Refinement and Validation of an Alternative Bioassay for Potency Testing of Therapeutic Botulinum Type A Toxin

Abstract

The type A neurotoxin produced by Clostridium botulinum is a potent neuromuscular blocking agent which causes paralysis by preventing the release of neurotransmitter from motor neurones. This property has led to the use of the toxin in the treatment of a number of neuromuscular diseases involving muscle spasms. At present, the only recognised assay with the specificity and sensitivity to estimate accurately the potency of botulinum toxin in clinical preparations is bioassay, in which lethality is used as the end point. Refinement of this assay, with respect to the end point, was explored on the basis of the development of flaccid paralysis of muscles following subcutaneous injection of the toxin at the inguinocrural region. Potency estimates, relative to in house reference preparations, for different therapeutic preparations obtained using flaccid paralysis as a scored response gave excellent agreement with estimates obtained in independent assay using the currently required control method. This study demonstrates that an alternative, more humane bioassay for potency testing of clostridia neurotoxins gives valid estimates equivalent to those currently in use.

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

“…This method can estimate BoNT/A quantities ranging from as little as 0.075 mouse LD 50 to an upper detection limit of about 38 LD 50 s (sensitivity over nearly 3 logs) with just one animal. A similar method based on assessing the severity of flaccid paralysis was developed to analyze low concentrations of therapeutic samples of BoNT/A without the necessity of a terminal end point [31]. In another related method, toxin dilution is injected intravenously and time-to-death is correlated to toxin concentration [32].…”

Laboratory Diagnostics of Botulism

“…Another type of mouse assay with a more humane endpoint of local muscle paralysis as a result of subcutaneous injection of botulinum toxin type A has been explored for testing the potency of botulinum neurotoxins for therapeutic use (186). The nonlethal mouse assay is equal to the conventional bioassay in sensitivity and specificity, but it does not cause signs of distress or impaired movements in the animals (186). However, the assay has been targeted to potency testing of purified neurotoxins, and thus it has not been validated for microbiological laboratories investigating complex sample matrices.…”

Bioassays for evaluation of medical products derived from bacterial toxins

“…Introduction of relative potency provided an opportunity to replace non-flexible assay design dependent on defined systemic effects of neurotoxin, determined typically after 96 or 72 hours injection, with 24 or 48 hours observation of local effects (e.g. flaccid paralysis [12]; digit abduction score [13]; grip strength [14] or compound muscle action potential [15 ] after injection of sublethal doses of toxin. The dose of toxin, injected locally will be below lethal dose and will typically be in the range of 0.05-0.2 nominal LD50/dose [12].…”

“…flaccid paralysis [12]; digit abduction score [13]; grip strength [14] or compound muscle action potential [15 ] after injection of sublethal doses of toxin. The dose of toxin, injected locally will be below lethal dose and will typically be in the range of 0.05-0.2 nominal LD50/dose [12]. In these assays potency can be calculated from observation of the local signs of paralysis, digit score, grip strength or compound muscle potential expressed relative to same effect induced by a product specific reference preparation of known activity and stability.…”