Metabolic and bioprocess engineering of production cell lines for recombinant protein production

Dissolved carbon dioxide (pCO₂) has been identified as an important process parameter affecting cell growth, productivity and product quality (e. g. glycosylation) of recombinant proteins when exceeding critical levels, occurring especially in industrial large-scale cell culture processes due to the increased hydrostatic pressure. As CO₂ can easily pass the cellular membrane and thereby influence intracellular pH (pH₊i), important cellular processes (e. g. cell cycle regulation, enzymes of TCA cycle) are directly influenced by pCO₂ and dependent bicarbonate concentration. Consequently, process control strategies attend to keep pCO₂ within physiological range. In a metabolic engineering approach an antibody producing CHO cell line stably expressing human carbonic anhydrase (hCAII), the enzyme that catalyzes the equilibrium of CO₂ in aqueous solutions, was generated and used to characterize CO₂ effects in simulated CO₂ acid load and high CO₂ levels as they occur in large scale mammalian cell culture. The cell line expressing active hCAII showed more rapid initial re-alkalinization of cytoplasm after induced CO₂ acid load. Results also suggest that cellular pH₊i fine tuning was performed by the Cl⁻/HCO₃⁻ exchanger (AE) and Na--dependent Cl⁻/HCO₃⁻ exchanger (NCBE) instead of the Na⁺/H⁺ exchanger (NHE1).