Making Peptide Mapping Routine with the Agilent 6545XT AdvanceBio LC/Q-TOF

Importance of Peptide Mapping in Monoclonal Antibody Analysis

Peptide mapping plays a pivotal role in the biopharmaceutical sector by offering detailed confirmation of antibody primary sequences and identifying post-translational modifications. Rapid and reliable characterization of monoclonal antibodies (mAbs) supports quality control, regulatory compliance, and accelerates research and development activities.

Objectives and Study Overview

This application note describes a streamlined protocol for routine peptide mapping of a reference mAb standard. The main aims are to integrate automated sample preparation, rapid chromatographic separation, accurate mass spectrometric detection, and automated data interpretation into a continuous workflow that delivers high sequence coverage with minimal hands-on time.

Methodology and Instrumentation

Sample preparation and analysis combine:

• Automated digestion and cleanup using Agilent AssayMAP Bravo liquid-handling robot.
• 15-minute peptide separation on Agilent 1290 Infinity II UHPLC with AdvanceBio Peptide Mapping column.
• High-resolution mass measurements using Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Jet Stream ESI.
• Data processing and peptide/PTM identification in Agilent MassHunter BioConfirm B.08 software.

Main Results and Discussion

The workflow achieved 99.4 % sequence coverage of both heavy and light chains of the NIST mAb within a 15-minute gradient. Mass accuracy consistently fell under 1 ppm for the majority of peptides. MS/MS spectra clearly distinguished native peptides from modified forms, for example:

• Methionine oxidation identified by a +15.99 Da shift in b-ion fragments.
• Asparagine deamidation confirmed by a +0.98 Da shift in b-ion fragments.

Automated scoring in BioConfirm streamlined review of peptide matches, sequence maps, and relative abundances, reducing data-processing bottlenecks.

Benefits and Practical Applications

Integrating robotics and rapid LC/MS accelerates peptide mapping from hours to under one hour per sample. The automated software reduces manual interpretation errors and supports high-throughput labs in both R&D and quality control environments. The concise method is readily transferable to other therapeutic proteins and biosimilars.

Future Trends and Opportunities

Emerging developments may include:

• Deeper integration of machine learning for PTM prediction and batch comparison.
• Faster chromatography using sub-2 µm particles or microflow formats.
• Cloud-based data sharing and standardized reporting for regulatory submissions.

Conclusion

The described workflow combines automated sample handling, short UHPLC gradients, high-accuracy MS, and intelligent software to convert peptide mapping into a routine, high-throughput operation. This approach delivers rapid, reproducible, and comprehensive characterization of monoclonal antibodies, meeting the demands of modern biopharmaceutical analysis.

Reference

1. Automation for LC/MS Sample Preparation: High Throughput In-Solution Digestion and Peptide Cleanup Enabled by the Agilent AssayMAP Bravo Platform. Agilent Technologies, publication 5991-2957EN.
2. Fast and Efficient Peptide Mapping of a Monoclonal Antibody (mAb): UHPLC Performance with Superficially Porous Particles. Agilent Technologies, publication 5991-3585EN.
3. High Resolution and Rapid Peptide Mapping of Monoclonal Antibody Using an Agilent 1290 Infinity UHPLC and an Agilent 6550 iFunnel Q-TOF LC/MS System. Agilent Technologies, publication 5991-3600EN.