The Experessio, Purfication And Biocativy Of Recombination Protein TRAIL-Fc In CHO Cells

TRAIL (tumor necrosis factor related apoptosis inducing ligand) was first cloned and identified to induce the tumor cells’apoptosis as a new member of TNF family by Wiley in 1995. The feature of TRAIL, selectively inducing tumor cells’apoptosis while having less cytotoxicity to normal cells, made it to be a hot-spot in cancer therapy research and potential for a new kind of anti-tumor drug.Object:The recombination TRAIL-Fc protein was obtained by high density fermentation of CHO cells,and the purification pilot process was established. The physicochemical property and bioactivity of the protein was investigated to provide the experimental information for the clinical research.Methods:(1) CHO-TFc cell line was adapted to serum-free medium by gradually-reducing method or directly reducing serum concentration from 10% to zero. Optimization condition of serum- free medium was performed by the additive for improving the cell density and viability.(2) The characteristics of CHO-TFC culture temperature, pH, DO(dissolved oxygen) and metabolism were investigated in 5L bioreactor fed-batch cultures to establish fermentation process. The recombinant TRAIL-Fc proteins were purified by Protein-A affinity chromatography and CM ion-exchange chromatography. The physicochemical property of the protein was determined.(3) The vitro activity experiments: The growth inhibitory of 7 kinds of tumor cell lines by TRAIL-Fc was detected by MTT method. The inhibitory effect in vivo was measured on the H22 tumor-bearing mouse model.Results:(1) CHO-TFc cell line was adapted to serum-free medium by gradually-reducing method.(2) 20 ng/ml of IGF-1 was added to the serum-free medium for optimization(3) In 5L bioreactor, the glucose concentration and cell density should be controlled for feeding strategy 2×10~6 cells/ml was inoculated to bioreactor, and the concentration of glucose was maintained around 2 g/L. When the density of the cells was 6-8×10~6cells/ml, the temperature was controlled to 32℃, and the concentration of glucose was maintained around 0.5g/L. The concentration of the protein in supernatant was 80-100mg/ml。(4) The stable purification procedure was established and the degree of purity was up to 90%. Mass spectrometry and western blot showed that the recombinant protein had expected molecular weight and immunogenicity. The experiment in vitro and in vivo showed that TRAIL-Fc had inhibitory effect on tumor cells and prolonged half-life in vivo.Conclusion:The engineering CHO-TFc cells was successfully adapted to serum-free medium, established a 5L bioreactor fed-batch fermentation technology and a high purity purification process. The activity of the target protein was estimated in vivo and vitro. The potential antitumor-therapy value of TRAIL-Fc protein was verified in this study.