生物反应器培养Vero细胞和H1N1流感病毒
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摘要
目的建立利用生物反应器制备Vero细胞流感H1N1全病毒灭活疫苗的新工艺。方法利用Vero细胞作为流感病毒增殖的细胞基质,分别以无血清培养液Pro VERO和含血清DMEM培养液,利用5 g/L微载体cytodex-1在3 L硅化生物反应器中进行培养,培养温度(37±0.5)℃,溶解氧(50±20)%,p H 7.2±0.2,搅拌速度(50±20)r/min,待细胞在微载体上长成单层后,以0.1 MOI接种流感病毒Vero细胞高产适应株H1N1/JD/Va,将收获的病毒液经Sepharose 4FF和Capto Q两级纯化,灭活后制备疫苗原液及成品,按照《中国药典》三部(2015版)方法对其进行检定。结果使用无血清细胞培养液培养Vero细胞24 h贴壁率约83%,含血清细胞培养液培养24 h贴壁率为112%;灌流培养方式培养至第6天时,细胞均长至单层,无血清细胞培养液最终细胞数达到(133.4±2.0)×10~4个/m L,含血清细胞培养液最终细胞数达到(353.4±5.9)×10~4个/m L。无血清病毒维持液培养,第66 h收获病毒液血凝效价为388,单位细胞产毒比例为2.9;含血清病毒维持液培养,第66 h收获病毒液血凝效价为891,单位细胞产毒比例为2.5。用含血清细胞培养液培养Vero细胞接种病毒,收获病毒液制备的疫苗原液及成品经检测,各项指标均符合《中国药典》三部(2015版)相关要求。结论建立了3 L生物反应器含血清细胞培养液培养Vero细胞和H1N1流感病毒的新工艺,为进一步放大生产规模奠定了基础。
Objective To develop a new process for preparation of inactivated influenza H1 N1 vaccine by culture of influenza H1 N1 virus in Vero cells using a bioreactor. Methods Vero cells as a cellular matrix for proliferation of influenza virus were cultured in serum-free medium Pro VERO and serum-containing DMEM respectively in a 3 L bioreactor using5 g/L Cytodex-1 microcarriers at a temperature of(37 ± 0. 5) ℃,a dissolved oxygen concentration of(50 ± 20) %,a p H value of(7. 2 ± 0. 2)and a stirring rate of(50 ± 20)r/min. Vero cells-adapted high-producing influenza virus H1 N1/JD/Va strain was inoculated onto Vero cells at a MOI of 0. 1 after a monolayer was formed on microcarriers. The harvested virus liquid was purified by Sepharose 4 FF and Capto Q chromatography and inactivated to prepare the bulk and final product of vaccine which was subjected to control tests according to the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Results The adherence rate of Vero cells cultured in serum-free medium for 24 h was about83%,while that in serum-containing medium was 112%. All the cells on day 6 after perfusion culture formed a monolayer. The final counts of cells in serum-free medium was(133. 4 ± 2. 0) × 10~4 cells/m L,while that in serumcontaining medium was(353. 4 ± 5. 9)× 10~4 cells/m L. The hemagglutination titer and virus production ratio in unit cells of virus after culture in serum-free maintenance medium for 66 h were 388 and 2. 9,while those in serum-containing maintenance medium were 891 and 2. 5,respectively. All the quality indexes of bulk and final product of vaccine prepared with the virus liquid from Vero cells culture in serum-containing medium met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Conclusion A new process for culture of Vero cells and influenza H1 N1 virus in serum-containing medium by using 3 L bioreactor was developed,which laid a foundation of further scale-up of production scale.
