Development and Validation of an ADA-Tolerant Assay for Quantification of an Exatecan-Based ADC in Monkey Plasma.

ABSTRACT

BACKGROUND: The development of an anti-drug antibody (ADA)-tolerant pharmacokinetic (PK) assay is important when the drug exposure is irrelevant to toxicity in the presence of ADA. We aimed to develop and validate an ADA-tolerant assay for an exatecan-based antibody-drug conjugate (ADC) in monkey plasma. RESULTS: The assay tolerated 5.00 µg/mL of ADA at 12 µg/mL of ADC. Its accuracy and precision results satisfied the acceptance criteria. Furthermore, the assay was free from hook and matrix effects and exhibited good dilutional linearity. Additionally, the ADC in plasma samples was stable under different storage conditions. METHOD: An ADA-tolerant ADC assay was configured with an anti-payload antibody for capture, and a drug-target protein combined with a horseradish peroxidase (HRP)-labeled antibody against a drug-target-protein tag for detection. Samples were firstly acidified to dissociate drug and ADA complexes, and to convert the carboxylate form to the lactone form of exatecan molecules; then, the ADAs in the samples were removed with a naked antibody-coated microplate. The treated samples were further incubated with coated anti-payload antibody and captured ADC molecules were quantified by the detection reagent. The developed assay was optimized and validated against regulatory guidelines. CONCLUSIONS: The assay met both methodological and sample-related ADA tolerance requirements, and was applicable to a nonclinical study in cynomolgus monkeys.