Study On Cell Culture Process Of CHO Cells Expressing Fusion Protein HSA/IL-2

Interleukin2(Interleukin-2,IL-2)is a multifunctional immune regulation of cytokines and limited in clinical application due to its short half-life in the human body.In previous study,the fusion protein of human serum albumin and human interleukin-2(HSA/IL-2)has expressed both in Pichia pastoris and CHO cells successfully.CHO cells solved some problems of protein degradation and glycosylation existing in Pichia,the following study is(1)As a result of unstable nature of the medium containing some proteins,the medium is hard to preserve;(2)small scale of CHO culture in shake flasks and low expression which limit its further study and industrialization of HSA/IL-2.In this study,medium selection and optimization,CHO cell culture technology in stirring bioreactor were researched.To solve the problem of medium,four commercial serum-free medium were chose to study.M1.was the best basal medium.In fed-batch culture,feed medium(F3)was the best one.In addition,different protein hydrolysates(yeast extract,soy peptone,tryptone)and small molecule enhances(sodium butyrate,valproic acid,DMSO)were used to increase the cell density and fusion protein expression respectively.M1 supplemented with 4mM valproic acid can obviously improve the expression level of HSA/IL-2 to 72 mg/L,1.24 times to the control group.Different concentration of hydrolysate have effect on cell growth and protein expression differently,M1 supplemented with 5g/L YE reached the best results.Different hydrolysates affect cell growth and protein expression differently,5.0 g/L YE could significantly promote cell growth and enhance the expression level of HSA/IL-2.The maximum viable cell density was 9.95×106 cells/m L,1.43 times to the control group.And the expression level of HSA/IL-2 reached 100 mg/L,1.72 times to the control group.In addition,YE could increased the rate of G1 phase and protecting cells against apoptosis.CHO cell culture technology was established in stirring bioreactor technology,realizing the level of scale up from shaking flask to bioreactor.The shear force produced by hydrodynamic including foam,aeration,bubbles has a significant effect on cell growth.Therefore,bubble problem ventilation and stirring speed were studied.Results showed that:(1)bubbles were commonly produced in cell culture which can be solved by adding antifoam,the optimal range of the concentration of antifoam is less than 0.05%;(2)ventilation and stirring speed affect cell growth distinctly,the optimum ventilation range of 0.05 L/min~0.1L/min and the optimum mixing speed is 200 rpm;(3)when CHO cells were cultured in stirring bioreactor with fed-culture process,the highest density is 7.4×106 cells/mL,below the level of shaker,the maximum fusion protein expression level is 120 mg/L;(4)based on the former study,adding 5 g/L YE to optimize the process of cell culture,the highest cell density was improved to 9.9×106 cells/mL and the final expression is 130 mg/L.Protein band was existing around 82 kDa by SDS-PAGE.Expressed protein was detected by both antibodies of HSA and IL-2 by western-blot.HSA/IL-2 has biological activity determined by CTLL-2/MTT method.