摘要
针对重组汉逊酵母乙肝表面抗原(HBsAg)在传统纯化过程中稳定性差的问题,采用静态光散射、荧光光谱和动态光散射等分析手段,从颗粒完整性角度研究其在不同pH条件下的稳定性变化。以其为指导,采用硅胶吸附/解吸附纯化HBsAg,建立该过程中关键因素的响应面模型,并与疏水层析联用,进一步纯化HBsAg,分析纯化效果以及纯化后疫苗颗粒完整性。采用透射电镜观察纯化后疫苗形貌,用高效分子排阻色谱法(HPSEC)分析颗粒稳定性。结果表明在酸性溶液下,pH接近HBsAg等电点时,抗原颗粒间静电斥力减小,颗粒容易聚集;碱性条件下,抗原颗粒内部疏水基团暴露,造成颗粒解聚。建立响应面模型,以活性收率为响应值时,最佳纯化工艺为吸附pH=7.43,洗脱pH=10.48,洗脱温度55.4℃,此时活性收率最高为39.1%;以纯化倍数为响应值时,吸附pH=7.16,洗脱pH=10.52,洗脱温度55.1℃,此时纯化倍数最高为1.90。进一步对洗脱液进行疏水层析纯化,活性收率为49.73%,颗粒完整性为85.79%,透射电镜观察到抗原颗粒粒径为20~40 nm。与传统疏水层析方法相比,采用硅胶吸附/解吸附与疏水层析联用的纯化方法,疫苗活性收率提高31.99个百分点,颗粒完整性提高20.90个百分点,颗粒稳定性提高22.93个百分点。该研究为高效纯化重组HBsAg及提高疫苗纯化过程的颗粒完整程度等提供新思路。
Low stability was one of the biggest problems during traditional purification process of recombinant hepatitis B surface antigen(rHBsAg).A combination process of silica gel adsorption/desorption and hydrophobic interaction chromatography was proposed in this work.The effect of pH on HBsAg stability was studied from the particle integrity of view using static light scattering,fluorescence spectrum and dynamic light scattering,respectively.HBsAg was purified by silica gel adsorption/desorption using response surface methodology for experimental design,followed by hydrophobic interaction chromatography,and both its morphology and particle integrity were studied.In acidic solution,the electrostatic repulsion of HBsAg particles decreased leading to aggregation when pH of solution was close to the isoelectric point of HBsAg.In alkaline solution,hydrophobic patches inside HBsAg particles were likely to be exposed leading to disaggregation.The silica gel adsorption/desorption process was optimized.When HBsAg activity recovery was taken as the response value,the optimum operating condition was as follows:adsorption pH was 7.43,desorption pH was 10.48 and desorption temperature was 55.4℃,and HBsAg activity recovery was 39.1%.When purification fold was taken as the response value,the optimum operating condition was as follows:adsorption pH was 7.16,desorption pH was 10.52 and desorption temperature was 55.1℃,and purification fold was 1.90.After hydrophobic interaction chromatography,HBsAg activity recovery was 49.73%and the particle integrity was 85.79%respectively.Compared with conventional hydrophobic interaction chromatography,the efficiency of HBsAg purification was improved greatly using this combination method,etc.,HBsAg activity recovery increased by 31.99%,the particle integrity by 20.90%,and the particle stability by 22.93%,respectively.It provided a new idea for both high efficient recombinant HBsAg purification and improvement of antigen particle integrity.
作者
胡胜杰
黄永东
赵岚
朱凯
苗壮
王飞
晋洪超
李建
杨珺
汪和睦
马光辉
袁洪水
Shengjie HU;Yongdong HUANG;Lan ZHAO;Kai ZHU;Zhuang MIAO;Fei WANG;Hongchao JIN;Jian LI;Jun YANG;Hemu WANG;Guanghui MA;Hongshui YUAN(College of Life Sciences,Hebei Agricultural University,Baoding,Hebei 071001,China;State Key Laboratory of Biochemical Engineering,Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190,China;School of Chemical Engineering,Beijing Institute of Petrochemical Technology,Beijing 102617,China;Tianjin Hemu Jianmin Biotechnology Co.,Ltd.,Tianjin 300384,China;School of Chemical Engineering,University of Chinese Academy of Sciences,Beijing 100049,China)
出处
《过程工程学报》
CAS
CSCD
北大核心
2020年第10期1198-1209,共12页
The Chinese Journal of Process Engineering
基金
国家重点研发计划资助项目(编号:2018YFC0311101
2016YFF0202304)
中国科学院重点部署项目(编号:KFZD-SW-218)
北京市自然科学基金资助项目(编号:2172054)
北京市自然科学基金-海淀原始创新联合基金(编号:19L2017)
关键词
乙肝表面抗原
颗粒完整性
硅胶吸附/解吸附
响应面法
疏水层析
hepatitis B surface antigen
particle integrity
silica gel adsorption/desorption
response surface methodology
hydrophobic interaction chromatography
