多种方法结合去除冻干人用狂犬病疫苗中Vero细胞残留DNA  

Removal of residual Vero cell DNA in freeze-dried rabies vaccine for human use by combination of various methods

作　　者：刘杰[1] 刘辉[1] 杨会强[1] 康庄[1] 孙艳[1] 毛川成 何敏[1] 李玉华[2] 

机构地区：[1]成都生物制品研究所有限责任公司病毒疫苗研究室,四川成都610023 [2]中国食品药品检定研究院虫媒病毒疫苗室,北京100050

出　　处：《中国生物制品学杂志》2013年第12期1827-1830,共4页Chinese Journal of Biologicals

摘　　要：目的采用多种方法结合去除冻干人用狂犬病疫苗(Vero细胞)中Vero细胞残留DNA。方法采用滤芯(滤板)澄清、鱼精蛋白处理、核酸酶处理、Sepharose 4FF层析纯化或澄清、核酸酶、Sepharose 4FF层析纯化结合的方法去除Vero细胞培养的狂犬病病毒液中的Vero细胞DNA,检测Vero细胞DNA残留量、抗原含量、核酸酶残留量及疫苗效价。结果 0.45μm UB玻璃纤维澄清病毒液,抗原损失率较小;鱼精蛋白去除Vero细胞DNA,抗原损失量较大;核酸酶处理后,病毒液中Vero细胞DNA残留量仍高于1 ng/ml;经Sepharose 4FF层析纯化,能有效去除浓缩后经核酸酶处理的病毒液中的核酸酶及一定量的Vero细胞DNA;采用多种方法结合去除病毒液中Vero细胞残留DNA,DNA残留量<100 pg/ml,疫苗效价≥4.0 IU/ml,均达到《中国药典》三部(2010版)要求。结论多种方法结合能有效去除冻干人用狂犬病疫苗(Vero细胞)中Vero细胞DNA残留量,且疫苗效力达到《中国药典》三部(2010版)要求。Objective To remove the residual Vero cell DNA in freeze-dried rabies vaccine for human use by combination of various methods. Methods The residual Vero cell DNA in rabies virus liquid cultured in Vero cells was removed by the combination of clarification with filter element （filter plate）, treatment with protamine, treatment with endonuclease and Sepharose 4FF chromatography or the combination of above-mentioned procedures except treatment with protamine, based on which the residual Vero cell DNA content, antigen content, residual endonuclease content and vaccine potency were determined. Results The loss rate of antigen in virus liquid clarified with 0. 45 μm UB glass fiber was low. However, the loss rate of antigen in virus liquid in which Vero cell DNA was removed by protamine was high. The residual Vero cell DNA content in virus liquid after treatment with endonuclease was still more than 1 ng/ml. Sepharose 4FF chromatography removed the endonuclease and Vero cell DNA in virus liquid after concentration and endonuclease treatment. After removal of residual Vero cell DNA by combination of various methods, the residual Vero cell DNA content was less than 100 pg/ml, while the vaccine potency of not less than 4. 0 IU/ml, which met the requirements in Chinese Pharmacopoeia （Volume Ⅲ, 2010 edition）. Conclusion The residual Vero cell DNA content in freeze-dried rabies vaccine for human use was removed effectively by combination of various methods, and the vaccine potency met the requirements in Chinese Pharmacopoeia （Volume Ⅲ, 2010 edition）.

关 键 词：狂犬病疫苗 VERO细胞 DNA 核酸酶 

分 类 号：R373.9[医药卫生—病原生物学] R392-33[医药卫生—基础医学]