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

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）.