INTRODUCTION The success of a robust bioprocess relies on the ability to accurately and effectively control critical process parameters. In the field of single-use bioreactors (SUB) these evaluations still remain rare and proper control strategies are not clearly outlined. As a result, this study was aimed to provide a rigorous understanding towards developing methods for optimal O 2 supply, lower dCO 2 built up and establishing a pH control strategy in large scale SUB. As a first step, mass transfer capabilities were studied using the static gassing out method for determining the volumetric mass transfer coefficient (kLa) for O 2 by varying the size and number of microspargers in the SUB. To evaluate the effects and possible interactions of several critical factors such as agitation rate, sparge rate and working volumes, design of experiments with full factorial design was conducted. The response surfaces with second degree polynomial were used to select the optimal controlled parameters with efficient kLas for the cell culture studies. The kLa’s were determined across different working volumes, agitation and O 2 sparge rates. The air sparge for CO 2 stripping in SUB was evaluated via a drilled holes in a sparger and a pipe. Both the strategies, drilled holes in a sparger or in a pipe, are efficient ways to remove CO 2 from the SUB. However, the process pH increases upon removal of CO 2 and the rate of increase in pH was observed to be dependent on the strategy used for CO 2 removal. Thus, in order to establish a better control on this pH deviation in SUB, the PID parameters in the pH-PID loop were tuned and CO 2 cascade feedback strategies (microspargers vs drilled holes) were also evaluated and optimized for the cell culture studies. Following the optimization of the aforementioned physical characteristics in the SUB, two CHO cell line based 200L fed-batch processes, one with minimal and the other with improved control strategies, were conducted in SUB. This study successfully demonstrated the advantages of improved pO 2 , pH and pCO 2 control in a SUB system. Establishing improved O 2 supply, lower dCO 2 built up and pH control in large scale single-use bioreactors. Shahid Rameez , Sigma S. Mostafa and Abhinav A. Shukla KBI Biopharma, 1101 Hamlin Road, Durham, NC 27704 LARGE SCALE SINGLE-USE BIOREACTORS (SUB) OPTIMAL SUPPLY FOR O 2 CONCLUSION Our aim was to develop a robust O 2 supply, dCO 2 removal and pH control strategy for large-scale fed-batch processes in SUB. The dissolved O 2 concentration in a suspension of cell culture depends on the rate of O 2 transfer from the gas phase to the liquid, rate at which O 2 is transported into the cells, and on O 2 uptake by the cells for growth, maintenance and production. The gas–liquid mass transfer was evaluated with different size sparger discs. A significant impact on supply of O 2 was observed changing the size and number of sparger discs. For CO 2 removal, our approach was to first conduct stripping rate studies using spargers and pipes with drilled holes to determine rate of removal of CO 2 from SUB. Supply of CO 2 on demand to control deviations in process pH was evaluated using the microspargers. In addition, the PID parameters in the pH-PID loop were tuned from default manufacturer’s settings to establish a faster response on these pH deviations. Finally using the best strategy for the proof of concept run at 200-L scale we demonstrated reduction in dCO 2 built up during a CHO cell culture process producing an monoclonal antibody. O 2 transfer is often the rate-limiting step in the aerobic bioprocess due to the low solubility of O 2 in the cell culture medium. The measurement and/or prediction of the volumetric mass transfer coefficient, (kLa), is a crucial step in the design and scale-up of SUBs. This work aimed at measurement of kLas using different size and number of sparger discs to provide a better knowledge about the selection, design, scale-up in SUBs. SUBs are widely used in mammalian cell culture processes and are rapidly replacing conventional stirred tank bioreactors. SUBs have impellers like conventional bioreactors, however, with different designs and sizes. Moreover, the impellers are integrated into the plastic bag. The plastic bag and the integrated impeller are pre-sterilized. Prior to onset of cell culture process the sterilized bag is mounted in the bioreactor vessel and the impeller is connected, mechanically or magnetically, to a driver connected to a motor. SUB enhance the biological and process safety during manufacturing by reducing cleaning and sterilization demands and risk of cross contamination. To be representative of the process, troubleshooting experiments were carried in a salt solution to outline a control strategy. The physical properties of this salt solution more closely mimic the physical properties of the cell culture medium. The solution was controlled at pH 6.85 and a high CO 2 was artificially built up in the SUB. While using drilled holes for removal of CO 2 in SUB we evaluated two strategies to attenuate pH deviations caused by CO 2 removal. Strategy 1: CO 2 on demand was passed through the micro-sparger instead of pipe with drilled holes. Hence, better mass transfer for CO 2 and thus better control on the pH deviation. Strategy 2: During the stripping for CO 2 , pH-PID parameters were tuned for faster response to gain better control on deviation in pH from process pH setpoint. Although, the results from such studies are from a salt buffer solution instead of medium with cells, these experiments provide for a platform to design a control strategy which is eventually employed in a cell culture process. Results: The kLa is dependent on the O 2 transfer from the gas sparge rates to the liquid, working liquid volumes in the SUB and the stirrer speeds (agitation rate). Figures on the right show experimental and predicted kLa values, under different operational conditions, using full factorial DOE based design in a 200L SUB . Working volume and gas flow rates were observed as significant factors in regulating kLas. kLas decreased when working at lower gas flow rates as a consequence of the O 2 transport limitation. Results: Changing the Size and number of sparger discs can result in significant impact on supply of O 2 CO 2 STRIPPING IN SUB FOR A CELL CULTURE PROCESS. One of the recurrent issues that is observed in industrial mammalian cell culture especially in large-scale bioreactors is accumulation of dissolved CO 2 (dCO 2 ). The impact of dCO 2 on cell culture has been studied in detail. It has been shown to have effect on cell growth rate, specific productivity, decrease in cell density, decrease in glucose, lactate, and glutamine specific metabolite rates and pH-dependent enzymatic reactions in the cell. In small-scale bioreactors majority of dCO 2 is stripped via surface aeration. However, in large-scale bioreactors, the liquid surface-to-volume ratio decreases and thus other strategies for dCO 2 removal have to be designed. In SUBs these evaluations remain rare and proper stripping strategies are not clearly outlined. Both excessive stripping or accumulation of dCO 2 are detrimental to cell growth thus an optimal level of dCO 2 has to be maintained for cell culture. This optimal value will vary with cell line and product of interest. CO 2 stripping causes the pH in the system to rise over time. The stripping rate and control on process pH deviations will depend on type of stripping strategy employed (drilled holes in sparger/pipe). Case Study: 200L Cell Culture run, where high CO 2 built up was expected due to addition of a basic feed which led to addition for high amounts of CO 2 to control pH increase in the culture. Problem: The CO 2 removal which could be achieved was minimal. The pH started to increase rapidly as soon as air was introduced in the culture to achieve CO 2 removal. Results: Supplying the CO 2 on demand through the microsparger established better control on the pH deviation in the salt solution. Furthermore, tuning the pH-PID parameters increased the response to control pH deviation within dead band of the pH set point in the solution. Results: Employing aforementioned gassing strategies in a 200L CHO cell culture process producing a monoclonal antibody, approximately 40% reduction in dCO 2 built up was achieved. O 2 SUPPLY CONSIDERATIONS In SUBs high values of mass transfer rates and excellent mixing can be achieved with proper O 2 delivery strategy. Many factors such as agitation, type and number of spargers , gas flow rates etc, have to be evaluated in detail in order to achieve optimal O 2 supply. The correct measurement and/or prediction of kLa, serves as benchmark during the design, operation and scale-up for O 2 delivery. Implementation of aforementioned strategy in a 200L CHO cell culture process