Carbon dioxide control in bioreactors and evaluating its effect on mammalian cell culture properties

Zohreh Ahleboot,1,* Mahdi khorshidtalab,2 Rasoul mahboudi,3 Shaian maleknia,4 Morteza asghari,5

1. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
2. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
3. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
4. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
5. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran

Abstract


Introduction

Control of carbon dioxide within the optimum range is important in mammalian bioprocess regarding cell growth, high protein production and consistent quality attributes.

Methods

In this study 4 bioreactor runs were performed. two different overlay flow rates (5 and 15 lpm) and two different flux of exhaust outputs were used. flux of exhaust output in two bioreactor runs that were considered as control experiments (lower overlay flow of 5 lpm) kept lower while in other two ones in which their overlay flow rate was about 15 lpm the flux of exhaust output was kept higher. the output of the exhaust was opened to adjust the pressure to 0.3 bar for all 4 bioreactor experiments. these batches were run in 30l bioreactor for evaluation the effect of increased aeration on the co2 removal, averages of ph values, expression and quality of protein thirty l bioreactors were inoculated to a working volume of 26 l, at a seeding density of 0.65×106 cells/ml and viability greater than 97%. the cultures were carried out using fc-16 medium, supplemented to 6 mm glutamine 2 g/l glucose. feeding strategy was chosen 2% f-2a (based on the initial volume of the bioreactor) and 0.2% feed beta till day 4, after that 3% till day 13, first feed from 48 hr. glucose levels were maintained to 2 ± 0.2 g/l, with a 40 g/l glucose stock solution. cell cultures were harvested at 60 ± 10% viability, which occurred at approximately 360 hours (day 15).

Results

With considering increased aeration and/or lower backpressure above the bioreactor culture media, in principle, it is expected to reduce the dissolved co2 concentration. hence, the following equilibrium with the bicarbonate ion would tend to displace itself to the right: h3o+ + hco3- ↔ 2h2o + co2. this phenomenon is accompanied by a tendency in ph increase. in experiments with more overlay flow (15 lpm), foam was better controlled. in addition, gas exhausting was followed by more co2 removal and finally, it resulted in better conditions for bioreactors. protein production was increased by 50%, while maxim,um cell density was increased by 30 % and the ph average was higher during the batches that were run with this strategy.

Conclusion

This experiment has shown that pco2 level affects a cell culture process even in 30l bioreactor. therefore, differences in pco2 profiles between different scales can potentially affect the process performance.

Keywords

Carbon dioxide, overlay flow rate, protein production, cell culture