Construction Of The Stable CHO Cell Line Expressing HSA-IFNα2b And Optimization Of Serum Free Medium

Interferon alpha 2b(IFNα2b) is a multifunctional cytokine for the treatment of viral diseases and tumor diseases. However, short half-life of IFNα2b in vivo limits the clinical applications. Our group previously designed the drug development platform of prolonged half-life based on human serum albumin(HSA) fusion protein. In this platform, HSA was fused to the N terminus of IFNα2b and fusion protein(HSA-IFNα2b) was expressed in Pichia pastoris. The obtained HSA-IFNα2b had great biological activity; however, some disadvantages were also existed, including instability, easy degradation and inhomogeneous product. Currently, Chinese hamster ovary(CHO) expression system was commonly used to express recombinant therapeutic proteins, which had advantages of low immunogenicity, good compatibility and high activity. In this study, fusion protein(HSA-IFNα2b) was expressed in CHO cells and the culture medium and conditions were optimized to enhance its production.Fusion protein encoding gene was inserted to plasmid and the obtained expression plasmid pMH3/HSA-IFNa2 b was transfected to CHO cells by electroporation. After selecting by G418 pressure and expression level, fusion protein expressing cell line, CHO/pMH3/HSA-IFNa2 b, was obtained and identified by western blot(WB). Results of WB showed that expressed fusion protein was detected by both antibodies of IFNα2b and HSA. The expression level of fusion protein was analyzed in suspension culture and the highest production was 31 mg/m L. After comparing CHO cell lines of different generations, fusion expression and cell growth had no obviously difference and thus a stable HSA-IFNa2 b expression CHO cell lines was obtained in this study.To optimize the culture medium, HSA-IFNa2 b expression and viable cell density were analyzed under 17 commercial serum-free medium(SFM) in suspension culture. Depending on results, BM and F4 were chosen as culture and feed medium, respectively. Meanwhile, some chemicals, including sodium butyrate, linoleic acid, ethanol amine and Sura, were added to culture medium for optimizing HSA-IFNa2 b production. Results showed that culture medium containing sodium butyrate was suitable for HSA-IFNa2 b expression. In fed-batch culture, fed medium F4 supplemented to culture broth of CHO cells as cell density exceeding 8×106 cells/mL and the highest cell density achieved 10×106 cells/m L. Meanwhile, culture time was extending to 10 days and thus HSA-IFNa2 b expression was enhanced to 61.5 mg / L. Moreover, HSA-IFNa2 b expression was further improved to 81 mg/L after adding 2 mM sodium butyrate. After analyzing cell cycle and transcription level, mRNA of HSA-IFNa2 b enhanced 2.6 times and the percentage of cells in G1 phase was increased from 41 % to 62.9 % without influencing cell growth.Finally, CHO/pMH3/HSA-IFNa2 b cells were respectively cultured in 3 L shake flasks and 5 L AP-20 disposable bioreactor for testing aeration and agitation. Cell growth and HSA-IFNa2 b was relatively consistent in two incubators and the highest HSA-IFNa2 b expression was 137 mg/L. Fusion protein was purified by blue affinity dye chromatography and hydrophobic chromatography, and the purity and total protein yield were 96.8 % and 22.3 %, respectively. Moreover, the specific activity of HSA-IFNa2 b expressed by CHO cells was 4.16 × 106 IU/mg and similar to purified fusion protein expressed by P. pastoris.