表达长效重组人生长激素-Fc融合蛋白的CHO细胞培养工艺的优化及放大

Optimization and scale-up of culture procedure of CHO cells for expression of long-acting recombinant human growth hormone-Fc fusion protein

目的优化表达长效重组人生长激素-Fc(recombiant human growth hormone-Fc,rhGH-Fc)融合蛋白的CHO细胞培养工艺,并进行初步工艺放大。方法采用7 L生物反应器培养表达rhGH-Fc融合蛋白的CHO细胞,操作参数:转速200 r/min,温度37℃,pH 7.2±0.1,溶氧值(DO)40%。通过改变对数增长期的pH(7.2±0.1降至6.9±0.1)及平台期降温幅度(37℃降至30及33℃),检测培养过程中细胞密度、细胞活率、培养液渗透压、葡萄糖及乳酸水平、以蛋白表达量为指标,优化7 L生物反应器培养工艺。应用优化的最佳工艺进行14 L线性放大,通气量分别设置为0.5、0.1及0.03 SLPM,检测蛋白表达量。结果7 L生物反应器培养工艺最佳pH为6.9±0.1,最佳温度为33℃,该工艺下蛋白表达量高达1200 mg/L;14 L生物反应器培养工艺最佳通气量为0.03 SLPM,该工艺下蛋白表达量为880 mg/L。结论成功优化了表达rhGH-Fc融合蛋白的CHO细胞7 L生物反应器的培养工艺,并实现了14 L生物反应器的放大,本实验为后续rhGH-Fc融合蛋白的大规模生产奠定了基础。

Objective To optimize and preliminarily scale up the culture procedure of CHO cells for expression of longacting recombinant human growth hormone-Fc(rhGH-Fc)fusion protein.Methods The CHO cells for expression of rhGH-Fc were cultured in 7 L bioreactor at a rotation rate of 200 r/min,a temperature of 37℃,a pH value of 7.2±0.1 and a dissolved oxygen(DO)value of 40%.The culture procedure was optimized by decreasing the pH value at logarithmic growth phase for 7.2±0.1 to 6.9±0.1 and temperature at platform phase from 37 to 30 and 33℃,using the cell density,cell survival viability,the osmotic pressure,glucose and lactic acid contents of medium and the expression level of protein as indicators.The optimized procedure was subjected to linear scale-up in a 14 L bioreactor,and the above-mentioned indicators were tested,serving the ventilation as 0.5,0.1 and 0.03 SLPM respectively.Results The optimal pH value and temperature for culture in 7 L bioreactor were 6.9±0.1 and 33℃respectively,and the protein expression level in the cells cultured by the procedure was 1200 mg/L.However,the optimal ventilation for culture in 14 L bioreactor was0.03 SLPM,under which the protein expression level was 880 mg/L.Conclusion The culture procedure of CHO cells for expression of rh GH-Fc in 7 L bioreactor was successfully optimized and scaled-up to 14 L bioreactor,which laid a foundation of large-scale production of rhGH-Fc fusion protein.