Quantification for Antibody-Conjugated Drug in Trastuzumab Emtansine and Application to In Vitro Linker Stability and In Vivo Pharmacokinetic Study in Rat Using an Immuno-Affinity Capture Liquid Chromatography-Mass Spectrometric Method

Abstract

Trastuzumab emtansine (T-DM1, brand name: Kadcyla®) is the first FDA-approved antibody-drug conjugate (ADC) for metastatic human epidermal growth factor receptor 2 positive (HER2+) breast cancer. It consists of three components: trastuzumab, an anti-HER2 monoclonal antibody, maytansinoid (DM1) as a cytotoxic drug, and maleimidomethyl cyclohexane-1-carboxylate (MCC) as a linker. In particular, the MCC linker is known as a non-cleavable linker and has a feature of being conjugated to DM1 by a covalent thioether bond. In this study, we developed an immuno-affinity capture liquid chromatography-mass spectrometric (LC-MS/MS) assay for quantifying the antibody-conjugated drug (acDrug) component of T-DM1. To quantify acDrug, desulfurated DM1 was prepared using a chemical desulfuration pretreatment and quantified as an acDrug. A quadratic regression (weighted 1/concentration), with equation y = ax2 + bx + c, was used to fit the calibration curves over the concentration range of 17.09~1709.44 ng/mL for the acDrug of T-DM1. The quantification run met the in-house acceptance criteria of ±25% accuracy and precision values for the quality control (QC) samples. In conclusion, an immuno-affinity capture LC-MS/MS assay was successfully developed to quantify acDrug of T-DM1 and applied to evaluate in vitro plasma linker stability and preclinical pharmacokinetic (PK) study in rats. This assay could be helpful when applied to other ADCs with the same linker-cytotoxic drug platform.

Generation of anti-SN38 antibody for loading efficacy and therapeutic monitoring of SN38-containing therapeutics

“…Three main parameters must be evaluated for ADME and PK studies of the ADCs: total antibody, drug conjugated to the antibody, and unconjugated drug. The drug conjugated to the antibody is the same active form of ADC and plays a crucial role in the dose-response analysis of ADCs [ 62 ]. The drug conjugated to the antibody is calculated using the average number of drugs conjugated to each antibody (DAR).…”