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目的验证重组人尿激酶原(Prourokinase,Pro-uk)纯化工艺的病毒去除效果。方法采用模拟Pro-uk纯化工艺中阴离子和阳离子交换层析的scale-down模型,以水泡性口炎病毒(Vesivular stomatitis virus,VSV)、脑心肌炎病毒(Encephalo-myocarditis virus,EMCV)、人单纯疱疹病毒1型(Herpes simplex virus type-1,HSV-1)为指示病毒,采用细胞病变法评价离子交换层析的病毒去除效果。结果阴离子交换层析对脂包膜病毒VSV和HSV-1的去除能力强,对无脂包膜病毒EMCV的去除能力较弱;阳离子交换层析对VSV和HSV-1的去除能力较弱,对EMCV的去除能力较强。经离子交换层析纯化后,3种指示病毒的累计去除效率均>99.99%(大于4 log10)。结论 Pro-uk纯化工艺中的离子交换层析是控制病毒安全性的有效步骤,其去除效率达到了《生物组织提取制品和真核细胞表达制品的病毒安全性评价技术审评一般原则》规定的安全标准。
Objective To verify the virus removal efficiency of Prourokinase (Pro-uk) purification process. METHODS: A scale-down model was established to simulate anion and cation exchange chromatography in the Pro-uk purification process. The vesicular stomatitis virus (VSV), encephalo-myocarditis virus (EMCV), human herpes simplex Herpes simplex virus type-1 (HSV-1) was used as the indicator virus, and cytopathic method was used to evaluate the virus removal efficiency of ion exchange chromatography. Results The results of anion-exchange chromatography showed that the ability of removal of lipid-enveloped virus (VSV) and HSV-1 was stronger than that of lipid-free enveloped virus (EMCV). Cation exchange chromatography (LC-MS) EMCV removal ability is stronger. The cumulative removal efficiencies of the three indicated viruses were> 99.99% (greater than 4 log10) after purification by ion exchange chromatography. Conclusion Ion exchange chromatography in Pro-uk purification process is an effective step to control the safety of virus, and its removal efficiency has reached the standard of “General Principles of Technical Review of Virus Safety Evaluation of Biological Tissue Extracts and Eukaryotic Expression Products” safety standard.