SBIR Phase II: De novo assays for detection of the proteolytic activity in botulinum neurotoxin-based pharmaceuticals

This Small Business Innovation Research (SBIR) Phase II project proposes to complete development of a universal, in vitro assay for determining the enzymatic activity of botulinum neurotoxin (BoNT)-based pharmaceuticals. This assay, called BoTest Matrix A Assay, will enable standardized comparisons of different BoNT preparations, regardless of manufacturer. BoNTs are used in many different cosmetic and pharmaceutical applications due to their exquisite specificity for motor neurons and their long-lasting effects. Currently, the strength and quality of BoNT-based pharmaceuticals is assessed using a mouse bioassay where BoNT is injected into dozens of mice and a unit of activity depends on the rate or proportion of mouse deaths. Beyond the obvious concerns about animal testing, the variability of the mouse bioassay and the lack of suitable standards and uniform protocols among manufacturers put patient safety and clinical outcomes at risk. The proposed assay(s) will reduce the use of animals and will provide a method to uniformly assess the activity of BoNT-based pharmaceuticals, thus increasing patient safety and the likelihood of successful treatment. The broader/commercial impacts of this research are improved procedures and protocols that would greatly increase patient safety while reducing animal testing. The BoTest Matrix Assay would be applicable to all stages of BoNT-based drug manufacturing, including quality control, quantification, stability testing, and decontamination testing at manufacturing sites?all applications that are currently performed with animal assays. In addition, the assay would be applicable for authenticity testing (against counterfeit drugs) at border control points and inoffice testing for high-dose procedures where toxin activity concentration is critical for clinical outcomes. The proposed technology would be also the first of its kind on the commercial market. Other commercially available assays for detecting BoNT activity are not suitable for assessing BoNT-based pharmaceuticals, because the stabilizers added to the pharmaceutical formulations interfere with those assays. The proposed technology could be further adapted for testing BoNT activity in highly complex matrices (e.g., blood, food, water). Thus, the assay's utility would extend into food, biodefense, and environmental testing.