(M1030-01-07) Validation of Anti-Drug Antibody Assay for Multi-Domain Biologics for Human Clinical Studies

Purpose: This study aimed to validate an anti-drug antibody (ADA) method for detecting ADAs in human serum and characterizing their specificity toward one or both domains of a fusion protein biologic. One of the domains resembles an endogenous protein and the method also evaluates potential cross-reactivity with the endogenous counterpart. A six-tiered assessment strategy was employed, and the method was performed in singlicate to enhance throughput. This approach may serve as a reference model for ADA detection in multi-domain biologics.

Methods: The validated method comprises six assays: screening, drug confirmatory, titer, two domain-mapping assays, and a cross-reactivity assay. Screening and confirmatory assays identify ADA-positive samples, while the remaining assays characterize ADA specificity. All assays utilize a bridging format, where ADA bridges biotinylated and ruthenium-labeled drug molecules. To mitigate interference from circulating drug, samples were acid-treated to dissociate ADA-drug complexes. Two in-house positive controls were developed and assessed, an affinity-purified rabbit polyclonal antibody targeting domain 1, and a recombinant mouse monoclonal antibody specific to domain 2.

Results: The bridging ADA method met industry and regulatory validation standards. Cut point factors were established for all six assay tiers. Screening and confirmatory assay sensitivities met FDA requirements ( < 100 ng/mL). Drug tolerance at 100 ng/mL ADA levels was 140 µg/mL (domain 1) and >750 µg/mL (domain 2) for the screening assay, and 90 µg/mL (domain 1) and 569 µg/mL (domain 2) for the confirmatory assay—sufficient to cover trough drug levels. Calculated cut points were screening (1.07), confirmatory (22.2%), domain 1 mapping (15.9%), domain 2 mapping (12.4%), cross-reactivity (22.9%), and titer (1.40). The method also met criteria for intra- and inter-assay precision, hook effect, specificity, matrix interference, selectivity, recovery, robustness, and control stability. Assay acceptance criteria were established and applied to support multiple clinical trials.

Conclusion: The ADA method was successfully validated for clinical use, including screening, confirmatory, and titer assessments. Given the biologic’s dual-domain structure this streamlined singlicate assay approach, incorporating domain mapping and cross-reactivity assessments, enables comprehensive ADA characterization. The method complies with regulatory guidance and is applicable to ongoing clinical studies. Moreover, it offers a robust reference framework for ADA assessment in other dual-specific or multi-domain biologics.

References: 1. FDA: Immunogenicity testing of therapeutic protein products – developing and validating assays for anti-drug antibody detection. Guidance for industry. U.S. Department of Health and Human Services Food and Drug Administration. Center for Drug Evaluation and Research (CDER). Center for Biologics Evaluation and Research (CBER). 2019. https://www.fda.gov/media/119788/download 2. FDA: Draft Guidance for Industry: Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products. 2016. https://www.fda.gove/media/77796/download

Acknowledgements: The authors would like to thank the analysts, study leader, and the support personnel with Specialty Bioanalytics (West Chester, PA) for generation of the data utilized within this study, and also thank the Lab Operation Group, the Critical Reagents Group in the Specialty Bioanalytic department (West Chester, PA) and the Discovery Group (Sydney) for their efforts in generating the reagents needed for this work.