Three unique bioanalytical challenges of antibody-drug conjugates

The bioanalysis of antibody-drug conjugates (ADCs) is vital for determining the safety and efficacy of this rising pharmaceutical class of drugs. Here, we outline three factors that make the bioanalysis of ADCs so complex and cover some of the main analytical techniques used.

ADCs exist as multiple components

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ADCs consist of cytotoxic small molecule drugs that are conjugated to an antibody, via a chemical linker. The antibody is specific for a tumor-associated antigen that has restricted expression on normal cells.

Cleavable chemical linkers are designed to be stable in the circulation and release the cytotoxic drug (often referred to as the ‘payload’) in response to certain elements within the target cell. These “releasing” triggers may be a defined pH range, high glutathione concentrations, or proteolytic cleavage.1 The resulting free drug can directly kill and then exit the target cell to cause “bystander killing” of neighboring cells.

In contrast, non-cleavable linkers lack an obvious proteolytic cleavage site1, and the cytotoxic payload is retained within the cell. ADCs with non-cleavable linkers release the cytotoxic payload upon degradation of the antibody, rather than the linker.2

In practice, ADCs are present as a mixture of multiple components. In some instances, the cytotoxic drug could be effluxed from the cell and metabolized into smaller components that contain different anticancer activity to the parent drug.3 Bioanalysis of ADCs generally includes measures of intact ADCs, free drug molecules, conjugated antibody, and total antibody.4 This complex mixture of species presents unique bioanalytical challenges, and there is a need to determine which techniques are suitable for the different stages of research and development.

ADC components are heterogenous

The number of drugs attached to each antibody is one of the primary sources of variability. Expressed as the average number of drugs conjugated to each antibody (usually between 0 and 8), the drug-to-antibody ratio (DAR) affects stability, antigen binding,5 and ultimately, ADC potency.6 A low DAR can reduce ADC potency, while high drug loading can increase clearance, increase risk of aggregation and premature release of the toxic payload in the circulation.5,7

Another source of variability derives from the conjugation site at which the payload is conjugated to the antibody. Efforts are underway to create more homogenous ADCs by controlling the site and number of drugs conjugated to the antibody, but analytical methods must account for this variability in the meantime.8

ADCs transform in vivo

The characterization of ADC biotransformations is a unique challenge for bioanalytical assay development. Although ADCs are designed to be stable until they reach the tumor cells, they can show instability in the circulation and are subject to various catabolic processes upon internalization into cancer cells. Assessing the released products is a key step in reliable pharmacokinetic assessment and predicting toxicity.9 Possible modifications include linker deconjugation, drug loss, partial drug loss, adduct formation, and hydrolysis.10 Understanding the pathways of catabolism is an important part of developing appropriate bioanalytical methods.

Key techniques in ADC bioanalysis

Overall, the changing catabolism of multiple and heterogeneous components creates unique challenges for in-depth bioanalysis of ADCs, and multiple bioanalytical methods are needed. Liquid chromatography mass spectrometry (LC-MS) analysis has been used extensively in ADC characterization and quantification.11 Quadrupole-time-of-flight mass spectrometry with electrospray ionization has been widely used for characterization of intact ADCs. DAR has been obtained using several techniques including UV/Vis spectroscopy, hydrophobic interaction chromatography, reverse phase high performance liquid chromatography and LC-MS.12 Lysosomal extracts can be used to monitor the efficiency of payload release from the linker13,14, and potential instabilities of the payload linker can be identified in serum and under conjugated conditions through analytical tests such as UV/VIS spectroscopy, reverse phase high performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry.9,13

Limitations such as time-consuming sample preparation and data processing are creating a need for methods that are better suited to the industry.12 Maximizing multiplexing capabilities is one approach that could help researchers save time and sample usage. An experimental comparison of analytical techniques suggested while the determined DAR was comparable across techniques, the accuracy of molecular weight analysis varied more extensively.15 The choice of mass analyzer will likely be affected by the screening stage and measurement of focus.

Absorption Systems has a great deal of experience navigating the complexities of ADC bioanalysis. To learn more about our capabilities, speak to one of our scientists.


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