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

Antibody-drug conjugates - ADCs - are formed by attaching a target-specific monoclonal antibody with cytotoxic drugs via suitable linkers. The stochastic conjugation process creates ADCs with different numbers of drug moieties.

The ADC species are characterized by their drug-to-antibody ratio or DAR, which determines their therapeutic efficacy. The hydrophobicity of ADCs makes them suitable candidates for hydrophobic interaction liquid chromatography or HIC-based characterization.

To begin, assemble a chromatography column containing an agarose matrix-based stationary phase. The matrix is cross-linked to butyl moieties - the hydrophobic alkyl functional groups.  Equilibrate the column with a high salt binding buffer. Follow it by loading the ADC solution onto the column.

Salt ions in the buffer displace water molecules around ADCs, exposing their hydrophobic regions. This enables ADCs to bind to the butyl ligands via hydrophobic interactions. ADCs with a higher number of bound drug moieties show stronger interaction with the column.

Slowly replace the high salt buffer with the elution buffer having a low salt concentration. This process of progressively decreasing the salt concentration is termed as gradient elution.

The decrease in salt ions re-enables water molecules to surround ADCs, disrupting their interaction with ligands. ADCs with the lowest number of bound drug moieties are eluted first, followed by species with higher DAR.

Monitor the elution to characterize the ADCs according to their DAR.

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.