Analyzing ADCs by HIC

Hello again, everybody. This week my colleague Andy is back to talk about HIC method development for ADCs. As if mAbs weren’t tough enough, ADCs present another level of complexity, and require special consideration to be sure that you get accurate results.


ADC’s often post additional challenges beyond mAbs – learn what to watch out for and how to adjust your method.

The incorporation of hydrophobic drug molecules into a monoclonal antibody (mAb) forming an antibody drug conjugate (ADC) results in numerous different molecular species with varying degrees of hydrophobicity. This means hydrophobic interaction chromatography (HIC) is an ideal technique for analyzing the all-important drug to antibody ratio (DAR).

Figure 1

However, the range of hydrophobicity presents certain important challenges:

(1) Sample preparation must result in complete dissolution of the sample.
(2) HIC gradient conditions must result in complete elution of all variants.

Too much ammonium sulfate in the sample solution could result in DAR 6 and DAR 8 variants not being fully dissolved which may result in an underestimated DAR value. Similarly, the gradient conditions need to ensure elution of all DAR species. This elution is achieved by including propan 2 ol as organic modifier.

Start by dissolving the sample in the dilute aqueous mobile phase buffer and then add the high ammonium sulfate mobile phase to dilute the sample to the required level. Do this in a clear container so that any signs of cloudiness (indicating sample precipitation) can be prevented by adding more of the high aqueous mobile phase buffer as required

Following the recommendations in the Quick Start Guide, use a gradient of 1–0 M ammonium sulfate with a simultaneous gradient of 5–25 % propan-2-ol. This will ensure the complete elution of all species. Because of the high eluent viscosity, a lower flow rate will be beneficial. A 10 cm column will give maximum resolution; a 3 cm column will enable faster separations

By choosing the appropriate mobile phase and gradient conditions, it is possible to separate ADC DAR variants in a very short analysis time using new Agilent AdvanceBio HIC columns (Application Note 5994-0149EN has more details).


Thanks again to Andy and Sandeep! I’ll be back in two weeks to wrap up our HIC series with some tips and reminders to keep HIC running smoothly.

If you have any questions, contact the author