CHO-DG44 Cell Line Development by FLP-Targeting – High Level Glycoprotein Expression with Significantly Decreased Time Lines

Complex therapeutic glycoproteins are typically produced in mammalian cells. The development of pharmaceutical cell lines is an unpredictable and time-consuming effort, requiring the identification of rare clones which combine integration of the transgenes into highly active genomic loci with superior folding, processing and secretion capabilities. Further, this intensive investment has to be repeated with every new product candidate. To overcome some of these obstacles, we designed an enhanced gene targeting system for cell line generation based on Flp recombinase mediated cassette exchange (RMCE). This strategy comprises a primary recombination event to ensure locus accessibility for further Flp targeting, elimination of heterologous tandem repeats, as well as isolation of favourable genomic loci. Resulting clones were screened for high level expression of the introduced serin protease inhibitor human alpha1-antitrypsin (hAAT). Complete replacement was confirmed by absence of the primary GFP reporter and correct resistance phenotype. Further RMCE targeting of selected clones by different transgenes resulted in high level expression of the newly introduced proteins. Unexpectedly, despite complete replacement in primary targeting hAAT expression persisted. Surprisingly, in FISH analysis chosen hAAT starter clones displayed multiple chromosomal integration sites randomly distributed throughout the genome. This indicates simultaneous recombination at multiple sites during primary targeting, which was not enforced during further RMCE application. Co-expression of the protease inhibitor does no reduce the value of the targeting system: Titers can match those obtained after high effort advanced pharmaceutical cell line development at much reduced development time.