Optimization of the culture temperature of recombinant CHO-DG44 cells expressing tumor necrosis factor receptor-Fc / 中国药学杂志

ABSTRACT

OBJECTIVE: To optimize the culture temperature of recombinant CHO-DG44 cells expressing tumor necrosis factor receptor-Fc fusion protein and to determine the bioactivity of expressed protein at the best culture temperature. METHODS: Recombinant CHO cells were batchly cultured at three different temperatures (37°C, 37°C shifting to 31°C and 31°C). Samples were daily tested for cell densities, viabilities, glucose concentration, lactic acid concentration and TNFR-Fc fusion protein concentration, then the best culture temperature was chosen to scale up the fermentation volume (10, 50, 200 mL, and 2 L). TNFR-Fc fusion protein was purified with Protein A affinity chromatography, determined for relative molecular mass, purity and neutralizing activity by SDS-PAGE, SE-HPLC and WST-8 separately. RESULTS: The culture condition of 37°C shifting to 31°C resulted in the maximum viable cell density, high viability and long culture time of the cells as well as high TNFR-Fc protein productivity, and this culture procedure could be successfully applied to the scale-up of recombinant CHO cells. The purified fusion protein, with a relative molecular mass about 150 000, reached the purity of more than 93% and neutralized the cytotoxic effect of TNFα. CONCLUSION: Compared to the traditional culture temperature of 37°C, the culture temperature of 37°C shifting to 31°C can obviously improve the TNFR-Fc fusion protein output by recombinant CHO-DG44 cells. And this technique can be applied to scale-up, which will lay a foundation of large scale industrial production.