Simulation And Experimental Research On The Key Properties Of Orbitally Shaken Bioreactors

Bio-pharmaceutical is the process of producing the aimed recombinat protein by cultivating cells,which is a promising industry in the 21 st century.The bioreactors are the key equipments for providing a suitable environment for cells cultivated.During the process of cells cultivating,cells need the nutrient substance from the liquid environment and are very sensitive to the liquid shear stress,which indicates that the fluid dynamics of flow field in bioreactors will large ly affect on cells behavior.However,the complex of the flowing field in bioreactors caused that most of cells cultivation technique had been based on the strong cells line and high-property of medium.Recently,with the development of computed fluid dynamics(CFD)technique,it became possible to investigated the cells behavior from the fluid dynamics veiw.Nowadays,most of reseach work have been done based on the stirred tank s(STRs).Due to the difference of mixing and oxygen supply,the orbitally sha ken bioreators(OSRs)which are promising in future,are lack of investigations.Therefore,we used the techniques of CFD,cells cultivation in suspension,PIV measuring liquid velocity and moving liquid wave capturing with high-speed camera to investigate the mixing,oxygen supply and cells damage issues in OSRs.The flow field in the bioreactor can reflect the fluid environment around cells basically.To analyze the flow field in the OSRs,the first work is to build up the simulation strategy for the flowing field using the techniques of CFD,PIV measuring fluid velocity and moving liquid wave capturing with high-speed camera.In the process of simulation,we optimized the time-step,meshing number and turbulent model and found that the computing process is more stable and efficient using the gravity method for orbital shaking load.Moreover,the liquid volume,shaking speed and vessel structure had stronge effect on flow field of OSRs.Effective mixing is main purpose for the bioreactor and the nutrient gr adient will do harm to cells growth and lead to secondary metabolite increasing.To evaluate the mixing efficiency of OSRs,we build up the ‘transient method' and ‘dynamic frozen method' to simulate mixing time.By the analyzation of mixing time at different liquid volume and shaking speed,we found that mixing time will not change when liquid volume smaller than a critical value or shaking speed higher than a critical value.We named the critical value as ‘saturated liquid volume' or ‘saturated shaking speed' which will occur when the height of liquid wave is 1.5 times higher than liquid height at static.The another aim of mixing is to keep cells suspended in the moving liquid,but there is no efficient method to predict whether cells is suspended or not before conducting cells cultivation.We found that cell sediment probably happen when the average axial fluid velocity is lower than 0.04 m/s.The oxygen transfer rate(OTR)is very important for mammalian cells because of their aerobic respiration.We studied the operating condition effect on the volumetric oxygen transfer coefficient(k La)firstly.We found that the oxygen supply rate of a bioreactor is based on Reynold number of flow region and Froude number near the vessel wall.Based on the simulated and measured k La,we built up a volume-and size-independent k La predicting model to analyze the effect of shaking speed,filling volume,shaking diameter and vessel diameter on oxygen transfer rate of orbitally shaken bioreactors.Cell damage issue is important but difficult to analyze in bioreactors.In the end of this paper,we investigated cell damage from the aspects of power input,turbulent scale and fluid shear stress,respectively.Based on the surface analysis for power input,a suitable range of operating condition was found,which was validated by CHO cells cultivation.Then,we simulated the turbulence characteristic scales in orbitally shaken bioreactors to evaluate the minimum vortex length,and found that fatal cell damage will happen when the minimum vortex length of flow field was located in the range of cell diameter.At last,based on the result of simulation and cells suspension cultivation,we found that the high shear stress(0.4-1.0 Pa)was not mortal to CHO cells but its volumetric proportion will influence the titer of the protein produced.