Abstract
Productivity in a CHO perfusion culture reactor was maximized when pCO2 was maintained in the range of 30–76 mm Hg. Higher levels of pCO2 (> 150 mm Hg) resulted in CHO cell growth inhibition and dramatic reduction in productivity. We measured the oxygen utilization and CO2 production rates for CHO cells in perfusion culture at 5.55×10-17 mol cell-1 sec-1 and 5.36×10-17 mol cell-1 sec-1 respectively. A simple method to directly measure the mass transfer coefficients for oxygen and carbon dioxide was also developed. For a 500 L bioreactor using pure oxygen sparge at 0.002 VVM from a microporous frit sparger, the overall apparent transfer rates (kLa+kAA) for oxygen and carbon dioxide were 0.07264 min-1 and 0.002962 min-1 respectively. Thus, while a very low flow rate of pure oxygen microbubbles would be adequate to meet oxygen supply requirements for up to 2.1×107 cells/mL, the low CO2 removal efficiency would limit culture density to only 2.4×106 cells/mL. An additional model was developed to predict the effect of bubble size on oxygen and CO2 transfer rates. If pure oxygen is used in both the headspace and sparge, then the sparging rate can be minimized by the use of bubbles in the size range of 2–3 mm. For bubbles in this size range, the ratio of oxygen supply to carbon dioxide removal rates is matched to the ratio of metabolic oxygen utilization and carbon dioxide generation rates. Using this strategy in the 500 L reactor, we predict that dissolved oxygen and CO2 levels can be maintained in the range to support maximum productivity (40% DO, 76 mm Hg pCO2) for a culture at 107 cells/mL, and with a minimum sparge rate of 0.006 vessel volumes per minute.
A = volumetric agitated gas-liquid interfacial area at the top of the liquid, 1/m
B = cell broth bleeding rate from the vessel, L/min
CER = carbon dioxide evolution rate in the bioreactor, mol/min
[CO2] = dissolved CO2 concentration in liquid, M
[CO2]* = CO2 concentration in equilibrium with sparger gas, M
[CO2]** = CO2 concentration in equilibrium with headspace gas, M
CO2(1) = dissolved carbon dioxide molecule in water
[CT] = total carbonic species concentration in bioreactor medium, M
[CT]F = total carbonic species concentration in feed medium, M
D = bioreactor diameter, m
DI = impeller diameter, m
Db = the initial delivered bubble diameter, m
F = fresh medium feeding rate, L/min
HL = liquid height in the vessel, m
kA = carbon dioxide transfer coefficient at liquid surface, m/min
k supOinfA = oxygen transfer coefficient at liquid surface, m/min
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aiba S, Humphery AE and Millis NF (1973) Biochemical Engineering (2nd edition), Chapter 6, Academic Press, New York.
Aunins JG, Glazomitsky K and Buckland BC (1991) Aeration in pilot scale vessels for animal cell culture, presentation, AIChE Annual meeting, Los Angeles, CA, Nov. 17–22.
Backer MP, Metzger LS, Slaber PL, Nevitt KL and Boder GB (1988) Large Scale Production of Monoclonal Antibodies in Suspension Culture., Biotechnol. and Bioeng. 32: 993–1000.
Bavarian F, Fan LS and Chalmers JJ (1991) Microscopic visualization of insect cell-bubble interactions I: Rising bubbles, air-medium interface, and the foam layer., Biotechnol. Prog. 7: 140–150.
Blumen T, AIChE Mtg Nov. 1993
Bonarius HPJ, de Gooijer CD, Tramper J and Schmid G (1995) Carbon dioxide evolution rates in animal cell culture in bicarbonate buffered and bicarbonate free medium. In: RE Spier et al. (eds.), Animal Cell Technology. Proceedings of 13th ESACT Meeting, Butterworth-Heinemann Wiltshire UK.
Chapman CM, Nienow AW and Middleton JC (1980) Surface aeration in a small, agitated, and sparged vessel. Biotechnol. and Bioeng. 22: 981–993.
Drapeau D, SIM Annual Meeting, Orlando Florida, August 1990.
Ganz MB, Boyarski G, Sterzel RB and Boron WF (1989) Arginine vasopressin enhances pHi regulation in the presence of HCO sup-inf3 by stimulating three acid-base transport systems, Nature 337: 648–651.
Ingham J, Piehl H, Dittmar KEJ and Lehmann J (1984) Repeated Fed-Batch Cultivation of Lymphocytic Cells. Proceedings of the Third Congress on Biotechnology, Munich 10–14 September 1994.
Itagaki A and Kimura G (1974) Tes and HEPES buffers in mammalian cell cultures and viral studies: problem of carbon dioxide requirement Exp. Cell Res. 83(2), (p. 351–61).
Jones RP and Greenfield PF (1982) Effect of carbon dioxide on yeast growth and fermentation. Enzyme Microb. Technol. 4: 210–22.
Krapf R, Berry CA, Alpern RJ and Rector FC Jr (1988) Regulation of cell pH by ambient bicarbonate, carbon dioxide tension, and pH in the rabbit proximal convoluted tubule. J. Clin. Invest. 81: 381–389.
Lovrecz G and Gray PP (1994) Use of on-line gas analysis to monitor recombinant mammalian cell cultures.
Madshus IH (1988) Regulation of intracellular pH in eukaryotic cells, Biochem. J. 250: 1–8.
Maiorella B (1994) Recombinant Sub Unit Vaccines, Eng Found, Cell Cult. Eng., San Diego March 7–12 1994.
Murhammer DW and Goochee CF (1988) Scale up of Insect Cell Cultures: Protective effects of Pluronic F-68, Bio/Technology 6: 1411–1418.
Onken U and Liefke E (1989) Effect of total and partial pressure (Oxygen and Carbon Dioxide) on aerobic microbial processes. Advances in Biochemical Engineering/Biotechnology, 40, p. 137–169, Ed A. Fiechte.
Perrin DD and Dempsey B (1974) Buffers for pH and Metal Ion control. Chapman and Hall, London.
Radlett PJ, Telling RC, Whitside JP and Maskell MA (1972) The supply of oxygen to submerged cultures of BHK 21 Cells. Biotechnol. and Bioeng. 14: 437–445.
Roos A and Boron WF (1981) Intracellular pH. Physiological Reviews, 61, p. 296–434, April 1981.
Stumm W and Morgan JJ (1981) Aquatic Chemistry, Chapter 4. John Wiley & Sons, New York.
Urlaub G and Chasin A (1980) Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. USA, 77, p. 4216–4220.
Weiss SA, Peplow D, Smith GC, Vaughn JL and Doughery E (1985) Biotechnical aspects of a large scale process for insect cells and baculoviruses, in Techniques in the Lifesciences, Cell Biology—Volume C1 (pp. 1–16) Elsevier.
Yoon S-J and Konstantinov K (1994) Continuous real-time monitoring of oxygen uptake rate (OUR) in animal cell bioreactors. Biotechnol. Bioeng. 44 (p. 170–177).
Author information
Authors and Affiliations
Additional information
Nomenclature
Rights and permissions
About this article
Cite this article
Gray, D.R., Chen, S., Howarth, W. et al. CO2 in large-scale and high-density CHO cell perfusion culture. Cytotechnology 22, 65–78 (1996). https://doi.org/10.1007/BF00353925
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00353925