抗破伤风毒素单克隆抗体细胞培养基的筛选与优化

Screening and optimization of cell culture medium for monoclonal antibody against tetanus toxin

目的对抗破伤风毒素单克隆抗体细胞培养基进行筛选与优化。方法首先通过评估多种商业无血清培养基中抗破伤风毒素单克隆抗体工程细胞株CHO-K1-G2的生长及代谢参数,筛选出一种进行组分优化的基础培养基;再利用试验设计(Design of Experiments,DOE)的方法从23种培养基添加物中筛选出能够显著促进细胞蛋白表达量的物质;最后用筛选出的基础培养基进行扩大培养,亲和纯化后获得目的蛋白,并对蛋白质性质进行初步分析。结果最终确定2号培养基作为后续优化的基础培养基;6种物质可作为培养基添加物,按照影响作用大小依次为次黄嘌呤、牛磺酸、乙醇胺、卵磷脂、柠檬酸铁和天冬氨酸。细胞表达量最优拟合值为238.22 mg/L。扩大培养后所得抗体效价为500 IU/mg,亲和常数为1.33×10^(-9)/M。结论建立了一种可促进CHO-K1-G2细胞株生长及抗体表达的"个性化培养基",为最终建立经济高效的抗体药物生产工艺奠定了基础。

Objective To screen and optimize the cell culture medium for monoclonal antibody(McAb)against tetanus toxin.Methods A basic medium with optimal components was screened by evaluation of growth and metabolism parameters of engineered CHO-K1-G2 cell strain for McAb against tetanus toxin in various serum-free commercial media.The additives significantly promoting cell protein expression were screened from 23 kinds of additives by Design of Experiments(DOE).The screened basic medium was used for expand culture,and the target protein was purified by affinity chromatography and analyzed for properties.Results No.2 medium was defined as the basic medium for further optimization.In the turn of effect,the six screened additives were hypoxanthine,taurine,ethanolamine,lecithin,ferric citrate and aspartic acid.The optimal fitting value of cell protein expression level was 238.22 mg/L.The antibody titer obtained by expand culture was 500 IU/mg,while the affinity constant was 1.33×10-9/M.Conclusion A“personalized culture medium”that can promote the growth of CHO-K1-G2 cell strain and antibody expression was established,which laid a foundation of the establishment of an economical and efficient antibody drug production process.