Peptide Mapping of Monoclonal Antibody (mAb) Using LCMS-9030 (Q-TOF) Mass Spectrometerwith a Shim-pack GISS-HP Column

Importance of the Topic

Monoclonal antibody (mAb) biotherapeutics represent a rapidly expanding class of drugs, with peptide mapping serving as a cornerstone for confirming primary structure and ensuring product quality. Fast and reliable peptide mapping workflows are crucial for biosimilar development, regulatory compliance, and routine quality control.

Objectives and Study Overview

This study aimed to optimize a peptide mapping protocol for a bevacizumab biosimilar, reducing analysis time while maintaining comprehensive sequence coverage. Building on previous workflows requiring 120 minutes per run, the authors targeted a method under 45 minutes that still delivers accurate mass identification and full sequence confirmation.

Methodology and Instrumentation

The workflow comprises the following key steps:

• Sample preparation: Denaturation and reduction of 5 mg/mL bevacizumab biosimilar in Tris-HCl buffer, alkylation with iodoacetamide, and overnight trypsin digestion.
• Chromatography: Separation on a Shim-pack GISS-HP C18 column (3 µm, 150 × 3.0 mm) using mobile phases of 0.1% formic acid/0.01% TFA in water (A) and acetonitrile (B), at 0.5 mL/min with a 45-minute gradient.
• Mass spectrometry: Analysis on a Shimadzu LCMS-9030 Q-TOF with heated ESI in positive mode, scanning m/z 100–2000.

Instrumentation:

• Nexera X2 UHPLC system
• Shim-pack GISS-HP column, 3 µm particle size
• Shimadzu LCMS-9030 Q-TOF mass spectrometer

Main Results and Discussion

The optimized workflow delivered the following performance:

• Run time shortened from 120 to 45 minutes without loss of data quality.
• Excellent repeatability over six injections: retention time RSD <0.1%, peak area RSD <3%.
• Identification of 19 light-chain and 42 heavy-chain tryptic peptides, achieving 100% sequence coverage for the light chain and 99.8% for the heavy chain.
• Accurate mass measurements within 3 ppm of theoretical values.
• Detection of expected missed cleavages at KR, RR, and KK sites, and observation of C-terminal lysine truncation in heavy chain peptides.

Benefits and Practical Applications

The rapid, high-resolution peptide mapping method supports accelerated biosimilar characterization and robust quality control. It enables full primary sequence confirmation in less than one hour, improving laboratory throughput and confidence in product integrity.

Future Trends and Potential Applications

Emerging directions include:

• Integration with automated sample preparation platforms for higher throughput.
• Application of advanced data analysis tools and cloud-based workflows to speed up sequence assignment.
• Extension to other biotherapeutics, including multi-specific antibodies and fusion proteins.
• Combining peptide mapping with sub-unit analysis for comprehensive structural characterization.

Conclusion

The optimized 45-minute peptide mapping protocol on the Shimadzu LCMS-9030 Q-TOF provides high reproducibility, full sequence coverage, and precise mass accuracy. It represents a valuable tool for routine mAb characterization in biopharmaceutical research and quality control.

References

1. Shimadzu (Asia Pacific), “Peptide Mapping of Monoclonal Antibody (mAb) Using Nexera Bio with Q-TOF Mass Spectrometer for Full Sequence Confirmation”, Application News No. AD-0176, 2018.
2. Skyline software, MacCoss Lab, University of Washington.