Abstract：
Formaldehyde detoxification is a process for converting tetanus toxin (TT) and diphtheria toxin (DT) into tetanus toxoid (TTd) and diphtheria toxoid (DTd), respectively. The mechanism of this detoxification process has been investigated by several previous studies based on lab-scale toxoids. To obtain greater insights of the effects induced by formaldehyde, industrial TTd and DTd batches obtained from different detoxification processes were studied in this work. Using liquid chromatography-mass spectrometry (LC-MS), 15 and 20 repeatable formaldehyde-induced modification sites of TTd and DTd were identified, respectively. Toxoid which had a higher formaldehyde-induced modification rate observed by LC-MS, also had larger bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Aggregates which were observed on size exclusion chromatogram (SEC) were confirmed by SDS-PAGE and LC-MS. Formaldehyde detoxification also led to a decrease of isoelectric point (pI) values and an increase of retention on weak anion exchange (WAX) column. Specific toxicity tests were conducted to evaluate toxicity of the TTd and DTd samples obtained with different detoxification conditions. Results from the specific toxicity tests showed that all toxoids used in this study were qualified, including toxoids obtained from mild and drastic detoxification conditions. However, obtained from mild detoxification conditions had less aggregates and may lead to a higher degree of glycosylation in conjugate vaccines than the ones obtained from drastic detoxification conditions. Thus, we suggest that mild detoxification conditions should be used to obtain TTd and DTd. Furthermore, as well as studying the formaldehyde-induced modifications and toxicity in TTd and DTd, the effects of the detoxification process on foreign proteins were also investigated. An increase in foreign proteins were observed in the aggregate than in the monomer of the toxoids. Additionally, some foreign proteins in the monomer of the toxins transferred to the aggregate of toxoids due to the formation of cross-linking. To eliminate the risk of cross-linking foreign proteins to toxoids in vaccination programs, a purification process is necessary before the detoxification process and/or the use of toxoids in vaccines.
摘要：
甲醛解毒是将破伤风毒素（TT）和白喉毒素（DT）转化为破伤风类毒素（TTd）和白喉类毒素（DTd）的过程，分别。这种解毒过程的机制已经通过先前的基于实验室规模类毒素的若干研究进行了研究。为了更好地了解甲醛引起的影响，在这项工作中，研究了从不同脱毒过程中获得的工业TTd和DTd批次。使用液相色谱-质谱(LC-MS),确定了TTd和DTd的15和20个可重复的甲醛诱导修饰位点，分别。通过LC-MS观察到具有较高甲醛诱导修饰率的类毒素,在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上也有较大的条带。通过SDS-PAGE和LC-MS确认在尺寸排阻色谱（SEC）上观察到的聚集体。甲醛解毒还导致等电点（pI）值降低，并增加在弱阴离子交换（WAX）柱上的保留。进行特定毒性测试以评估在不同解毒条件下获得的TTd和DTd样品的毒性。特定毒性试验的结果表明，本研究中使用的所有类毒素均合格，包括从温和和剧烈的解毒条件下获得的类毒素。然而,与从剧烈的解毒条件获得的疫苗相比，从轻度解毒条件获得的疫苗具有更少的聚集体，并且可能导致缀合疫苗中的糖基化程度更高。因此，我们建议应使用温和的解毒条件来获得TTd和DTd。此外，以及研究甲醛在TTd和DTd中引起的修饰和毒性，还研究了解毒过程对外源蛋白的影响。与类毒素的单体相比，聚集体中观察到外源蛋白的增加。此外,由于交联的形成，毒素单体中的一些外源蛋白质转移到类毒素的聚集体中。为了消除疫苗接种计划中外来蛋白与类毒素交联的风险，在解毒过程和/或在疫苗中使用类毒素之前,纯化过程是必要的。
