Quantitative Analysis of ADC Components - Lig

There are both large and small ADC molecules. A variety of biological analysis methods and platforms are needed to analyze the diversity of ADCs. Conventional LC-MS/MS small-molecule assays are often used to analyze uncoupled payloads and DAR distributions over time, while ligand binding assays (LBA) and LC-MS/MS can also be used to analyze total antibodies (TAb) and total ADCs. The choice of analysis platform depends on the availability of key reagents, the required analytical sensitivity, and the different problems that need to be addressed at various stages of drug development. In this article, the classical LBA and LC-MS/MS methods for quantitative analysis of ADC components are briefly introduced. 1. Ligand binding assay (LBA) LBA is the gold standard for macromolecular bioanalysis. LBA is typically used to measure TAb and total ADC, utilizing the mAb or payload component of the ADC, respectively. 1.1 LBA determination of total antibody (TAb) Total Antibody (TAb) detection monitors antibody concentrations regardless of whether the payload is attached to the antibody, i.e., fully coupled, partially coupled, and completely uncoupled ADCs. This analysis is used for the overall PK evaluation of the ADC. In TAb analysis, specific reagents are used, such as recombinant target proteins, as well as general reagents, such as antibodies against the whole human IgG framework region or the (Fab')2 or Fc region, or against the light chain (LC) or the H+L chain region. These reagents bind to the antibody component of ADC regardless of its DAR value. Although these reagents do not directly bind to the payload, the payload can indirectly affect the binding of these reagents to the Ab component of the ADC due to steric hindrance. The interference is more pronounced in the species with higher DAR. Therefore, the analysis may not accurately calculate all of the expected DAR classes in the systemic circulation, thus affecting the overall PK characteristics of the ADC observed.