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. 1999 Nov;31(3):243–254. doi: 10.1023/A:1008026613975

Analysis of the use of fortified medium in continuous culture of mammalian cells

Anshu Gambhir 1, Chun Zhang 1, Anna Europa 1, Wei-Shou Hu 1,✉
PMCID: PMC3449539  PMID: 19003148

Abstract

Continuous culture is frequently used in the cultivation of mammalian cells for the manufacturing of recombinant protein pharmaceuticals. In such operations a large volume of medium is turned over each day, especially in the case where cell recycle, or perfusion cultivation, is practiced. In principle, the volumetric throughput of medium can be reduced by using a more concentrated feed while maintaining the same nutrient provision rate. Overall, the medium components are divided into two categories: ‘consumable nutrients' and ‘unconsumable inorganic bulk salts’. In such fortified medium, the concentrations of consumable nutrients, but not bulk salts, are increased. With a stoichiometrically-balanced medium, the large amount of nutrients fed into the culture is largely consumed by cells to give rise to residual concentrations of these nutrients in their optimal range. However, unless care is taken to initiate the continuous culture, overshoot of nutrients may occur during the transient period. The high nutrient concentration during overshoot may be inhibitory by itself, or the resulting high osmolality may retard the growth. Using a mathematical model that incorporates the growth inhibitory effect of high osmolality we demonstrate such a potentially catastrophic effect of nutrient and osmolality overshoot by simulation. To avoid overshoot a controlled nutrient feeding scheme should be devised at the initiation of continuous culture.

