Automated Switching between Peptide and Glycan Mapping with the Agilent 1290 Infinity II LC System

Importance of the topic

The structural complexity of monoclonal antibodies (mAbs) demands high-resolution analytical methods for comprehensive characterization. Glycan mapping reveals critical post-translational modifications at the carbohydrate level, while peptide mapping provides sequence confirmation and identification of modifications such as deamidation or oxidation. Integrating both analyses on a single platform without manual intervention enhances laboratory productivity and data consistency.

Study objectives and overview

This application demonstrates an automated workflow using the Agilent 1290 Infinity II LC system for sequential glycan and peptide mapping of therapeutic mAbs in one continuous sequence. The approach leverages column switching and a unified detection scheme (FLD, DAD, Q-TOF MS) to analyze multiple mAb samples—including trastuzumab and rituximab originators and biosimilars—highlighting method robustness, precision, and flexibility.

Methodology and Instrumentation

The system configuration includes:

• Agilent 1290 Infinity II High-Speed Pump, Multisampler, Multicolumn Thermostat, column switching valve, DAD, FLD
• Agilent 6545 LC/Q-TOF MS with Jet Stream ESI source
• Columns: AdvanceBio Glycan Mapping (2.1×150 mm, 2.7 µm HILIC) and AdvanceBio Peptide Plus (2.1×150 mm, 2.7 µm RPLC)
• Agilent InstantPC N-glycan release and labeling kit for rapid deglycosylation and fluorescent tagging

Sample preparation:

• Glycan mapping: mAb denaturation, PNGase F release, 1-minute InstantPC labeling, cleanup via HILIC SPE
• Peptide mapping: denature/reduce/alkylate (guanidine-HCl, DTT, IAA), desalting, overnight trypsin digest (1:25 enzyme:protein)

Chromatographic parameters:

• HILIC gradient at 0.5 mL/min, 40 °C, FLD Ex 285/Em 345 nm, MS m/z 600–3 000
• RPLC gradient at 0.3 mL/min, 50 °C, DAD monitoring 214/220/280 nm, MS positive mode, auto MS/MS

Key results and discussion

• Glycan mapping of an N-glycan library yielded baseline separation of isomeric glycans (e.g., G1F[3]/G1F[6]) with FLD- and MS-based relative standard deviations (RSDs) < 2% retention, < 5% area.
• Trastuzumab originator versus biosimilar profiles revealed under-galactosylation in the candidate product; feeding galactose, uridine, and MnCl₂ successfully improved G1F/G0F ratios.
• Rituximab originator and two biosimilars showed qualitatively consistent glycan species with minor quantitative differences, illustrating method applicability in comparability studies.
• Peptide mapping achieved > 98.5% sequence coverage for trastuzumab, with reliable detection of pyroglutamate formation, deamidation, oxidation, N-terminal cyclization, and C-terminal lysine clipping (area RSD < 1%).
• Automated method switching required only a short conditioning step between glycan and peptide analyses, eliminating manual valve changes and enhancing throughput.

Benefits and practical applications of the method

• High throughput: combined workflows reduce total analysis time and operator involvement.
• Flexibility: single-injection dual mapping supports QA/QC, clone selection, comparability, and biosimilar development.
• Data consistency: unified platform ensures reproducible retention, accurate quantitation, and robust MS identification.

Future trends and potential applications

Advances in automation and multi-attribute method development will drive further integration of glycopeptide analysis and real-time data evaluation. Coupling high-throughput workflows with AI-driven data processing will enhance structural elucidation, support regulatory compliance, and accelerate biotherapeutic development.

Conclusion

The Agilent 1290 Infinity II LC system with column switching, FLD/DAD, and Q-TOF MS delivers a robust, reproducible platform for automated peptide and glycan mapping of mAbs. This integrated approach streamlines workflows, ensures high data quality, and supports a broad range of biopharma analytical needs.

Used Instrumentation

• Agilent 1290 Infinity II High-Speed Pump, Multisampler, Multicolumn Thermostat, column switching valve, DAD (G7117B), FLD (G1321A)
• Agilent 6545 LC/Q-TOF MS with Jet Stream ESI
• AdvanceBio Glycan Mapping and Peptide Plus columns, InstantPC glycan prep kit

References

1. Sandra K., Vandenheede I., Sandra P. Modern Chromatographic and Mass Spectrometric Techniques for Protein Biopharmaceutical Characterization. J. Chromatogr. A 2014, 1335, 81–103.
2. Fekete S. et al. Chromatographic, Electrophoretic, and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals. Anal. Chem. 2016, 88(1), 480–507.
3. Beck A. et al. Characterization of Therapeutic Antibodies and Related Products. Anal. Chem. 2013, 85(2), 715–736.
4. D’Atri V. et al. Hydrophilic Interaction Chromatography for the Characterization of Therapeutic Monoclonal Antibodies at Protein, Peptide, and Glycan Levels. LC-GC Europe 2017, 30(8), 424–434.
5. Guile G. R. et al. A Rapid High-Resolution High-Performance Liquid Chromatographic Method for Separating Glycan Mixtures and Analyzing Oligosaccharide Profiles. Anal. Biochem. 1996, 240(2), 210–226.
6. Martosella J. et al. A Novel HILIC Column for High Speed N-linked Glycan Analysis. Agilent Technologies application note 5991-4886EN, 2017.
7. Yan J., Jones A. Streamlined Workflows for N-Glycan Analysis of Biotherapeutics Using Agilent AdvanceBio Gly-X InstantPC and 2-AB Express Sample Preparation with LC/FLD/MS. Agilent Technologies application note 5994-1348EN, 2019.
8. Potter O., Qin V. Separation of Deamidated Peptides with an Agilent AdvanceBio Peptide Plus Column. Agilent Technologies application note 5994-2971EN, 2021.
9. Agilent Technologies. AdvanceBio Glycan Standards InstantPC, 2-AB, 2-AA, APTS, InstantAB, InstantQ, Unlabeled. Flyer 5994-2202EN, 2019.
10. Dumont E. et al. Cell Clone Selection Using the Agilent Bio-Monolith Protein A Column and LC/MS. Application note 5991-5124EN, 2014.
11. Dumont E. et al. Cell Culture Optimization Using an Agilent Bio-Monolith Protein A Column and LC/MS. Application note 5991-5125EN, 2014.
12. Gramer M. J. et al. Modulation of Antibody Galactosylation Through Feeding of Uridine, Manganese Chloride, and Galactose. Biotechnol. Bioeng. 2011, 108(7), 1591–1602.