Analysis of Botulinum Neurotoxin A (BoNT/A) in Pharmaceutical Products based on Proteolytic Activity and Signature Markers

Yiu-chung Wong, Hong Kong Government Laboratory, Hong Kong, China (ycwong@govtlab.gov.hk)

Botulinum neurotoxins (BoNT) are produced by the anaerobic bacterium Clostridium botulinum and are one of the most lethal known poisons (LD₅₀ = 0.8 mg for a 70 kg human by inhalation). Despite its high lethal toxicity, BoNT have been used to treat spasms and other muscle problems. Most recently, BoNT serotype A (BoNT/A) gains its worldwide popularity in cosmetic surgery to prevent development of facial wrinkles. Clinical testing of BoNT/A is conventionally relied on in vivo mouse lethality assay in accordance with pharmacopieal and AOAC methods.

Identification of BoNT/A in pharmaceutical products by chemical means is always a challenge due to its complex structure, ultra-trace level and the interference from excipients. Howvere, to address intense public pressure to the inhumane testing, sensitive non-anaimal assays are necessary. This work reports a mass spectrometry study on BoNT/A in pharmaceutical injection samples. BoNT/A was isolated using magnetic beads immunoprecipitation, followed by on-beads digestion and was characterized by peptide mass finger printing using high-resolution tandem mass spectrometry coupled with ion mobility and Q-ToF in MS^E mode. The activity of the toxin was confirmed by its proteolytic activity towards specially designed synthetic SNAP-25 substrate. The specific cleaved peptide fragments obtained from SNAP-25 substrate correlated well with its proteolytic activity in linear range from 10 – 100 MLD₅₀/uL (r² > 0.99). Recent work on direct measurement of BoNT/A in pharmaceutical injection samples by mass spectrometry will also be reported. The study is based on ten potential signature markers, seven from the light chain and three from the heavy chain. Pharmaceutical samples that claimed to contain BoNT/A was trypsin digested under optimal conditions and its identity was examined by the presence of the concerned signiture markers. Detection limits were at 0.05 ppm level which is sensitive enough to detect registered BoNT pharmaceuticals in the market. The methods has been used to examine a few brands of BoNT/A pharmaceutical injections commonly available in the Asia Pacific region.

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Abstract Reference & Short Personal Biography of Presenting Author

Dr. Wong is a senior chemist of the Government Laboratory of Hong Kong. He is interested in the measurement of trace organic and inorganic toxicants in environment, food, dietary supplement and pharmaceutical matrices. He also organized a series of of international proficiency testing programmes and developed several certified reference materials in the food and herbal medicine fields. Recently, Dr. Wong is the co-editor of the two-volume book “Toxins and Toxicants in Food” published by Wiley and the contributing chapter author of the 3rd Edition Encyclopedia of Analytical Science by Elsevier.