Abstract
Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.
Highlights
Clostridial neurotoxins bind to nerve terminals and deliver their zinc-endopeptidase (Light Chain, LC) [1] inside the cytosol, where they cleave one of the soluble N-ethylmaleimidesensitive factor attachment receptor (SNARE) proteins leading to inhibition of neuroexocytosis [2,3,4,5,6]
Monoclonal 2E2A6 is highly specific for SNAP25197 with no detectable cross-reactivity to SNAP25206 in Western blot (Figure 1A), and ELISA (OD405 = 0.892 for SNAP25134–197 vs. OD405 = 0.036 for SNAP25134–206 peptides). 2E2A6 has been characterized using Surface Plasmon Resonance (SPR) analysis and compared with a commercial antibody, MC-6053 (Research & Diagnostic Antibodies), claiming to be specific for SNAP25197, but that was found to bind SNAP25206 with a KD of 240 nM in the SPR analysis demonstrating some cross-reactivity
The replacement cell-based potency assays (CBPAs) for Botulinum neurotoxin serotype A (BoNT/A) potency testing has a sensitivity similar to the mouse bioassay (EC50,1-0.4 U/well) and is specific for BoNT/A, by the use of monoclonal antibodies specific for SNAP25197
Summary
Clostridial neurotoxins bind to nerve terminals and deliver their zinc-endopeptidase (Light Chain, LC) [1] inside the cytosol, where they cleave one of the soluble N-ethylmaleimidesensitive factor attachment receptor (SNARE) proteins leading to inhibition of neuroexocytosis [2,3,4,5,6]. Botulinum neurotoxin serotype A (BoNT/A) causes prolonged, reversible muscle weakness by entering motor nerve terminals and cleaving 9 amino acids from the C-terminus of the SNARE protein SNAP25 (SNAP25206) to yield SNAP25197 [7], disrupting exocytosis and blocking neurotransmitter release [5,8,9]. The mLD50 is highly sensitive (7– 20 pg/mL) and has been adopted by all BoNT-based products manufacturers to test drug product potency. The mouse bioassay presents several challenges including assay time required, inability to differentiate between serotypes, sample capacity, and need for highly trained personnel and special animal facilities. Ex vivo alternatives include the rat or mouse phrenic nerve diaphragm [22] and the rat intercostal muscle strips assays [23,24] that allow several tests from tissues of a single animal. A sensitive cellbased potency assay (CBPA) is the preferred alternative [16,17,18,19,25]
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