An Integrated Workflow for Peptide Mapping of Monoclonal Antibodies

Significance of the topic

Peptide mapping of monoclonal antibodies (mAbs) is essential for confirming amino acid sequences and monitoring post-translational modifications (PTMs) that can affect drug safety and efficacy. High sensitivity and reproducibility are critical in biopharmaceutical development and quality control.

Objectives and overview of the study

This study presents an integrated, high-throughput workflow combining automated sample preparation, high-resolution liquid chromatography–tandem mass spectrometry (LC–MS/MS), and automated data analysis to streamline mAb peptide mapping. The goal was to improve assay reproducibility, scalability, and throughput while maintaining comprehensive sequence coverage and accurate PTM quantification.

Methodology

Sample preparation was carried out on an automated liquid-handling platform using Agilent AssayMAP Bravo with in-solution digestion and peptide cleanup applications. The process included protein denaturation, reduction, alkylation, Trypsin/Lys-C digestion in a 96-well format, and desalting on C18 cartridges.

Instrumentation

• Agilent AssayMAP Bravo liquid-handling robot
• Agilent 1290 Infinity II LC system with AdvanceBio Peptide Mapping column (2.1×150 mm, 2.7 μm)
• Agilent 6550 iFunnel Q-TOF LC/MS system with Dual JetStream ESI source
• Agilent MassHunter BioConfirm software v. B.08

Main results and discussion

The workflow achieved over 95% average protein sequence coverage across eight replicates for sample loads ranging from 0.5 to 100 μg. LC injection replicates showed coefficient of variation (CV) values around 1–2%, and full sample-preparation replicates exhibited CVs averaging 6% for peptide peak areas. The method reliably identified and localized methionine oxidation and asparagine deamidation sites with site-specific MS/MS fragment assignment and quantified relative modification levels. Dynamic exclusion and extended dynamic range acquisition enabled comprehensive PTM analysis.

Benefits and practical applications of the method

• High throughput and walk-away automation reduce hands-on time and improve laboratory efficiency
• Scalable reproducibility supports large-scale studies such as clone selection and stability testing
• Comprehensive sequence and PTM analysis enhances biopharmaceutical characterization and comparability

Future trends and opportunities

Integration of affinity purification modules (e.g., protein A/G) to expand applicability to complex matrices, coupling with next-generation mass spectrometers for deeper PTM coverage, and linking with laboratory information management systems (LIMS) and AI-driven data interpretation to further accelerate antibody therapeutic development.

Conclusion

The described integrated workflow unites automated sample prep, advanced LC–MS/MS, and software-driven analysis into a reproducible platform delivering high sequence coverage and precise PTM quantitation for mAb peptide mapping. This approach enhances throughput and consistency, supporting robust biopharmaceutical development and quality control.

References

1. Greer FM. MS Analysis of Biopharmaceutical Products. Innov Pharm Technol. 2001:83–90.
2. 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.
3. Robinson NE. Protein Deamidation. Proc Natl Acad Sci U S A. 2002;99(8):5283–5288.
4. 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.