Search tips
Search criteria 


Logo of cytotechspringer.comThis journalToc AlertsSubmit OnlineOpen Choice
Cytotechnology. 2003 May; 42(1): 35–45.
PMCID: PMC3449505

Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates


Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium feed rates with a bioreactor cell concentration of ~5 × 106 cells mL-1. Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell-1 day-1. Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen activator concentration was maximized (~40 mg L-1) at 0.2 nL cell-1 day-1. The volumetric protein productivity (~60 mg L-1 day-1 was maximized above 0.3 nL cell-1 day-1. The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion cultures.

Keywords: Cell specific perfusion feed rates, CHO, Control, Optimization, t-PA, Viable cell probe

Full Text

The Full Text of this article is available as a PDF (176K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Cerckel I, Garcia A, Degouys V, Dubois D, Fabry L., Miller AOA. Dielectric spectroscopy of mammalian cells 1. Evaluation of the biomass of HeLa-and CHO cells in suspension by low-frequency dielectric spectroscopy. Cytotechnology. 1993;13:185–193. doi: 10.1007/BF00749814. [PubMed] [Cross Ref]
  • Cohen SA, Strydom DJ. Amino acid analysis using phenylisothiocyanate derivatives. Anal Biochem. 1988;174:1–16. doi: 10.1016/0003-2697(88)90512-X. [PubMed] [Cross Ref]
  • Degouys V, Cerckel I, Garcia A, Harfield J, Dubois D, Fabry L, Miller AOA. Dielectric spectroscopy of mammalian cells 2. Simultaneous in situ evaluation by aperture inpedance pulse spectroscopy and low-frequency dielectric spectroscopy of the biomass of HTC cells on Cytodex 3. Cytotechnology. 1993;13:195–202. doi: 10.1007/BF00749815. [PubMed] [Cross Ref]
  • Dowd JE, Kwok KE, Piret JM. Increased t-PA yields using ultrafiltration of product from CHO fed-batch culture. Biotechnol Prog. 2000;16:786–794. doi: 10.1021/bp000079b. [PubMed] [Cross Ref]
  • Dowd JE, Kwok KE, Piret JM. Glucose-based optimization of CHO cell perfusion culture. Biotechnol Bioeng. 2001;75:252–256. doi: 10.1002/bit.10013. [PubMed] [Cross Ref]
  • Dowd JE, Weber I, Rodriguez B, Piret JM, Kwok KE. Predictive control of hollow-fiber bioreactors for the production of monoclonal antibodies. Biotechnol Bioeng. 1999;63:484–492. doi: 10.1002/(SICI)1097-0290(19990520)63:4<484::AID-BIT12>3.0.CO;2-5. [PubMed] [Cross Ref]
  • Ducommun P, Bolzonella I, Rhiel M, Pugeaud P, Von Stockar U, Marison IW. On-line determination of animal cell concentration. Biotechnol Bioeng. 2001;72:515–522. doi: 10.1002/1097-0290(20010305)72:5<515::AID-BIT1015>3.0.CO;2-Q. [PubMed] [Cross Ref]
  • Ducommun P, Kadouri A, Von Stockar U, Marison IW. On-line determination of animal cell concentration in two industrial high-density culture processes by dielectric spectroscopy. Biotechnol Bioeng. 2002;77:316–323. doi: 10.1002/bit.1197. [PubMed] [Cross Ref]
  • Fann CH, Guirgis F, Chen G, Lao MS, Piret JM. Limitations to the amplification and stability of human tissue-type plasminogen activator expression by Chinese Hamster Ovary cells. Biotechnol Bioeng. 2000;69:204–212. doi: 10.1002/(SICI)1097-0290(20000720)69:2<204::AID-BIT9>3.0.CO;2-Z. [PubMed] [Cross Ref]
  • Fehrenbach R, Comberbach M, Pêtre JO. On-line biomass monitoring by capacitance measurement. J Biotech. 1992;23:303–314. doi: 10.1016/0168-1656(92)90077-M. [PubMed] [Cross Ref]
  • Guan Y, Evans PM, Kemp RB. Specific heat flow rate: An on-line monitor and potential control variable of specific metabolic rate in animal cell culture that combines microcalorimetry with dielectric spectroscopy. Biotechnol Bioeng. 1998;58:87–94. doi: 10.1002/(SICI)1097-0290(19980605)58:5<464::AID-BIT2>3.0.CO;2-B. [PubMed] [Cross Ref]
