不同通气方式对抗破伤风毒素抗体工程细胞株生长及蛋白表达的影响

Effects of different ventilation methods on growth of recombinant CHO cell line and expression of tetanus toxin antibody

目的探讨生物反应器不同通气方式对抗破伤风毒素抗体的CHO工程细胞株生长、代谢及目的蛋白表达的影响。方法在3个3 L生物反应器中,分别选用微泡、大泡和膜供氧的通气方式,以相同的初始密度接种CHO种子细胞,设置相同的温度、pH、搅拌速度、溶氧(dissolved oxygen,DO)等关键培养参数,采用补料分批培养模式进行培养。每日取样,分别检测培养液的活细胞密度(viable cell density,VCD)、细胞活率、渗透压及葡萄糖、乳酸、NH4+和抗体蛋白浓度。筛选细胞生长较好及乳酸等代谢副产物累积较少且蛋白表达量高的一种通气方式。结果微泡通气方式下,CHO细胞生长缓慢,最高VCD为7.63×10^6个/mL,接种后细胞活率一直低于90%且下降较快,培养8 d,抗体蛋白浓度为76 mg/L。大泡通气方式下,最高VCD为12.45×10^6个/mL,培养1~6 d细胞活率均高于90%,培养第8天乳酸浓度开始下降,培养10 d,抗体蛋白浓度为534 mg/L。膜供氧通气方式下,最高VCD为13.54×10^6个/mL,培养1~8 d细胞活率均高于90%,培养第8天乳酸浓度开始下降,培养10 d,抗体蛋白浓度为755 mg/L。结论大泡及膜供氧通气方式下细胞生长和蛋白表达情况均较好,选取易进行工艺放大的大泡通气方式进行后续培养工艺的建立。

Objective To explore the effects of different ventilation methods on the growth,metabolism and protein expression of recombinant CHO cell line for tetanus toxin antibody.Methods Seed CHO cells were inoculated into three 3 L bioreactors at the same initial densities and cultured at the same temperatures,pH values,stirring speeds,dissolved oxygen(DO)and other metabolic parameters by fed-batch mode,while micro bubble,large bubble and membrane ventilations were adopted respectively.Samples were taken every day and determined for viable cell density(VCD),cell viability,osmotic pressure as well as the concentrations of glucose,lactate,ammonium ion and antibody protein.A ventilation method with well cell growth,less accumulation of metabolic byproducts such as lactic acid and high protein expression was screened to establish the subsequent culture process.Results Under the condition of microbubble ventilation,while the cells grew slowly,the VCD was 7.63×10^6 cells/mL at most,the cell viability was less than 90% and decreased rapidly since inoculation,the total culture time was 8 d,and the antibody protein concentration was 76 mg/L.Under the condition of large bubble ventilation,the highest VCD was 12.45×10^6 cells/mL,and the cell viability was more than 90% in 1~6 d,while the lactate concentration decreased on day 8 after culture.The total culture time was 10 d,while the antibody protein concentration was 534 mg/L.However,under the condition of membrane ventilation,the highest VCD was 13.54×10^6 cells/mL,and the cell viability was more than 90%in 1~8 d,while the lactate concentration decreased on day 8 after culture.The total culture time was 10 d,while the antibody protein concentration was 755 mg/L.Conclusion The cell growth and protein expression are satisfactory under conditions of large bubble and membrane ventilations.The mode of large bubble ventilation which is easy to be scaled up is selected for subsequent culture process.