Butyl-Dependant Hydrophobic Interaction Liquid Chromatography: A Technique to Characterize Antibody-Drug Conjugates Based on Hydrophobic Interactions

To analyze the conjugate, equilibrate the HPLC-HIC column with 100% high-salt buffer for 5 minutes. Then, mix 15 microliters of a 1 mg/mL solution of trastuzumab linked to MMAE, with 15 microliters of 2X high-salt buffer in a vial. Inject the mixture onto the HPLC column. Elute in an isocratic 1 mL/min flow with 100% high-salt buffer for one minute.

Follow this with a 15-minute gradient from 100% to 0% of high-salt buffer in low-salt buffer. Monitor the elution at 280 nanometers. Integrate the peaks on the chromatogram with retention times of 7.5, 9.2, and 11.5 minutes, which correspond to trastuzumab with zero, one, and two conjugated toxins respectively.

Now, calculate the DAR by adding up the areas of the peaks at 9.2 minutes, which has a weight of 1, and 10.5 minutes, which has a weight of 2, and divide by the total area of the three peaks.