Yiu-chung Wong
Hong Kong Government Laboratory, Hong Kong
Title: Analysis of Botulinum Neurotoxin A (BoNT/A) in pharmaceutical products using MALDI-TOF and LC-MS/MS
Biography
Biography: Yiu-chung Wong
Abstract
Botulinum neurotoxins (BoNT) are produced by the anaerobic bacterium Clostridium botulinum and are one of the most lethal known poisons (LD50 = 0.8 g 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. In response to increased public pressure to the inhumane testing, sensitive non-anaimal assays are of urgent need. 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. This work reported 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 MSE 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). Proteolytic activities of BoNT/A in different excipient matrices were also investigated. The method has been validated across few brands of BoNT/A pharmaceutical injections commonly available in the Asia Pacific and was found to produce reliable results.
