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Antibody-drug conjugates (ADCs) are composed of cytotoxic drugs and antibodies linked together by a linker. The complexity and heterogeneity of ADC structures, along with the low concentrations of cytotoxic drugs released in vivo, pose significant challenges for ADC bioanalysis. Understanding the pharmacokinetic behavior, PK/PD, and TK of ADCs is essential for successful development. Therefore, accurate ADC bioanalytical methods are required to evaluate the intact ADC, total antibody, released small-molecule cytotoxins, and related metabolites. The choice of appropriate ADC bioanalytical methods for comprehensive analysis largely depends on the nature of the cytotoxic drug, the chemical linker, and the attachment site. Due to advancements in ligand-binding assays and mass spectrometry-related technologies, our understanding of the overall pharmacokinetics of ADCs has become more comprehensive and thorough.
Compared to antibody drugs, ADCs have more complex and heterogeneous structures. The design of ADCs aims to deliver the payload to target tissues or target cells via the specificity of monoclonal antibodies, and subsequently release it, while exposing normal tissues less, in hopes of improving the therapeutic dose and safety window for tumor treatment. ADCs specifically bind to high-expressed proteins (target antigens) on the surface of tumor cells via monoclonal antibodies to form ADC-antigen complexes, which are then internalized into cells through clathrin-mediated endocytosis, releasing the drug inside tumor cells and killing them.
Ligand-binding assays (LBAs) are the most commonly used strategy for quantifying the antibody portion in ADC drugs, including the quantification of total antibody and conjugated antibody. This ADC bioanalysis method offers high sensitivity, high throughput, and stability at relatively low experimental and equipment costs. LBAs can meet different testing needs by designing specific assay formats.
The final time point of this reaction shows the number of analytes monitored in different biological samples. Analytes in biological samples can be captured and detected by most LBAs using antibodies as key reagents. Enzyme-linked immunosorbent assays (ELISAs) are the gold standard for LBAs to detect different analytes in biological samples. Monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) are the key reagents of choice in LBAs.
The efficacy and toxicity of ADCs are related to the antibody-drug conjugates, free drugs, and total antibodies, which should all be monitored. ADC bioanalysis typically uses LC-MS/MS for analyzing small-molecule toxins and LBAs for analyzing larger molecules such as total antibodies or antibody-drug conjugates.
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