The potency testing of botulinum neurotoxin products.

I should like to strongly endorse the article by Andy Pickett,1 published in the September issue of ATLA, in which he criticises the lack of published information relating to a new in vitro method developed by Allergan Inc., which has received regulatory approval as a replacement for the LD50 mouse bioassay for the potency testing of a botulinum neurotoxin (BoNT) series of products. Clearly, there is much interest in developing alternatives to the mouse LD50 potency test for BoNT, which is understandable, in view of the highly severe endpoint and the large numbers of animals involved.2–11 For example, cell-based assays have been devised that use either continuous cell lines, such as neuro-2a, PC12, and SK-NSH cells, or primary neurons derived from chicken, mouse and rat spinal cord cells.12 However, none of these methods have been fully developed, validated and accepted by regulatory authorities. It was therefore with great interest that I learned that Allergan Inc. (which currently produces the vast majority of the pharmaceutically-used BoNT products, as BOTOX® [onabotulinumtoxin A]) had developed a replacement potency test, which has received regulatory approval.13 Until June 2011, the mouse LD50 potency assay was the only method approved by regulatory authorities, anywhere in the world. Allergan announced that the FDA had approved a fully in vitro, cell-based assay for the potency testing of its products (BOTOX® [onabotulinumtoxin A] and BOTOX® Cosmetic) for stability and potency. The company stated that the new assay is specifically applicable to its botulinum toxin type A product, and that the newly-approved test was to be implemented immediately for the release of product for sale in the US. This approval does not extend to botulinum toxins made by other manufacturers. It is estimated that use of the new assay will eventually reduce Allergan’s animal-based assay testing by up to 95% over the next three years. It was also reported that the UK’s Medicines and Healthcare Products Regulatory Agency (MHRA) has approved the assay for BOTOX® vials sold in the UK. In addition, it was noted that the first positive opinion, from Agence Française de Sécurité Sanitaire des Produits de Santé (AFSSAPS), relating to VISTABEL®, paves the way for approval in 29 countries in the European Union. The second positive opinion was granted by the Irish Medicines Board (IMB) for BOTOX®, and covers 14 European Union countries involved in the Mutual Recognition Process. Since then, Hong Kong and Switzerland have also approved use of the new assay, and, apparently, registrations are ongoing in several other countries worldwide. On the face of it, this would seem to be very good news, and the development and acceptance of the assay has been welcomed by several scientific and animal welfare bodies. The method has apparently been well-documented in regulatory submissions, and has been subjected to a validation study by Allergan. However, as Spielmann noted,14 it is essential for the scientific basis of the new cellbased potency assay and the validation data to be put into the public domain, in order for the method to be available for use by other toxin producers. It is also important for all of this information to be published, to permit independent scrutiny and assessment of the validation data obtained, and for other scientists to be able to see if the method is reliable and relevant when performed in their laboratories, as is standard practice with the validation of other novel replacement safety testing methods. 15,16 In this context, it should be noted that Allergan is discussing how to license the technology “to other parties that share its commitment to implementing non-animal alternatives to animal-based assays in the manufacture of their medical products”.13 So, what do we know about the new assay from the paucity of details publicly available? Two US patents, filed by the company in June 2012, contain potentially relevant information on the development of methodology to screen for cell lines responsive to BoNT/A intoxication. One of these17 discloses details of methods for making and detecting the binding of antibodies against the epitope of the carboxyl-terminus at the P1 residue of the alpha-SNAP-25 BoNT/A protein cleavage product. The other patent18 describes work that appears to have paved the way for the development of an in vitro cytotoxicity luminescent gene reporter assay involving geneticallyengineered neuronal cell lines (e.g. H1), highly sensitive to very low amounts of BoNT/A activity. This is coupled with immunological detection of the SNAP-25 cleavage product, presumably by using the specific antibodies described in the other patent. Toxin potency in the assay is expressed as a ratio of BoNT/A activity to cell viability. ATLA 40, 343–348, 2012 343