摘要
目的 评价终浓度为100 μg/ml甲醛、2～8℃、10d灭活工艺对2022年四价流感病毒裂解疫苗生产用By(B/Phuket/3073/2013/BVR-1B)病毒毒株灭活的适用性.方法 病毒收获液(鸡胚病毒培养产物)经过澄清过滤,控制蛋白浓度＜800 µg/ml,温度2～8℃,添加甲醛溶液至终浓度100 µg/ml,对病毒收获液进行灭活.在灭活第0、1、2、3、4、5、10天取样,采用鸡胚半数感染剂量(median infective dose,EID50)法检测病毒滴度,采用Reed-Muench法计算lgEID50/ml;采用直接血凝法检测流感病毒血凝滴度;对灭活10d的样品进行病毒灭活验证试验;灭活10d后的病毒液经超滤、离心、层析等工艺提纯,取样进行透射电镜观察病毒颗粒完整性.结果 病毒收获液灭活第0、1、2、3、4、5、10天的病毒滴度分别为(8.5±0.4)、(4.2±0.5)、(1.3±0.4)、0、0、0、0 lgEID50/ml,血凝滴度结果均在 1∶256～1∶512;病毒灭活验证试验显示,经过10d的灭活,病毒均已全部灭活;透射电镜观察显示,所得的病毒均为完整病毒颗粒.结论 终浓度100 µg/ml甲醛、2～8℃灭活10 d的灭活工艺可用于By(B/Phuket/3073/2013/BVR-1B)流感病毒的灭活处理.
Abstract
Objective To evaluate the applicability of a 10-day inactivation process using 100 μg/ml formaldehyde at 2-8 ℃ on the By(B/Phuket/3073/2013/BVR-1B)virus strain used in the production of quadrivalent influenza vaccine(split virion,inactivated)in 2022.Methods Virus harvest(cultured in chick embryo)were clarified and filtered,and the protein concentration was regulated to under 800 μg/ml.The temperature was kept at 2-8 ℃,then formaldehyde solution was added until reaching a final concentration of 100 μg/ml to inactivate the virus harvest.Samples were collected on day 0,1,2,3,4,5 and 10 in the course of inactivation.The virus titer was determined using the method of median embryo infective dose(EID50),the lgEID50/ml was calculated through the Reed-Muench method.The influenza virus hemagglutinin titer was determined with direct hemagglutination.The samples collected after 10 day's inactivated were tested to validate virus inactivation.The virus fluid inactivated for 10 days was purified through ultrafiltration,centrifugation,and chromatography,then sampled and observed under the transmission electron microscope(TEM)to detect virus particles.Results The virus titers of the 7 batches of virus harvest on day 0,1,2,3,4,5,and 10 of inactivation were 8.5±0.4,4.2±0.5,1.3±0.4,0,0,0,0 lgEID50/ml,respectively,and the hemagglutinin titers were in the range of 1∶256-1∶512.Validation assay showed that after 10 days of inactivation,the virus was fully inactivated,and all the virus particles under TEM were intact.Conclusion The inactivation process using formaldehyde at final concentration of 100 μg/ml at the temperature of 2-8 ℃ for 10 days can effectively inactivate the By strain of influenza virus(B/Phuket/3073/2013/BVR-1B).
基金项目
"重大新药创制"科技重大专项(2015ZX09101044)
河南省科技重大专项(171100310400)
新乡市科技重大专项(ZD16007)
NETL
NSTL
万方数据