, Volume 22, Issue 1–3, pp 3–16 | Cite as

Engineering challenges in high density cell culture systems

  • Sadettin S. Ozturk
Special Issue


High density cell culture systems offer the advantage of production of bio-pharmaceuticals in compact bioreactors with high volumetric production rates; however, these systems are difficult to design and operate. First of all, the cells have to be retained in the bioreactor by physical means during perfusion. The design of the cell retention is the key to performance of high density cell culture systems. Oxygenation and media design are also important for maximizing the cell number. In high density perfusion reactors, variable cell density, and hence the metabolic demand, require constant adjustment of perfusion rates. The use of cell specific perfusion rate (CSPR) control provides a constant environment to the cells resulting in consistent production. On-line measurement of cell density and metabolic activities can be used for the estimation of cell densities and the control of CSPR. Issues related to mass transfer and mixing become more important at high cell densities. Due to the difference in mass transfer coefficients for oxygen and CO2, a significant accumulation of dissolved CO2 is experienced with silicone tubing aeration. Also, mixing is observed to decrease at high densities. Base addition, if not properly done, could result in localized cell lysis and poor culture performance. Non-uniform mixing in reactors promotes the heterogeneity of the culture. Cell aggregation results in segregation of the cells within different mixing zones. This paper discusses these issues and makes recommendations for further development of high density cell culture bioreactors.

