Benzonase核酸酶在Vero细胞流感病毒灭活疫苗中的应用研究

Application of Benzonase Nuclease in Manufacture of Vero Cell Inactivated Influenza Virus Vaccine

连续细胞系用于生物制品生产可能会造成宿主成分传递给受种者的问题。为了评估Benzonase核酸酶在细胞流感病毒灭活疫苗中降解宿主细胞残余DNA的效果。本研究采用Vero细胞培养流感病毒H3N2,获得病毒原液后,经“Benzonase核酸酶+300kD超滤膜包+Capto Core 700”(实验组)、“300kD超滤膜包+Capto Core 700”(方案一)和“300kD超滤膜包+Capto Core 700+Capto Q”(方案二)三种纯化方案分别对病毒原液进行纯化,取样检测各步骤的血凝效价、血凝素含量、总蛋白含量、宿主蛋白含量、宿主DNA含量评估对流感病毒的纯化效果,并通过透射电镜观察病毒颗粒形态。灭活验证通过后,按血凝素HA含量为15μg/剂(体积0.5mL)皮下免疫BALB/c小鼠2次,免后14d后分离血清检测抗体滴度水平评价免疫原性。结果显示,实验组最终纯化液病毒的回收率为(66.70±4.87)%,配置为单价制剂后各项指标符合药典要求;方案一最终纯化液病毒回收率为(50.00±3.41)%,rcDNA含量为(28366.67±1006.65)pg/mL,配置为单价制剂后rcDNA含量不符合药典要求(<100 pg/剂);方案二对比方案一增加了阴离子交换,纯化后病毒回收率为(46.83±1.62)%,配置为单价制剂后各项指标符合药典要求。透射电镜结果显示病毒颗粒不会受到核酸酶的影响。免疫原性评价结果显示小鼠血清抗体滴度与对照组无显著差异,表明核酸酶对流感病毒的免疫原性没有影响。另外,Vero细胞DNA经核酸酶处理后,使用毛细管电泳未检测到具有大小的DNA片段,说明核酸酶可以将DNA降解为核苷酸。综上,本研究初步确定Benzonase核酸酶用于Vero细胞为基质的流感病毒灭活疫苗的应用。

The use of continuous cell lines for biologics production may cause problems with the transmission of host components to the recipient.To evaluate the effect of benzonase nuclease in degrading residual host cell DNA(rcDNA)in cellular influenza virus inactivated vaccines produced with Vero cell line,influenza H3N2 virus was cultured in Vero cells to obtain viral stocks,which were purified by three purification protocols,"Benzonase nuclease+300 kD ultrafiltration+capto core 700"(Experimental group),"300 kD ultrafiltration+capto core 700"(Protocol I),and"300 kD ultrafiltration+capto core 700+capto Q"(Protocol II),Hemagglutinin titer,hemagglutinin content,total protein content,host protein content,and rcDNA content at each step were sampled to evaluate the purification effect of influenza virus,And virus particle morphology was assessed by transmission electron microscopy(TEM).After the inactivation validation was completed,BALB/c mice were immunized twice subcutaneously with hemagglutinin HA at 15μg/dose(0.5mL),and the serum was isolated 14 days after immunization to determine antibody titer levels.The results showed that the recovery yield of virus in the experimental group was(66.70±4.87)%,and each prameter meets compendial requirements after configuration as a monovalent preparation.With protocol I the viral recovery was(50.00±3.41)%and the rcDNA was(28366.67±1006.65)pg/mL,and the rcDNA did not meet the compendial requirements(<100pg/dose)after configuration as a monovalent preparation.With protocol II the recovery rate of virus was(46.83±1.62)%,and each indicator meets compendial requirements after configuration as a monovalent preparation.In addition,TEM results showed that virus particles were not affected by nucleases.Immunogenicity evaluation showed no significant differences in mouse serum antibody titers compared to controls,indicating that the benzonase nuclease did not affect the immunogenicity of the influenza virus.Moreover,Vero cell DNA was subjected to benzonase nuclease treatment and capillary electrophoresis was used to detect residual DNA fragment size.Results showed that intact fragmented DNA was not detected,indicating that the DNA had been degraded into nucleotides.Taken together,this study preliminarily identifies the use of benzonase nuclease for Vero cell⁃based production of inactivated vaccines against influenza virus.