High-Resolution Separation of Cysteine-Linked Antibody-Drug Conjugate Mimics Using Hydrophobic Interaction Chromatography

Significance of the Topic

Monoclonal antibody-drug conjugates (ADCs) combine the targeting ability of antibodies with potent cytotoxic agents, offering enhanced therapeutic indices in oncology and other fields. Accurate characterization of drug-to-antibody ratio (DAR) distribution is essential for ensuring product safety, efficacy, and batch consistency.

Objectives and Study Overview

This study evaluates a polymer-based MAbPac HIC-Butyl column for high-resolution separation of cysteine-linked ADC mimics. The goal is to demonstrate reliable resolution of species with DAR values ranging from 0 to 8, supporting robust monitoring during ADC development.

Methodology and Instrumentation

The analysis was performed on a Thermo Scientific Dionex UltiMate 3000 BioRSLC system using a MAbPac HIC-Butyl column (4.6 × 100 mm, 5 µm). Mobile phase A comprised 1.5 M ammonium sulfate and 50 mM sodium phosphate (pH 7.0) with 5 % 2-propanol, while mobile phase B contained 50 mM sodium phosphate (pH 7.0) with 20 % 2-propanol. A gradient from 100 % A to 100 % B over 15 minutes at 1.0 mL/min and 25 °C was applied. Detection was carried out at 280 nm, and data processing used Chromeleon 6.8.

Used Instrumentation

• Thermo Scientific Dionex UltiMate 3000 BioRSLC system
• SR-3000 Solvent Rack
• LPG-3400RS Biocompatible Pump
• WPS-3000TBRS Autosampler
• TCC-3000RS Column Compartment
• VWD-3400RS Variable Wavelength Detector
• MAbPac HIC-Butyl column (4.6 × 100 mm)

Main Results and Discussion

Separation of cysteine-conjugated ADC mimics revealed distinct peaks for DAR 0 through DAR 8, with retention time increasing alongside drug load. Optimal resolution was achieved with 5 % 2-propanol in mobile phase A and 20 % in B at 25 °C. Absence or excess of organic modifier impaired peak shape or induced denaturation. Notably, the DAR 6 species exhibited four hydrophobic variants, underscoring intravariant heterogeneity. Low, moderate, and high drug-load samples produced expected distributions, confirming sensitivity to DAR variations.

Benefits and Practical Applications

• Enables precise DAR distribution profiling for ADC quality control
• Serves as an orthogonal technique to size-exclusion and ion-exchange chromatography
• Resolves minor hydrophobic variants for stability and comparability studies
• Facilitates development of GMP-compliant analytical methods

Future Trends and Potential Applications

Emerging directions include coupling high-resolution HIC with mass spectrometry for direct variant identification, designing advanced stationary phases for improved selectivity, and automating workflows for high-throughput QC. Real-time process monitoring and multi-attribute method integration promise enhanced ADC characterization throughout development and manufacturing.

Conclusion

The MAbPac HIC-Butyl column delivers robust, high-resolution separation of cysteine-linked ADC mimics across a broad DAR range. Optimized mobile phase composition yields sharp, well-resolved peaks and reveals complex variant profiles, supporting comprehensive ADC analysis.

References

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