To accelerate process development and to enhance mammalian cell production process performance, small-scale higher-throughput bioreactor systems are being developed. A 24-well plate based system with independent feedback control in each well of temperature, pH and dissolved oxygen (DO) concentration was investigated. The system is operated on a shaking platform that houses a 24-well cassette. Each well has a total volume of 10 mL (7 mL working volume) and sensors for temperature, pH and DO. The flow rate of CO2 controls the pH while DO is maintained by addition of air and/or stripping by nitrogen. As in stirred bioreactors, base additions were required to maintain pH control, and this was achieved by an algorithm that used the drop in CO2 flow rate to time manual additions of sodium bicarbonate when needed. This system provides multiple instrumented parallel cultures to optimize process parameters for recombinant protein production.

The culture system has been used to successfully grow Chinese Hamster Ovary (CHO) cells producing recombinant t-PA as well as Human Embryonic Kidney (HEK)-293 cells expressing GFP. Viable cell densities over 3.5×106 cells/mL were obtained. This system readily accommodated temperature and pH factorial design studies in duplicate wells with CHO cells at pH set points of 6.8, 7.1 and 7.4 while the temperature was set at 33, 35 and 37°C. The DO in all wells was set at 75% air saturation and the system independently controlled the temperature and pH in each well regardless of the conditions imposed. The main advantages compared to shake flask cultures were the flexible pH and temperature control. Comparisons with shake flask and stirred tank results will be discussed as well as the application to selecting temperature and pH shift strategies.

We gratefully acknowledge the assistance of Stephen Boyer of MicroReactor Technologies and John Poppleton of Applikon Biotechnology.