Keywords: continuous culture, growth inhibition, osmolality, perfusion culture

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References

  1. Batt BC, Davis RH, Kompala DS. Inclined sedimentation for selective retention of viable hybridomas in a continuous suspension bioreactor. Biotechnol Prog. 1990;6:458–64. doi: 10.1021/bp00006a600. [DOI] [PubMed] [Google Scholar]
  2. Bibila TA, Ranucci CS, Konstantin G, Buckland BC, Aunins JG. Monoclonal Antibody Process Development using Medium Concentrates. Biotechnol Prog. 1994;10:87–96. doi: 10.1021/bp00025a011. [DOI] [PubMed] [Google Scholar]
  3. Birch JR, Lambert K, Thompson PW, Kenny AC, Wood LA. Antibody Production with Airlift Fermentors. In: Lydersen BK, editor. Large Scale Cell Culture Technology. New York: Hanser; 1987. [Google Scholar]
  4. Bree MA, Dhurjati P, Geoghegan RF, Jr., Robnett B. Kinetic Modelling of Hybridoma Cell Growth and Immunoglobulin Production in a Large-Scale Suspension Culture. Biotechnol Bioeng. 1988;32:1067–1072. doi: 10.1002/bit.260320814. [DOI] [PubMed] [Google Scholar]
  5. Bushell ME, Bell SL, Scott MF, Spier RE, W J.N., Sanders PG. Enhancement of Monoclonal Antibody yield by Hybridoma fed-batch culture, resulting in extended maintenance of Viable Cell population. Biotechnol Bioeng. 1994;44:1099–1106. doi: 10.1002/bit.260440911. [DOI] [PubMed] [Google Scholar]
  6. Comer MJ, Kearns MJ, Wahl J, Munster M, Lorenz T, Szperalski B, Koch S, Behrendt U, Brunner H. Industrial Production of Monoclonal Antibodies and Therapeutic Proteins by Dialysis Fermentation. Cytotechnology. 1990;3:295–299. doi: 10.1007/BF00365493. [DOI] [PubMed] [Google Scholar]
  7. de la Broise D, Noiseux M, Lemieux R, Massie B. Long Term Perfusion Culture of Hybridoma: a “Grow or Die” Cell Cycle System. Biotechnol Bioeng. 1991;38:781–787. doi: 10.1002/bit.260380712. [DOI] [PubMed] [Google Scholar]
  8. Fleischacker R. Practical matters in instrumentation for mammalian cell cultures. In: Thilly WJ, editor. Mammalian Cell Technology. London: Butterworth; 1986. [Google Scholar]
  9. Fleischaker RJJ, Sinskey A. Oxygen demand and supply in cell culture. Eur J Appl Microbiol Biotechnol. 1981;12:193–197. doi: 10.1007/BF00499486. [DOI] [Google Scholar]
  10. Fleischaker RT. An experimental study in the use of instrumentation to analyze metabolism and product formation in cell culture. Cambridge, MA: Massachusetts Institute of Technology; 1982. [Google Scholar]
  11. Frame K. A Kinetic Study of Hybridoma Growth and Monoclonal Antibody Production in a CSTR. Minneapolis, MN: University of Minnesota; 1988. [Google Scholar]
  12. Frame KK, Hu W-S. Kinetic study of hybridoma cell growth in continuous culture. I. A model for non-producing cells. Biotechnol Bioeng. 1991;37:55–64. doi: 10.1002/bit.260370109. [DOI] [PubMed] [Google Scholar]
  13. Glacken MW. Bioreactor Control and Optimization. In: Ho CS, Wang DIC, editors. Animal Cell Bioreactors. Oxford: Butterworth-Heinemann; 1991. pp. 373–404. [DOI] [PubMed] [Google Scholar]
  14. Glacken MW, Adema E, Sinskey AJ. Mathematical descriptions of hybridoma culture kinetics: I. Initial metabolic rates. Biotechnol Bioeng. 1988;32:491–506. doi: 10.1002/bit.260320412. [DOI] [PubMed] [Google Scholar]
  15. Glacken MW, Fleischaker RJ, Sinskey AJ. Reduction of waste product excretion via nutrient control: Possible strategies for maximizing product and cell yields on serum in cultures of mammalian cells. Biotechnol Bioeng. 1986;28:1376–1389. doi: 10.1002/bit.260280912. [DOI] [PubMed] [Google Scholar]
  16. Hu WS, Oberg MG. Monitoring and control of animal cell bioreactors: biochemical engineering considerations. Bioprocess Technology. 1990;10:451–81. [PubMed] [Google Scholar]
  17. Hu WS, Piret JM. Mammalian cell culture processes. Curr Opin Biotechnol. 1992;3:110–4. doi: 10.1016/0958-1669(92)90137-8. [DOI] [PubMed] [Google Scholar]
  18. Hu W-S, Peshwa MV. Animal cell bioreactors. Recent advances and challenges to scale-up. Can J Chem Eng. 1991;69:409–420. doi: 10.1002/cjce.5450690203. [DOI] [Google Scholar]
