Characterization of SNARE cleavage products generated by formulated botulinum neurotoxin type-a drug products.

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.

Potency evaluation of a formulated drug product containing 150-kd botulinum neurotoxin type A.

OBJECTIVE: To assess the potency of a formulated drug product containing 150-kd botulinum toxin type A (BoNT/A) as the active pharmaceutical ingredient. METHODS: Potencies of 3 unexpired lots of a commercially available BoNT/A drug product, reportedly devoid of complexing proteins (Xeomin), were determined using an approved in-house potency bioassay by injecting mice intraperitoneally and recording percent-mortality across dilutions. For each test session, duplicate sets of dilutions were performed for each lot alongside a 900-kd BoNT/A (BOTOX) potency reference standard. A relative potency for each 150-kd BoNT/A preparation was determined using this potency reference standard. A standard normalized potency estimate for each lot of 150-kd BoNT/A was calculated by multiplying the relative potency by the nominal value for the reference standard. The average potency for each 150-kd BoNT/A lot was calculated using a weighted combination of assay results and compared against the labeled potency of 100 U per vial. Similar follow-on testing was performed 1 year later to assess stability. RESULTS: The average potencies for the 3 lots of 150-kd BoNT/A product were 69 (95% confidence interval [CI], 65-73), 75 (95% CI, 70-80), and 78 (95% CI, 70-87) U per vial. Follow-on testing produced even lower potency results for all 3 lots. CONCLUSIONS: The potency of the drug product containing 150-kd BoNT/A (Xeomin) measured substantially lower than the labeled 100 U per vial when tested in a potency assay approved for release testing of an established drug product containing 900-kd BoNT/A (BOTOX).