Glukoselimitierung als Strategie zur Steigerung der Produktion von MUC1 und anderen rekombinanten Glykoproteinen mit CHO-Zellen

Many researchers are interested in glucose-limited perfusion culture in order to improve cellular metabolism towards more efficient substrate consumption and reduced production of cytotoxic metabolites (e.g. lactate). This work presents the development of a highly reproducible glucose-limited perfusion process using a recombinant CHO cell line producing human MUC-1 glycoprotein (currently under evaluation for immunotherapy of breast cancer) in a standard stirred vessel bioreactor with cell retention by spin filter using serum-free medium. The strategy is easy to perform and does not need any online glucose or other invasive measurement making the process highly stable against disturbances. Using this strategy, glucose consumption rate decreases, glucose concentration in the bioreactor drops and remains below detectable levels and the cells do not longer produce any lactate, while cell specific productivity and space time yield increase 5-fold and 4-fold respectively (250mg/L/day). Viable cell density remains very high at 1-2*E7 cells/mL. Process time can easily be extended to more than 1000h. The glycosylation pattern ofthe product is unaffected, as shown by detailed glycoanalysis. Based on preliminary results from substrate bilancing and 13C-lactate feeding experiments which prove a switch in metabolism towards an increased TCA-cycle activity and which indicate gluconeogenesis, a metabolic model has been set up and is discussed in detail. Finally this process strategy has been transferred successfully to a second recombinant CHO cell line secreting another mucin