Collaborative study for the BINACLE assay for in vitro determination of botulinum neurotoxin activity

Abstract

The muscle-relaxing effects of botulinum neurotoxin (BoNT) serotypes A and B are widely utilized in clinical and aesthetic medicine. While the mouse bioassay remains the gold standard for evaluating the potency of pharmaceutical BoNT preparations, a non-animal alternative would be preferable to comply with European Directive 2010/63/EU. The Binding and Cleavage (BINACLE) assay, which measures BoNT activity based on the receptor-binding and proteolytic properties of the toxin, represents a promising alternative. Previous studies showed that this method allows reliable and sensitive activity determination. Here, we describe a collaborative study involving ten international laboratories, which aimed to characterize the applicability of the BINACLE assay for the potency testing of pharmaceutical BoNT products. Our results demonstrate that the BINACLE assay enables reproducible relative potency determinations for BoNT/A, the most pharmaceutically relevant serotype, with variability within acceptable limits. Reproducible relative potency calculations were also demonstrated for BoNT/B, while the data highlighted opportunities for refinement of the BoNT/B BINACLE protocol to further enhance the robustness of the method. Overall, this work confirms the utility of the BINACLE assay as a reliable and sensitive method for determining BoNT product potency, thereby substantiating its applicability as an alternative to the mouse bioassay.

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Cite

BibTeX RIS

BibTeX

@misc{kegel2026collaborative,
  title = {{Collaborative study for the BINACLE assay for in vitro determination of botulinum neurotoxin activity}},
  author = {B. Kegel and K.-M. Hanschmann and U. Bonifas and J. Klimek and H. A. Behrensdorf-Nicol},
  howpublished = {bioRxiv},
  year = {2026},
  month = sep,
  doi = {10.64898/2026.09.01.748470},
  url = {https://www.biorxiv.org/content/10.64898/2026.09.01.748470v1},
  note = {CC BY-NC-ND},
}

RIS

TY  - UNPB
TI  - Collaborative study for the BINACLE assay for in vitro determination of botulinum neurotoxin activity
AU  - B. Kegel
AU  - K.-M. Hanschmann
AU  - U. Bonifas
AU  - J. Klimek
AU  - H. A. Behrensdorf-Nicol
PY  - 2026
DA  - 2026/09/01
PB  - bioRxiv
DO  - 10.64898/2026.09.01.748470
UR  - https://www.biorxiv.org/content/10.64898/2026.09.01.748470v1
AB  - The muscle-relaxing effects of botulinum neurotoxin (BoNT) serotypes A and B are widely utilized in clinical and aesthetic medicine. While the mouse bioassay remains the gold standard for evaluating the potency of pharmaceutical BoNT preparations, a non-animal alternative would be preferable to comply with European Directive 2010/63/EU. The Binding and Cleavage (BINACLE) assay, which measures BoNT activity based on the receptor-binding and proteolytic properties of the toxin, represents a promising alternative. Previous studies showed that this method allows reliable and sensitive activity determination. Here, we describe a collaborative study involving ten international laboratories, which aimed to characterize the applicability of the BINACLE assay for the potency testing of pharmaceutical BoNT products. Our results demonstrate that the BINACLE assay enables reproducible relative potency determinations for BoNT/A, the most pharmaceutically relevant serotype, with variability within acceptable limits. Reproducible relative potency calculations were also demonstrated for BoNT/B, while the data highlighted opportunities for refinement of the BoNT/B BINACLE protocol to further enhance the robustness of the method. Overall, this work confirms the utility of the BINACLE assay as a reliable and sensitive method for determining BoNT product potency, thereby substantiating its applicability as an alternative to the mouse bioassay.
ER  -