| Presently,influenza virus vaccines in China are produced by chicken embryo.However,due to the limitation of the number of chicken embryos and the difficulty of expanding the production of chicken embryo technology,once the pandemic comes,the production of influenza with chicken embryo will be far from enough to meet the needs of the people.However,the production of influenza vaccine based on cells is not limited by the number of chicken embryos,which is easier to scale up and can be applied to people who are allergic to egg protein.MDCK cells are one of the most popular substrates for influenza vaccine production,so it is of great practical significance to develop the large-scale technology of MDCK cell culture.The culture of cell-based viral vaccines is usually divided into two phases,namely,cell growth and virulent production.In order to develop a more efficient and cost-effective vaccine manufacturing process,we first simulated the basic process and process optimization on a laboratory-scale using shaker and shake flask.The primary and clear parameter is how to subculture the cells,the cells were inoculated according to the gradient density（2×106,1.5×106,1×106,7×105and 5×105 cells/mL）in the shaker.By drawing the cell growth curve of different cell inoculation density,and monitoring the cell viability and cell metabolism of each experimental group at the same time,It was found that at 48h,each gradient density inoculation was in the logarithmic growth period of cell growth and division,and the maximum cell growth reached 8.3×106 cells/mL,also,it was found that the inoculation of each density reached the platform stage in 60 hours,most of which reached tens of millions.There was no significant difference in cell density,cell viability and metabolism between groups.In order to ensure that the cells are in the logarithmic growth period,the passage time is set as 48h.G1 phase cell ratio was measured by sampling the 5density gradients at 48h,and the cell proportion of 1.5×106and 7×105 cells/mL cells was the highest at the G1 stage at 48h,and the cell density of 1.5×106 cells/mL cells was the highest at the cell growth curve at 48h,which was 8.3×106 cells/mL.Therefore,the condition for cell passage was determined to be 1.5×106 cells/mL inoculation and the cell passage was conducted at 48h.In addition,the rotation speed of the shaker（160、150、140、130、120、110 RPM/min）and the temperature of the cell growth stage（38,37,35,33℃）were explored in gradient,and the range of the optimal rotation speed was 130-150 RPM/min and the optimal cell growth temperature was 37℃.On the basis of cell growth stage,the parameters of virus inoculation（MOI,cell density of virus vaccination and TPCK-trypsin concentration）were explored.The gradient range of MOI of H7N9 avian influenza virus infection was(5×10-2、2.5×10-2、5×10-3、10-3、5×10-4、2.5×10-4、1.25×10-4、2.5×10-5),the gradient range of TPCK trypsin was 0,0.5,1,1.5,2 and 2.5μg/mL,the gradient selection of cell density（7×106、6×106、5×106、4×106和3×106cells/mL）when H7N9avian influenza virus was inoculated.The optimal range or values of these 3 factors were explored step by step,and the optimal parameters of virus producing period were obtained:the cell density of inoculating virus was 4×106 cells/mL,the MOI of H7N9 virus inoculation was 10-3,the optimal range of TPCK trypsin was 1-1.5μg/mL,these parameters could make the hemagglutination titer of H7N9 avian influenza virus reach 1:26.5.After determining the basic conditions of the virus production period,the virus value-added curve was drawn,and the best virus collection time was 48h.However,the hemagglutination is low,so appropriate optimization is extremely necessary.Based on the basic process parameters,this study optimizes the process from the perspective of the use of nutrients or additives.Glutamine is one of the key enzymes in tricarboxylic acid cycle.According to the parameters determined in the above study,glutamax was added at different dilute concentrations（1、2、4、8 mM）.No effect of adding glutamax on improving the hemagglutination titer of influenza virus was found,indicating that the commercial medium used has been optimized to the best on this parameter.TPCK trypsin will be completely degraded within 24 hours after it is added in the process of virus production.Therefore,TPCK trypsin is added regularly in groups（three groups are added every 12 hours,only once in 12 hours and every 24hours）to verify its promotion of influenza virus proliferation.No significant effect of TPCK trypsin supplementation on virus production is found.Using different commercial medium（medium A,B,C）ratio,medium A:medium B with 1:1 ratio,the cell density can be increased by 2×106 cells/mL in 48h without affecting the virus production.In the stage of virus production,the fresh culture medium was replaced every 24 hours,the cells grew continuously and expressed virus,which indicated that the cells had reached the state of virus carrying.After 48 hours of cell lysis with triton-X100,the hemagglutination titer of virus in the culture medium increased sharply,reaching 1:29 HA units/50μL.Based on the process parameters of the scale of the laboratory,the 7.5L bioreactor was enlarged to explore the stirring speed,pH,temperature and dissolved oxygen,and to determine the best parameters of cell growth period.The results showed that the optimal pH condition for cell growth was 7.0,dissolved oxygen was50%,temperature was 37℃,and stirring speed was 70-110 RPM/min.Through the research of this paper,the high-efficiency production process of H7N9 avian influenza virus vaccine based on suspension MDCK cells was developed,which laid a foundation for the industrial production of H7N9 avian influenza virus vaccine. |
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