Objective To develop a new process for preparation of inactivated influenza H1 N1 vaccine by culture of influenza H1 N1 virus in Vero cells using a bioreactor. Methods Vero cells as a cellular matrix for proliferation of influenza virus were cultured in serum-free medium Pro VERO and serum-containing DMEM respectively in a 3 L bioreactor using5 g/L Cytodex-1 microcarriers at a temperature of(37 ± 0. 5) ℃,a dissolved oxygen concentration of(50 ± 20) %,a p H value of(7. 2 ± 0. 2)and a stirring rate of(50 ± 20)r/min. Vero cells-adapted high-producing influenza virus H1 N1/JD/Va strain was inoculated onto Vero cells at a MOI of 0. 1 after a monolayer was formed on microcarriers. The harvested virus liquid was purified by Sepharose 4 FF and Capto Q chromatography and inactivated to prepare the bulk and final product of vaccine which was subjected to control tests according to the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Results The adherence rate of Vero cells cultured in serum-free medium for 24 h was about83%,while that in serum-containing medium was 112%. All the cells on day 6 after perfusion culture formed a monolayer. The final counts of cells in serum-free medium was(133. 4 ± 2. 0) × 10~4 cells/m L,while that in serumcontaining medium was(353. 4 ± 5. 9)× 10~4 cells/m L. The hemagglutination titer and virus production ratio in unit cells of virus after culture in serum-free maintenance medium for 66 h were 388 and 2. 9,while those in serum-containing maintenance medium were 891 and 2. 5,respectively. All the quality indexes of bulk and final product of vaccine prepared with the virus liquid from Vero cells culture in serum-containing medium met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Conclusion A new process for culture of Vero cells and influenza H1 N1 virus in serum-containing medium by using 3 L bioreactor was developed,which laid a foundation of further scale-up of production scale.
引文
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[2]GENG X L,LIAO G Y.Progress in research on live attenuated influenza vaccine[J].Chin J Bioloicals,2017,30(1):103-105.(in Chinese)耿兴良,廖国阳.流感病毒减毒活疫苗的研究进展[J].中国生物制品学杂志,2017,30(1):103-105.
[3]GUO Q,LIAO G Y.Progress in research on avian influenza H7N9 vaccine for human use[J].Chin J Biologicals,2017,30(2):210-214.(in Chinese)郭琦,廖国阳.人用H7N9禽流感疫苗的研究进展[J].中国生物制品学杂志,2017,30(2):210-214.
[4]GENZEL Y,DIETZSCH C,RAPP E,et al.MDCK and Vero cells for influenza virus vaccine production:a one-to-one comparison up to lab-scale bioreactor cultivation[J].Appl Microbiol Biotechnol,2010,88(2):461-475.
[5]XU G F,GENG X L,SONG S H,et al.Optimization of condition for removal of vero cell DNA in influenza H5N1 vaccine with Benzonase nuclease[J].Chin J Biologicals,2017,30(5):540-545.(in Chinese)徐国峰,耿兴良,宋绍辉,等.Benzonase酶去除流感疫苗中Vero细胞DNA条件的优化[J].中国生物制品学杂志,2017,30(5):540-545.
[6]YU W,YANG F,YANG J,et al.Construction high-yield candidate influenza vaccine viruses in Vero cells by reassortment[J].J Med Virol,2016,88(11):1914-1921.
[7]ZHOU J,YANG F,YANG J,et al.Reassortment of high-yield influenza viruses in vero cells and safety assessment as candidate vaccine strains[J].Hum Vaccin Immunother,2017,13(1):111-116.
[8]ARIFIN M A,MEL M,ABDUL KARIM M I,et al.Production of Newcastle disease virus by Vero cells grown on cytodex 1microcarriers in a 2-litre stirred tank bioreactor[J].J Biomed Biotechnol,2010,2010:586363.doi:10.1155/2010/586363.
[9]MARKET RESEARCH REPORTS.BIZ.Bioreactors and Fermenters Market-Global Industry Analysis,Size,Share,Volume,Growth,Trends,and Forecast 2016-2024[R/OL].(2017-05-10)[2017-11-11].https://www.mrrse.com/bioreactors-fermentersmarket.
[10]LIU C C,GUO M S,LIN F H,et al.Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system[J].PLo S One,2011,6(5):e20005.doi:10.1371/journal.pone.0020005.
[11]ROUROU S,RIAHI N,MAJOUL S,et al.Development of an in situ detachment protocol of Vero cells grown on Cytodex1microcarriers under animal component-free conditions in stirred bioreactor[J].Appl Biochem Biotechnol,2013,170(7):1724-1737.
[12]TAPIA F,VOGEL T,GENZEL Y,et al.Production of hightiter human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor[J].Vaccine,2014,32(8):1003-1011.