  19. Hu W-S, Zhou W, Europa LF. Controlling mammalian cell metabolism in bioreactors. Korean J Microbiol Biotechnol. 1998;8:8–13. [Google Scholar]
  20. Kitano K, Shintani Y, Ichimori Y, Tsukamoto K, Sasai S, Kida M. Production of human monoclonal antibodies by heterohybridomas. Appl Microbiol Biotechnol. 1986;24:282–286. doi: 10.1007/BF00257050. [DOI] [Google Scholar]
  21. Konstantinov K, Chuppa S, Sajan E, Tsai Y, Yoon S, Golini F. Real-time biomass-concentration monitoring in animal-cell cultures. Trends Biotechnol. 1994;12:324–333. doi: 10.1016/0167-7799(94)90049-3. [DOI] [PubMed] [Google Scholar]
  22. Konstantinov KB. Monitoring and Control Of the Physiological State Of Cell Cultures. Biotechnol Bioeng. 1996;52:271–289. doi: 10.1002/bit.260520203. [DOI] [PubMed] [Google Scholar]
  23. Konstantinov KB, Nishio N, Seki T and Yoshida T (1991) Physiologically motivated strategies for control of the fed-batch cultivation of recombinant Escherichia coli for phenylalanine production. J Ferm Bioeng: 350-355.
  24. Konstantinov KB, Pambayun R, Matanguihan R, Yoshida T, Perusich CM, Hu W-S. On-line monitoring of hybridoma cell growth using a laser turbidity sensor. Biotechnol Bioeng. 1992;40:1337–1342. doi: 10.1002/bit.260401107. [DOI] [PubMed] [Google Scholar]
  25. Konstantinov KB, Tsai YS, Moles D, Matanguihan R. Control Of Long-Term Perfusion Chinese Hamster Ovary Cell Glucose Auxostat. Biotechnol Prog. 1996;12:100–109. doi: 10.1021/bp950044p. [DOI] [PubMed] [Google Scholar]
  26. Kurokawa H, Park YS, Iijima S, Kobayashi T. Growth Characteristics in Fed-Batch Culture of Hybridoma Cells with Control of Glucose and Glutamine Concentrations. Biotechnol Bioeng. 1994;44:95–103. doi: 10.1002/bit.260440114. [DOI] [PubMed] [Google Scholar]
  27. Kurokawa H, Tatsuya O, Kamihira M, Park YS, Iijima S, Kobayashi T. Kinetic study of Hybridoma Metabolism and Antibody production in Continuous Culture using Serumfree media. J Ferm Bioeng. 1993;76:128–133. doi: 10.1016/0922-338X(93)90069-K. [DOI] [Google Scholar]
  28. Kyung Y-S, Peshwa MV, Gryte DM, W-S H. High cell density culture of mammalian cells with dynamic perfusion based on on-line oxygen uptake rate measurements. Cytotechnology. 1994;14:183–190. doi: 10.1007/BF00749615. [DOI] [PubMed] [Google Scholar]
  29. Laporte TL, Shevitz J, Kim YS, Wag SS. Hybridoma Perfusion system using a sedimentation device. Biotechnol Techniques. 1995;9:837–842. doi: 10.1007/BF00159411. [DOI] [Google Scholar]
  30. Matanguihan RM, Pambayun R, Izuishi T, Konstantinov KB, Yoshida T, Gryte DM, Hu WS, et al. Development of instrumentation and control strategy in bioreactor culture of hybridoma cells. In: Kaminogawa S, et al., editors. Animal Cell Technology: Basic and Applied Aspects. Dordrecht: Kluwer Academic Publishers; 1993. pp. 501–507. [Google Scholar]
  31. Merten OW, Palfi GE, Staheli J, Steiner J. Invasive infrared sensor for the determination of the cell number in a continuous fermentation of hybridomas. Dev Biol Standard. 1987;66:357–360. [PubMed] [Google Scholar]
  32. Miller WM, Wilke CR, Blanch HW. A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: Effect of nutrient concentration, dilution rate, and pH. Biotechnol Bioeng. 1988;32:947–965. doi: 10.1002/bit.260320803. [DOI] [PubMed] [Google Scholar]
  33. Nicholson ML, Hampson BS, Pugh GG, Ho CS. Continuous cell culture. In: Ho CS, Wang DIC, editors. Animal Cell Bioreactors. Oxford: Butterworth-Heinemann; 1991. pp. 269–303. [Google Scholar]
  34. Oh SKW, Chua FKF, Choo ABH. Intracellular responses of productive hybridomas subjected to high osmotic pressure. Biotechnol Bioeng. 1995;46:525–535. doi: 10.1002/bit.260460605. [DOI] [PubMed] [Google Scholar]
  35. Oyaas K, Ellingsen TE, Dyrset N, Levine DW. Hyperosmotic hybridoma cell cultures: increased monoclonal antibody production with addition of glycine betaine. Biotechnol Bioeng. 1994;44:991–998. doi: 10.1002/bit.260440816. [DOI] [PubMed] [Google Scholar]
