Boundary of oxidative and overflow metabolism (boom) controller for CHO cell feed control

There is limited literature for CHO cell cultures with low batch glucose concentrations (Gowtham et al. 2017; Lu et al. 2005; Wong et al. 2005). Work like Xu et al. (2016) and Berry et al. (2016) have shown positive results for controlled fed-batch cultures at low glucose concentrations following standard high glucose (5-6 g/L) batch cultures. However, the Xu et al. (2016) and Berry et al. (2016) approaches still accumulate lactate. Controlling glucose earlier could potentially avoid lactate accumulation and lead to even greater improvements in culture outcomes. The objective of this project was to develop an advanced feed controller for CHO cell cultures that maximizes cell growth by maintaining the culture in a state of maximal oxidative metabolism while minimizing overflow metabolism. The Boundary of Oxidative and Overflow Metabolism (BOOM) controller periodically manipulates the feed rate while monitoring online signals to gauge the remaining oxidative “space”, in order to decide whether feed can be increased while remaining in oxidative metabolism. The Oxygen Uptake Rate (OUR) is the primary signal of interest, since it plateaus when a culture shifts from oxidative to overflow metabolism, encoding vital information about metabolic state. This project’s approach is different from past work in that the batch glucose concentrations is much lower (on the order of 1 g/L), the glucose and/or glutamine feeding begins very early in the process, and glucose feed is triggered/controlled by the off-gas sensing of the metabolic state instead of a targeted glucose concentration. During early runs several chemistry effects were observed directly due to the bolus feed additions interfering with the media-dissolved gas equilibrium. For example, a bolus feed that only contained 5 mM bicarbonate, resulted in an observed short sharp decrease in CO2 off-gas as the feed absorbed CO2 from the 5% CO2 sparge gas. Continuous feeding was introduced in subsequent runs as a means to mitigate disrupting the media-dissolved gas-equilibrium and disturbing the off-gas sensing. In order to have effective continuous feeding, the feed pump used a pulse width modulation (PWM) with a 10-minute period to allow extremely low effective feed rates required for the 1-L vessel. Two runs were used to demonstrate that the PWM feed pump could provide these very low pump feed rates for the 1-L vessel containing as little as 500 mL media. Initial glucose concentrations between 0.6 to 2.0 g/L were used (compared to 8 g/L glucose in the standard media formulation). Feedings have started between 6- and 20-hour post-inoculation. Distinct qualitative and quantitative differences have been observed in the corresponding oxygen uptake rate (OTR) responses due to the feeding spikes, suggesting that metabolic state can be detected. The development of the state estimator to control glucose feeding will be presented

Intermittent Estimation of Volumetric Mass Transfer Coefficient Using Offgas Sensor Data for Continuous OUR Estimation for CHO Cell Culture

For the last several years drugs based on monoclonal antibodies have been manufactured using Chinese Hamster Ovaries (CHO) cells by the bio-pharmaceutical industries to treat cancer and other autoimmune diseases. Several control strategies are used to increase the productivity and efficiency in bio-pharmaceutical manufacturing. Cell growth can be controlled by adjusting the feed rate based on oxygen uptake rate of the cells in the bioreactor. Determining the volumetric mass transfer coefficient and oxygen saturation concentration is vital in correctly estimating oxygen uptake rate. Thus, a robust and efficient method to determine volumetric mass transfer coefficient and oxygen saturation concentration, which uses common industrial sensors, is desired. In this thesis, a new method to determine volumetric mass transfer coefficient is proposed and implemented on simulated and laboratory experiments. Using this method, volumetric mass transfer coefficient can be calculated independently of oxygen saturation concentration. The fitting parameters required to estimate volumetric mass transfer coefficients are estimated using only the estimated oxygen mole ratio of input gas, the measured oxygen mole ratio of the off-gas and the dissolved oxygen concentration in the bioreactor. A modified version of Savitzky-Golay filtering is used to determine the change in oxygen concentration in the bioreactor liquid. Another algorithm is used to reduce the variations between estimated OUR ( OUR ) and OURlinfit signal to estimate the oxygen saturation concentration in the liquid. Finally, both these signals are used to estimate final OUR signal. The performance of these algorithms were validated by simulated experiments and lab experiments. A Simulink model was used to simulate bioreactor experiments and the values obtained after implementing the algorithm on simulated experiment data were compared with known values from the Simulink model to verify algorithm accuracy. High accuracy was obtained in all the simulated experiments even in presence of noise. The variation and noise in estimated OUR was significantly reduced when these algorithms were employed. The algorithm could also be used in cases when there were sudden gas mix changes by estimating OUR using parameters estimated just prior to the gas mix change. The algorithm was applied to laboratory experiments and it showed consistent results over short periods of time. Since the oxygen saturation concentration is important information required to estimate OUR and control the growth rate of cells, these algorithms have the potential of proving useful in implementing robust controller to increase the productivity and efficiency of the monoclonal antibody manufacturing process using CHO cells