Purpose: Assay sensitivity is the lowest concentration at which the antibody preparation consistently produces either a positive result or a readout equal to the cut-point determined for that assay. Sensitive immunogenicity assays for ADA detection are critical during biological drug development. As per Guidance for Industry, Immunogenicity Testing of Therapeutic Protein Products – Developing and validating Assays for anti-Drug Antibody Detection, U.S. Department of Health and Human Services, Food and Drug Administration, January 2019, the assessment of assay sensitivity is expected to be performed in the presence of circulating levels of therapeutic drug. Hence, there is a need to develop a statistically derived and physiologically relevant specificity criterion focusing on screening and confirmatory assay performance. The purpose is to focus on the clinical relevant measurements of ADA.
Methods: Positive control antibody was titrated until the last dilution crosses the screening cut point over two days. Six independent results obtained in the absence of therapeutic drug (Screening assay) were used for statistical derivation of assay sensitivity using the formula, mean + t0.05,df x SD considering 5% false positive rate. The assay sensitivity when applied in the confirmatory assay did not inhibit the response more than the generated confirmatory assay cut point as the response was very low. Assay sensitivity can be derived using interpolation method or the intercept method. But what is more relevant?
Results: Interpolation method depends on the dose response curve and the curve fitting. However, in the intercept method, calculation of assay sensitivity is independent of the curve fitting. In case1, statistically derived sensitivity was 3.37ng/mL and in case 2 it was 3.08ng/mL. This assay sensitivity when applied in the confirmatory assay did not inhibit the response as the response was very low. Based on the confirmatory tier assay sensitivity was considered to be 6ng/mL and 5ng/mL for case 1 and case 2 respectively.
Conclusion: Assay sensitivity should be derived in the presence of high concentration of drug (Confirmatory) to understand the relation of ADA with safety and efficacy. Thus, a physiologically relevant and effective strategy to confirm sensitivity is through the intercept method. Additionally, whenever assay sensitivity cannot be calculated using non-linear regression (4PL) due to improper curve fitting, interpolation of antibody concentration corresponding to cut point was not possible statistically, then forecast function can be used to calculate assay sensitivity and determining the new LPC levels. Case to case evaluation and assessment should be done to consider the correct model to determine the assay sensitivity with proper justification.