Effect Of CO₂ On CHO Cell Growth And Productivity And Elucidating Its Intrinstic Regulation Mechanism

With the huge market demand for protein drugs,large-scale and high-density processes for mammalian cells have been established as the major means for manufacturing commercial products in the biopharmaceutical industry.However,carbon dioxide(CO2)over-dose accumulation become one of the crucial parameters in CHO cell scale-up processes,especially with high cell density.Since the extremely pCO2 not only has adverse effects on CHO cell growth,metabolism,productivity and quality,but also deteriorates culture environment by changing the process parameters like pH and osmolality,many process control solutions have been investigated to release the pressure of pCO2.With the limiting of comprehensive investigation about the effects of CO2 on cell metabolism and protein producing processes,to date the pCO2 control requirements and control consequences of the culture processes are not clear.Fully exploration of accurate effects and relative mechanisms of extremely pCO2 on CHO cells is necessary for guiding the rational experimental design and realizing the stable and controllable scale-up process.In this study,CHO cell lines were chosen as the object,and the characteristics of CHO cell growth,metabolism and protein production was firstly investigated under different pCO2 conditions.Details of intracellular status were unveiled by transcriptome and metabolome analysis,and key enzyme activity evaluation as well.Moreover,intermediate metabolites supplement was performed to examine the compensation effects on the metabolic deficiency.Finally,a mathematical model had already been developed to describe the relationship among lactate,CO2,pH and base addition.By means of this relationship,we could calculate pCO2 for taking suitable operation methods to control the pCO2 in the culture processes.Firstly,based on the previously fed-batch culture processes,the importance of pCO2 in large-scale culture process was preliminarily identified.The results of four fed-batch cultures showed that the elevated pCO2 caused by the insufficient CO2 removal ability of large reactors may be the main reason for the decrease of cell growth and productivity and the change of lactic acid metabolism.The effects of pCO2(10-360 mmHg)on cell growth,metabolism and productivity were further investigated.The results showed that excessive pCO2 was not only harmful for the cell growth and productivity,but also promoted the formation of lactic acid and reduced the utilization of glucose.At the same time,with low pCO2(10 mmHg),CHO cells can barely grow,and most of the consumed glucose was converted into lactic acid,which caused the decrease of the glucose utilization.Basically,too high or low pCO2 would do harm to the cell growth,changed cell metabolism of glucose and lactate and finally reduced the utilization of glucose.In order to investigate the effect and instinct mechanism of elevated pCO2(160 mmHg)on cell grow and productivity,we firstly analyzed the influence of elevated pCO2 on the metabolism of cells in maintenance phase.The results indicated that elevated pCO2 could indicate insufficient central carbon metabolism,especially significantly changed the metabolism of pyruvate and TCA cycle of CHO cells in maintenance culture phase.Secondly,by using transcriptome method to investigate the bottleneck of certain carbon metabolism,the transcription levels of LDHc and IDH2 were changed with elevated pCO2 conditions which could influence the metabolic phenotype of CHO cells.Thirdly,?-ketoglutarate and pyruvate were added to the culture with elevated pCO2 condition to verify the bottleneck of certain carbon metabolism,respectively.With added ?-ketoglutarate,the growth and titer of CHO cells in elevated pCO2 condition were significant recovered to those in normal pCO2 condition.Meanwhile,compared with that in elevated pCO2 condition,the mitochondrial function was also improved,and the lactate was shifted to be consumed again.These results indicated that the regulated metabolism node of elevated pCO2 was intracellular ?-ketoglutarate.Finally,by detection the activities of intracellular IDH,the excessive CO2/HCO3-could cause feedback inhibition to IDH3 and inhibit the regular metabolic process of TCA cycle.Beside that,the rising of CO2 concentration promoted the reductive carboxylation of ?-ketoglutarate,which also inhibited the regular metabolic process of TCA cycle.These changes could decrease the content of intracellular ?-ketoglutarate and further induced the metabolism disorder in CHO cells with elevated pCO2 condition.The research data showed that low pCO2(10-20 mmHg)caused by high headspace aeration rates could inhibited the cell growth and productivity.In order to investigate the effect and instinct mechanism of low pCO2 on cell grow and productivity,we firstly analyzed the influence of low pCO2 on the metabolism of cells in maintenance phase.The results indicated that low pCO2 could indicate insufficient central carbon metabolism,especially significantly decreased the flux of pyruvate to acetyl-CoA and TCA cycle by 31%and 40-50%of CHO cells in maintenance culture phase,respectively.Secondly,by means of untargeted metabolome method,we fully compared the differences of intracellular metabolites between low pCO2 and normal pCO2 conditions.The results shed light on the impact of low pCO2 levels on cell metabolism from the two following aspects:the central carbon metabolism and antioxidant synthesis pathway.In the central carbon metabolism,the concentrations of TCA cycle related metabolites and glutamate decreased significantly under low pCO2 condition,while intracellular lactate increased.Thirdly,to verify the bottleneck of certain carbon metabolism,pyruvate,glutamate and TCA cycle related metabolites were added to the culture with low pCO2 condition.With added pyruvate,the growth and titers of CHO cells under low pCO2 condition were significantly increased,and the mitochondrial function was also improved.These results indicated that the intracellular pyruvate could be a regulated node of low pCO2.Finally,by detecting the activities and transcription levels of intracellular LDH,the increase of transcription levels of LDHa and the activities of LDHi would be the main reasons for the inhibition of lactate consumption in the low pCO2 condition,which further inhibited the regular metabolic process of TCA cycle and induced the metabolism disorder in CHO cells with low pCO2 condition.With investigating the effect of pCO2 on cell growth,metabolism and productivities,we set up a mathematic equation to describe the relationship between pCO2,lactate,base and pH(L+0.051×pCO2×10(pH-6.38)=Bi+Bfed+Bbase)in order to calculate and control the pCO2 in culture processes.By means of this relationship,we could calculate pCO2 for taking suitable operation methods to control the pCO2 in the culture processes.Through this research,the effect and instinct mechanism of pCO2 on cell grow and productivity during cell culture process was comprehensively investigated,the understanding of the metabolism of CHO cells was also enriched,which laid the foundation for the establishment of reasonably scale-up cell culture process and effective control of pCO2.