Validation approach of an LC-MS/MS assay for ADA detection, applied to a Von Willebrand factor-targeted biotherapeutic

BACKGROUND: The efficiency of ligand binding assay (LBA)-based immunogenicity assessment can be compromised by the presence of high concentrations of soluble pharmacological target. This is particularly challenging for therapies targeting highly abundant multimeric plasma proteins, such as Von Willebrand Factor (VWF). METHOD: We developed and validated a hybrid LBA-LC-MS/MS assay in which a VWF-directed monoclonal antibody is immobilized to selectively extract anti-drug antibodies (ADAs) from plasma. Nonspecific binding was minimized by immunopurification and the ADAs were digested using trypsin. Semi-quantitative LC-MS/MS analysis of signature peptides unique to seven ADA isotypes (IgG1-4, IgM, IgA, and IgE) was performed against isotype-specific reference standards. RESULTS: The method validation was conducted in alignment with current regulatory expectations for immunogenicity assessment, addressing all critical performance characteristics, including cut-point determination, sensitivity, specificity, and selectivity as well as precision, reproducibility, and robustness. The validated assay was successfully implemented as the primary method for assessing immunogenicity in clinical trial samples. CONCLUSION: This study demonstrates that hybrid LBA-LC-MS/MS enables single tier isotype-specific immunogenicity assessment in the presence of extreme target concentrations. The approach provides a viable alternative to traditional LBA-based strategies and can serve as the primary technique for immunogenicity assessment for regulatory-compliant evaluation of ADA responses.