去除百日咳毒素、丝状血凝素中内毒素的工艺探索

Procedure for removal of endotoxin from pertussis toxin and filamentous hemagglutinin

目的探讨无细胞百日咳疫苗纯化工艺中百日咳毒素(pertussis toxin,PT)、丝状血凝素(filamentous hemagglutinin,FHA)的内毒素去除问题。方法选用Triton法和离子交换层析法,分别对百日咳杆菌发酵液上清和经Capto SP Imp Res柱纯化的发酵液进行内毒素去除,按《中国药典》三部(2015版)相关方法检测纯化样品的蛋白含量、内毒素含量、抗原含量,并进行SDS-PAGE分析。结果使用Triton法和离子交换层析法均可以将FHA和PT这两种成分的内毒素在脱毒前阶段降低至200 EU/mg以下。在合适的纯化条件下使用离子层析去除内毒素的能力高于Triton法,而在对抗原的回收率上Triton法又稍占优势。结论使用Triton法和离子交换层析法均可解决FHA和PT去除内毒素的问题,两类工艺下对目的蛋白活性的影响有待进一步研究。

Objective To investigate the removal of endotoxin from pertussis toxin（PT） and filamentous hemagglutinin（FHA）. Methods The endotoxin in fermentation supernatant and fermentation liquid purified by Capto SP Imp Res column chromatography were removed by using Triton and IEX separately. According to the requirements in Chinese Pharmacopoeia（Volume Ⅲ, 2015 edition）, the purified samples were determined for the contents of protein, endotoxin and antigen, and analyzed by SDS-PAGE. Results Both Triton and IEX methods decreased the endotoxin content in FHA and PT before the stage of detoxification to 200 EU/mg below. However, under the suitable condition for purification, the ability of IEX in removal of endotoxin was superior to that of Triton, while the ability of Triton method in recovery of antigen was superior to that of IEX. Conclusion Both the methods may solve the problem of endotoxin removal from PT and FHA effectively. However, the effect of two procedures on protein activity needs to be further studied.