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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。
Objective To analyze the removal efficiency of endotoxin in each purification process of hepatitis B surface antigen (HBsAg) expressed by recombinant Hansenula. Methods The detection of HBsAg expression in Hansenula polymorpha was studied by the method of 鲎 reagent. The medium-volume process was validated. The samples of various processes such as broken cell, microfiltration, ultrafiltration, silica gel adsorption, chromatography, Endotoxin content, calculate the endotoxin removal rate, analyze the relationship between the purification process and the endotoxin removal rate, and use the recombinant Saccharomyces cerevisiae to express the samples in each purification process of HBsAg as a control. Results The endotoxin content of HBsAg in recombinant Hansenula was reduced from above 241 EU / ml in cell-broken samples to less than 1.0 EU / ml in sterilized samples. The average removal rates of endotoxin in microfiltration and ultrafiltration samples were 56% and 86%, respectively, accounting for over 90% of the total purification process. CONCLUSION: The recombinant HBsAg can be effectively removed by microfiltration, ultrafiltration and hydrophobic chromatography. The removal of endotoxin by ultrafiltration is the most effective method for the removal of endotoxin by the purification of Hansenula polymorpha Parameters and experimental basis.