Detection of Antibody-Conjugate Payload in Cynomolgus Monkey Serum By A High Throughput Capture LC-MS/MS Bioanalysis Method

Abstract

Antibody-drug conjugated (ADC) plays an important role in oncology indications. The efficacy and toxicity of ADC generally depend on the concentration of the drugs in the body system, and physiologically-based pharmacokinetics (PK) is a quantitative tool to understand the drug concentration in the body. Understanding the PK of the ADC requires a sophisticated bioanalytical approach to carefully characterize the whole drug. ADC bioanalysis generally needs multiple analysis strategies, which can accurately quantify total antibody (TAb), antibody-drud conjugated (ADC), antibody-conjugated payload, free payload. In this work, we mainly described and validated a high throughput capture Liquid Chromatography tandem-Mass Spectrometry (LC-MS/MS) bioanalysis method to detect the concentrations of antibody-conjugated payload. This allowed for the determination of the DAR (Drug to Antibody Ratio) for in vivo samples by of antibody-conjugated payload/ of n TAb. In addition, the method significantly improved throughput by pre-coated antibody on 96-well plate and then the antibody-conjugated payload (ac-MMAE) was released by Cathepsin B. The method had no interference or carryover in endogenous substances and showed linearity (R2≥0.99) in the concentration range within 15.6 - 2000.0 ng/mL. The inter-run accuracy ranged from 75.8 to 120.0%, and precision was within ≤ 20.0%. Meanwhile, selectivity and the benchtop stability of the method were also validated. This optimization method has already been successfully applied to the change of average DAR in PK study.