Calibration of replacement international standard and European pharmacopoeia biological reference preparation for tetanus toxoid, adsorbed.

Here we report the characterisation of a preparation of tetanus toxoid, adsorbed, and its calibration by 27 laboratories in 19 countries in a joint international collaborative study co-sponsored by World Health Organization (WHO) Expert Committee of Biological Standardization (ECBS) and the European Biological Standardisation Programme of European Directorate for the Quality of Medicines (EDQM), Council of Europe. Calibration was in terms of the Second International Standard (I.S.) for Tetanus Toxoid, Adsorbed, by the established WHO/European Pharmacopoeia (Ph Eur) challenge methods. The replacement standard preparation was found to have a unitage of 469 IU/ampoule on the basis of its calibration in guinea-pigs and 496 IU/ampoule on the basis of its calibration in mice. Assessment, both within the collaborative study and as part of candidate characterisation, indicated satisfactory stability of the candidate preparation. This study also provided some information on the effect of mouse strain on potency testing of tetanus vaccines. A limited assessment of the impact of the replacement standard on testing of current production batches of vaccines was also carried out by four manufacturers. This study did not directly address the serological approaches to potency testing. However, one laboratory offered data from mouse serology assay, which gave comparable estimates to in vivo mouse bioassay.Based on the results of this study and with the agreement of participants, the candidate standard was established as the Third International Standard for Tetanus Toxoid, Adsorbed (coded 98/552) by the WHO Expert Committee of Biological Standardization (ECBS) in November 2000. The same preparation was also established as the second Ph Eur Biological Reference Preparation (Ph Eur BRP, batch no. 2) by the Steering Committee of the Biological Standardisation Programme of the EDQM and approved by the European Pharmacopoeia Commission.     

Optimization of tetanus toxoid ammonium sulfate precipitation process using response surface methodology

Tetanus toxoid (TTd) is a highly immunogenic, detoxified form of tetanus toxin, a causative agent of tetanus disease, produced by Clostridium tetani. Since tetanus disease cannot be eradicated but is easily prevented by vaccination, the need for the tetanus vaccine is permanent. The aim of this work was to investigate the possibility of optimizing TTd purification, i.e., ammonium sulfate precipitation process. The influence of the percentage of ammonium sulfate, starting amount of TTd, buffer type, pH, temperature, and starting purity of TTd on the purification process were investigated using optimal design for response surface models. Responses measured for evaluation of the ammonium sulfate precipitation process were TTd amount (Lf/mL) and total protein content. These two parameters were used to calculate purity (Lf/mgPN) and the yield of the process. Results indicate that citrate buffer, lower temperature, and lower starting amount of TTd result in higher purities of precipitates. Gel electrophoresis combined with matrix-assisted laser desorption ionization–mass spectrometric analysis of precipitates revealed that there are no inter-protein cross-links and that all contaminating proteins have pIs similar to TTd, so this is most probably the reason for the limited success of purification by precipitation.     

精制破伤风类毒素产毒及精制工艺的改进

用超滤、硫酸铵二段盐析法取代等电点沉淀法后,精制破伤风类毒素（精破类）的纯度由８０７Ｌｆ／ｍｇＰＮ提高到１８８３Ｌｆ／ｍｇＰＮ,纯度提高一倍以上。使用双胨培养基取代酪素培养基后,产毒水平由４７Ｌｆ／ｍｌ提高到８８Ｌｆ／ｍｌ（ｔ＝６．４６,ｐ＜０．００１）;用新法精制后,精破类纯度分别为１９４９Ｌｆ／ｍｇＰＮ及１７８５Ｌｆ／ｍｇＰＮ（ｔ＝０．３３４,ｐ＞０．０５）,引用双胨培养基后可提高产毒水平,但不影响精破类的纯度。     

一种提高破伤风类毒素马匹免疫成功率的方法

采用抗原加倍方法用于成率率低于705群的巴匹免疫,结果显示,进一步免疫成功的马匹明显多于常规免疫（P＜0．05）,免疫效价和死亡率无明显变化（P＞0．05）。     

Simplified active test of estimating tetanus toxoid]

The main purpose of these investigations was the comparison of two potency tests of Tetanus Toxoid. It was found that two doses test with reduced number of animals can be used in assay of different vaccines containing Tetanus component. One dose test is agreeable with requirements of WHO. It is credible and not complicated method of estimation Diphtheria and Tetanus Toxoids. This test required minimal number of animals.     

Physicochemical and immunochemical assays for monitoring consistent production of tetanus toxoid

The detoxification of tetanus toxin by formaldehyde is a crucial step in the production of tetanus toxoid. The inactivation results in chemically modified proteins and it determines largely the ultimate efficacy and safety of the vaccine. Currently, the quality of tetanus toxoid lots is evaluated in potency and safety tests performed in animals. As a possible alternative, this article describes a panel of in vitro methods, which provides detailed information about the quality of tetanus toxoid. Ten experimental lots of tetanus toxoid were prepared using increasing concentrations of formaldehyde and glycine to obtain tetanus toxoids having differences in antigenicity, immunogenicity, residual toxicity and protein structure. The structural properties of each individual toxoid were determined using immunochemical and physicochemical methods, including biosensor analysis, ELISA, circular dichroism, TNBS assay, differential scanning calorimetry, fluorescence and SDS-PAGE. The quality of a tetanus toxoid lot can be assessed by these set of analytical techniques. Based on antigenicity, immunogenicity and residual toxicity data, criteria are formulated that tetanus toxoids lot have to meet in order to have a high quality. The in vitro methods are a valuable selection of techniques for monitoring consistency of production of tetanus toxoid, especially for the detoxification process of tetanus toxin.