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Antibody Drug Conjugates: Bioanalytical Assay Challenges and Strategies

Antibody-drug conjugates (ADCs) combine the high specificity of a monoclonal antibody (mAb) for a target antigen with the highly potent cytotoxic activity of small-molecule drugs. Although the concept behind ADCs is relatively simple, the successful design and development of ADCs is a complex task. Similar to other biotherapeutics, the understanding of Pharmacokinetics (PK), Pharmacodynamics (PD), and Absorption, Distribution, Metabolism, and Excretion (ADME) properties of ADCs is critical for their successful development.

ADCs are complex molecules containing heterogeneous mixtures of species with different drug-to-antibody ratios (DAR) and different sites of conjugation, arising from different conjugation chemistry approaches. The heterogeneity in starting reference material may evolve further in vivo due to biotransformation, spontaneous or environment induced deconjugation of payload from the ADC, and/or differences in the clearance rate of various DAR species.

The inherently heterogeneous and dynamic characteristics of ADCs require additional consideration when evaluating their pharmacology as well as bioanalytical and PK properties. For instance, the ADC structural complexity and heterogeneity drives the need to monitor multiple analytes (conjugated antibody, conjugated drug, total antibody, unconjugated drug and its metabolites, DAR distribution etc.) for understanding the in vivo fate of these complex molecules. Currently there is limited industry experience on what ADC analytes provide the best correlation for exposure-response relationship for safety and efficacy of ADCs in the clinic. Furthermore, due to the lack of regulatory guidelines related to ADC bioanalysis and limited industry wide experience, the assay validation guidelines for both large and small molecules are applied for ADC bioanalysis.

Because of its structural complexity, and dynamic and heterogeneous nature, ADCs require unique bioanalytical strategies to characterise and quantify the most relevant ADC analytes. Depending on the information sought (such as ADC stability, clearance, exposure, safety etc.) at a given phase of the drug development, analytical assay platforms such as ligand binding assays (LBA), liquid chromatography separation coupled with mass spectrometry detection (LC-MS), and combination of both platforms (e.g. hybrid ligand-binding LC-MS) are employed for analysing diverse ADC analytes.

However, as ADC candidates progress from different phases of development, the bioanalytical assay strategies (assay formats, assay reagents, analytical platform) may evolve from a flexible “fit-for-purpose” approach used in early discovery to validated assays at late phases of development. In addition, the DAR-sensitivity of bioanalytical assays may differ across the development phases of a program. While a DAR-sensitive assay tries to measure ADC analyte concentration based on the number of small molecule drugs attached to the mAb moiety, the DAR-insensitive assay attempts to measure various DAR components of the ADC equally, and hence is not biased towards the drug load of the ADC.

The evolution of the bioanalytical assay (e.g. from DAR sensitive to DAR insensitive) could result in differences in observed PK profile and calculation of key PK parameters over the course of an ADC candidate development. However, by applying rational scientific understanding of what each assay format, assay reagent and analytical platform is measuring, an appropriate interpretation of observed data can be made. Moreover, once enough understanding of the most relevant ADC analyte and DAR species is built, keeping the assay formats, assay reagents, and analytical platform similar in late stages of drug development, such as between IND-enabling and first-in-human studies, might help simplify interpretation of the observed results.

Seema Kumar

 

Seema Kumar is the Associate Director at Merck KGaA. At the upcoming Biologics & Biosimilars Congress, Seema will present on PK assay continuity between drug discovery and development.


View the agenda for the 4th Biologics & Biosimilars Congress: Europe.

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