Biologics undergo extensive characterization to demonstrate their safety, purity, and efficacy. Jennifer Lawson (product manager for cell line, media, and testing solutions at Sartorius) highlighted the role of potency assays in that process. Because they reflect the complexity of biological systems, scientists must develop robust assays that will provide sufficient data for good-practice (GxP) applications. Lawson pointed out milestones in the bioassay life cycle and explored ways to help ensure method suitability.
Development Criteria: Potency assay development requires consideration of several factors in addition to typical bioassay parameters. A potency assay must be functionally relevant to a productâs mechanism of action (MoA) â balanced against the inherent variability of bioassays. Moreover, assays with multiple steps, primary cell types, and/or complex reagents are likely to hinder routine, GxP-compliant testing.
Lawson noted that cells are key sources of assay variation. Using well- prepared, well-characterized master and working cell banks based on immortalized/genetically engineered cell lines helps to limit such variability. Although continuous culture can provide a reliable source of cells for routine assays, âthaw-and-useâ strategies also should be investigated to control variability further.
Microplate layouts should be randomized to prevent position effects, but excessive randomization introduces risk for error during manual operations. Sample preparation also merits attention. Each replicate requires an independent sample dilution. Using the same dilution to fill multiple wells can yield artifically precise data and lead to assay failures. Lawson recommended beginning method development using a broad dilution series, then honing it after establishing other parameters.
Statistical analysis is an important factor that should not be neglected. The four-parameter logistic model is generally suitable for bioassay data, but other models can be assessed for suitability in a data-driven process. Consideration also should be given to an assayâs endpoint, which must be robust and suitable for GxP applications.
Lawson showed how to apply such criteria by describing the development of Sartoriusâs platform assays. She featured an enzyme-linked immunosorbent assay (ELISA) designed for potency testing of a commercialized nivolumab product. Knowing that the product inhibits binding of PD-1 to associated ligands PD-L1 and PD-L2, Sartorius designed a competition ELISA using plates coated in PD-1, constant concentrations of PD-L1, and a dilution series of the nivolumab product. Modde data analytics software enabled a design of experiments (DoE) approach to evaluating key assay parameters.
In a second example, Sartorius developed a cell-based platform to measure product inhibition of target proliferation â here, the ability of an antibodyâdrug conjugate (ADC) to inhibit proliferation mediated through binding to HER2. Such assays must be controlled tightly because they require extended incubation periods. Analysts applied the iQue Screener Plus flow cytometer to evaluate different cell lines and seeding densities. Those studies helped to demonstrate the Sartorius platformâs applicability to other HER2-targeting biologics and to protein products with similar MoAs.
Beyond Development: Before clinical studies, drug developers should evaluate assay system performance during system suitability studies to establish tentative assay acceptance criteria and determine reportable values. Critical raw materials should be evaluated for variability across lots to anticipate problems with assay accuracy and reproducibility.
A potency assay typically undergoes qualification for GMP compliance on advancing to phase 1 to allow release of material for clinical trials. Robustness must be assessed as part of validation, which is required as a molecule reaches final clinical evaluation. A GMP-validated assay must be in place for commercial lot release. Whereas qualification determines a methodâs performance in terms of its specificity, linearity, accuracy, and precision, validation confirms those capabilities against predetermined criteria.
Reducing Development Time: Bioassays consistently top the list of activities that drug developers outsource. Sartorius has incorporated potency assay development into its biologics platform to help companies accelerate testing and prevent costly delays at late clinical stages. The platform holds promise for reducing assay-development times by half.
Questions and Answers
How do you approach identification of endpoints? They must be relevant to a productâs MoA and suitable for demonstrating regulatory compliance.Note that some technologies are optimized for drug discovery but not for GxP applications. Other considerations include data integrity and handling.
What is an optimal number of replicates? That differs by method and depends on an assayâs variability. Analysts typically perform at least three independent replicates. High assay variability might necessitate additional independent replicates.