Selection and stability for a recombinant CHO cell line expressing human GM-CSF in gene amplification

We investigated the relationship between the specific production rate and the concentration of the toxic drug methotrexate (MTX), inhibitory to nucleic acid synthesis in the medium, during the construction of recombinant CHO cells. The results of selection of MTX tolerant cells when the MTX concentration was increased under 10 different stepwise conditions showed that the number of days required to obtain cells resistant to 1000 nM MTX depended on the selection conditions. Tolerant cells were obtained most rapidly at an MTX concentration of 10nM. With increasing MTX concentration, the specific production rate increased, while the specific growth rate gradually decreased. In the case of a gradual increase in the MTX concentration, the specific production rate reached a higher level. We also investigated the stability of the specific production rate during cultivation over a long period with or without MTX. In long-term cultivation, the specific production rate decreased, while the specific growth rate remained at the same level. The specific production rate in a culture without MTX decreased more rapidly than that in a culture with MTX.