Investigation of Antibody Drug Conjugate’s Fast Clearance: Assay or Biology?

The presentation will provide an overview of the analytical techniques, development, and validation of assays for ADC bioanalysis. The application of these methods in support of a pre-clinical study enabling FIH to characterize the toxicokinetics (TK) and immunogenicity of the ADC will be described. Results of the study indicating much accelerated clearance of the ADC after the second dosing (on Day 22) led to an investigation whether antidrug antibodies (ADA) interfered with the ligand binding assay (LBA) or if the observation truly describes the biology and the pharmacokinetics of the ADC. Numerous experiments were designed to conduct the investigation and an orthogonal method, i.e., LC-MS/MS was developed and validated to determine the ADC concentrations. This indirect method to quantify ADC by measuring the total payload (conjugated and free payload, knowing that there was insignificant free payload in the samples) was based on enzymatic digestion (papain) to release the conjugated payload, and was validated and used to re-analyze study samples. Results of this sample re-analysis did not change the overall conclusions of the TK study, and there was excellent correlation of LBA and LC-MS/MS data in all sample with only low or no ADA. ADA interference with the LBA TK assay was observed in cases of high level of ADA formation (after 2nd dosing on Day 22), and hence a switch to the LC-MS/MS platform for future study support (FIH) was undertaken.