consistent relative abundances compared to subunit data.

Benefits and Practical Applications of the Method

• The multi‐level approach allows cross‐validation of glycan profiles and site‐specific information to support R&D, process development and QC.
• High throughput automation reduces sample handling time and improves reproducibility.
• Mass accuracy and resolution of the 6545XT Q-TOF enable unambiguous glycoform assignment in complex mixtures.

Future Trends and Opportunities

• Integration of ion mobility separation to distinguish glycan linkage and stereoisomers.
• Advanced informatics combining machine learning for automated glycoform annotation and quantitation.
• Expansion to other biotherapeutic formats such as antibody–drug conjugates and fusion proteins.

Conclusion

The presented workflows using Agilent’s AdvanceBio LC/Q-TOF system and BioConfirm software deliver comprehensive, reproducible and high‐throughput glycosylation analysis of mAbs at multiple structural levels. This integrated solution addresses both regulatory and research needs for detailed glycan profiling.

Reference

• Rademacher T.W., Williams P., Dwek R.A. Agalactosyl glycoforms of IgG autoantibodies are pathogenic. Proc. Natl. Acad. Sci. U.S.A. 1994;91:6123–6127.
• Agilent Technologies. Precise Characterization of Intact Monoclonal Antibodies by the Agilent 6545XT AdvanceBio LC/Q-TOF. Publication 5991-7813EN.
• Agilent Technologies. Making Peptide Mapping Routine with the Agilent 6545XT AdvanceBio LC/Q-TOF. Publication 5991-7815EN.
• Agilent Technologies. A Comprehensive Approach for Monoclonal Antibody N-linked Glycan Analysis from Sample Preparation to Data Analysis. Publication 5991-8550EN.