Botulinum neurotoxin type-A (BoNT/A), as onabotulinumtoxinA, is approved globally for 11 major therapeutic and cosmetic indications. While the mechanism of action for BoNT/A at the presynaptic nerve terminal has been established, questions remain regarding intracellular trafficking patterns and overall fate of the toxin. Resolving these questions partly depends on the ability to detect BoNT/A’s location, distribution, and movement within a cell. Due to BoNT/A’s high potency and extremely low concentrations within neurons, an alternative approach has been employed. This involves utilizing specific antibodies against the BoNT/A-cleaved SNAP25 substrate (SNAP25₁₉₇) to track the enzymatic activity of toxin within cells. Using our highly specific mouse monoclonal antibody (mAb) against SNAP25₁₉₇, we generated human and murine recombinant versions (rMAb) using specific backbone immunoglobulins. In this study, we validated the specificity of our anti-SNAP25₁₉₇ rMAbs in several different assays and performed side-by-side comparisons to commercially-available and in-house antibodies against SNAP25. Our rMAbs were highly specific for SNAP25₁₉₇ in all assays and on several different BoNT/A-treated tissues, showing no cross-reactivity with full-length SNAP25. This was not the case with other reportedly SNAP25₁₉₇-selective antibodies, which were selective in some, but not all assays. The rMAbs described herein represent effective new tools for detecting BoNT/A activity within cells.

Differences in botulinum neurotoxin manufacturing, formulation, and potency evaluation can impact dose and biological activity, which ultimately affect duration of action. The potency of different labeled vials of incobotulinumtoxinA (Xeomin^®; 50 U, 100 U, or 200 U vials; incobotA) versus onabotulinumtoxinA (BOTOX^®; 100 U vial; onabotA) were compared on a unit-to-unit basis to assess biological activity using in vitro (light-chain activity high-performance liquid chromatography (LCA-HPLC) and cell-based potency assay (CBPA)) and in vivo (rat compound muscle action potential (CMAP) and mouse digit abduction score (DAS)) assays. Using LCA-HPLC, incobotA units displayed approximately 54% of the protease activity of label-stated equivalent onabotA units. Lower potency, reflected by higher EC₅₀, ID₅₀, and ED₅₀ values (pooled mean ± SEM), was displayed by incobotA compared to onabotA in the CBPA (EC₅₀: incobotA 7.6 ± 0.7 U/mL; onabotA 5.9 ± 0.5 U/mL), CMAP (ID₅₀: incobotA 0.078 ± 0.005 U/rat; onabotA 0.053 ± 0.004 U/rat), and DAS (ED₅₀: incobotA 14.2 ± 0.5 U/kg; onabotA 8.7 ± 0.3 U/kg) assays. Lastly, in the DAS assay, onabotA had a longer duration of action compared to incobotA when dosed at label-stated equivalent units. In summary, onabotA consistently displayed greater biological activity than incobotA in two in vitro and two in vivo assays. Differences in the assay results do not support dose interchangeability between the two products.