Periodic operation of continuous recombinant cultures improves antibiotic selection

While extrachromosomal insertion of plasmid DNA in suitable hosts allows for overproduction of recombinant proteins, sustained production of these over long duration is not possible due to reversion of recombinant cells to unproductive plasmid-free cells. Application of antibiotic selection pressure, the most effective measure to overcome this problem, at a constant rate is prohibitively expensive. Continuous recombinant culture operations where the feed composition is varied periodically (antibiotic being fed only when the culture is dominated by plasmid-free cells) are considered in this work. A mathematical model that accounts for limiting substrate, recombinant and plasmid-free cells, and antibiotic is employed. The two types of antibiotic action considered are: (i) death of plasmid-free cells and (ii) repression of growth of plasmid-free cells. Three types of steady states (periodic states) are admissible in steady state (forced periodic) operations, with only one type being the desired one. Specific forms of kinetics are considered as illustrations, with the cycle-average antibiotic feed concentration being considered the bifurcation parameter. Conditions for existence and multiplicity of various steady states are obtained analytically. Stability characteristics of periodic states are examined via Floquet stability theory. Two different periodic feeding policies are considered in numerical illustrations. One of these policies, the on-off addition of antibiotic and limiting substrate, results in retention of recombinant cells in continuous cultures over a broader region of operating conditions, increased productivity of recombinant cells, and increased cost-effectiveness. The effects of key operating parameters on the system performance are studied in detail.