基于Box-Behnken实验设计与响应面法优化禽流感疫苗生产工艺?
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摘要
为优化基于MDCK细胞无血清悬浮培养生产禽流感病毒的工艺以提高禽流感病毒血凝素(HA)滴度,采用部分析因实验设计考察了病毒生产工艺中接毒时细胞密度、TPCK-胰酶浓度、培养温度、感染复数和病毒收获时间对病毒HA滴度的影响,确定了接毒时细胞密度、TPCK-胰酶浓度、和病毒收获时间作为影响病毒HA滴度的显著因子,并进一步采用3因素3水平的Box-Behnken实验设计和响应面法,对3个显著因子作用条件进行了优化,获得了最优的工艺条件。结果显示:当接毒时细胞密度为7.0×10~6 cells×m L~(-1)、TPCK-胰酶浓度为17 mg×L~(-1)、病毒收获时间为60 h时,病毒HA滴度最高可达10.85 log_2 HAU/25μL。以上研究结果为禽流感疫苗高效大规模工业化生产提供了有力的数据支持。
In order to optimize production processes of avian influenza vaccine based on MDCK cell serum-free suspension culture, and improve avian influenza virus Hemagglutinin(HA) titer, Effect of cell density at infection, TPCK-trypsin concentration, Temperature post infection, multiplicity of infection and virus harvesting time were investigated by fractional factorial design. Cell density at infection, TPCK-trypsin concentration, and virus harvesting time were determined as significant factors affecting the virus HA titer, and Box-Behnken experiment design with three factors and three levels together with response surface methodology were further applied. Virus HA titer of 10.85 log_2 HAU/25 μL can be obtained under the optimum condition of cell density at infection = 7.0 × 10~6 cells×m L~(-1), TPCK-trypsin concentration = 17 mg×L~(-1) and virus harvesting time = 60 h. These results provide fundamental data for efficient and large-scale industrial production of avian influenza vaccine.
In order to optimize production processes of avian influenza vaccine based on MDCK cell serum-free suspension culture, and improve avian influenza virus Hemagglutinin(HA) titer, Effect of cell density at infection, TPCK-trypsin concentration, Temperature post infection, multiplicity of infection and virus harvesting time were investigated by fractional factorial design. Cell density at infection, TPCK-trypsin concentration, and virus harvesting time were determined as significant factors affecting the virus HA titer, and Box-Behnken experiment design with three factors and three levels together with response surface methodology were further applied. Virus HA titer of 10.85 log_2 HAU/25 μL can be obtained under the optimum condition of cell density at infection = 7.0 × 10~6 cells×m L~(-1), TPCK-trypsin concentration = 17 mg×L~(-1) and virus harvesting time = 60 h. These results provide fundamental data for efficient and large-scale industrial production of avian influenza vaccine.
引文
[1]BARDIYA N,BAE J.Influenza vaccines:recent advances in production technologies[J].Applied Microbiology and Biotechnology.2005,67(3):299-305
[2]TRILLA A,TRILLA G,DAER C.The 1918"Spanish flu"in Spain[J].Clinical Infectious Diseases,2008,47(5):668-673.
[3]WONG,S Y.Highly pathogenic avian influenza(H5N1)in Hong Kong,1997-2014:towards an urban biopolitical immunology[D].Manchester:The,University of Manchester(United Kingdom),2015.
[4]MONDOR M,AKSAY S,DROLET H,et al.Of chickens and men:avian influenza in humans[J].Current Molecular Medicine,2009,9(2):131-51.
[5]LAI S,QIN Y,COWLING B J,et al.Global epidemiology of avian influenza A H5N1 virus infection in humans,1997-2015:Asystematic review of individual case data[J].Lancet Infectious Diseases,2016,16(7):e108-e118.
[6]HORIMOTO T,KAWAOKA Y.Influenza:lessons from past pandemics,warnings from current incidents[J].Nature Reviews Microbiology,2005,3(8):591-600.
