Critical Bioanalytical Challenges of ADCs

In reality, ADCs are present as a combination of multiple components. In some instances, the cytotoxic drug can be discharged from the cell and metabolized into smaller parts containing different parent drug anticancer activity.

FREMONT, CA: Bioanalysis of ADCs is critical to determine the rising drug class's safety and efficacy. Below are three reasons that make Antibody-Drug Conjugates’ (ADC) bioanalysis so complicated and the fundamental analytical techniques used.

ADCs Exist as Multiple Components

ADCs comprise cytotoxic small molecule drugs that are conjugated to an antibody through a chemical binder. The antibody is unique to a tumor-associated antigen that has a minimal expression on normal cells. Cleavable chemical linkers are engineered to be stable in circulation and release cytotoxic drugs (often referred to as payloads) in response to some aspects inside the target cell. These release triggers can include a given pH spectrum, high glutathione concentrations, or proteolytic cleavage. The resulting free drug can explicitly destroy and then leave the target cell to kill the surrounding cells.

On the other hand, the non-cleavable linkers lack a robust proteolytic cleavage site, and the cytotoxic payload is retained inside the cell. ADCs with non-cleavable linkers release the cytotoxic payload when the antibody is degraded, rather than the linker. In reality, ADCs are present as a combination of multiple components. In some instances, the cytotoxic drug can be discharged from the cell and metabolized into smaller parts containing different parent drug anticancer activity. ADC bioanalysis typically involves tests of intact ADCs, free drug molecules, conjugated antibodies, and complete antibodies. 

This complex mixture of species poses specific bioanalytical challenges, and it is essential to evaluate which techniques are sufficient for the various stages of research and development.

ADC Components are Heterogenous

One of the critical causes of variability is the number of drugs attached to each antibody. Expressed as the average number of drugs conjugated to each antibody (typically between 0 and 8), The Drug-To-Antibody Ratio (DAR) affects stability, antigen binding, and eventually, the potency of ADC. Low DAR can reduce ADC potency, whereas high drug loading can increase clearance, increase the likelihood of aggregation and early release of toxic payloads into circulation.

Another source of variability is the conjugation site, where the payload is conjugated to the antibody. Efforts are underway to establish more homogeneous ADCs by monitoring the site and the number of antibody-conjugated drugs, but analytical methods must account for this variability in the meantime.

ADCs Transform in Vivo

The classification of ADC biotransformations is a significant challenge for the development of bioanalytical assays. While ADCs are designed to be stable before they enter tumor cells, they can show circulation instability and are subject to various catabolic processes after internalization into cancer cells. The evaluation of the published products is a crucial step in a reliable pharmacokinetic assessment and predictive toxicity. Possible modifications include connection deconjugation, partial drug loss, drug loss, adduct formation, and hydrolysis. Understanding the paths of catabolism is an integral part of developing effective bioanalytical methods.

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