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.

甲醛解毒是将破伤风毒素（TT）和白喉毒素（DT）分别转化为破伤风类毒素（TTd）和白喉类毒素（DTd）的过程。先前基于实验室规模类毒素的几项研究已经研究了这种解毒过程的机制。为了更深入地了解甲醛引起的影响，本工作研究了从不同解毒过程中获得的工业 TTd 和 DTd 批次。使用液相色谱-质谱 (LC-MS)，分别鉴定了 15 个和 20 个可重复的甲醛诱导的 TTd 和 DTd 修饰位点。LC-MS观察到的类毒素具有较高的甲醛诱导修饰率，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) 值降低和在弱阴离子交换 (WAX) 柱上的保留增加。进行了特定的毒性测试，以评估在不同解毒条件下获得的 TTd 和 DTd 样品的毒性。特定毒性测试的结果表明，本研究中使用的所有类毒素均合格，包括从温和和剧烈解毒条件下获得的类毒素。然而，从温和的解毒条件获得的聚集体较少，并且可能导致结合疫苗中的糖基化程度高于从剧烈的解毒条件获得的那些。因此，我们建议应使用温和的解毒条件来获得 TTd 和 DTd。此外，除了研究甲醛诱导的 TTd 和 DTd 修饰和毒性外，还研究了解毒过程对外源蛋白的影响。与类毒素单体相比，在聚集体中观察到外来蛋白质的增加。此外，由于交联的形成，毒素单体中的一些外源蛋白转移到类毒素的聚集体中。为了消除在疫苗接种计划中将外源蛋白质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需要进行纯化过程。还研究了解毒过程对外来蛋白质的影响。与类毒素单体相比，在聚集体中观察到外来蛋白质的增加。此外，由于交联的形成，毒素单体中的一些外源蛋白转移到类毒素的聚集体中。为了消除在疫苗接种计划中将外源蛋白质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需要进行纯化过程。还研究了解毒过程对外来蛋白质的影响。与类毒素单体相比，在聚集体中观察到外来蛋白质的增加。此外，由于交联的形成，毒素单体中的一些外源蛋白转移到类毒素的聚集体中。为了消除在疫苗接种计划中将外源蛋白质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需要进行纯化过程。