Diphtheria antitoxin potency assay: an in vitro alternative to the in vivo subcutaneous toxin neutralization test

Diphtheria is an acute infectious disease that can be fatal due to the action of diphtheria toxin (DT). Diphtheria antitoxin (DAT) remains essential for treatment and must be administered within two days of symptom onset to effectively neutralize circulating DT. Immediate availability of DAT is critical, especially during outbreaks. Potency testing is required before lot release, yet current pharmacopeial methods still rely on in vivo assays, such as the subcutaneous toxin neutralization test (TNT-SC) or intradermal TNT (TNT-ID). In response to efforts to reduce animal use, this study aimed to validate an in vitro potency assay for DAT using Vero cells, as an alternative to the in vivo TNT-SC for lot release. Twelve DAT samples were tested using the in vivo method and the alternative in vitro assay. Diagnostic performance (sensitivity, specificity, accuracy), agreement (Lin's CCC, Bland-Altman analysis), linearity, precision, and selectivity were evaluated. The in vitro assay showed high concordance with the in vivo method (CCC >0.90) and correctly classified all samples regarding conformity status. The assay demonstrated acceptable linearity and precision (gCV% ≤ 30 %). These results support the Vero cell assay as a relevant and reliable full replacement for in vivo TNT-SC in DAT potency determination.