| Recombinant human tumor necrosis factor receptor type II-antibody fusion protein(rhTNFR-Fc) has a significant effect in the treatment of rheumatoid arthritis, rheumatoidarthritis, etc.,increasingly shows its important role and broad market prospects. With thedevelopment of cell fusion technology and genetic engineering, large-scale animal cellculture technology has become biopharmaceutical key to industrialization. However, Theexpression levels of the product,production scale and culture expression patterns hinderthe promotion of the drug market in China.The topics to CHO cells expressingrhTNFR-Fc for the study, choose nutritious medium and establish reasonable controlstrategy.There by increasing cell density and protein production in CHO cells.Recovery test in CHO cells, the choice of the method first recoveried under40℃andthen dissolved in water bath at37℃,speeding up the thawing time frozen cells,Foundthrough experiments,cryopreservation agents to choose different CHO cellcryopreservation and recovery equally influential. Frozen cells compared withconventional liquid nitrogen method,2℃~8℃refrigerator CHO cell suspension was storedin a short time, there is a big advantage in terms of rapid recovery growth state of the cell,in the production of tight situations can be used as an auxiliary method to protect thecontinuation of cell passage.After a gradual reduction in serum domesticated, state of CHO cells were better thanthat no serum domesticated cells, the cell density can reach to2.51×106cells/mL,celldensity than serum domesticated nearly1.6times higher.During acclimation cell suspension,adding cysteine hydrochloride can promote cellgrowth, the expression levels showed adding cysteine hydrochloride can increase theexpression of cell, the expression level compared with the control group by about1.7times. Meanwhile, the medium was supplemented with dextran help reduce cell clumping,while increasing the cell density, and to determine the optimal concentration of dextransulfate was added50μg/mL.A research to optimized the basal medium,it wsa found that CHO cells grown onBD,GIBCO,LONZA and E001basic medium, cells were cultured for5days andpurified,E001medium expression of41.94mg/L,while the expression of the productLONZA medium was29.96mg/L.At the time of CHO cells expressing age,the temperature controlled at37℃is higher than that when the temperature at32℃. By expression of osmotic pressure wasfound toimprove the osmotic pressure can effectively improve protein expression,the highestexpression when osmolality430mOsm time in24h.Taking into account the osmotic effecton cells at48h on a microscopic analysis of cells,The highest density of the control groupbut to maintain the osmotic pressure in the poor state of270mOsm,maintaining osmoticpressure at350to430,the cell state is good, but the density decreased significantlycompared with the control,the osmotic pressure is maintained at470mOsm,and thedensity difference between the state of cells decreased significantly.Comprehensiveanalysis,the osmotic of the pressure of the cells at around390mOsm.Experiments to optimize training methods indicates that, supplied with F001To theculture medium is better able to promote growth and expression, Ultimately determine thatCHO cells appropriated the fed-batch culture methods, basal medium choice E001, feedmedium choice F001.By studying this subject, expressing recombinant human tumor necrosis factorreceptor type II-Fc fusion protein in CHO cells suspended domestication and cultivationprocess optimization for improving product yield and cell density has a very importantrole and significance. lay the foundation for industrial production for rhTNFR-Fc fusionprotein. |
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