无血清悬浮MDCK细胞培养流感病毒纯化工艺的初步建立

Preliminary development of purification process for influenza vaccine cultured in serum-free suspended MDCK cells

目的建立无血清悬浮MDCK细胞培养流感病毒的下游纯化工艺。方法采用40 L反应器,收获无血清悬浮MDCK细胞培养的流感病毒,经澄清浓缩后,添加不同终浓度(1、10、30、50 U/mL)的Benzonase■核酸酶,置37℃酶解不同时间(2、4、6、8 h),优化核酸酶去除细胞残留DNA的条件。采用两步层析法,探讨不同线性流速(60、150、300 cm/h)和上样缓冲液盐离子浓度(150、500、1 000 mmol/L)对复合介质纯化效果的影响;探讨不同线性流速(60、150、300 cm/h)对离子层析介质纯化效果的影响。根据确定的参数进行3批次纯化工艺样品的检测。结果病毒浓缩液经10 U/mL Benzonase?核酸酶37℃酶解6 h后,DNA去除率即可达99%以上。确定复合介质的流速为60 cm/h,上样缓冲液组成为150 mmol/L NaCl+50 mmol/L Tris,pH 7.4,确定离子交换介质流速为150 cm/h。3批纯化工艺样品去除了99.94%的宿主细胞残留蛋白和99.99%的残留DNA,残留DNA达欧盟标准,不高于10 ng/剂。结论初步建立了无血清悬浮MDCK细胞流感疫苗的下游纯化工艺。

Objective To develop a downstream purification process for influenza vaccine cultured in serum-free suspended MDCK cells.Methods Influenza virus cultured in serum-free suspended MDCK cells in a 40 L bioreactor was harvested,clarified,concentrated,added with Benzonase■ nuclease at various final concentrations(1,10,30 and 50 U/mL)and incubated at 37 ℃ for various time durations(2,4,6 and 8 h)to optimize the condition for removal of residual DNA.The virus was further purified by two step chromatography,and the influences of linear flow rate(60,150 and 300 cm/h) and sodium chloride concentration(150,500 and 1 000 mmol/L) in the loading buffer on the purification effect of complex medium,as well as the influence of linear flow rate(60,150 and 300 cm/h)on that of ion chromatographic medium, were evaluated.Three batches of samples were purified by the developed process and determined.Results The DNA removal rate of the concentrated virus solution was more than 99% after digestion with10 U/mL Benzonase? nuclease at 37 ℃ for 6 h.The optimal flow rate of complex medium was 60 cm/h,while the optimal loading buffer consisted of 150 mmol/L sodium chloride + 50 mmol/L Tris,pH 7.4,and optimal flow rate of ion chromatographic medium was 150 cm/h.The removal rates of residual host cell protein and residual DNA in three batches of purified samples were 99.94% and 99.99% respectively.The residual DNA content was not more than10 ng/dose,which met the standard in European Union.Conclusion A downstream purification process of influenza vaccine derived from serum-free suspended culture of MDCK cells was preliminarily developed.