The Effect Of Calcium Ion In The Production Of Adenovirus Vector By Perfusion Culture Of HEK293 Cells

At present,the spread of various infectious diseases has had a serious impact on human life,health,and work life,increasing the pressure on disease prevention and control.Vaccines can stimulate the body’s immune system,protect people from infectious diseases,and are the most economical and safe way to purify and control infectious diseases.Recombinant adenovirus vectors have been widely used in various vaccine production and gene therapy fields due to their advantages of efficient transduction of target genes,low potential pathogenicity,low immune response,and no host chromosome integration.However,based on the analysis of the current use of high-dose adenovirus vectors in clinical practice,the issue of improving the production efficiency of large-scale manufacturing of recombinant adenovirus vectors still needs to be addressed urgently.Taking HEK293 cells as an example to produce recombinant adenovirus vectors,the"cell density effect"is one of the main factors limiting its production efficiency,that is,when the cell density exceeds a certain critical value,the single cell yield decreases with the increase of cell density.And perfusion culture is an effective measure to solve this problem.In addition,research has shown that many trace elements such as vitamins,calcium ions,and growth factors are crucial for improving the production efficiency of adenovirus vectors,and calcium ions play a crucial role as the second messenger of cells in intracellular signal transduction.Therefore,this study attempts to optimize the serum free culture medium by exploring the effect of calcium ion concentration in the culture medium on the production efficiency of adenovirus vectors,while adopting a perfusion culture mode to further improve the viral titer of adenovirus vectors.The main research content and results are as follows:（1）Explore the effect of calcium ion concentration in the culture medium on cell growth.Simulate perfusion culture of HEK293 cells using CD293 serum-free medium with different calcium ion concentrations at shake flask level.The results showed that increasing the calcium ion concentration in the culture medium from 0.1 mmol·L^-1 to 0.5 mmol·L^-1 and 1 mmol·L^-1would cause cell clustering,and the higher the calcium ion concentration in the culture medium,the more severe the cell clustering.Therefore,to ensure cell density,the calcium ion concentration in the culture medium should not be increased during the cell growth stage.（2）Study the effects of adjusting calcium ion concentration strategies on culture methods,medium types,and toxin production under different virus species conditions.The results showed that the virus titer was 4.66×10⁹IFU·m L^-1 when using simulated perfusion culture,which is 1.64 times the titer of virus in batch culture,indicates that perfusion culture has the advantage of producing virus.In addition,the strategy of adjusting the concentration of calcium ions to increase virus titer shows a tendency towards the culture medium and virus species.CD293 serum-free medium performs best when producing adenovirus vector herpes zoster vaccine,with a virus titer of 2.87×10⁹IFU·m L^-1,with a 50%increase in virus titer compared to the control group,but the specific process still needs further optimization.（3）Optimization of the production process of herpes zoster vaccine adenovirus vector using HEK293 cell simulation perfusion culture.The key process parameters for process production are the cell density at the time of infection,the number of infections,the concentration of calcium ions in the culture medium,and the time to increase the concentration of calcium ions in the culture medium.Determine the response value as 2 dpi virus titer and cell yield,and analyze the robustness set point using MODDE software as follows:CCI is5.06×10⁶cells·m L^-1,with a MOI of 3.6,the time to increase the calcium ion concentration in the culture medium was 10.4 hpi,and the calcium ion concentration in the culture medium was0.22 mmol·L^-1.The software predicts that the 2 dpi virus titer can reach 1.17×10¹⁰IFU·m L^-1,cell yield can reach 2313.8 IFU·cells^-1.And successfully verified the prediction results and optimized the design space.Under the process conditions of this robustness set point,the virus titer is 1.08×10¹⁰IFU·m L^-1,with a 70%increase in virus titer compared to the control group under these conditions.The process conditions were successfully validated at the bioreactor level.The virus titer is 1.5×10¹⁰IFU·m L^-1increases the horizontal titer by 50%compared to the shaking bottle.（4）Analysis of the principle of adjusting the concentration of calcium ions in the culture medium to increase the titer of adenovirus vectors.Calcium ion regulatory proteins are the main regulatory factors for calcium ion dependent signaling.Therefore,the expression levels of calcium ion regulatory proteins in high and low yield samples were detected,and the results showed that the expression levels of calcium ion regulatory proteins in cells with high viral titers were higher,3.1 times higher than those in the control group.Calcium ion regulatory proteins and their related pathways may be the key to improving the viral titer of adenovirus vectors in this strategy.