响应面法建立层析纯化去除百日咳丝状血凝素中内毒素工艺

Establishing the chromatographic process of endotoxin removal from Pertussis filamentous hemagglutinin by response surface methodology

【背景】无细胞组分百日咳疫苗在人群中接种后不良反应发生率大大降低,是未来百日咳疫苗的发展方向,但是新的抗原纯化方式需要工艺中加入内毒素的去除。【目的】使用响应面法优化层析纯化法去除无细胞百日咳疫苗中百日咳丝状血凝素(Filamentous hemagglutinin,FHA)中内毒素的工艺。【方法】通过单因素试验,确定响应面设计范围;根据响应面法设计原理,使用Mini TAB软件,以FHA的回收率和收获的FHA蛋白浓度,同时兼内毒素合格为考察指标,对上样样品量、样品p H、样品电导Cond进行优化,最终确定去除FHA内毒素的层析纯化工艺。【结果】使用目前的层析纯化条件获得的Capto adhere去除FHA的内毒素的最佳工艺条件:p H 5.3,Cond 9.6,Mass 3.0。【结论】用响应面法优化了去除百日咳丝状血凝素中内毒素的层析纯化工艺,这种方法效率高、耗时少,为后续生物制品工艺扩大再生产提供参考。

[Background] The future development of pertussis vaccine is acellular pertussis（component） vaccine, which could significantly reduce the incidence of adverse events after inoculation. This new type of vaccine needs to remove the endotoxin in the process of production. [Objective] In our study, the removal process of the endotoxin from pertussis filamentous hemagglutinin（FHA） was investigated by optimized chromatography purification with response surface methodology. [Methods] The process of chromatographic purification was verified by the single factor experiment, to ensure the range of response surface methodology; the method of response surface analysis was adopted according to the CCD experimental design principles by using Mini TAB software： the variable factors were p H, Cond, and Mass; Response values were considered the concentration of FHA protein and recovery of FHA, and finally combined the value of endotoxin. [Results] These results showed that the optimum conditions for chromatographic process of endotoxin removal from pertussis filamentous hemagglutinin were as follows： p H 5.3, Cond 9.6, and Mass 3.0. [Conclusion] The optimum processes of chromatographic purification on endotoxin removal were screened successfully by response surface methodology. This method has advantages of higher efficient and lower energy consumption, which will lay a good foundation for the subsequent biological process of expanded reproduction.