ADC Analysis by Hydrophobic Interaction Chromatography

  • Ryan FlemingEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2078)


Hydrophobic interaction chromatography (HIC) is a traditional technique used for the separation, purification, and characterization of proteins. As the number of antibody–drug conjugates (ADCs) continues to increase in clinical trials, HIC and other orthogonal methods utilizing changes in hydrophobicity are being used for ADC characterization and analysis. Unlike other techniques, HIC uniquely allows for protein analysis under mild nondenaturing conditions that preserve the native structure and activity of the molecules. Analysis of the ADC in its native form is advantageous. Herein, we describe a generic HIC protocol for the screening, analysis, and characterization of ADCs using an ammonium sulfate buffer and a high-pressure liquid chromatography system. Parameters affecting data quality and interpretation are addressed. In addition, several recommendations are included for method optimization and troubleshooting.

Key words

Hydrophobic interaction chromatography (HIC) Antibody–drug conjugates (ADCs) Hydrophobicity Retention time Drug-to-antibody ratio (DAR) Payload 



I thank Dr. Nazzareno Dimasi, Dr. Amit Kumar, and Benjamin Ruddle for useful discussion, critical review, and comments.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Antibody Discovery and Protein Engineering, Biologic Therapeutics, AstraZenecaGaithersburgUSA

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