Limitations to the amplification and stability of human tissue-type plasminogen activator expression by Chinese hamster ovary cells

Chinese hamster ovary cell production of recombinant tissue-type plasminoge n activator (t-PA) was increased by amplification of cotransfected dihydrof olate reductase cDNA using stepwise adaptation to increasing methotrexate ( MTX) concentrations. The highest producing clones were isolated at 5 mu M M TX and yielded 26,000 U/10(6) cells/day t-PA (43 mu g/10(6) cells/day). Abo ve 25 mu M MTX, cell specific t-PA production rates became increasingly var iable and the cDNA copynumbers decreased. No apparent correlation between t he cell specific t-PA production rate and the growth rate was observed upon subcloning of the amplified cells. When MTX selection was removed, the t-P A production rate decreased up to tenfold within 40 days; this was accompan ied by an up to 60% drop in cDNA copynumber. Subclones isolated after 108 d ays of culture in the absence of MTX were, on average, sixfold more stable than their parental cells. In culture without MTX, the maximum stable t-PA production rate obtained (over 250 days) was 7000 +/- 750 U/10(6) cells/day (similar to 2 mu g/10(6) cells/day), approximately threefold lower than th e maximum unstable levels of production reached under selective pressure. T aken together, these results define a wide range of the highest t-PA expres sion rates obtained under MTX selection, for which stable expression withou t selection has not been reported. (C) 2000 John Wiley & Sons, Inc.