Antibody-drug conjugates (ADCs) are oncology therapies consisting of a monoclonal antibody (mAb) linked to cytotoxic small molecule payload. Measuring pharmacokinetics (PK) and other safety characteristics of ADCs is particularly challenging due to the complexity of the molecules and their environment. Recent ADC developments, including optimized mAb designs, improved conjugation strategies, and novel linker-payloads with alternative mechanisms of action, require assessment of the PK and safety. In order to do this, several strategies are employed, often simultaneously, for bioanalysis of in vivo samples. For digestion-based and intact/reduced assays, bead-based immunocapture anti-human IgG capture is performed on ADCs from plasma and homogenized tissue. Digestion-based LC-MS/MS follows a sequential workflow in which immunocaptured ADCs are digested using trypsin and/or linker-cleaving enzyme to measure the total antibody (TAB) and total ADC (TADC), respectively. Quantitative measurements inform PK parameters such as Cmax, Tmax, clearance, and half-life. Furthermore, correlations between TAB and TADC measurements inform molecular stability in vivo. Intact/reduced LC-MS assays demonstrate intact ADC drug-to-antibody ratio (DAR) change throughout an in vivo study. Changes in ADC profile due to payload deconjugation, biotransformation, adduction, and more can be elucidated. Combining the sensitivity of digestion-based assays and the breadth of intact/reduced assays creates a more comprehensive view of an ADC’s DMPK. Additionally, free payload assays quantitate payload concentrations in plasma and tumor, providing information about ADC stability, targeted delivery, and toxicity. Case studies are presented in which the lower limit of quantitation (100ng/mL for TAB/TADC and 20pg/mL for free payload) is established while minimizing necessary in vivo sample volumes. Additionally, intact/reduced LC-MS approaches are used for intact DAR and linker-payload stability monitoring in vivo, and an overview of such approaches will be shared in a DMPK context. Harnessing the capabilities of MS, examples of multiple bioanalytical strategies are shared to address challenges in ADC bioanalysis.

Iliana Levesque

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