High-Throughput Multispecies Parallelized PlasmaStability Assay for the Determination and Characterization of Antibody–DrugConjugate Aggregation andDrug Release

摘要

The stability of antibody–drug conjugates (ADCs) in circulation is critical for maximum efficacy and minimal toxicity. An ADC reaching the intended target intact can deliver the highest possible drug load to the tumor and reduce off-target toxicity from free drug in the blood. As such, assessment of ADC stability is a vital piece of data during development. However, traditional ADC stability assays can be manually intensive, low-throughput, and require large quantities of ADC material. Here, we introduce an automated, high-throughput plasma stability assay for screening drug release and aggregation over 144 h for up to 40 ADCs across five matrices simultaneously. The amount of ADC material during early drug development is often limited, so this assay was implemented in 384-well format to minimize material requirements to <100 μg of each ADC and 100 μL of plasma per species type. Drug release and aggregation output were modeled using nonlinear regression equations to calculate formation rates for each data type. A set of 15 ADCs with different antibodiesand identical valine–citrulline–p-aminobenzylcarbamate–monomethylauristatinE linker-drug payloads was tested and formation rates were comparedacross ADCs and between species, revealing several noteworthy trends.In particular, a wide range in aggregation was found when alteringonly the antibody, suggesting a key role for plasma stability screeningearly in the development process to find and remove antibody candidateswith the potential to create unstable ADCs. The assay presented herecan be leveraged to provide stability data on new chemistry and antibodyscreening initiatives, select the best candidate for in vivo studies,and provide results that highlight stability issues inherent to particularADC designs throughout all stages of ADC development.