Immuno-based botulinum toxin serotype a activity assays

Abstract

The present specification discloses SNAP-25 compositions, methods of making α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, methods of detecting BoNT/A activity, and methods of detecting neutralizing α-BoNT/A antibodies.

1 . (canceled) 
     
     
         2 . A liquid pharmaceutical composition comprising a Clostridial toxin. 
     
     
         3 . The liquid pharmaceutical composition of  claim 2 , wherein the Clostridial toxin is a non-complexed about 150 kDa botulinum toxin serotype A (BoNT/A). 
     
     
         4 . The liquid pharmaceutical composition of  claim 2 , wherein the liquid pharmaceutical composition comprises a dose of 1 ng or less of BoNT/A. 
     
     
         5 . The liquid pharmaceutical composition of  claim 2 , wherein the liquid pharmaceutical composition comprises water. 
     
     
         6 . The liquid pharmaceutical composition of  claim 2 , wherein the concentration of the BoNT/A is from about 0.9 pM to about 8.3 pM. 
     
     
         7 . The liquid pharmaceutical composition of  claim 2 , wherein the concentration of the BoNT/A is about 2.8 pM. 
     
     
         8 . The liquid pharmaceutical composition of  claim 2 , wherein the liquid pharmaceutical composition is suitable for use as a human cosmetic BoNT/A therapy. 
     
     
         9 . A process for manufacturing a pharmaceutical product comprising the liquid pharmaceutical composition of  claim 2 , comprising:
 (i) contacting a sample of non-reconstituted, aqueous composition comprising a Clostridial toxin with differentiated Neuro-2a cells susceptible to intoxication by about 10 pM or less of the Clostridial toxin, wherein the susceptibility to intoxication is the lower limit of quantification of cleaved Clostridial toxin substrate as determined by Western blot; and   (ii) releasing the liquid composition as an injectable pharmaceutical product.   
     
     
         10 . The process of  claim 9 , further comprising a step of determining the Clostridial toxin activity in the sample and releasing the pharmaceutical product if the Clostridial toxin activity in the sample matches the Clostridial toxin activity of an accepted reference. 
     
     
         11 . The process of  claim 10 , wherein determining the Clostridial toxin activity in the sample is accomplished by detecting disappearance of an intact synaptosomal-associated protein of 25 kDa (SNAP-25) polypeptide in the differentiated Neuro-2a cells. 
     
     
         12 . The process of  claim 11 , wherein the SNAP-25 polypeptide is a non-naturally occurring SNAP-25 polypeptide comprising a fluorophore or a fluorescent protein. 
     
     
         13 . The process of  claim 9 , wherein the differentiated Neuro-2a cells are susceptible to intoxication by about 1 pM or less of the Clostridial toxin. 
     
     
         14 . The process of  claim 9 , wherein the pharmaceutical composition comprises a dose of the Clostridial toxin suitable for use as a human cosmetic or medicinal therapy. 
     
     
         15 . The process of  claim 9 , wherein the Clostridial toxin is a non-complexed about 150 kDa botulinum toxin serotype A (BoNT/A).