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.
Highlights
Seven serotypes (A to G) of Clostridium botulinum are known
The suitability of the method was exemplified through analysis of three distinct BoNT serotype A (BoNT/A) drug products
IncobotulinumtoxinA drug product generated a secondary SNAP cleavage product not produced at significant levels by any other BoNT/A drug product or BoNT/A bulk toxin tested
Summary
Seven serotypes (A to G) of Clostridium botulinum are known. Each serotype synthesizes a 150-kD neurotoxin (BoNT) along with a group of nontoxic neurotoxin-associated proteins (NAPs) that form complexes [1]. BoNT is a zinc-endopeptidase that is composed of a 100-kD heavy-chain and a 50-kD light-chain, connected by a disulfide bond. The heavy-chain is responsible for binding (C-terminal domain) and translocation (N-terminal domain), whereas the light-chain is responsible for proteolytic activity. The light-chain of each serotype cleaves one or more soluble N-ethylmaleimide—. The endopeptidase activities of the toxins are unique in that they require the reduction of the disulfide bond between the light and heavy chains in order for enzymatic activity to occur. The light-chain of BoNT serotype A (BoNT/A) cleaves SNAP-251–206 at position Gln197–Arg198 [2]. This specific cleavage is an intrinsic property of the serotype
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