[7]GEORGE M,FAROOQ M,DANG T,et al.Production of cell culture(MDCK)derived live attenuated influenza vaccine(LAIV)in a fully disposable platform process[J].Biotechnology&Bioengineering,2010,106(6):906-917.
[8]MAHARANI U,MARSELINA T,ERNAWATI G,et al.The expression of essential components for human influenza virus internalisation in Vero and MDCK cells[J].Cytotechnology,2013,1(1):1-6
[9]陶源,胡又佳,朱宝泉.基于哺乳动物细胞培养的流感疫苗生产[J].世界临床药物,2011,32(9):539-542.TAO Y,HU Y J,ZHU B Q.Progress in production of infl uenza vaccine by mammalian cells[J].World Clinical Drugs,2011,32(9):539-542.
[10]黄锭,刘旭平,赵亮,等.MDCK细胞无血清悬浮培养法扩增流行性感冒病毒[C]//第六届全国化学工程与生物化工年会论文集.长沙:中国化工学会,2010:1-5.HUANG D,LIU X P,ZHAO L,et al.Propagation of influenza virusesased on suspension culture of MDCK cells[C]//Chinese National Chemical BiochemicalEngineering Annual Meeting.Changsha:The Chemical Industry and Engineering Society of China2010.1-5.
[11]AGGARWAL K,JING F,MARANGA L,et al.Bioprocess optimization for cell culture based influenza vaccine production[J].Vaccine.2011,29(17):3320-3328.
[12]ISKEN B,GENZEL Y,REICHL U.Productivity,apoptosis,and infection dynamics of influenza A/PR/8 strains and A/PR/8-based reassortants[J].Vaccine.2012,30(35):5253-5261.
[13]BOCK A,SCHULZE-HORSEL J,SCHWARZER J,et al.High-density microcarrier cell cultures for influenza virus production[J].Biotechnology Progress.2011,27(1):241-250.
[14]YOUIL R,SU Q,TONER T,et al.Comparative study of influenza virus replication in Vero and MDCK cell lines[J].Journal of Virological Methods.2004,120(1):23-31.
[15]李春艳,肖晶,李曦,等.微载体规模化培养MDCK细胞增殖H9N2亚型禽流感病毒的研究[J].中国人兽共患病学报.2009,25(12):1149-1153.LI C Y,XIAO J,LI X,et al.Research on multiplication of the H9N2 subtype avian influenza virus in large-scale microcarrier-based MDCK cell culture system[J].Chinese Journal of Zoonoses.2009,25(12):1149-1153.
[16]袁丽红,邵辉,顾永明,等.盾叶薯蓣中水解原位提取薯蓣皂甙元[J].高校化学工程学报,2007,21(3):538-544.YUAN L H,SHAO H,GU Y M,et al.Simunaneous extraction and hydrolysis in situ for the recovery of diosgenin from Dioscorea zingiberensis[J].Journal of Chemical Engineering of Chinese Universities,2007,21(3):538-544.
[17]樊铖,展侠,陈剑,等.响应面优化法在PDMS/PVDF膜制膜条件优化中的应用[J].高校化学工程学报,2012,26(3):374-381FAN C,ZHAN X,CHEN J,et al.Application of response surface methodology(rsm)to optimize the preparation conditions of PDMS/PVDF composite membranes[J].Journal of Chemical Engineering of Chinese Universities,2012,26(3):374-381.
[2]TRILLA A,TRILLA G,DAER C.The 1918"Spanish flu"in Spain[J].Clinical Infectious Diseases,2008,47(5):668-673.
[3]WONG,S Y.Highly pathogenic avian influenza(H5N1)in Hong Kong,1997-2014:towards an urban biopolitical immunology[D].Manchester:The,University of Manchester(United Kingdom),2015.
[4]MONDOR M,AKSAY S,DROLET H,et al.Of chickens and men:avian influenza in humans[J].Current Molecular Medicine,2009,9(2):131-51.
