| Popis: |
It is crucial to have a high producing stable cell line to be able to produce recombinant proteins from mammalian cells in large scale for clinical trials. The aim of this study has been to evaluate steps in a process platform for development of high producing sable cell lines with respect to time and titer. Gene amplification through addition of methotrexate (MTX) based on Invitogen’s OptiCHO system have been studied. In this study 17 strategies of MTX amplification have been evaluated on three antibody expressing Chinese hamster ovary (CHO) cell pools. Protein production has been monitored in supernatants from 14-days-batch in shake flask after each MTX level. This study has shown that it is possible to increase protein production through amplification from 1 mg/L to 190 mg/L. The amplification strategy which can be recommended with respect to time and titer is stepwise increasing MTX concentration from 500 nM to 1.000 nM. This amplification process took 47 days to proceed and gave titers of IgG up to 150 mg/L. To get an indication if the most preferable clones for large scale production can be selected in an earlier stage a small fed-batch study was preformed. An initial addition of Efficient Feed B to batch could be an option to increase the possibility to select the most productive clone for scale up. The development process for high producing stable cell lines can most likely be shortened while protein production can be increased. Further studies remain to prove this statement. |
