摘要
在填充床生物反应器用含5%FBS的DMEM:F12培养基培养产重组人促红细胞生成素(rHuEPO)的细胞C28~10d后,使用自制的无血清生产培养基(SFMp)生产rHuEPO。SFMp培养基既能维持细胞生长,又能生产EPO,也便于纯化分离rHuEPO。使用填充床生物反应器培养细胞,能维持培养20~25d,rHuEPO表达水平达12~284mg/L之间,反应器的产率达到710mg/L/d,比滚瓶的产率增加12~14倍。葡萄糖最高消耗量达到21g/L/d,细胞培养密度最高达到30×107/ml以上,每次可收无血清培养上清80~87L。由于细胞被固定在聚酯片上,培养上清中脱落细胞很少。观察了反应器的乳酸和氨的含量,其结果表明乳酸和氨含量分别低于35g/L和5mmol/L,不影响产物的表达。经过多批培养和生长rHuEPO的结果表明,自行配制的SFMp培养基在该反应器能有效地维持细胞生长和生产rHuEPO。
Recombinant CHO(C 2) cells producing human erythropoietin (rHuEPO) were cultured with 5%FBS DMEM:F12 medium for 8~10 days in packed bed bioreactor,then rHuEPO was produced with self made serum free medium (SFM p).SFM p medium can support the growth of C 2 cells and the production of rHuEPO.Moreover,rHuEPO was easily separated from the culture supernatant.Cell culture in the packed bed basket system using SFM p was maintained in a stable condition for 20~25 days.The expression level of rHuEPO was 12~28.4mg/L.The bioreactor productivity was 71.0mg/L·d and increased 12~14 fold over that of the roller bottle.Glucose consumption rate was 21g/L·d.At the end of 30 days of continuous perfusion culture,a final cell density of over 3.0×10 7/ml of culture volume was achieved.Since the cells were entrapped in the polyester disks,the culture supernatant contained a few of detachment cells.Variations in lactate and ammonia production between the reactor and roller bottle were observed,which results showed that lactate and ammonia production of bioreactor were 3.5g/L and 5mmol/L respectively,and didn't affect the expression of interest protein.This experiment demonstrates that SFM p is suitable to the growth and rHuEPO production of recombinant C 2 in the packed bed bioreactor.
出处
《生物工程学报》
CAS
CSCD
北大核心
1997年第4期375-379,共5页
Chinese Journal of Biotechnology