Metz Bernard, Jiskoot Wim, Hennink Wim E, Crommelin Daan J A, Kersten Gideon F A
Laboratory for Product and Process Development, The Netherlands Vaccine Institute (NVI), Bilthoven, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands.
Vaccine. 2003 Dec 12;22(2):156-67. doi: 10.1016/j.vaccine.2003.08.003.
The most critical step in the production of diphtheria vaccines is the inactivation of the toxin by formaldehyde. Diphtheria toxoid (DTx) is produced during this inactivation process through partly unknown, chemical modifications of the toxin. Consequently, diphtheria vaccines are difficult to characterise completely and the quality of the toxoids is routinely determined with potency and safety tests. This article describes the possibility of monitoring the quality in diphtheria vaccine production with a selection of physicochemical and immunochemical tests as an alternative to established in vivo tests. To this end, diphtheria toxin was treated with increasing formaldehyde concentrations resulting in toxoid products varying in potency and residual toxicity. Differences in the quality of the experimental toxoids were also assessed with physicochemical and immunochemical techniques. The results obtained with several of these analyses, including SDS-PAGE, primary amino group determination, fluorescence spectroscopy, circular dichroism (CD) and biosensor analysis, showed a clear correlation with the potency and safety tests. A set of criteria is proposed that a diphtheria toxoid must comply with, i.e. an apparent shift of the B-fragment on SDS-PAGE, a reduction of primary amino groups in a diphtheria molecule, an increased resistance to denaturation, an increased circular dichroism signal in the near-UV region and a reduced binding to selected monoclonal antibodies. In principle, a selected set of in vitro analyses can replace the classical in vivo tests to evaluate the quality of diphtheria toxoid vaccines, provided that the validity of these tests is demonstrated in extensive validation studies and regulatory acceptance is obtained.
白喉疫苗生产中最关键的步骤是用甲醛使毒素失活。在此失活过程中，通过部分未知的毒素化学修饰产生白喉类毒素（DTx）。因此，白喉疫苗难以完全表征，类毒素的质量通常通过效力和安全性测试来确定。本文描述了通过一系列物理化学和免疫化学测试监测白喉疫苗生产质量的可能性，以此作为既定体内测试的替代方法。为此，用递增浓度的甲醛处理白喉毒素，得到效力和残留毒性各异的类毒素产品。还通过物理化学和免疫化学技术评估了实验类毒素质量的差异。包括SDS-PAGE、伯氨基测定、荧光光谱、圆二色性（CD）和生物传感器分析在内的几项分析结果，与效力和安全性测试显示出明显的相关性。提出了一套白喉类毒素必须符合的标准，即SDS-PAGE上B片段的明显迁移、白喉分子中伯氨基的减少、对变性的抗性增加、近紫外区域圆二色性信号增强以及与选定单克隆抗体的结合减少。原则上，一组选定的体外分析可以替代经典的体内测试来评估白喉类毒素疫苗的质量，前提是这些测试的有效性在广泛的验证研究中得到证明并获得监管认可。