  36. Ozturk SS, Palsson BO. Effect of Medium Osmolarity on Hybridoma Growth, Metabolism, and Antibody Production. Biotechnol Bioeng. 1991;37:989–993. doi: 10.1002/bit.260371015. [DOI] [PubMed] [Google Scholar]
  37. Riley MR, R M., ZX J., AM A., MD W. Simultaneous measurement of glucose and glutamine in insect cell culture media by near infrared spectroscopy. Biotechnol Bioeng. 1997;55:11–15. doi: 10.1002/(SICI)1097-0290(19970705)55:1<11::AID-BIT2>3.0.CO;2-#. [DOI] [PubMed] [Google Scholar]
  38. Seamans TC, Hu W-S. Kinetics of Growth and Antibody Production by a Hybridoma Cell Line in a Perfusion Culture. J Ferm Bioeng. 1990;70:241–245. doi: 10.1016/0922-338X(90)90056-3. [DOI] [Google Scholar]
  39. Takamatsu H, Hamamoto K, Ishimaru K, Yokoyama S, Tokashiki M. Large-Scale Perfusion Culture Process For Suspended Mammalian That Uses a Centrifuge With Multiple Settling Zones. Appl Microbiol Biotechnol. 1996;45:454–457. doi: 10.1007/BF00578455. [DOI] [PubMed] [Google Scholar]
  40. Takazawa Y, Tokashiki M. High Cell Density Perfusion Culture of Mouse-Human Hybridomas. Appl Microbiol Biotechnol. 1989;32:280–284. doi: 10.1007/BF00184974. [DOI] [Google Scholar]
  41. Tokashiki M, Arai T, Hamamoto K, Ishimaru K. High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge. Cytotechnology. 1990;3:239–44. doi: 10.1007/BF00365487. [DOI] [PubMed] [Google Scholar]
  42. Vits H, Hu W-S. Fluctuations in Continuous Mammalian Cell Bioreactors with Retention. Biotechnol Prog. 1992;8:397–403. doi: 10.1021/bp00017a004. [DOI] [PubMed] [Google Scholar]
  43. Wang G, Zhang W, Jacklin C, Eppstein L, Freedman D. High Cell Density Perfusion Culture of Hybridoma Cells for Production of Monoclonal Antibodies in the Celligen Packed Bed Reactor. In: Wang G, Zhang W, Jacklin C, Eppstein L, Freedman D, editors. Animal Cell Technology: Basic and Applied Aspects. Dordrecht: Kluwer Academic Publishers; 1993. pp. 463–469. [Google Scholar]
  44. Xie L, Wang DI. Integrated Approaches to the Design of Media and Feeding Strategies for Fed-batch Cultures of Animal Cells. Trends Biotechnol. 1997;15:109–13. doi: 10.1016/S0167-7799(97)01014-7. [DOI] [PubMed] [Google Scholar]
  45. Xie L, Wang DIC. Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies. Biotechnol Bioeng. 1994;43:1175–1189. doi: 10.1002/bit.260431123. [DOI] [PubMed] [Google Scholar]
  46. Xie LZ, Wang DIC. High Cell Density and High Monoclonal Antibody Production through Medium Design and Rational Control In a Bioreactor. Biotechnol Bioeng. 1996;51:725–729. doi: 10.1002/(SICI)1097-0290(19960920)51:6<725::AID-BIT12>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  47. Zhang S, Handa-Corrigan A, Spier RE. Kinetics of Cell Metabolism and Antibody Production in High Concentration Perfusion Cultures. In: Zhang S, Handa-Corrigan A, Spier RE, editors. Animal Cell Technology: Basic and Applied Aspects. Dordrecht: Kluwer Academic Publishers; 1993. pp. 309–324. [Google Scholar]
  48. Zhong J-J, Konstantinov KB, Yoshida T. Computer-aided on-line monitoring of physiological variables in suspended cell cultures of Perilla frutescens in a bioreactor. J Ferm Bioeng. 1994;77:445–447. doi: 10.1016/0922-338X(94)90023-X. [DOI] [Google Scholar]
  49. Zhou W, Hu W-S. On-line characterization of a hybridoma cell culture process. Biotechnol Bioeng. 1994;44:170–177. doi: 10.1002/bit.260440205. [DOI] [PubMed] [Google Scholar]
  50. Zhou W, Hu W-S. Effect of insulin on a serum-free hybridoma culture. Biotechnol Bioeng. 1995;47:181–185. doi: 10.1002/bit.260470209. [DOI] [PubMed] [Google Scholar]
  51. Zhou WC, Rehm J, Hu WS. High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding. Biotechnol Bioeng. 1995;46:579–587. doi: 10.1002/bit.260460611. [DOI] [PubMed] [Google Scholar]
  52. Zhou W-C, Rehm J, Europa A, Hu W-S. Alteration of mammalian cell metabolism by dynamic nutrient feeding. Cytotechnology. 1997;24:99–108. doi: 10.1023/A:1007945826228. [DOI] [PMC free article] [PubMed] [Google Scholar]

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