表达长效生长激素的CHO细胞培养工艺的优化及放大

Optimization and scale-up of culture process of CHO cells for expression of long-acting recombinant human growth hormone

目的在NewBrunSwick31014 L生物反应器中培养表达重组人长效生长激素(recombinant human growth hormone Fc fusion protein,rhGH-Fc)的CHO细胞,优化工艺参数以提高表达量,并进行NewBrunSwick31014 L到New-BrunSwick51040 L生物反应器的工艺放大。方法在原有14 L培养工艺基础上,调节转速、通气速率等参数,并对参数调整时期进行工艺优化。通过对渗透压、乳酸、铵离子、钾离子等代谢过程产物的检测来确保代谢正常,利用HPLC法测定表达量。再根据经验公式算出可线性放大的体积依赖型参数,保持原有培养方案及补料策略不变的情况下,结合不同放大经验方法确定操作窗口,从而进行工艺放大。结果14 L生物反应器优化工艺乳酸累积量低于20 mmol/L、最终铵离子累积量为8 mmol/L、钾离子累积量为14 mmol/L、渗透压为425 osmol/kg,降温后比耗糖速率为0.12 g/(10^(9) cells·d),表达量为0.9 g/L。经检测,放大至40 L生物反应器,整个培养过程耗糖曲线及代谢产物均与14 L生物反应器中一致,表达量为1.0 g/L。结论成功优化了rhGH-Fc工程细胞大规模培养的工艺,并进行了规模放大,为rhGH-Fc的后续开发奠定了基础。

Objective To culture CHO cells for expression of long-acting growth hormone(rhGH-Fc protein)in New-BrunSwick31014 L bioreactor,optimize the process parameters to improve expression level and realize the scale-up of process from NewBrunSwick31014 L to NewBrunSwick31040 L bioreactor.Methods The original culture process was optimized by adjusting the parameters such as stirring blade speed and ventilation rate as well as the time duration for adjusting.The metabolic process products such as osmotic pressure,lactate,ammonium ion and potassium ion were determined to ensure the normal metabolism.The expression level was determined by HPLC.The volume-dependent parameters which can be linearly enlarged was calculated according to the empirical formula and,on the basis of unchanged original culture scheme and feeding strategy,the operation window was determined by combining different empirical methods for scale-up.Results In the optimized process in 14 L bioreactor,the lactate accumulation was less than 20 mmol/L,while the final ammonium ion accumulation was 8 mmol/L,the final potassium ion accumulation was 14 mmol/L,the final osmotic pressure was 425 osmol/kg,the specific rate of glucose consumption after cooling was 0.12 g/(10^(9) cells·d),and the expression level was 0.9 g/L.After scale-up to 40 L bioreactor,the glucose consumption curve and metabolites of in the entire cultivation process were consistent with those in 14 L bioreactor,and the expression level was 1.0 g/L.Conclusion The process for culture of rhGH-Fc engineered cells were successfully optimized and scaled-up,which laid a foundation of further development of rhGH-Fc.