Development of the CEMAX system for cell line development based on site-specific integration of expression cassettes
Bielefelder Schriften zur Zellkulturtechnik, Bd. 3
169 Seiten, Erscheinungsjahr: 2012
Preis: 39.00 EUR
CEMAX, site-directed integration, gene targeting, homing endonuclease, I-SceI
The random nature of transgene integration requires an intensive clone screening process for the establishment of a production cell employing high yield and quality of biopharmaceutical, recombinant proteins. This thesis was focused on the development of an expression system that makes use of site-specific integration of the product gene in CHO-K1 cells combined with serum-free cultivation. Common barriers in protein production from stable cell lines should thus be overcome.
The goal was achieved by the development of the CEMAX system that allows site-specific integration of expression cassettes at highly active sites of transcription. Producer cells were generated using a DNA double-strand break induced homologous recombination mechanism based on recombinant CEMAX host cells. This enabled the rapid and reproducible establishment of stable producer cell lines with known characteristics. Productivities of up to 10 pg per cell and day could be achieved even for the production of highly glycosylated proteins. In a separate aspect of this work CHO-K1 host cells growing in suspension in serum-free medium were improved to reach viable cell densities of 107 cells per ml an allow cloning efficiencies up to 71 % in serum-free medium.