Glycopeptide Characterization for Various Monoclonal Antibodies Using the Agilent 6545XT AdvanceBio LC/Q-TOF

Significance of the Topic

Monoclonal antibodies (mAbs) are key biotherapeutics whose glycosylation patterns critically influence efficacy, safety and immunogenicity. Comprehensive profiling of glycopeptides at the peptide level provides site-specific and quantitative glycoform information essential for QC, comparability and process optimization in biopharmaceutical development.

Objectives and Study Overview

This application note describes the development of an automated, high-throughput LC/MS workflow for level 3 mAb glycopeptide characterization. The study compares reversed-phase (RP-C18) and hydrophilic interaction (HILIC) separations of tryptic mAb digests and evaluates automated data processing to achieve robust identification and relative quantitation of glycopeptides from three representative IgG1 samples (NISTmAb, Trastuzumab and a CHO-derived mAb).

Methodology and Instrumentation

Sample preparation was performed on the Agilent AssayMAP Bravo platform with reduction, alkylation and Trypsin/Lys-C digestion. Separation and detection details include:

• Agilent 1290 Infinity II LC
• AdvanceBio Peptide Mapping column (RP-C18, 2.1×150 mm, 2.7 μm) at 60 °C or AdvanceBio Glycan Mapping column (HILIC, 2.1×150 mm, 2.7 μm) at 50 °C
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Agilent Jet Stream source
• MS/MS acquisition (Extended Dynamic Range, 2 GHz) and separate MS-only runs for quantitation
• Data processing with Agilent MassHunter BioConfirm 10.0, employing Peptide Feature Extraction and automated glycopeptide matching

Main Results and Discussion

HILIC separation delivered markedly improved retention and resolution of hydrophilic glycopeptides compared to RP-C18, where glycoforms co-eluted in a narrow early gradient window. Relative quantitation of six major glycoforms on the TKPREEQYNSTYR peptide showed comparable percentages between RP and HILIC methods (<2 % SD for HILIC vs ~0.6 % SD for RP), with HILIC offering superior accuracy.
Comparative analysis of three mAbs revealed distinct glycan profiles: NISTmAb displayed balanced G0F/G1F levels, Herceptin was enriched in G0F (>65 %), and the CHO-derived mAb exhibited trace hybrid glycans. Automated processing facilitated rapid batch analysis, yielding peptide sequences, PTM locations and glycopeptide quantitation in a single workflow.

Benefits and Practical Applications

The integrated workflow combines high-throughput sample prep, orthogonal LC separation and accurate MS detection with streamlined software analysis. It supports detailed site-specific glycoform mapping, consistent quantitation and efficient comparison across multiple mAbs, aiding bioprocess monitoring, comparability studies and quality control in pharmaceutical environments.

Future Trends and Applications

Emerging directions include deeper glycoproteome coverage via ion mobility, integration of machine-learning algorithms for automated annotation, miniaturized workflows for limited samples, and extension to multi-PTM characterization. Coupling glycopeptide data with functional assays and real-time analytics will further refine biotherapeutic design and manufacturing.

Conclusion

This study presents a robust, reproducible LC/MS workflow for automated glycopeptide characterization of mAbs, leveraging Agilent’s AssayMAP Bravo, 1290 Infinity II LC, 6545XT LC/Q-TOF and MassHunter BioConfirm. The approach delivers site-specific identification, accurate relative quantitation and high throughput, fulfilling critical analytical requirements in biopharmaceutical research and QA/QC.

References

1. Rademacher TW, Williams P, Dwek RA. Agalactosyl glycoforms of IgG autoantibodies are pathogenic. Proc Natl Acad Sci. 1994;91:6123–6127.
2. Agilent Technologies. Precise Characterization of Intact Monoclonal Antibodies by the Agilent 6545XT AdvanceBio LC/Q-TOF. Publication 5991-7813EN.
3. Agilent Technologies. Making Peptide Mapping Routine with the Agilent 6545XT AdvanceBio LC/Q-TOF. Publication 5991-7815EN.
4. Dong Q, et al. In-Depth Characterization and Spectral Library Building of Glycopeptides in the Tryptic Digest of a Monoclonal Antibody Using 1D and 2D LC-MS/MS. J Proteome Res. 2016;15:1472–1486.
5. Agilent Technologies. A Comprehensive Approach for Monoclonal Antibody N-linked Glycan Analysis from Sample Preparation to Data Analysis. Publication 5991-8550EN.
6. Agilent Technologies. Automation for LC/MS Sample Preparation: High Throughput In-Solution Digestion and Peptide Cleanup Enabled by the Agilent AssayMAP Bravo Platform. Publication 5991-2957EN.
7. Agilent Technologies. Highly Confident Peptide Mapping of Protein Digests Using Agilent LC/Q-TOFs. Publication 5991-8552EN.