| 摘要 |
<p>1. A method for producing a heterologous secreted protein from CHO cells grown on microcarriers, comprising the steps of: inoculating a cell bioreactor, containing microcarriers as a solid support for adherent cell growth and serum-free cell culture medium supplemented with trace elements, with an inoculum of CHO host cells transformed to express a heterologous secreted protein, wherein the inoculum was prepared by growing the transformed CHO host cells in serum-containing cell culture medium; culturing the inoculated CHO host cells in adherent cell culture without perfusion of cell culture medium for a period in the range of about one to three days before initiating feed; introducing improvements meaning that initiating feed of serum-free cell culture medium supplemented with insulin, ferric citrate (III), selenium and trace elements to the cell bioreactor via continuous perfusion; increasing the feed flow rate of serum-free cell culture medium supplemented with trace elements in stages over about a six to ten day period until a final feed flow rate in a range of about 1 to 3.5volume/volume/day is reached to produce and secrete a heterologous protein from the CHO host cells into the serum-free cell culture medium; and culturing the CHO host cells in adherent cell culture at the final feed flow rate. 2. The method according to claim 1, wherein the period is about 2 days. 3. The method according to claim 1, wherein the feed initiating step initiates a feed at a flow rate in the range of about 0.1 to 0.4 volume/volume/day of serum-free cell culture medium supplemented with insulin, ferric citrate (III), selenium and trace elements. 4. The method according to claim 3, wherein the feed is initiated at a feed flow rate in the range of about 0.2 to 0.3 volume/volume/day. 5. The method according to claim 3, wherein the feed is initiated at a feed flow rate of about 0.2 volume/volume/day. 6. The method according to claim 1, wherein the feed flow rate is increased in stages over about a seven to nine day period. 7. The method according to claim 1, wherein the feed flow rate is increased in stages over about an eight day period. 8. The method according to claim 1, wherein the final feed flow rate is in the range of about 1.5 to 2.5 volume/volume/day. 9. The method according to claim 1, wherein the final feed flow rate is about 2 volume/volume/day. 10. The method according to claim 1, further comprising a step of recovering the heterologous protein from the serum-free cell culture medium. 11. The method according to claim 1, wherein the heterologous protein is soluble interferon alpha/beta receptor (sIFNAR2). 12. The method according to claim 1, wherein the microcarriers are Cytopore 2. 13. The method according to claim 1, wherein the serum-free cell culture medium is DMEM/F12.</p> |
