Properties of Drug Linkers Affecting Hydrophobic Interaction Chromatography for Determining the Drug-to-Antibody Ratio of Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) are a fascinating modality in cancer therapy that combine the specificity of monoclonal antibodies (mAbs) with the cytotoxicity of potent drugs. To ensure the product quality of ADCs, the drug-to-antibody ratio (DAR), a critical quality attribute (CQA), must be evaluated at quality control (QC) testing. Hydrophobic interaction chromatography (HIC) is considered the gold standard for DAR determination due to advantages such as the ability to evaluate drug load distribution under native conditions. For some ADCs, however, DAR cannot be determined by HIC analysis due to poor peak separation of DAR variants, but the cause of this poor peak separation is unclear. In this study, we investigated which ADC properties impact the peak separation of DAR variants in HIC. HIC analyses of ADCs with different mAb and drug linker (DL) combinations indicated that peak separation was impacted by DL properties rather than mAb properties. A systematic comparison using model ADCs prepared with trastuzumab and various drug linkers revealed that the linker length is a critical structural determinant of peak separation. We hypothesized that a linker that was too short restricted the steric accessibility of drug moieties on ADCs, resulting in weak retention on the stationary phase of the HIC column. Accordingly, we investigated the correlation between linker length and steric accessibility using an enzyme-linked immunosorbent assay (ELISA) to evaluate the binding affinity of biotin, which is conjugated to ADCs as a model drug, for streptavidin. The results supported the notion that short linkers are less preferable for the steric accessibility of the drug moieties. Our findings will aid in understanding the mechanism of HIC peak separation and contribute to the selection of appropriate analytical methods based on ADC structures, even within the accelerated development timelines required to supply new drugs to patients in need.