A cell-based alternative to the mouse potency assay for pharmaceutical type E botulinum antitoxins

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Eran Diamant
Amram Torgeman
Eyal Epstein
Adva Mechaly
Alon Ben David
Lilach Levin
Arieh Schwartz
Eyal Dor
Meni Girshengorn
Ada Barnea
Ohad Mazor
Ran Zichel

Abstract

The pharmacopeia mouse neutralization assay (PMNA) is the standard method for determining the potency of pharmaceutical botulinum antitoxins. However, a PMNA requires a large number of mice, and thus, an alternative in vitro method to replace it is needed. Herein, we developed an in vitro SiMa cell line-based neutralization assay (SBNA), compatible with a PMNA design, for therapeutic antitoxins against type E botulinum neurotoxin (BoNT/E). The SBNA measures the residual cellular activity of BoNT/E following antitoxin neutralization using a specific quantitative sandwich ELISA for its cleaved cellular target protein, i.e., SNAP-25. The potencies of pharmaceutical antitoxin preparations were determined by applying two different quantification approaches: (1) a cutoff value, in accordance with the pharmacopeia concept, and (2) nonlinear regression of a standard curve generated by serial dilutions of a standard antitoxin. Both approaches achieved accurate potencies compared to the PMNA (average %RE of ~16%). Furthermore, the SBNA was able to determine in vitro, for the first time, the accurate neutralizing activity (%RE ≤20) of next-generation equine and nonequine therapeutic antitoxins. Collectively, a high correlation between SBNA and PMNA results was obtained for all antitoxin preparations (r = 0.99, P < 0.0001 for the standard curve approach, and r = 0.97, p < 0.0001 for the cutoff approach). In conclusion, the SBNA can potentially replace the PMNA and markedly reduce the need for laboratory animals for the approval of botulinum antitoxin preparations.

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Diamant, E., Torgeman, A., Epstein, E., Mechaly, A., Ben David, A., Levin, L., Schwartz, A., Dor, E., Girshengorn, M., Barnea, A., Mazor, O. and Zichel, R. (2021) “A cell-based alternative to the mouse potency assay for pharmaceutical type E botulinum antitoxins”, ALTEX - Alternatives to animal experimentation. doi: 10.14573/altex.2105251.
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