High-Resolution Mapping of Drug Conjugated Peptides in an ADC Digest

Importance of the Topic

Antibody–drug conjugates represent a growing class of biotherapeutics that combine monoclonal antibodies with cytotoxic agents. High‐resolution peptide mapping is critical for locating conjugation sites and ensuring product quality in ADC development and manufacturing.

Study Objectives and Overview

This study evaluates the use of the Agilent AdvanceBio Peptide Mapping Column and an Agilent 1290 Infinity LC system for detailed peptide mapping of Trastuzumab and its drug conjugate. By optimizing chromatographic conditions, the goal was to distinguish and identify peptides modified by cytotoxic payloads.

Methodology and Instrumentation

Tryptic digestion of the antibody and ADC was performed after reduction and alkylation under denaturing conditions. Peptides were separated using reversed‐phase LC with a 2.1×250 mm, 2.7 µm AdvanceBio column. Mobile phases consisted of 0.1% TFA in water (A) and 0.08% TFA in 90% acetonitrile (B). A 60‐minute gradient from 3% to 50% B at 0.5 mL/min and column temperature of 60 °C provided optimum resolution. UV detection was conducted at 210 nm and 252 nm.

Used Instrumentation

• Agilent 1290 Infinity Binary Pump with integrated vacuum degasser
• Agilent 1290 Infinity Autosampler
• Agilent 1290 Infinity Thermostatted Column Compartment
• Agilent 1290 Infinity Diode Array Detector with Max-Light flow cell

Main Results and Discussion

Optimization experiments revealed a peak capacity of 354 under the chosen gradient and flow conditions, significantly improving resolution compared to shorter columns. The peptide map of the ADC displayed distinct late‐eluting peaks between 40 and 60 minutes that were absent in the unconjugated antibody, corresponding to hydrophobic, drug‐linked peptides. Retention time and area RSD values were below 2%, demonstrating high method precision.

Benefits and Practical Applications

• Accurate localization of conjugation sites in ADCs
• Enhanced resolution of hydrophobic, drug‐modified peptides
• High reproducibility supports quality control and comparability studies

Future Trends and Applications

Emerging needs for higher throughput and deeper characterization will drive integration of mass spectrometry and automated data analysis. Novel stationary phases and AI‐powered workflows may further enhance ADC peptide mapping sensitivity, resolution, and speed.

Conclusion

The Agilent AdvanceBio Peptide Mapping Column coupled with the 1290 Infinity LC system delivers high‐resolution, reproducible peptide maps for ADC characterization. This approach enables clear identification of drug‐conjugated peptides, supporting robust analytical workflows in biotherapeutic development.

References

1. Gudihal R, Waddell K. Identification of Oxidation Sites on a Monoclonal Antibody Using an Agilent 1260 Infinity HPLC-Chip/MS System Coupled to an Accurate-Mass 6520 Q-TOF LC/MS. Agilent Technologies Application Note, 5990-8768EN, 2011.
2. Kim MT et al. Statistical Modeling of the Drug Load Distribution on Trastuzumab Emtansine (Kadcyla), a Lysine-Linked Antibody Drug Conjugate. Bioconjugate Chem. 2014;25:1223–1232.