Preparation And Characterization Of Fc Fusion Protein Expressed By CHO Cell

Fc fusion protein means fusing the biological activity protein or peptide to the IgG's Fc fragment by using genetic engineering technology,with the aim to produce a new recombinant protein.With the wide application of antibodies' biopharmaceutical,it's of great significance to solve the key downstream process issues about Fc fusion protein preparation.This thesis focuses on investigation and optimization of the cell culture medium and feeding strategy,the purification through the Protein A affinity chromatography,as well as the anion exchange chromatography.The study involves:1.Carry out medium mixing experiment through shake flask culture to optimize the medium component.The selected mediums are commercialized and suitable for CHO cell growth.The aime of this step is to inspect the influences of different mediums on the cell growth and protein expression,at the same time to reduce the cost.Compare with single seed medium,the formula of seed medium(Mab-Works medium C):basic medium(CD OptiCHO)=1:1 ratio increases cell density up to 161.8%and increase protein expression level up to 100%.Further experiment designed for saving cost indicated that the formula of seed medium(Mab-Works medium C):basic medium(CD OptiCHO):basic medium(CDM4Mab)= 2:1:1 is better than that of seed medium(Mab-Works medium C):basal medium(CD OptiCHO)= 1:1 formula for cell culture.The cell density,protein expression level and the cost for the former is respectively 13.3%higher,18.1%higher,and 25%lower than those for the later.Besides,cells can maintain good viability for the 2:1:1 formula.2.Optimize the fed-batch strategy for cell culture.It is found that:strategy 2(the fed medium volume is 30%of the final volume,averagely fed at 3,6,9 days)is better than strategy 1(fed medium volume is 30%of the final volume and averagely fed at 2,4,6,8 days),with 30.9%increase of the cell density and 13.2%inctrase of protein expression.3.Succeed in scaling up cell culture in 5L bioreactor.The cell density and the taget protein expression is comparable and repeatable with the shake flask results.4.Determine the optimal operation for the Protein A affinity chromatography process.After loading the supernante of culture both,the Fc-fused protein was eluted by decreasing the buffer pH value to 3.3.The purity of the eluted protein is about 98.64%and the recovery is about 94.7%;5.Anion exchange chromatography is used to further remove other impurities.Based the determination of the PI of the Fc-fused protein,a passthrough chromatographic model was adopted at pH 5.0?5.5 and the buffer conductivity is below 2 mS/cm.