重组汉逊酵母表达的HBsAg纯化工艺中内毒素的去除效果

目的分析重组汉逊酵母表达的乙型肝炎表面抗原(HBsAg)各纯化工序样品中内毒素的去除效果。方法采用鲎试剂法检测重组汉逊酵母表达的HBsAg小量工艺探索、中量工艺验证、中试纯化工艺中破碎细胞、微滤、超滤、硅胶吸附、层析、除菌过滤各工序样品的内毒素含量,计算内毒素去除率,分析各纯化工序与内毒素去除率的关系,并以重组酿酒酵母表达HBsAg各纯化工序中样品作为对照。结果重组汉逊酵母表达的HBsAg纯化过程中内毒素含量由细胞破碎样品的241 EU/ml以上降至除菌过滤样品的1.0 EU/ml以下。微滤和超滤样品内毒素去除率平均可达56%和86%,占总体纯化工艺中内毒素去除率的90%以上。结论重组汉逊酵母表达HBsAg纯化工序微滤、超滤和疏水层析均可有效去除内毒素,以超滤去除内毒素效果最明显,为重组汉逊酵母纯化工艺去除内毒素的研究提供了技术参数和实验依据。

Removal of endotoxin in purification process of HBsAg expressed in recombinant Hansenula polymorpha

Objective To evaluate the removal effect of endotoxin in purification process of HBsAg expressed in recombinant Hansenula polymorpha.Methods The endotoxin contents in HBsAg expressed in recombinant H.polymorpha in steps of cell disruption,microfiltration,ultra-filtration,adsorption with silica gel,chromatography and sterile filtration in small and middle scales as well as pilot purification process were determined by limulus amoebocyte lysate(LAL) test,using those in the HBsAg expressed in recombinant Saccharomyces cerevisiae as control,based on which the removal rate of endotoxin was calculated,and the relationship between purification procedure and the removal rate was analyzed.Results During the purification of HBsAg expressed in recombinant H.polymorpha,the endotoxin content of samples decreased from 241 EU / ml in step of cell disruption to less than 1.0 EU / ml in step of sterile filtration.The removal rates of endotoxin in samples in steps of microfiltration and ultra-filtration were 56% and 86% respectively,accounting for more than 90% of removal rate of endotoxin in the whole purification process.Conclusion The steps of microfiltration,ultra-filtration and hydrophobic chromatography in purification process were effective for removal of endotoxin,of which ultra-filtration was the most effective.It provided technical parameters and an experimental basis for study on removal of endotoxin in purification process.