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.

OnabotulinumtoxinA Displays Greater Biological Activity Compared to IncobotulinumtoxinA, Demonstrating Non-Interchangeability in Both In Vitro and In Vivo Assays

“…The purpose of this study was to compare the potency of different labeled vials of incobotA (different toxin amounts/vial; 50 U, 100 U, and 200 U vials) versus onabotA (100 U vial) on a unit-to-unit basis to assess the biological activity of these products using both in vitro (light-chain activity high-performance liquid chromatography (LCA-HPLC) and cell-based potency assay (CBPA)) and in vivo (compound muscle action potential (CMAP) electrophysiology and digit abduction score (DAS)) assays. In agreement with previous findings [ 28 , 32 ], onabotA showed greater biological activity than incobotA in all assays, demonstrating that the potency units and performance of these two products in these pre-clinical assays are not interchangeable and each product should be administered as stipulated in product labeling.…”

“…The LCA-HPLC assay measures SNAP-25 cleavage specificity [ 28 ]. In the context of this study, the light chain of BoNT/A cleaves a commercially available truncated BoNT/A substrate (SNAPtide ® 520, List Biological Laboratories, Inc., Campbell, CA, USA) derived from synaptosomal associated protein, 25 kDa (SNAP-25).…”

“…Furthermore, European incobotA product labels (Xeomin and Bocouture ® ) now state “therapeutic equivalence” to onabotA for select indications [ 22 , 23 ]. However, other studies refute the claim of interchangeability between onabotA and incobotA (reviewed in [ 21 , 24 , 25 , 26 ]), and the discussion has also included preclinical testing [ 27 , 28 , 29 , 30 , 31 , 32 ].…”

A Label Free Colorimetric Assay for the Detection of Active Botulinum Neurotoxin Type A by SNAP-25 Conjugated Colloidal Gold

“…Before analysis samples were diluted in a digestion buffer (20 µL) of ZnCl 2 (0.5 mM), Dithiothreitol (2 mM), HEPES (50 mM) and Tween-20 (0.05%) at pH 7.4 and incubated for an hour at 37 °C [26]. This reduced any toxin present in the sample to the active form by separating the catalytic light chain from the heavy chain.…”

Editorial comment on ‘5 year experience with incobotulinumtoxinA (Xeomin®) the first botulinum toxin drug free of complexing proteins’

“…Hunt et al. [3] followed with additional data, which confirmed the reduced activity of incobotulinumtoxinA with an in vitro method and identified an unexplained SNAP25 cleavage product. These results support the regulatory statements, which caution the user on the comparability of units and the lack of interconversion.…”