Key words

cell culture process monitoring oxygenation CO2 transfer aggregation segregation diffusion, on-line monitoring 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aunins J and Henzler JH (1993) Aeration in cell culture Bioreactors, in bioprocessing, v.3 of Biotechnology: a multi-volume comprehensive treatise, 2nd ed., Stephanopoulos GN, Rehm H-J, Reed G (eds) in cooperation with Puhler A and Stadler P, VCH: Weinheim, Germany, pp. 223–281.Google Scholar
  2. Avgerinos G, Drapeau D, Socolow JS, Mao J, Hsiao K and Broeze R (1990) Spin filter perfusion system for high density cell culture: production of recombinant urinary type plasminogen activator in CHO cells. Biotechnology, 8, 54–58.Google Scholar
  3. Bailey J and Ollis DF (1986) Biochemical engineering fundamentals, McGraw Hill, New York.Google Scholar
  4. Berg GJ and Boedeker BGD (1988) Employing a ceramic matrix for the immobilization of mammalian cells in culture, in Animal Cell Technology, Vol. 3., Spier RE and Griffiths JB (eds), Academic Press, London, p. 321–335.Google Scholar
  5. Blumen T, Grammp G and Hettwer D (1993) paper presented at the ACS meeting in San Francisco, CA.Google Scholar
  6. Chiou TW, Murakami S and Wang DIC (1991) A fiber-bed bioreactor for anchorage-dependent animal cell cultures: part I. Bioreactor design and operations. Biotechnology and Bioengineering, 37, 775–761.Google Scholar
  7. Croughan MS et al. (1994) paper presented at the Cell Culture Engineering IV meeting in San Diego.Google Scholar
  8. Drapeau D, Luan Y-T, Whiteford JC, Lavin DP and Adamson SR (1990) Cell culture scale-up in stirred tank bioreactors, SIM Annual Meeting, Orlando, FL.Google Scholar
  9. Fenge C, Frauane E and Schugerl K (1992) Perfusion bioreactor performance at different cell bleed rates. Animal Cell Technology, 365–372.Google Scholar
  10. Fenge C, Klein C, Heuer C, Siegel U and Fraune E (1993) Agitation, aeration and perfusion modules for cell culture bioreactors. Cytotechnology, 11, 233–244.Google Scholar
  11. Fleischaker RJ and Sinskey A (1981) Oxygen demand and supply in cell culture. European Journal of Applied Microbiology and Biotechnology, 12, 193–193.Google Scholar
  12. Glacken MW, Adema E and Sinskey AJ (1989) Mathematical descriptions of hybridoma culture kinetics: I, Biotech Bioeng, 32, 491–506, 1988.Google Scholar
  13. Goochee CF and Murhammer DW (1990) Sparged animal cell bioreactors: mechanism of cell damage and pluronic F-68 protection. Biotechnology Prog, 6, 391–397.Google Scholar
  14. Gray D (1996) Paper presented at the Cell Culture Engineering V meeting in San Diego (previous paper in this proceeding).Google Scholar
  15. Griffiths JB (1990) Perfusion systems for cell cultivation, in Large scale mammalian cell culture technology, Lubiniecki AS and Dekker Marcel (eds) New York, p. 217–250.Google Scholar
  16. Hansen H, Damgaard B and Emborg C (1993) Enhanced antibody production associated with altered amino acid metabolism in a hybridoma high density perfusion culture established by gravity separation. Cytotechnology, 11, 155–166.Google Scholar
  17. Henzler HJ and Kauling DJ (1993) Oxgenation of cell cultures. Bioprocess Engineering, 9, 61–75.Google Scholar
  18. Hulscher M, Scheiber U and Onken U (1992) Biotech. Bioeng, 39, 442–446.Google Scholar
  19. Karkare SB, Philips PG, Burke DH, Dean RC and Runstadler PW Jr. (1985) In: Large-scale mammalian cell culture, Veder J and Tolbert WR (eds). Academic Press, New York., 127–149.Google Scholar
  20. Kyung Y-S, Peshwa MV, Gryte DM and Hu W-S (1994) High density culture of mammalian cells with dynamic perfusion based on on-line oxygen uptake measurements, Cytotechnology, 14, 183–190.Google Scholar
  21. Lubiniecki AS (1990) Large scale mammalian cell culture technology, Marcel Dekker, New York.Google Scholar
  22. Micheals J and Papautsakis ET (1995) In: Animal cell Culture technology: developments towards the 21th Century, E.C. Beuvery et al., Kluwer Academic Publishers, The Netherlands.Google Scholar
  23. Moreira JL and Feliciano AS (1993) Hydrodynamic control of suspended natural animal cell aggregates. Animal Cell Technology Products of Today, prospects for Tomorrow.Google Scholar
  24. Moreira JL, Alves PM, Aunins JG and Carrondo MJT (1994) Changes in animal cell natural aggregates in suspended batch cultures. Applied Microbiology Biotechnology, 41, 203–209.Google Scholar
  25. Moreira JL, Alves PM, Rodrigues JM, Cruz PE, Aunins JG and Carrondo MJT (1994b) Studies of baby hamster kidney natural cell aggregation in suspended batch cultures. Annals of the New York Academy of Sciences, 745, 122–133.Google Scholar
