Development of a Metabolically Optimized Fermentation Process Based on Glucose-Limited CHO Perfusion Culture

Abstract

Many researchers are interested in glucose-limited perfusion culture in order to improve cellular metabolism towards more efficient substrate consumprtion and reduced production of cytotoxic metabolites (e.g. lactate). We developed 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 bioreator with cell retention by spin filter using serum-free culture 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 5fold and 4fold respectivly (250 mg/L/day). Cell density remains very high at 1-2*10(7) cells/mL. Process time can easily be extended to more than 1000 hours. The glycosylation of the product is unaffected as shown by glycoanalysis. Preliminary results from metabolite quantification by LC-MS and from C-13-lactate feeding experiments proove a switch in metabolism towards an increases TCA-cycle activity and indicate that gluconeogenesis takes place. Confirmation experiments are underway. This strategy has already been transferred successfully to a second recombinant CHO cell line secreting another human Mucin. Other CHO cell lines producing an enzyme and a humanized antibody and a murine hybridoma cell line are under investigation