Peptide Mapping of an Antibody-Drug Conjugate Using PeptideWorks Tryptic Protein Digestion Kit

Importance of the Topic

Peptide mapping of antibody-drug conjugates (ADCs) is essential for confirming protein sequence integrity and monitoring conjugation sites. ADC analysis supports quality assessment and structural characterization in biopharmaceutical development.

Study Objectives and Overview

This study evaluates Waters PeptideWorks Tryptic Protein Digestion Kit for streamlined ADC peptide mapping. It compares maps of trastuzumab and its ADC (T-DM1), highlighting workflow automation and reproducibility.

Methodology and Instrumentation

Sample preparation used the PeptideWorks kit with chemically optimized recombinant RapiZyme™ Trypsin in a manual or automated (Andrew+ Pipetting Robot) workflow under 2.5 hours. Digests were separated on an XSelect Premier Peptide CSH C18 column (2.1×150 mm, 2.5 µm) with a 0.5–50% acetonitrile gradient (0.1%/0.08% formic acid) at 60 °C, 0.2 mL/min. Peptide detection by UV at 252 nm and Xevo G2 XS mass spectrometer (ESI+, m/z 50–2000) with waters_connect software.

Main Results and Discussion

ADC samples exhibited additional late-eluting peaks absent in the native mAb, corresponding to hydrophobic drug-conjugated peptides. Mass spectrometry confirmed diastereomer pairs and identified conjugation at lysine residues through extracted ion chromatograms (m/z 616.27, +3; 923.90, +2) and MS/MS fragment ions (y1, y2, b2, b3). Sequence coverage improved from 92% (mAb) and 95% (ADC) to 98% when accounting for missed cleavages at conjugation sites.

Practical Benefits and Applications

The PeptideWorks kit enables rapid, automatable sample preparation, yielding reproducible peptide maps and detailed insight into drug conjugation patterns. This approach supports quality control, comparability studies, and biotherapeutic characterization workflows.

Future Trends and Potential Applications

Integration with high-throughput robotics and advanced mass spectrometry platforms will further accelerate ADC characterization. Emerging informatics tools will enhance data analysis, enabling deeper structural insights and broader application to diverse bioconjugates.

Conclusion

Waters PeptideWorks Tryptic Protein Digestion Kit offers a fast, reliable solution for ADC peptide mapping. The workflow delivers high sequence coverage, clear identification of conjugation sites, and compatibility with automation, making it a valuable asset in analytical biochemistry of ADCs.

References

• Hanna CM et al. Quick and Robust Sample Preparation for Tryptic Peptide Mapping With the PeptideWorks Kit Using Simple, Automatable Workflows. Waters Application Note 720008019. July 2023.
• Waters. PeptideWorks Tryptic Protein Digestion Kit Care and Use Manual. Document 720007980. October 2024.
• Waters. RapiZyme Trypsin Care and Use Manual. Document 720007851. February 2023.
• Chen L et al. In-depth structural characterization of Kadcyla (ado-trastuzumab emtansine) and its biosimilar. mAbs 8(7):1210–1223, 2016.
• Luo Q et al. Structural Characterization of a Monoclonal Antibody–Maytansinoid Immunoconjugate. Anal. Chem. 88(1):695–702, 2016.