Two‐center validation of assays for the detection of binding and neutralizing anti‐factor VIII antibodies

Abstract

IntroductionPatients with hemophilia A treated with coagulation Factor VIII (FVIII) products are at risk for developing anti‐FVIII antibodies. The ABIRISK Consortium aimed to provide knowledge on the formation and detection of anti‐drug antibodies against biopharmaceutical products, including FVIII. Accordingly, standardized and validated assays for the detection of binding (total) and neutralizing antibodies are needed.AimTwo‐center validation of an ELISA for the detection of total FVIII‐binding IgG‐antibodies and Nijmegen‐Bethesda assays for the quantification of FVIII‐neutralizing antibodies according to consensus validation guidelines.MethodsValidation of assays at both sites was done according to published recommendations and included preanalytics, the determination of key assay parameters, including cut‐points, assay sensitivity, precision, and FVIII interference.ResultsThe validated assays reproducibly detected FVIII‐binding and ‐neutralizing antibodies with comparable performance in both laboratories. Floating screening cut‐points were established for both assays. Determined mass‐based sensitivity of both assays (all values ≤66 ng/mL) complied with the minimum sensitivity for the detection of anti‐drug antibodies as recommended by the FDA (<100 ng/mL). Intra‐ and inter‐assay coefficients of variation did not exceed 25%. Assay validation further revealed that pre‐analytical heat treatment led to potentially false‐positive ELISA results, while up to 0.15 IU/mL, residual FVIII showed no significant impact. Overall, good agreement of results was found for patient samples analyzed at both study sites.ConclusionComprehensive validation of different anti‐FVIII‐antibody assays in two laboratories gave novel insights into the impact of pre‐analytical sample treatment as well as the comparability of test results generated by the use of methodically different assays.