Peptide Mapping of Tryptic Digests for mAbs using a novel ECD cell on the 6545XT AdvanceBio LC/Q-TOF Mass Spectrometer

Significance of the Topic

Monoclonal antibodies (mAbs) are widely used as biotherapeutics, and post-translation modifications (PTMs) such as glycosylation critically influence their safety, efficacy, and target binding. Reliable mapping of these labile modifications is essential for ensuring product consistency, regulatory compliance, and therapeutic performance.

Objectives and Study Overview

This study evaluates a novel electron-based dissociation (ExD) cell installed on the Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer for peptide mapping of tryptic digests from NIST mAb and trastuzumab. The goal is to compare ExD against conventional collision-induced dissociation (CID) to demonstrate improved glycopeptide preservation and sequence coverage.

Methodology

Tryptic digests were prepared using Agilent AssayMAP Bravo with either in-solution or SP3 protocols. Peptides were reconstituted at ~0.5 µg/µL in 0.1% formic acid. LC separation employed a water/acetonitrile gradient with 0.1% formic acid over 100 min at 0.3 mL/min and a column temperature of 40 °C. MS data were acquired in Extended Dynamic Range (2 GHz) over 100–3000 m/z, using a top-5 data-dependent method at 3 spectra/s for both MS1 and MS/MS.

Used Instrumentation

• Agilent AssayMAP Bravo system for automated digestion
• Agilent 1290 Infinity II LC system
• Agilent 6545XT AdvanceBio LC/Q-TOF with ExD cell
• MassHunter BioConfirm 12.1 software for data analysis

Main Results and Discussion

• Glycopeptides contributed under 10% of the base peak intensity but achieved ~99% sequence coverage with ExD for both NIST and trastuzumab samples.
• ExD fragmentation preserved intact glycan attachments, yielding abundant c- and z-type fragment ions that differentiate glycoforms such as G0F and G1F.
• CID fragmentation predominantly produced the HexNAc oxonium ion at m/z 204.086 and fewer backbone fragments, limiting the characterization of intact glycopeptides.

Benefits and Practical Applications

Electron-based dissociation enhances structural insight into mAb glycosylation by retaining labile PTMs and providing near-complete peptide coverage. This capability supports rigorous quality control, comparability assessments, and regulatory documentation in biopharmaceutical development.

Future Trends and Possibilities

• Incorporation of ExD-enabled workflows into high-throughput process monitoring and release testing.
• Development of advanced bioinformatics tools for automated identification and quantitation of glycoforms.
• Application of electron-based fragmentation to other sensitive PTMs, including phosphorylation.
• Further instrument refinement to boost sensitivity, scan speed, and multiplexed analysis of complex glycoproteins.

Conclusion

The Agilent 6545XT AdvanceBio LC/Q-TOF with ExD cell demonstrates superior performance for glycopeptide mapping of monoclonal antibodies, delivering intact glycan retention and exceptional sequence coverage compared to CID. This approach offers a robust platform for detailed mAb characterization.

References

[1] Zheng K., Bantog C., Bayer R. The impact of glycosylation on monoclonal antibody conformation and stability. MAbs. 2011;3(6):568–576.