title: Characterization of SNARE cleavage products generated by formulated botulinum neurotoxin type-a drug products.
abstract: The study evaluated substrate cleavage product(s) generated by three botulinum neurotoxin serotype A (BoNT/A) medicinal drug products utilizing a novel and highly specific, light-chain activity, high-performance liquid chromatography (LCA-HPLC) method. Samples were reacted with a commercially available BoNT/A fluorescent substrate derived from the SNAP-25 sequence. Reaction products were separated by reversed-phase HPLC. The method detected an atypical cleavage pattern by one of the formulated drug products. IncobotulinumtoxinA produced two cleavage fragments rather than the single fragment typically generated by BoNT/A. Identification confirmed the secondary cleavage at a position corresponding to SNAP-25 Arg198-Ala199 (normal BoNT/A cleavage is Gln197-Arg198). Arg198-Ala199 is also the cleavage site for trypsin and serotype C toxin. Normal cleavage was observed for all other BoNT/A drug product samples, as well as 900-kD and 150-kD bulk toxin BoNT/A. The reason for this unexpected secondary cleavage pattern by one formulated BoNT/A drug product is unknown. Possible explanations include a contaminating protease and/or damage to the 150-kD type-A toxin causing nonspecific substrate recognition and subsequent cleavage uncharacteristic of type-A toxin. The BoNT/A drug products were also analyzed via the LCA-HPLC assay using a commercial BoNT/C fluorescent substrate derived from the syntaxin sequence. Cleavage of the serotype C substrate by incobotulinumtoxinA was also confirmed whilst neither of the other drug products cleaved the syntaxin substrate.
authors: Allergan, Inc., 2525 Dupont Dr, Irvine, CA 92612, USA. hunt_terrence@allergan.com
authors: Terrence
authors: T
authors: Hunt
authors: David
authors: D
authors: Rupp
authors: Gary
authors: G
authors: Shimizu
authors: Karen
authors: K
authors: Tam
authors: Julia
authors: J
authors: Weidler
authors: Jack
authors: J
authors: Xie
mesh_terms: D019274:Botulinum Toxins, Type A / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D050600:SNARE Proteins / Q000737:chemistry*
chemical_list: D050600:SNARE Proteins; D019274:Botulinum Toxins, Type A; C542869:abobotulinumtoxinA; C545476:incobotulinumtoxinA