Convenient and Reliable Analysis of Antibody Drug Conjugates

Importance of the Topic

Antibody drug conjugates (ADCs) represent a transformative class of targeted cancer therapies. Precise measurement of drug-to-antibody ratio (DAR) is essential for ensuring consistent potency, safety, and batch-to-batch comparability in biopharmaceutical development.

Study Objectives and Overview

This application note describes a hydrophobic interaction chromatography (HIC) method using a ternary gradient with isopropanol as an organic modifier to determine the DAR of brentuximab vedotin. The goal is to achieve clear separation of DAR species and high reproducibility on the Agilent 1260 Infinity II Prime Bio LC system.

Methodology

• Sample Preparation: Brentuximab vedotin (Adcetris) dissolved at 100 mg/mL in a 50:50 mixture of water and buffer A.
• Buffer Systems: (A) 1.5 M ammonium sulfate in 50 mM phosphate buffer, pH 7; (B) 50 mM phosphate buffer, pH 7; (C) isopropanol.
• Gradient Conditions: 0–25 min from 55% A/40% B/5% C to 0% A/75% B/25% C; stop time 35 min; post time 10 min.
• Chromatographic Parameters: Flow rate 0.4 mL/min; column temperature 25 °C; detection at 280 nm; injection volume 15 µL; sample thermostat 10 °C.

Instrumentation

The system comprised an Agilent 1260 Infinity II Bio Flexible Pump (quaternary), 1290 Infinity II Bio Multisampler with thermostat, 1290 Infinity II Multicolumn Thermostat, and a 1290 Infinity II Variable Wavelength Detector with biocompatible micro flow cell. An Agilent AdvanceBio HIC column (3.5 µm, 4.6 × 100 mm) was employed, controlled by OpenLab CDS v2.5.

Results and Discussion

• Separation Performance: Five DAR species (D0–D8) corresponding to 0, 2, 4, 6, and 8 drug moieties were baseline resolved.
• Reproducibility: Seven consecutive injections yielded retention time RSDs < 0.055% and peak area RSDs < 0.46% (except D8 at 4.5%), demonstrating robustness under high-salt/organic-solvent mixing.
• DAR Calculation: Integration of species areas produced an average DAR of 3.3, aligning with literature values.

Benefits and Practical Applications

This HIC approach delivers a rapid, reproducible method for ADC characterization, supporting quality control, process development, and regulatory compliance in biopharmaceutical workflows.

Future Trends and Applications

Potential developments include:

1. Coupling HIC with mass spectrometry for detailed structural insights.
2. Automated sample handling to increase throughput.
3. Extension to diverse ADC constructs and complex protein conjugates.

Conclusion

Employing a ternary gradient with isopropanol on the Agilent 1260 Infinity II Prime Bio LC enables accurate, reproducible DAR determination of brentuximab vedotin. The system’s corrosion-resistant design and flexible pumping ensure reliable performance under challenging conditions.

References

• McCombs JR, Owen SC. Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry. J Am Assoc Pharm Sci. 2015;17(2):339–351.
• Rodriguez-Aller M, et al. Practical Method Development for Separation of Monoclonal Antibodies and Antibody-Drug-Conjugate Species in Hydrophobic Interaction Chromatography. J Pharm Biomed Anal. 2016;118:393–403.