Study On The Applicability Of Hyperosmotic Stress And Perfusion Culture Strategy To Increase The Production Of Recombinant Adenoviral Vector In HEK293 Cells

With various diseases ravaging internationally,the market demand for recombinant adenoviral vector（Re-Adv）vaccines has dramatically increased.There is an urgent need for methods to increase Re-Adv yield to meet the market demand,and process research is an effective solution to this problem.The perfusion culture process has been widely used for Re-Adv production.With the addition of sophisticated biopharmaceutical production equipment,Re-Adv’s yield per unit culture volume is much higher than that of traditional batch culture.Osmotic pressure is a critical physicochemical property parameter of mammalian serum-free medium.Applying hyperosmotic stress in cells before virus infection could increase the yield of Re-Adv in batch mode.Since the production mode of this hyperosmotic stress process remains in batch culture,which is limited by the so-called“cell density effect”,the percentage of yield increase is much less than that of perfusion culture.Combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Re-Adv at high cell density.In this study,shake flasks combined with semi-perfusion culture were used as a scaled-down model for bioreactor perfusion culture.The relationship between critical process parameters:viable cell density at the time of infection（TOI）,the multiplicity of infection（MOI）,p H,and virus harvest time（Har）and critical quality attribute:virus titer of Re-Adv at harvest time（IFU）,was determined through design of experiments.After obtaining the design space(p H of 7.13 to 7.20,TOI of 1.2×10⁷ to1.5×10⁷ cells·m L^-1,MOI of 2 to 4.8 IFU·cell^-1,Har of 48 to 56 hpi),subsequent experimental studies were performed using its robust setpoint(p H=7.15,TOI=1.26×10⁷ cells·m L^-1,MOI=3.6 IFU·cell^-1,Har=53.3 hpi).The results showed that using a perfusion culture process with hyperosmotic pressure medium（370 m Osm）during the cell growth phase and isosmotic pressure medium（300m Osm）during the virus production phase,effectively increased the yield of Re-Adv,which may due to increased expression of heat shock protein 70 during the late phases of virus replication.The Re-Adv titer in a 3 L bioreactor with such a process reached 3.2×10¹⁰IFU·m L^-1,three times higher than the isosmotic perfusion culture process.More importantly,this is the first time that the strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Re-Adv,and also reveals the reasons for the increased production of Re-Adv by the hyperosmotic stress process,which provides a reference for the process optimization of other viruses production by HEK293 cells.