Abstract：
During the production of clostridial vaccines large numbers of mice are used for various in-process control tests. Replacement in vitro assays had been developed for the testing of the toxins and toxoids of several clostridial species, but none of these assays had been assessed in an international collaborative study. Under the common aegis of the European Partnership for Alternative Approaches to Animal Testing (EPAA) and of the European Directorate for the Quality of Medicines & HealthCare (EDQM), a project on clostridial vaccines for veterinary use was started as part of the EDQM-co-ordinated Biological Standardisation Programme (BSP). Within the framework of this project (coded BSP130) a collaborative study was organised to evaluate Vero cell-based alternative methods to the current mouse tests used to measure: i) the toxicity of Clostridium septicum toxin, ii) the absence of toxicity of C. septicum toxoid and iii) the antigenicity of C. septicum toxoid. The principal aims of the study were to determine the repeatability and reproducibility of the in vitro assays and to demonstrate concordance of the in vitro and current in vivo tests. The study results demonstrated good concordance, but the information gathered through the study (later on called Part 1) and the participants\' workshop prompted the extension of the project in order to further optimise the in vitro protocols and improve their repeatability and reproducibility, which were comparable to but not better than those of the in vivo assays in Part 1. The 3 in vitro assays to be optimised in the extension of the BSP130 project were : i) the in vitro toxin neutralisation equivalence plus (TNE+), as a replacement for the in vivo minimum lethal dose (MLD) test for quantification of the toxicity of toxin; ii) the in vitro MLD, as a replacement for the in vivo MLD test for detection of residual toxicity associated with toxoid; iii) the in vitro total combining power (TCP), as a replacement for the in vivo TCP test for quantification of the antigenicity of toxoid. At this point, the Analytical Method Transfer Laboratory of Ceva-Phylaxia (Hungary), supported by the project management team, developed suitable SOPs for the 3 in vitro assays. These optimised methods were further assessed in BSP130 through a second international collaborative study (Part 2) aimed at defining repeatability and reproducibility in different laboratories and determining the levels of improvement compared with the original in vivo tests and the initial in vitro assays used in Part 1 of the project. Fourteen laboratories, comprising 4 public sector and 10 manufacturers\' medicines control laboratories, from 11 countries participated in the collaborative Part 2 study, each testing 6 different C. septicum toxins and 6 C. septicum toxoids. Improved repeatability and reproducibility were observed for the optimised assays. The results of this study confirm the suitability of these assays for in-process control of C. septicum vaccines, with better repeatability and reproducibility than their in vivo equivalents. It is expected that, with appropriate minor changes and the use of relevant reagents, these optimised in vitro assays could be used not only for the assessment of C. septicum toxins and toxoids but for all cytotoxin-based clostridial antigens. The development and implementation of such in vitro assays would offer a great opportunity to significantly reduce animal usage, shorten the duration of QC test procedures and increase the precision of toxicity and antigenicity assays in clostridial veterinary vaccine in-process control. This would also provide more accurate and reproducible dosing of antigens in the final vaccine products, help to promote compendial acceptance and to proffer a basis for improved international harmonisation across this area of product testing.
摘要：
在梭菌疫苗的生产过程中,大量小鼠用于各种过程中控制测试。已经开发了替代体外测定法，用于测试几种梭菌物种的毒素和类毒素，但在一项国际合作研究中，这些检测方法均未得到评估。在欧洲动物试验替代方法伙伴关系（EPAA）和欧洲药品质量与医疗保健局（EDQM）的共同主持下，作为EDQM协调的生物标准化计划（BSP）的一部分，开始了一个兽医用梭菌疫苗项目。在该项目的框架内（编码为BSP130），组织了一项合作研究，以评估基于Vero细胞的替代方法，以用于测量：i）败血梭菌毒素的毒性，ii)不存在败血症类毒素的毒性和iii)败血症类毒素的抗原性。该研究的主要目的是确定体外测定的可重复性和可重复性，并证明体外和当前体内测试的一致性。研究结果证明了良好的一致性，但是通过研究（后来称为第1部分）和参与者研讨会收集的信息促使项目的扩展，以进一步优化体外方案并提高其可重复性和再现性，与第1部分中的体内测定的那些相当但不优于这些。在BSP130项目的扩展中要优化的3种体外测定是：i）体外毒素中和等效性加上（TNE），作为体内最小致死剂量（MLD）测试的替代，用于量化毒素的毒性；ii）体外MLD，作为体内MLD测试的替代，用于检测与类毒素相关的残留毒性；iii）体外总结合能力（TCP），作为定量类毒素抗原性的体内TCP测试的替代。在这一点上,Ceva-Phylaxia（匈牙利）的分析方法转移实验室，在项目管理团队的支持下，为3种体外试验开发了合适的SOP。通过第二次国际合作研究（第2部分）在BSP130中进一步评估了这些优化的方法，该研究旨在定义不同实验室的可重复性和可重复性，并确定与原始体内测试和第1部分中使用的初始体外测定相比的改善水平。十四个实验室,包括4个公共部门和10个制造商\'药品控制实验室，来自11个国家参与了第二部分的合作研究，每个测试6种不同的败血症杆菌毒素和6种败血症杆菌类毒素。对于优化的测定,观察到改进的可重复性和再现性。这项研究的结果证实了这些检测方法对败血杆菌疫苗的过程控制的适用性，具有比体内等效物更好的可重复性和可重复性。预计,适当的微小变化和使用相关试剂，这些优化的体外测定不仅可用于评估败血梭菌毒素和类毒素,而且可用于所有基于细胞毒素的梭菌抗原。这种体外试验的开发和实施将为显著减少动物使用提供一个很好的机会。缩短QC测试程序的持续时间，并提高梭菌兽医疫苗过程控制中毒性和抗原性测定的准确性。这也将在最终疫苗产品中提供更准确和可重复的抗原剂量。有助于促进药典的接受度，并为产品测试这一领域的国际协调提供基础。
