30L生物反应器培养重组CHO细胞生产重组人组织型纤溶酶原激活剂  

Production of Recombinant Human Tissue Type Plasminogen Activator by Culture of Recombinant CHO Cells in 30 L Bioreactor

作　　者：梅建国[1] 沈志强[1] 庄金秋[1] 

机构地区：[1]山东省滨州畜牧兽医研究院,山东滨州256600

出　　处：《中国生物制品学杂志》2010年第4期428-430,共3页Chinese Journal of Biologicals

摘　　要：目的应用30L填充床生物反应器培养重组CHO细胞生产重组人组织型纤溶酶原激活剂(rht-PA)。方法将表达rht-PA的CHO细胞株用含10%胎牛血清的IMDM复苏并放大培养,接种至30L生物反应器中,并采用BiocommandPlus软件系统实时监控。先用含血清的培养基生长培养,再更换为无血清培养基进行表达培养。在整个培养过程中,采用灌流培养方式,每日采样测定培养上清中葡萄糖浓度,隔日测定rht-PA的表达水平及生物学活性。采用Lysine-Sepharose4B和Zn2+-Sepharose4B两步亲和层析法纯化rht-PA,并检测纯化产物的比活、产率及纯度。结果整个培养过程持续51d,包括生长培养6d,表达培养45d,平均日灌流量为46.7L,最高达60L,共收获表达培养液约2100L;rht-PA的平均表达水平为15.15mg/L,最高可达19.25mg/L,生物学活性平均约为8000IU/ml;表达培养至第13天时,葡萄糖消耗量达最高水平(15.97g/L·d);纯化的rht-PA比活达6×105IU/mg,产率为63%,纯度达99%以上。结论应用30L填充床生物反应器可实现重组CHO细胞的长时间连续培养及产物rht-PA的高效表达。Objective To produce recombinant human tissue type plasminogen activator（rht-PA）by culture of recombinant CHO cells in 30 L packed bed bioreactor. Methods The CHO cell strain for expression of rht-PA was resuscitated with IMDM containing 10% fetal bovine serum（FBS）, then amplified and inoculated to a 30 L bioreactor under real-time monitoring by Biocommand Plus software. Serum-containing medium was used for culture of CHO cells at first then was changed into serum-free one. Perfusion culture mode was adopted in the whole course of culture, during which the glucose concentration in culture supernatant was determined every day, and expression level and bioactivity of rht-PA every other day. The expressed rht-PA was purified by LysineSepharose 4B and Zn2＋-Sepharose 4B affinity chromatography and determined for specific activity, yield and purity. Results The whole culture course lasted for 51 d, consisting of serum-containing culture for 6 d for cell growth and serum-free culture for 45 d for rht-PA expression. The perfusion rate was 46. 7 L / d in average and 60 L / d at the most. About 2 100 L of culture supernatant was harvested. The expression level of rht-PA was 15. 15 mg / L in average and 19. 25 mg / L at the most. The mean bioactivity of rht-PA was about 8 000 IU / ml. The glucose consumed reached a peak value of 15. 97 g / L·d on day 13 after serum-free culture for expression. The specific activity, yield and purity of purified rht-PA were 6 × 105 IU / mg, 63% and more than 99% respectively. Conclusion A 30 L packed bed bioreactor was suitable for long-term continuous culture of recombinant CHO cells and high expression of rht-PA.

关 键 词：人组织型纤溶酶原激活剂 重组CHO细胞 生物反应器 灌流培养 

分 类 号：Q813.11[生物学—生物工程]