Detection of antibody-conjugate payload in cynomolgus monkey serum by a high throughput capture LC–MS/MS bioanalysis method

Abstract

Antibody-drug conjugate (ADC) plays a vital 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 pharmacokinetic (P.K.) is a quantitative tool to understand the drug concentration in the body. To characterize the whole drug carefully, sophisticated bioanalysis was required. ADC bioanalysis generally needs multiple analysis strategies, which can accurately quantify total antibody (TAb), antibody-drug conjugate (ADC), antibody-conjugate payload (ac-payload), and 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 ac-payload (such as MMAE) in cynomolgus monkey serum. This method was allowed to determinate the Drug to Antibody Ratio (DAR), obtained by n of ac-payload/ n of TAb. In addition, the technique could significantly improve the throughput of the pre-coated antibody on a 96-well plate. Besides, this 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 was successfully applied to the change of average DAR in P.K. study.