[5]LAI S,QIN Y,COWLING B J,et al.Global epidemiology of avian influenza A H5N1 virus infection in humans,1997-2015:Asystematic review of individual case data[J].Lancet Infectious Diseases,2016,16(7):e108-e118.
[6]HORIMOTO T,KAWAOKA Y.Influenza:lessons from past pandemics,warnings from current incidents[J].Nature Reviews Microbiology,2005,3(8):591-600.
[7]GEORGE M,FAROOQ M,DANG T,et al.Production of cell culture(MDCK)derived live attenuated influenza vaccine(LAIV)in a fully disposable platform process[J].Biotechnology&Bioengineering,2010,106(6):906-917.
[8]MAHARANI U,MARSELINA T,ERNAWATI G,et al.The expression of essential components for human influenza virus internalisation in Vero and MDCK cells[J].Cytotechnology,2013,1(1):1-6
[9]陶源,胡又佳,朱宝泉.基于哺乳动物细胞培养的流感疫苗生产[J].世界临床药物,2011,32(9):539-542.TAO Y,HU Y J,ZHU B Q.Progress in production of infl uenza vaccine by mammalian cells[J].World Clinical Drugs,2011,32(9):539-542.
[10]黄锭,刘旭平,赵亮,等.MDCK细胞无血清悬浮培养法扩增流行性感冒病毒[C]//第六届全国化学工程与生物化工年会论文集.长沙:中国化工学会,2010:1-5.HUANG D,LIU X P,ZHAO L,et al.Propagation of influenza virusesased on suspension culture of MDCK cells[C]//Chinese National Chemical BiochemicalEngineering Annual Meeting.Changsha:The Chemical Industry and Engineering Society of China2010.1-5.
[11]AGGARWAL K,JING F,MARANGA L,et al.Bioprocess optimization for cell culture based influenza vaccine production[J].Vaccine.2011,29(17):3320-3328.
[12]ISKEN B,GENZEL Y,REICHL U.Productivity,apoptosis,and infection dynamics of influenza A/PR/8 strains and A/PR/8-based reassortants[J].Vaccine.2012,30(35):5253-5261.
[13]BOCK A,SCHULZE-HORSEL J,SCHWARZER J,et al.High-density microcarrier cell cultures for influenza virus production[J].Biotechnology Progress.2011,27(1):241-250.
[14]YOUIL R,SU Q,TONER T,et al.Comparative study of influenza virus replication in Vero and MDCK cell lines[J].Journal of Virological Methods.2004,120(1):23-31.
[15]李春艳,肖晶,李曦,等.微载体规模化培养MDCK细胞增殖H9N2亚型禽流感病毒的研究[J].中国人兽共患病学报.2009,25(12):1149-1153.LI C Y,XIAO J,LI X,et al.Research on multiplication of the H9N2 subtype avian influenza virus in large-scale microcarrier-based MDCK cell culture system[J].Chinese Journal of Zoonoses.2009,25(12):1149-1153.
[16]袁丽红,邵辉,顾永明,等.盾叶薯蓣中水解原位提取薯蓣皂甙元[J].高校化学工程学报,2007,21(3):538-544.YUAN L H,SHAO H,GU Y M,et al.Simunaneous extraction and hydrolysis in situ for the recovery of diosgenin from Dioscorea zingiberensis[J].Journal of Chemical Engineering of Chinese Universities,2007,21(3):538-544.
[17]樊铖,展侠,陈剑,等.响应面优化法在PDMS/PVDF膜制膜条件优化中的应用[J].高校化学工程学报,2012,26(3):374-381FAN C,ZHAN X,CHEN J,et al.Application of response surface methodology(rsm)to optimize the preparation conditions of PDMS/PVDF composite membranes[J].Journal of Chemical Engineering of Chinese Universities,2012,26(3):374-381.