Study On Culture Process Of CHO Cell Expressing Fusion Protein (GLP-1^A2G)₂-HSA

Glucagon like peptide-1(GLP-1)is a drug for the treatment of type II diabetes.However,it has a short half-life in vivo and lot-to-lot expression instability,which limit its clinical application.In our early study,the modified fusion protein NGGH(6×His-tag+Ek+2×GLP-1A2G+HSA)was successfully expressed in CHO cells using vector pMH3.This design not only improved the half-life of GLP-1 in vivo,but also solved the problem of lot-to-lot expression instability.Therefore,the research of high expression strategy and cell culture process was very necessary.This research studied on some small molecular added in medium to improve the growth rate of CHO cells and expression of NGGH,and the then optimized the recombinant CHO cells culture process in 5 L Single-Use Bioreactor.In this paper,We stuedied on the effect of different concentrations of valproic acid and suramin on the growth of CHO cells and protein yield,the screened out the optimal concentrations of suramin and valproic acid were 50 ?M and 0.75 m M,respectively.We found that suramin increased NGGH yield 50% by enhancing CHO cells growth and protecting them against apoptosis and valproic acid increasing protein yield 70% by inhibiting CHO cells cycle and increasing the level of mRNA.The mixture of them two compared with the control group increased protein yield 70% and suramin inhibited the toxicity of valproic acid to the cells in a certain degree,so that the viability of CHO cells was higher at the end of culture and purification was easy.CHO cells cuiture conditions(dissolved oxygen,pH and feeding strategy)in 5 L Single-Use Bioreactor were optimized.The result showed that the optimal conditions were pH 6.8~7.4,DO controlled in two-phase way and feeding F001 in whole phases.In addition,suramin and valproic acid were added in the first and third day.In these conditions,The maximum cell density reached 1.26×107 cells/mL and the yield of NGGH protein rearched 283 mg/L(1.35 times of that in flask)which achieved the amplification of recombinant CHO cells culture from shake flasks to bioreactor.Fusion protein was purified by blue affinity chromatography,hydrophobic chromatography,and ion chromatography.Then it was cut by Bovine Recombinant enterokinase and purified by Ni column to remove His-tag.The purity and total protein yield were 96.1% and 22.1%,respectively.CHO/GLP-1R-EGFP cell was used to detect activity of fusion protein GGH.The results showed that activity of the protein expressed in the optimital process was similar with that in shak flask without any small molecules added.In other words,the optimization of culture process of recombinant CHO cells did not affect the activity of the fusion protein.