几十年来,欧洲药典专论破伤风疫苗(吸附)(0452)和兽用破伤风疫苗(0697)要求每批破伤风类毒素的特定毒性和毒素的缺乏以及毒性的不可逆性必须通过在豚鼠中进行体内毒性测试,然后才能将其包括在人或兽用疫苗中。根据3Rs的替换概念,减少和改善动物实验,Paul-Ehrlich-Institut(PEI,德国)。这种方法,所谓的BINACLE(结合和裂解)测定,使用TeNT的受体结合和蛋白水解特性来特异性检测活性毒素分子。成功的内部验证研究以及小规模的可转移性研究表明,该方法可能代表药典体内毒性测试的合适替代方法。作为后续,欧洲药品和医疗保健质量局(EDQM)在其生物标准化计划(BSP)的主持下组织了一项国际合作研究,旨在验证BINACLE试验作为破伤风类毒素豚鼠毒性试验的潜在替代方法的适用性。在本研究的框架内,编码为BSP136的可行性阶段-也称为第1阶段-运行以选择和鉴定关键研究试剂和样品,并评估项目负责人开发的BINACLE标准操作程序的性能。然后是旨在评估BINACLE的国际合作研究,称为BSP136第2阶段,已开始。总共向19个国际实验室(包括疫苗制造商和国家控制实验室)提供了详细的检测方案,检测所需的关键试剂,三个样本由欧洲主要疫苗制造商捐赠的三种不同的散装破伤风类毒素和一种国际标准类毒素组成。要求每个参与者按照所提供的方案进行三个独立的BINACLE测定。结果的统计分析表明,大多数参与实验室能够根据提供的方案进行BINACLE测定。然而,参与者获得的结果差异很大,并不是所有的实验室都能够实现活性TeNT的灵敏检测。多种因素可能导致BSP136研究结果的变异性升高。从这些因素的分析,制定策略以帮助提高BINACLE测定的标准化,并在后续验证研究中获得更一致的结果,BSP136第3阶段(第2部分),为此,实验阶段发生在2023年。本手稿总结了构成BSP136项目第1部分的第1和第2阶段的结果。
For several decades the European Pharmacopoeia monographs Tetanus vaccine (adsorbed) (0452) and Tetanus vaccine for veterinary use (0697) required that Specific toxicity and Absence of toxin and irreversibility of the toxoidof each bulk of tetanus toxoids had to be tested by an in vivo toxicity test in guinea pigs before it could be included in vaccines for human or veterinary use. In line with the
3Rs concept of replacing, reducing and refining animal experiments, an in vitro method for the detection of active tetanus neurotoxin (TeNT) has been developed at the Paul-Ehrlich-Institut (PEI, Germany). This method, the so-called BINACLE (binding and cleavage) assay, uses the receptor-binding and proteolytic properties of TeNT for the specific detection of active toxin molecules. Successful in-house validation studies as well as a small-scale transferability study had demonstrated that this method may represent a suitable alternative to the compendial in vivo toxicity test. As a follow up, an international collaborative study aimed at verifying the suitability of the BINACLE assay as a potential alternative to the guinea pig toxicity test for tetanus toxoids was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) under the aegis of its Biological Standardisation Programme (BSP). Within the framework of this study, coded BSP136, a feasibility phase - also referred to as Phase 1 - was run to select and qualify critical study reagents and samples and to assess the performance of the BINACLE Standard Operating Procedure developed by the project leaders. Then the international collaborative study aimed at evaluating the BINACLE, referred to as BSP136 Phase 2, was started. A total of 19 international laboratories (comprising vaccine manufacturers as well as national control laboratories) were supplied with a detailed assay protocol, critical reagents required for the assay, three samples consisting of three different bulk tetanus toxoids donated by major European vaccine manufacturers and one international standard toxoid. Each of the participants was asked to perform three independent BINACLE assays following the provided protocol. The statistical analysis of the results showed that most of the participating laboratories were able to perform the BINACLE assay according to the provided protocol. However, the results obtained by the participants varied widely, and not all the laboratories were able to achieve a sensitive detection of active TeNT. Multiple factors may have contributed to the elevated variability of the BSP136 study results. From an analysis of these factors, strategies were developed to help increase the standardisation of the BINACLE assay and obtain more consistent results in a follow-up validation study, BSP 136 Phase 3 (Part 2), for which the experimental phase took place in 2023. The present manuscript summarises the outcome of Phases 1 and 2, which constitute Part 1 of the BSP136 project.