10 ADC candidates with an average DAR at 2 were screened for the lead nomination. A single dosing of each ADC candidate was given via i.v. injection to monkeys and serum samples were collected for DAR profiling, and the quantifications of total antibody, ADC and free payload (MMAE in this case). DAR profiling was performed to reveal the various biotransforms of ADC in vivo. The PK (pharmacokinetics) profiles of ADC, together with total antibody and free payload, are used to evaluate the dose-response relationship for efficacy and also safety.
While DAR profiling is completely dependent on HRMS, the quantification of ADC and total antibody are conventionally conducted on LBA platform. However, due to the high demanding for the specific reagent used for ADC quantification, LBA sometimes has long leading time, waiting for the reagent, and/or has interference problem, due to the poor quantify of the reagent. We established a multiplex MS-based assay in which one sample was used to produce three sets of quantitative data including total antibody, ADC and free payload. The LLOQs (lower limit of quantification) achieved were 200, 200, 0.01 ng/mL respectively. No specific reagents were needed resulting in a quick turn-around time. This is a big advantage at the early drug discovery stage. A comparison of LBS and MS-based platforms were discussed.
Learning Objectives:
Drug-antibody ratio (DAR) profiling in monkey serum samples was performed using IC (immunocapture) followed by middle-down HR-MS (high resolution mass spectrometry) analysis.
A multiplex method for quantifying total antibody, ADC (antibody-drug conjugate), free toxin in monkey serum samples for 10 ADC candidates was developed using bead-based IC and signature-peptide based LC-MS/MS analysis.
The advantage and limitation of using MS-based approach for the bioanalysis of ADC therapeutics comparing to LBA (ligand binding assay) were discussed.
