CGTs are often treatments for rare genetic mutations that affect endogenous enzyme production or function. Enzymatic activity assays are a critical part of the bioanalytical strategy to assess the pharmacokinetics and efficacy of gene therapies. There is limited regulatory guidance that pertains specifically to bioanalytical enzymatic assays and the general industrial consensus is to use the FDA BMV and Biomarker guidance documents as a framework for enzymatic assay development and validation. However, enzyme activity assays present a unique set of challenges including sensitivity to laboratory conditions, e.g., temperature, reaction time and pH, lack of a true reference material and enzyme stability. Normal laboratory and analytical conditions may lead to assay drift and require increased monitoring during the life cycle of an enzymatic assay. These challenges require solutions that are tailored to each assay and may be different to the process and system controls used for traditional chromatographic and ligand binding assays (LBA) used in the same context. Here, we present an overview of our experiences with regulated enzyme activity assay bioanalysis, including case studies, recommendations for enzyme assay characterization and life cycle management.
Enzyme activity assays generally use a non-detectable molecule that is specifically cleaved by the enzyme of interest into a detectable subunit of the parent molecule. The cleaved product is then detected using colorimetric or fluorescent spectrophotometry. Enzyme activity can be determined using either a calibration curve prepared from the enzyme cleavage product (static curve) or a calibration curve prepared from an enzyme stock of known activity (dynamic curve). Quality control samples in enzyme assays must be able to monitor both the assay system (system suitability controls) and represent patient samples (endogenous enzyme controls). Setting acceptance criteria for enzyme activity assays has been particularly challenging due to the dynamic nature of the assay system. The statistical approach used to set acceptance ranges should be statistically appropriate to the assay.
Enzymatic activity assays are a critical component to the bioanalytical package of CGT. Due to the dynamic nature of enzyme activity assays, close control and monitoring of assay performance is required. The lack of true reference material and complex assay system also require novel approaches to assay calibration material to ensure reliable long-term assay performance. Assay performance should be monitored using quality control samples that represent both the assay system and patient samples. Further, rational statistical approaches need to be used to set acceptance criteria and monitor assay performance. As the regulatory landscape of biomarker and enzymatic bioanalysis evolves, it will be important to incorporate the expertise of bioanalytical laboratories into new guidelines. This will allow for more reliable and reproducible bioanalytical results to support emerging cell and gene therapies.
Upon completion, participants will be able to understand the unique challenges associated with CGT-based enzyme bioanalysis.
Upon completion, participants will have statistical models for how to monitor enzyme assays over time under variable operational conditions.
Upon completion, participants will be able to have robust discussions on the best practices for using surrogate reference materials for regulated assays supporting CGT bioanalysis, specifically exposure and immunogenicity.