Antibody Drug Conjugates Require Thoughtful Bioanalysis
Bioanalytical support can reduce time to market for ADC drugs
With over 250 antibody-drug Conjugates (ADCs) currently under development there has been an increase in the interest and need for ADC bioanalysis. To keep up with this demand and ensure we are able to produce robust and relevant assays, it is essential that we understand the unique challenges and requirements that come with these targeted therapies.
To better appreciate these bioanalytical challenges, an understanding of the concept and basic composition of an ADC is essential.
ADCs are monoclonal antibodies (mAbs) linked to cytotoxic small molecules (often referred to as the payload). The mAbs provide selectivity that reduces the systemic toxicity of the cytotoxic small molecule. This composition means there is the potential for significant diversity within this drug class. The mAb, payload, linker and their ratios are all variables that can influence how the ADC behaves invivo. This variation within the drug class means not only do the bioanalytical challenges and requirements vary from other, more standard drug designs, but also significantly between individual ADCs.
For a given ADC there are further challenges that complicate bioanalysis. ADCs are complex heterogeneous mixtures of multiple species that can exist in a variety of forms in vivo: unconjugated payload, unconjugated antibody, varying levels of conjugation, conjugation with other proteins, etc. As each of these forms have the potential to have its own distinct impact on the safety or efficacy of the drug, determining bioanalytical requirements requires much more consideration than with more simple homogenous drugs.
This in vivo structural variation, combined with the lack of specific guidelines to support their bioanalysis, makes appropriate assay selection by far the greatest challenge of ADC bioanalysis. It is simply not feasible to develop and utilise every possible bioanalytical assay, in every study throughout drug development. The time and resources involved, along with an ethical need to minimise animal usage, mean that instead a more focused approach is required.
As with many other classes of molecules, it is review papers and previously successful applications that give us the best starting point when determining which assays may be required. Although some of these assays appear routinely, such as free payload conducted by LC-MS and total antibody by ligand binding assay (LBA), there is no one size fits all approach.
A good example of this comes in the form of ADCs with a cysteine-linked payload. With these types of ADC, it’s known that the payload can migrate from the ADC to other proteins within the blood. For some studies, like those designed to identify a lead candidate in biosimilar studies, it can be useful to quantify these non-ADC protein conjugates as it can help identify potentially critical differences in an ADCs behaviour. This mechanistic investigation is a particularly non-standard assay type, so identifying a need for it is only the first challenge.
The following questions of which analytical technique is most appropriate (LBA? MS? Hybrid LBA-MS?), along with what experiments are required to characterise the assay’s performance, provide an even more substantial challenge. The only way to efficiently overcome these challenges is to return to the fundamental principles of the guidelines:
- Does the method measure the intended analyte?
- What is the variability associated with these measurements?
- What is the range in measurements that provides reliable data?
- How do sample collection, handling, and storage affect the reliability of the data from the bioanalytical method?
It is only by developing experiments to robustly answer these questions, and by applying sound scientific principles, that meaningful data can be produced. To approach any assay with a mindset too narrowly linked to the either the chromatography or ligand binding guidance, would risk overlooking critical assay characteristics.
Overall ADC bioanalysis provides us with a great opportunity to not only expand our scientific knowledge but also encourages greater collaboration between traditionally distinct techniques like LBA and LC-MS. The fact that it forces us to go back to considering the core principles behind the guidance documents that we have become so familiar with also encourages us to reconsider what it really means for an assay to be ‘fit for purpose’. This reconsideration goes beyond ADC bioanalysis and applies to all classes of analyte. Ultimately this reconsideration provides an opportunity for us to improve the reliability and usefulness of bioanalytical data across the industry.