A Bilayer-Based In Vitro Assay for Botulinum Holotoxin Potency Assessments

Abstract

The standard procedure for botulinum toxin potency assessments is the mouse lethality assay by intraperitoneal injection. However, the mouse assay is laborious, expensive and ethically controversial. We have developed and are refining an in vitro, synthetic bilayer-based assay (BBA) for characterizing the potency of botulinum neurotoxin (BoNT) containing therapeutics, as a replacement for the mouse lethality assay. The BBA employs interfacial planar lipid bilayer, preconfigured to mimic a neuronal cell membrane, along with a nanopore-based endopeptidase assay for assessing the peptidase activity of the toxin. When the BoNT-containing sample is introduced into the assay, the BoNT heavy chain (HC) binds to its target receptors before forming a transmembrane channel that then transports the light chain (LC) across the lipid bilayer. Once the LC has traversed the membrane and is released from the HC, it then cleaves and inactivates its respective target protein, i.e., its soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) protein target. In the BBA, both the rate of HC insertions into the planar lipid bilayer and LC peptidase activity can be independently, but simultaneously, monitored, with the rate of SNARE protein cleavage being directly proportional to the potency of the sample introduced. While the assay is being specifically developed for BoNT testing, the platform will be generally applicable to a large number of other biomolecules with transmembrane activity for which there is currently no standard in vitro characterization technique.