Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

<div><p>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.</p> </div

Effects of selenium deficiency on West Nile virus replication and cytopathogenicity-1

E extracted from control, Se- and Se+ Vero and SK-N-SH cells and GPx1 enzyme activity was measured at days 3, 7 and 10 post-induction of Se deficiency by using the cGPx1 assay kit. Data are reported as mean ± SD of triplicate experiments. * p < 0.05, ** p < 0.005 compared to control cells. Analyses of GPx1 protein by Western blot. 50 μg of total protein extracted from Vero and SK-N-SH cells grown in control, Se- and Se+ media were separated on PAGE, followed by immunoblotting with anti-GPx1. Equal loading was confirmed by re-blotting the same membranes with anti-β-actin.<p><b>Copyright information:</b></p><p>Taken from "effects of selenium deficiency on West Nile virus replication and cytopathogenicity"</p><p>http://www.virologyj.com/content/5/1/66</p><p>Virology Journal 2008;5():66-66.</p><p>Published online 31 May 2008</p><p>PMCID:PMC2453119.</p><p></p

Development of a sensitive screening CBPA.

<p><b>A.</b> Protocols for the sensitive and screening (in italic) cell-based potency assays (CBPAs) utilizing differentiated SiMa cells and ECL-sandwich ELISA. <b>B.</b> Representative screening CBPA with SiMa cells treated with 0.014–10 nM BoNT/A (150 kDa) for 6 h followed by overnight incubation in toxin-free medium to allow for SNAP25₁₉₇ accumulation. Average EC₅₀ of 125 independent assays performed by three operators is shown. <b>C.</b> Dilutional linearity-recovery table with concentrations from 1.75 to 0.5 relative potencies (R.P.). The last column exemplifies how many individual assays produced data in which the confidence interval (C.I.) overlapped 1 making that dilution indistinguishable from the reference 1× preparation in that specific assay. Percent recoveries from 86 to 117% demonstrate excellent accuracy of the assay.</p

Effects of selenium deficiency on West Nile virus replication and cytopathogenicity-7

E extracted from control, Se- and Se+ Vero and SK-N-SH cells and GPx1 enzyme activity was measured at days 3, 7 and 10 post-induction of Se deficiency by using the cGPx1 assay kit. Data are reported as mean ± SD of triplicate experiments. * p < 0.05, ** p < 0.005 compared to control cells. Analyses of GPx1 protein by Western blot. 50 μg of total protein extracted from Vero and SK-N-SH cells grown in control, Se- and Se+ media were separated on PAGE, followed by immunoblotting with anti-GPx1. Equal loading was confirmed by re-blotting the same membranes with anti-β-actin.<p><b>Copyright information:</b></p><p>Taken from "effects of selenium deficiency on West Nile virus replication and cytopathogenicity"</p><p>http://www.virologyj.com/content/5/1/66</p><p>Virology Journal 2008;5():66-66.</p><p>Published online 31 May 2008</p><p>PMCID:PMC2453119.</p><p></p

Sandwich ELISA assay with chemiluminescence is as sensitive as the ECL assay.

<p><b>A.</b> Example of a representative experiment in which differentiated SiMa cells were treated with BoNT/A at concentrations from 0.03 to 25 pM for 24 h followed by 48 h incubation, and the lysates were evaluated in the optimized chemiluminescence read-out. The results obtained were similar to the ECL read-out (EC₅₀∼0.5 to 2 pM), excellent signal to background, and reproducibility of the replicates. <b>B.</b> Summary of optimized parameters for the chemiluminescence CBPA. Seven parameters comprising cell culture and ELISA read-out were specifically optimized for this assay by testing several conditions for each parameter.</p

SiMa cells are sensitive and specific for BoNT/A uptake.

<p><b>A.</b> Differentiated SiMa cells were treated with 1 nM BoNT/A complex from 1 to 60 min. Cell lysates were evaluated in the ECL-sandwich ELISA. Signal above background was observed at the earliest time points indicating high affinity binding (Error bars = std. dev.). <b>B.</b> Specificity of uptake by SiMa cells was demonstrated by comparing the uptake of BoNT/A (150 kDa) to recombinant LH_N/A (lacking binding domain but comprising the translocation domain and an active light chain) and inactive BoNT/A (iBoNT/A, inactive light chain). No cleavage of SNAP25 was detected with iBoNT/A. Non-specific uptake of LH_N/A was observed only at doses >10 nM. <b>C.</b> Graph comparing specific uptake of BoNT/A complex (at pM concentrations) to a full dose-response of recombinant LH_N/A (at pM to µM concentrations) with a highest dose of 50 µM. The EC₅₀ for the LH_N/A molecule was 2.1 µM versus 0.85 pM for the fully active BoNT/A.</p

System suitability-Sensitive Assay: Excellent accuracy and linearity.

<p>System suitability-Sensitive Assay: Excellent accuracy and linearity.</p

Characterization of anti-SNAP25₁₉₇ monoclonal antibody 2E2A6.

<p>Specificity demonstrated by lack of cross-reactivity towards SNAP25₂₀₆. <b>A.</b> Neuro-2a cells were treated with BoNT/A (150 kDa) from 0.01 to 10 nM (duplicate wells) for 24 h. Western blot was performed with 2E2A6 antibody (1 µg/mL). No cross-reactivity with SNAP25₂₀₆ (no bands observed in the 0 nM BoNT/A lanes) and no other non-specific bands were detected in the whole blot. <b>B & C.</b> Surface plasmon resonance (SPR) was used to characterize the 2E2A6 antibody and to compare the binding affinity and binding kinetics of 2E2A6 and MC-6053. 2E2A6 bound SNAP25₁₉₇ with high affinity and did not bind SNAP25₂₀₆ at any of the concentrations tested up to 10 µM. MC-6053 was able to bind SNAP25₂₀₆ with a K_D of 240 nM. <b>D.</b> The K_d for 2E2A6 is tenfold lower than the K_d for MC-6053 as shown in the normalized graph resulting in better affinity.</p

The CBPA can measure BoNT/A biological activity in BOTOX® vials.

<p><b>A.</b> A custom medium to reconstitute BOTOX® vials (the nominal value of 100 U was used) was designed to overcome the hypertonicity caused by NaCl present in the formulation. The matrix for the subsequent dilutions was kept constant. Performance of the assay was improved resulting in better sensitivity (EC₅₀ = 0.9 U/well) and higher efficacy of uptake. <b>B.</b> Two different lots of BOTOX® (the nominal value of 100 U was used) were evaluated in the CBPA. Data was analyzed in PLA 2.0 resulting in 0.82 relative potency (CI: 0.7–1.1) indicating similar potency of both lots. <b>C.</b> A single lot of BOTOX® was tested by two operators (n = 8 and n = 9 independent experiments respectively), four runs for each operator are shown. Relative potencies were similar for all the tests performed demonstrating consistent performance by two operators.</p

Assay Optimization and Standardization.

<p>Assay Optimization and Standardization.</p