General Commentary ARTICLE
Commentary: A Cell Line for Detection of Botulinum Neurotoxin type B
- 1Botulinum Research Center, University of Massachusetts Dartmouth, United States
The quest for an assay which is a viable, appropriate and validatable alternative to the mouse LD50 potency assay is something that researchers and companies, working in the field of botulinum toxin (BoNT), have engaged with for decades. The mouse potency assay has not only been used as the long-standing gold standard for BoNT identification and serotype determination, for example when cases of botulism food poisoning are suspected, but also for the routine assessment and quality release of BoNT commercial product batches used for clinical treatments worldwide.
The literature shows that so-called “alternative assays” have been available since the 1970s (Kozaki et al., 1979; Notermans et al., 1979). However, these early methodologies, together with many of those reported over the intervening 40 years, only detected the presence of the respective BoNT serotype protein, based upon ELISA technology. They detected none of the four key and essential activities of the BoNT molecule – receptor binding, endocytic internalisation, Light Chain translocation and enzymatic activity. In the last decade, other more modern assay methodologies have arrived, but these have assessed just one property of the BoNT molecule, typically the enzymatic activity alone (Lindstrom and Korkeala, 2006). However, this trend is changing and cell-based assays are now available which can determine all known properties of the BoNT molecule (Pellett, 2013).
The work of Rust and colleagues, describing an alternative cell-based assay for serotype B BoNT, presents a sound, scientifically determined methodology that could be applied to the single commercial type B product now available (Rust et al., 2017). The authors in fact conclude that “the assay may well be suited for measuring potency of pharmaceutical BoNT/B products and antitoxin antibodies in toxin neutralization testing”. Unfortunately, there are several key issues that must be addressed before any such assay is brought into use by a commercial company selling BoNT products. This is also true for similar assays relating to other serotypes, of which there are many.
Rust & colleagues (Rust et al., 2017) are not the first to engineer a cell line for such an application. The US company BioSentinel, Inc. has, for some years, marketed an engineered cell line and accompanying technology as a replacement assay for BoNT potency determinations (Atapattu et al., 2013). The assay system, termed BoCell®, has also been automated, making routine quality control use of the method much more straightforward (Burlingham et al., 2017). To some extent, the work of Rust and colleagues copies that of BioSentinel. Detailed patent evaluations and searches must be carried out before any exploitation of their work, to determine if there is any infringement of prior existing patents.
Assays, such as those developed by Rust and colleagues, need to go through rigorous development and validation before they could be considered acceptable for use by a commercial company marketing BoNT products (Harmonisation, 2005; Sharma et al., 2008). Also, comparative data with the mouse LD50 test must be generated and effective equivalence demonstrated between the methods. The authors have provided none of these data and so an evaluation of the assay in terms of commercial suitability and validity cannot be made. Statements that such an assay could be used commercially therefore fall far short of reality.
But there are several other key issues that must be addressed before such a method can be used.
Firstly, who owns the cell line? Rust and colleagues have engineered the SiMa cell line obtained from the German collection Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). But DSMZ are not the original owners of the line, they are simply a repository. The SiMa cell line was first reported by Marini and colleagues at the University of Tuebingen in Germany in 1999 (Marini et al., 1999). The line was derived in August 1991 from a Caucasian male, 20 months-old, with an adrenal neuroblastoma. This makes him the rightful owner. Has DSMZ obtained appropriate permissions to allow distribution and use of his cell line? Did Rust and colleagues have permission to modify the cell line and engineer as they described? The whole subject of cell line ownership has been highly controversial in recent years and completely clear permissions, coupled to commercial agreements for exploitation, must be obtained by any commercial company seeking to use these lines (Greely and Cho, 2013). In addition, the DSMZ ordering terms state clearly “Cell lines and their products shall not be sold or used for commercial purposes or utilized in any other type of commercial activity”: this represents a major problem for a commercial company seeking to use the line. Therefore, aspects about commercial use of the cell line, and even whether the engineered version is legal, are unclear. The authors should clarify the situation.
Secondly, who owns the antibodies used in the Rust et al work? The authors have raised a specific cleaved-VAMP antibody for their assay. Unfortunately, the company used (Davids Biotechnologie Germany) has strict conditions on use of the products they generate: “Unless otherwise specified all products and services from Davids Biotechnologie GmbH are sold for research use only”. A licensing agreement is therefore required (and may not be even possible) if the antibody is to be used for commercial release testing of, for example, BoNT type B products. A number of other primary and secondary antibodies have also been used by the group, with stated suppliers ranging from Synaptic Systems, Abcam and GE Healthcare to “in-house”. This number of companies involved in producing reagents would simply be a negotiation nightmare for a commercial company to deal with, in order to adopt such an assay. If any of these companies demands a royalty-bearing license agreement, then any benefit from t]having and implementing such an assay diminishes rapidly.
