Predictive control and optimization of bioprocesses for recombinant T-PA protein production by mammalian cells (2009)

Genetically engineered mammalian cells produce a large array of recombinant proteins for research, diagnostic and therapeutic applications. The relatively low cellular production rates in mammalian cells require intensification of the production methods to raise product concentrations and volumetric productivity. Recombinant human tissue plasminogen activator (t-PA) produced in Chinese Hamster Ovary (CHO) cells served as the basis for this investigation. Multiple model adaptive protocols were applied to control the extracellular environment with the goal of fed-batch and perfusion process optimization of protein titers and productivity. Fed-batch and perfusion bioreactors in various forms are widely used to produce recombinant proteins and monoclonal antibodies for therapeutic and diagnostic use. Better control of the cellular environment can lead to higher volumetric productivity, ensure product consistency and optimize medium utilization. The objective was to manipulate and control substrate concentrations in fed-batch and perfusion bioprocesses using predictive modeling and control. The goal of the predictive controller was to minimize future deviations from the set point concentration, by structuring the controller output. The appropriate structure for the future manipulated variable was specified using the selected model of uptake rate estimates. When there was a deviation from the set point value, the flow rates were adjusted to drive the process close to the set point value in a defined first order manner. The shape of the first order process response depended on the magnitude of the deviation from the set point value. With daily sampling, a feed rate profile (8 flow rates per day) was specified to control the bioprocess. The predictive control protocols have demonstrated glucose variation of less than 0.4 mM in transient conditions, and less than 0.2 mM in pseudo-steady-state conditions. The non-linear controller allows for rapid changes in set point concentrations (6 to 9 h) or a reference trajectory to be followed. Set point changes and reference trajectories were simulated and tested with real process data. Modeling error and measurement bias was simulated to have the greatest potential effect during exponential growth. With good model estimation of the process, predictive control was able to maintain the process at the set point with a level of variability approaching that of the glucose assay. Fed-batch operation for the production of t-PA using Chinese Hamster Ovary (CHO) cells was optimized using serial and parallel experimentation. The isotonic concentrate efficacy was improved to obtain 2- to 2.5-fold increases in integrated viable cell days versus batch. With a low glucose inoculum train, the viability index was increased up to 4.5-fold. Hydrolysates were substituted for the amino acid portion of the concentrate with no significant change in fed-batch results. The concentrate addition rate was based on a constant 4 pmol/cell-day glucose uptake rate that maintained relatively constant glucose concentrations (approximately 3 mM). Increased viable cell indices did not lead to concomitant increases in t-PA concentrations compared to batch. The fed-batch concentrate was tested in hybridoma culture, where a four-fold increase in viable cell index yielded a four-fold increase in antibody concentration. Instead, there appeared to be an extracellular t-PA concentration maximum at 30-35 mg/L. The half-life of t-PA decreased from 42 to 14 days with decreasing cell viability (&gt; 90% to ~ 70%), but this was not sufficient to explain the apparent t-PA threshold. Analysis of both the total and t-PA mRNA levels in dose response experiments revealed no response to extracellular t-PA concentrations. Instead, increasing intracellular t-PA levels revealed a secretory pathway limitation. A new reactor configuration used an acoustic filter to retain the cells in the reactor and an ultrafiltration module to strip the t-PA from the clarified medium and returned the permeate back to the reactor. By adding this harvesting step, the t-PA fed-batch production was increased over 2-fold, up to a yield of80mg/L. Perfusion cultures of CHO cells producing t-PA were performed using an acoustic filter to retain cells in the bioreactor as spent medium was removed. A robust off-line glucose analysis and predictive control protocol was developed that maintained the process within approximately 0.5 mM of the glucose set point without the need for a more fallible on-line sensor. Earlier onset of perfusion with a ramping glucose set point (1 to 2 mM/day) resulted in improved growth and consistency in the perfusion culture start-up. A medium formulation with elevated levels of glutamine resulted in significant increases in glutamine consumption and ammonium production, along with reductions in consumption rates of glucose and several amino acids. In contrast, elevated levels of glucose had no significant impact on the cellular metabolism. Amino acid analysis of the initial batch and early perfusion culture resulted in an improved medium formulation which resulted in increased medium residence times and increased t-PA concentrations. Glucose depletion was used as an indicator of the extent of overall medium utilization, to map acceptable ranges of operation and the edge of failure. Peak t-PA concentrations of over 90 mg/L were obtained by controlling at a glucose depletion of approximately 25 mM, but were not sustainable for more than 3 days. A consistent t-PA concentration of 40 mg/L was obtained at a glucose depletion of 22.5 mM. The variability in the t-PA concentrations increased gradually with increasing glucose depletion up to approximately 23 mM, then increased 3-fold between a glucose depletion of 23 and 25 mM.