Peptide Mapping on Monoclonal Antibodies Using Agilent 6545XT AdvanceBio LC/Q-TOF and Protein Metrics Byos Software

Importance of the Topic

Peptide mapping using liquid chromatography–mass spectrometry (LC/MS) is a cornerstone technique for detailed characterization of monoclonal antibodies and other biotherapeutics. It enables identification of sequence variants and post-translational modifications (PTMs) that influence product efficacy, safety, and stability. The evolution of multi-attribute methods has increased demand for robust, reproducible workflows that integrate sample preparation, high-resolution data acquisition, and streamlined data analysis.

Study Objectives and Overview

This application note presents a unified workflow for peptide mapping of NISTmAb reference material under control and chemically stressed conditions. The goals are to demonstrate compatibility between Agilent sample preparation and LC/Q-TOF instrumentation and vendor-neutral data processing with Protein Metrics Byos software; to achieve comprehensive sequence coverage; and to quantify key critical quality attributes such as deamidation and oxidation.

Methodology

Three NISTmAb sample sets were prepared: untreated control, high pH–induced deamidation (Tris-HCl, pH 8.7, 37 °C, six days), and hydrogen peroxide–induced oxidation (0.002% H2O2, room temperature, overnight). All samples were reduced, alkylated with iodoacetamide, and digested with trypsin on the Agilent AssayMAP Bravo Protein Sample Prep platform, followed by desalting.

Instrumentation

• Agilent AssayMAP Bravo Protein Sample Prep platform (G5571AA)
• Agilent 1290 Infinity II LC system with high-speed pump, multisampler with cooler, and thermostatted column compartment
• Agilent ZORBAX RRHD StableBond C18 column, 2.1 × 150 mm, 1.8 μm
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Jet Stream ESI source

Data Acquisition and Processing

Peptides were separated at 0.25 mL/min using a gradient from 1% to 35% organic over 70 minutes at 50 °C. MS data were acquired at up to 5 spectra/sec in AutoMS2 mode. Raw data were imported into Protein Metrics Byos (v3.10-52) using the preconfigured multi-attribute method workflows for peptide identification, PTM annotation, chromatogram alignment, and batch comparison.

Main Results and Discussion

Sequence coverage exceeded 98% for the heavy chain and 94% for the light chain. Byos automatically annotated PTM peptides and their unmodified counterparts, displaying extracted ion chromatograms, MS2 spectra, and mass error distributions. Chromatograms from control and stressed samples were aligned using retention time warping. Increased levels of Asn deamidation and specific Met oxidation sites were quantified and flagged as critical quality attributes.

Benefits and Practical Applications

• A fully automated sample preparation and analysis workflow reduces manual variability and increases throughput.
• Vendor-neutral data processing supports integration of data from multiple instrument platforms.
• Automated annotation and reporting streamline regulatory submissions and in-process monitoring.

Future Trends and Opportunities

Advances in data independent acquisition and artificial intelligence-driven data analysis are expected to enhance sensitivity and specificity of peptide mapping. Integration with real-time process analytical technology (PAT) and bioprocess control loops will improve in-line quality monitoring. Development of standardized multi-attribute method libraries may further harmonize data across labs and platforms.

Conclusion

The described LC/MS peptide mapping workflow combining Agilent sample preparation and LC/Q-TOF instrumentation with Protein Metrics Byos software delivers high sequence coverage, reliable PTM quantification, and streamlined reporting. This integrated solution supports robust characterization and quality control of monoclonal antibodies and other biotherapeutics.

References

1. Rogers RS et al. A View on the Importance of Multi-Attribute Method for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy. The AAPS Journal 2018, 20(7).
2. Automation of Sample Preparation for Accurate and Scalable Quantification and Characterization of Biotherapeutic Proteins Using the Agilent AssayMAP Bravo Platform. Agilent Technologies Application Note, publication 5991-4872EN, 2016.
3. Quantitation of Chemical-Induced Deamidation and Oxidation on Monoclonal Antibodies Using Agilent 6545XT AdvanceBio LC/Q-TOF and Agilent MassHunter BioConfirm Software. Agilent Technologies Application Note, publication 5994-0406EN, 2018.