The use of antibodies in these alternative assays also raises the issues of provenance of each antibody, together with batch-to-batch reproducibility. The Protein A coated plates from Thermo Fisher Scientific would also fall into this category, due to the biological nature of the Protein A. These data would be absolutely essential for any submission to a regulatory authority in any request to substitute the mouse potency assay for a cell-based alternative. The requirements for such data, which would be both historical and those that must be newly generated to support the reagent, will lead into deep commercial confidentiality issues with the suppliers. Suppliers can be reticent to supply such data and can demand commercial agreements to, for example, purchase the reagents solely from them, both in specified quantities and over long periods. Such demands negate one of the primary key supply tenants of pharmaceutical product manufacture and testing – second sourcing. Pharma companies strive always to have more than one supplier, especially for key raw materials or reagents, to ensure that, no matter what circumstances, they are able to continue product manufacture, testing and supply without pause.
Finally, the NanoLuc® detection system used by Rust and colleagues is a proprietary methodology owned by Promega Corporation, Madison WI, USA. Promega have similar statements in their terms and conditions of sale relating to non-commercial use of these products and also their NanoLuc® vectors. Licenses for even laboratory research use are required, in some circumstances. The legalities and Limited Use Label Licenses are extensive.
The search for an alternative BoNT potency assay to the mouse potency method has attracted considerable funding from many sources and over many years. This funding has been provided by a wide variety of organisations, agencies and government departments. No data exist on the sums involved. Often, the funding has been provided to an academic department for support of a student(s) studying for higher qualifications: this seems to be the case with the work of Rust and colleagues at the University of Sheffield. Inevitably, the applications for funding involve statements that, if the work proposed were to result in a new assay, then this assay would be available to (or more strongly, should be taken up by) commercial companies for exploitation as an alternative method. However, the funding and these activities are fruitless, and actually of no value whatsoever, if they are not exploited commercially: essentially, they are a waste of research funding which is both valuable and very scarce in this day and age. Yet in virtually none of these cases – and there are many - have the participants discussed with industry what they are doing and how they propose to develop their approach. They are, essentially, working for themselves alone. The group of Rust and colleagues included no industry representation and no indication that the group had been working with industry, which is yet again very disappointing. No matter how sound the science, or how good the results, if the work is designed to be for the benefit of industry then why isn’t industry involved?
To date, no alternative BoNT potency assay developed in a university laboratory or other institution has been submitted to and accepted by regulatory authorities. Of the two alternatives currently in existence (for Botox® and Xeomin®), both were developed in-house by the manufacturers. In fact, the estimated cost of developing the method used for Botox® has been cited as $65 million, an extraordinary amount and well beyond any grant programs for universities or institutions. The fact that companies have taken their own ways forward must surely be testimony enough to those seeking to develop these alternatives, that the commercial world cannot use what they think they can produce. Why the funding agencies are not seeing these real facts is a puzzle. Further monetary support still continues: this should cease. The world of alterative assays for BoNT has taken a completely different way ahead, dealing with the commercial realities that must be taken into account and no longer needs any input from academia, on any aspect.
Conflict of Interest
Andy Pickett is Director and Founder of Toxin Science Limited, Wrexham, UK; Adjunct Professor at the Botulinum Research Center, Institute for Advanced Sciences, North Dartmouth, USA; and was previously Senior Program Leader & Scientific Expert, Neurotoxins, Galderma Uppsala, Sweden. The opinions and statements expressed are those of the author and Toxin Science Limited only. No sponsorship was received for this work.
Keywords: Botulinum Toxin, Potency assay, Mouse assay, Alternative assay, Commercial issues
Received: 15 Aug 2018;
Accepted: 03 Sep 2018.
Edited by:Salvatore Salomone, Università degli Studi di Catania, Italy
Reviewed by:Joaquim Ferreira, Instituto de Medicina Molecular (IMM), Portugal
Copyright: © 2018 Pickett. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Dr. Andy Pickett, Botulinum Research Center, University of Massachusetts Dartmouth, North Dartmouth, United States, firstname.lastname@example.org