An AdvanceBio HIC Column for Drug‑to-Antibody Ratio (DAR) Analysis of Antibody Drug Conjugates (ADCs)

Importance of the Topic

Understanding drug-to-antibody ratio (DAR) is essential for evaluating the potency, safety, and efficacy of antibody–drug conjugates (ADCs). Hydrophobic interaction chromatography (HIC) offers a gentle separation technique capable of resolving minor hydrophobicity differences among ADC variants without denaturing proteins.

Study Objectives and Overview

This study evaluates the performance of the Agilent AdvanceBio HIC column coupled with the 1260 Infinity II Bio-Inert LC system for rapid and accurate DAR analysis of cysteine-linked ADCs. It compares separation speed, resolution, and accuracy on different column lengths and gradient conditions.

Methodology

• Mobile phases: 50 mM sodium phosphate (pH 7.0), 2 M ammonium sulfate in 50 mM sodium phosphate (pH 7.0), propan-2-ol, and water
• Gradient elution to reduce salt concentration and increase organic modifier, promoting sequential elution of DAR variants
• Column configurations: 4.6 × 100 mm (standard 31 min run) and 4.6 × 30 mm (fast 8 min run)
• Typical flow rates: 0.5 mL/min for longer column, 1.0 mL/min for shorter column
• Injection volume: 5 μL; column temperature: 25 °C
• Sample preparation: partial reduction of monoclonal antibody and conjugation with payload via cysteine residues

Used Instrumentation

• Agilent 1260 Infinity II Bio-Inert LC system
• Bio-inert pump (G5654A), multisampler with cooler (G5668A), multicolumn thermostat (G7116A), and diode array detector (G7115A)
• Milli-Q A10 water purification system

Main Results and Discussion

The AdvanceBio HIC column demonstrated high resolution of DAR0 through DAR8 variants under mild conditions. Integration of peak areas provided a calculated DAR of approximately 4.04, in agreement with expected values. Optimization of the propan-2-ol gradient was critical to prevent adsorption of high-DAR species. Fast separations on the 30 mm column (8 min) yielded DAR values identical to the longer run (24 min) on the 100 mm column, highlighting the trade-off between speed and resolution.

Benefits and Practical Applications

• Preserves native structure of ADCs by avoiding harsh organic solvents and ion-pair reagents
• Enables accurate quantification of DAR for quality control and biopharmaceutical development
• Offers flexibility in run time and throughput by selecting appropriate column length and gradient

Future Trends and Potential Applications

Future developments may include coupling HIC separations with mass spectrometry for structural confirmation, automation of sample preparation workflows, and adaptation to novel ADC formats. Advances in column technology and mobile-phase chemistries will further enhance throughput and sensitivity for routine process monitoring and regulatory compliance.

Conclusion

The AdvanceBio HIC column on the Agilent 1260 Infinity II Bio-Inert LC system provides a robust, gentle, and versatile platform for rapid DAR analysis of cysteine-linked ADCs. Optimization of gradient and column parameters enables consistent DAR measurement across different run times, supporting efficient biopharmaceutical workflows.

Reference

1. Zuo S., Measuring Drug-to-Antibody Ratio (DAR) for Antibody Drug Conjugates (ADCs) with UHPLC/Q-TOF, Agilent Technologies Application Note 5991-6559EN, 2016.
2. Schneider S., Analysis of Cysteine-Linked Antibody Drug Conjugates, Agilent Technologies Application Note 5991-8493EN, 2017.