用无血清培养基在填充床生物反应器生产 rHuEPO

Production of rHuEPO with a Serum free Medium in Packed Bed Bioreactor

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（ＳＦＭｐ）生产ｒＨｕＥＰＯ。ＳＦＭｐ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１２～２８４ｍｇ／Ｌ之间，反应器的产率达到７１０ｍｇ／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３０×１０７／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３５ｇ／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ＳＦＭｐ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.