  26. O'Connor GM, Sanchez-RieraFernando and Cooney Charles L (1992) Design and evaluation of control strategies for high cell density fermentations. Biotechnology and Bioengineering, 39, 293–304.Google Scholar
  27. Orton DR and Wang DIC (1991) Quantitative and mechanistic effects of gas sparging in animal cell bioreactors. Paper presented at 202nd ACS National Meeting, New York, NY, August 25–30.Google Scholar
  28. Ozturk S and Palsson BO (1991) Effect of medium osmolarity on hybridoma growth, metabolism, and antibody production. Biotechnology and Bioengineering, 37, 989–993.Google Scholar
  29. Ozturk SS, Ray NG and Runstadler PW Jr. (1991) in Symposium on Transport Processes in Bioreactors: Fundamentals and Applications, Annual Meeting of AIChE, Los Angeles, CA.Google Scholar
  30. Ozturk SS (1994) Scale-up and optimization of high density cell culture bioreactors, in Advances in Bioprocess Engineering, Galindo E and Ramirez OT, (eds) Kluwer Academic Publishers, The Netherlands.Google Scholar
  31. Ozturk SS, Blackie JD, Wu P, Figueroa C, Thrift JC and Naveh D (1995a) Investigation of high density cell culture kinetics using on-line cell density and metabolic rate measurements. In: Animal Cell Culture Technology: Developments towards the 21th Century, Beuvery EC et al. (eds) Kluwer Academic Publishers, The Netherlands, pp. 301–305.Google Scholar
  32. Ozturk SS, Blackie J, Thrift J and Naveh D (1995b) Perfusion rate control based on on-line monitoring of oxygen consumption rates in mammalian cell fermentation, ACS National Meeting, San Diego, CA.Google Scholar
  33. Ozturk SS, Blackie J, Thrift J and Naveh D (1995c) Analysis of oxygen mass transfer, CO2 accumulation, and pH control for mammalian cell bioreactors, ACS National Meeting, San Diego, CA.Google Scholar
  34. Ozturk SS, Blackie J, Thrift J and Naveh D (1996a) Real-time monitoring of mammalian cell fermentation for monoclonal antibodies using a computer controlled immunodetection (BioCad/RPM) System, Biotechnology and Bioengineering, 48, 95–101.Google Scholar
  35. Ozturk SS, Thrift JC, Balckie JD and Naveh D (1996b) Real-time monitoring and control of glucose and lactate concentrations in a mammalian cell perfusion reactor using a commercial YSI analyzer, Biotechnology and Bioengineering, in press.Google Scholar
  36. Ozturk SS (1996) Heterogeneities in high density cultures: aggregation induced segregation, Biotechnology and Bioengineering, submitted.Google Scholar
  37. Reuss M, Schmalzriedt and Jenne M (1994) Structured modelling of bioreactors, In: Scale-up and optimization of high density cell culture bioreactors, in Advances in Bioprocess Engineering, Galindo E and Ramirez OT, (eds) Kluwer Academic Publishers, The Netherlands.Google Scholar
  38. Runstadler PW, Ozturk SS and Ray NG (1992) In: Animal cell technology: basic applied aspects, Murakami H, (ed) Kluwer Academic Publishers, The Netherlands.Google Scholar
  39. Tokashiki (1990) High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge. Cytotechnology, 3, 239–244.Google Scholar
  40. Tyler JE (1990) Microencapsulation of mammalian cells, In: Large scale mammalian cell culture technology, Lubiniecki AS and Dekker Marcel, (eds) New York, p. 343–362.Google Scholar
  41. Wie VB, Tomas, Brouns M, Elliott ML (1991) A novel continuous centrifugal bioreactor for high density cultivation of mammalian and microbial cells. Biotechnology and Bioengineering, 38, 1190–1202.Google Scholar
  42. Wu P, Ozturk SS, Blackie JD, Thrift JC Figueroa C and Naveh D (1995) Evaluation and applications of optical cell density probes in mammalian cell bioreactors. Biotechnol. Bioeng, 42, 312–323, 1995.Google Scholar
  43. Xie L and Wang DIC (1994) Stoichiometric analysis of animal cell growth and its application in medium design. Biotechnology and Bioengineering, 43, 1164–1174.Google Scholar
  44. Zanghi JA, Knopi RH and Miller WM (1995) Effects of pCO2 on the polysialyation of neural cell adhesion molecule in small cell lung cancer, paper presented at the AIChE Annual Meeting, Miami, FL, November 1996.Google Scholar
  45. Zhang S, Handa-Corrigan A and Spier RE (1992) A comparison of oxygenation methods for high density perfusion cultures of animal cells. Biotechnology and Bioengineering, 41, 685–692.Google Scholar
  46. Zhou W and Hu WS (1994) On-line characterization of a hybridoma cell culture process. Biotechnology and Bioengineering 44, 170–177.Google Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Sadettin S. Ozturk
    • 1
  1. 1.Bayer Corporation, BiotechnologyBerkeleyUSA

Personalised recommendations