Fraction Analysis of Cysteine‑Linked Antibody-Drug Conjugates Using Hydrophobic Interaction Chromatography

Importance of the Topic

Hydrophobic interaction chromatography (HIC) is a key tool for characterizing the drug-to-antibody ratio and heterogeneity of cysteine-linked antibody-drug conjugates (ADCs). Accurate fractionation of individual drug load species supports safety, efficacy, and quality control in biopharmaceutical development.

Objectives and Study Overview

This application note demonstrates peak-based fraction collection of brentuximab vedotin using HIC on a metal-free Agilent 1260 Infinity II Bio-Inert LC system, followed by re-analysis of collected fractions via both HIC and reversed-phase chromatography to confirm purity and identity of each drug-to-antibody ratio species.

Methodology and Instrumentation

The workflow combined hydrophobic interaction chromatography and selective peak triggering for fraction collection. Key steps included

• HIC separation using a generic HIC column with ammonium sulfate gradient
• Automated, peak-based fraction collection using the Agilent 1260 Infinity II Bio-Inert Fraction Collector in volume slice recovery mode to avoid sample loss
• Re-analysis of fractions by the same HIC method without collection enabled
• Confirmation of species identity by reversed-phase chromatography on a PLRP-S column after TCEP reduction

Instrumentation Used

• Agilent 1260 Infinity II Bio-Inert Pump and Multisampler with sample cooler
• Multicolumn Thermostat with bio-inert heat exchanger
• Diode Array Detector WR with bio-inert flow cell
• 1260 Infinity II Bio-Inert Fraction Collector
• Agilent OpenLAB CDS ChemStation Edition software
• Columns: generic HIC, Agilent PLRP-S 300Å 2.1 × 50 mm, 3 µm

Main Results and Discussion

HIC separation of brentuximab vedotin yielded five distinct peaks corresponding to drug loads of 0, 2, 4, 6, and 8 MMAE molecules. Peak-based fraction collection precisely isolated each species. Re-analysis by HIC confirmed high purity of collected fractions by matching retention times. Subsequent reversed-phase chromatography of TCEP-reduced fractions on the PLRP-S column verified light-chain and heavy-chain species carrying the expected drug load increments, confirming correct fractionation and peak assignment.

Benefits and Practical Applications

• Metal-free flow path prevents corrosion from high-salt mobile phases in HIC
• Peak-based triggering with volume slice recovery ensures accurate, reproducible fraction collection and prevents sample loss
• Reversible fraction re-analysis enables robust validation of ADC heterogeneity for QA/QC and research laboratories

Future Trends and Possibilities

Advances may include integration of mass spectrometry detection for direct molecular weight confirmation, microfluidic peak collection for reduced solvent consumption, and automated coupling of HIC fractionation with downstream peptide mapping or glycan analysis to streamline ADC characterization workflows.

Conclusion

The Agilent 1260 Infinity II Bio-Inert LC system with peak-based fraction collection provides a reliable, corrosion-free platform for isolating and verifying individual ADC species by HIC and reversed-phase chromatography. This approach enhances the analytical toolbox for precise ADC drug-to-antibody ratio profiling.

Reference

1. Schneider S. Analysis of Cysteine-linked Antibody Drug Conjugates using Hydrophobic Interaction Chromatography on the Agilent 1260 Infinity II Bio-Inert LC, Agilent Technologies Application Note 5991-8493EN, 2017
2. Younes et al. Brentuximab Vedotin (SGN-35) for Relapsed CD30-Positive Lymphomas, New England Journal of Medicine 2010, 363, 1812–1821
3. Wakankar A. Analytical methods for physicochemical characterization of antibody drug conjugates, mAbs 2011, 3:2, 161–172