  • Guan YH, Kemp RB. On-line heat flux measurements improve the culture medium for the growth and productivity of genetically engineered CHO cells. Cytotechnology. 1998;30:107–120. doi: 10.1023/A:1008038515285. [PMC free article] [PubMed] [Cross Ref]
  • Hagen SR, Augustin J, Grings E, Tassinari P. Precolumn phenylisothiocyanate derivatization and liquid chromatography of free amino acids in biological samples. Food Chem. 1993;46:319–323. doi: 10.1016/0308-8146(93)90127-2. [Cross Ref]
  • Harris CM, Todd RW, Bungard SJ, Lovitt RW, Morris JG, Kell DB. The dielectric permittivity of microbial suspensions at radio frequencies; A novel method for the real-time estimation of microbial biomass. Enzyme Microb Technol. 1987;9:181–186. doi: 10.1016/0141-0229(87)90075-5. [Cross Ref]
  • Heidemann R, Zhang C, Qi H, Rule JL, Rozales C, Sinyoung P, Chuppa S, Ray M, Michaels J, Konstantinov K, Naveh D. The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells. Cytotechnology. 2000;32:157–167. doi: 10.1023/A:1008196521213. [PMC free article] [PubMed] [Cross Ref]
  • Hiller G, Clark D, Blanch H. Cell retention chemostat studies of hybridoma cells. Analysis of hybridoma growth and metabolism in continuous suspension culture on serum free medium. Biotechnol Bioeng. 1993;42:185–195. doi: 10.1002/bit.260420206. [PubMed] [Cross Ref]
  • Konstantinov K, Chuppa S, Sajan E, Tsai Y, Yoon S, Golini F. Real-time biomass-concentration monitoring in animal cell cultures. Trends Biotech. 1994;12:324–333. doi: 10.1016/0167-7799(94)90049-3. [PubMed] [Cross Ref]
  • 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. [PubMed] [Cross Ref]
  • Konstantinov KB, Tsai Y-S, Moles D, Matanguihan R. Control of long-term perfusion Chinese Hamster Ovary cell culture by glucose auxostat. Biotechnol Prog. 1996;12:102–109. doi: 10.1021/bp950044p. [PubMed] [Cross Ref]
  • Kurkela R, Fraune E, Vihko P. Pilot-scale production of murine monoclonal antibodies in agitated, ceramic-matrix or hollow-fiber cell culture systems. BioTechniques. 1993;15:674–683. [PubMed]
  • Kyung Y-S, Peshwa MV, Gryte DM, Hu W-S. High density culture of mammalian cells with dynamic perfusion based on on-line uptake rate measurements. Cytotechnology. 1994;14:183–190. doi: 10.1007/BF00749615. [PubMed] [Cross Ref]
  • Markx GH, Davey CL, Kell DB, Morris P. The dielectric permittivity at radio frequencies and the Bruggeman probe: Novel techniques for the on-line determination of biomass concentrations in plant cell cultures. J Biotech. 1991;20:279–290. doi: 10.1016/0168-1656(91)90337-U. [Cross Ref]
  • Maruhashi F, Murakami S, Baba K. Automated monitoring of cell concentration and viability using an image analysis system. Cytotechnology. 1994;15:281–289. doi: 10.1007/BF00762403. [PubMed] [Cross Ref]
  • Merten OW. Constructive improvement of the ultrasonic separation device ADI 1015. Cytotechnology. 2000;24:175–179. doi: 10.1023/A:1008147822625. [PMC free article] [PubMed] [Cross Ref]
  • Merten OW, Palfi GE, Staheli J, Steiner J. Invasive infrared sensor for the determination of the cell number in a continuous fermentation of hybridomas. Devel Biol Stand. 1985;66:357–360. [PubMed]
  • Miller WM, Blanch HW, Wilke CR. 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. [PubMed] [Cross Ref]
  • Ozturk S, Thrift J, Blackie J, Naveh D. Real time monitoring and control of glucose and lactate concentrations in a mammalian cell perfusion reactor. Biotechnol Bioeng. 1997;53:372–378. doi: 10.1002/(SICI)1097-0290(19970220)53:4<372::AID-BIT3>3.0.CO;2-K. [PubMed] [Cross Ref]
  • Pelletier F, Fonteix C, De Silva AL, Marc A, Engasser JM. Software sensors for the monitoring of perfusion cultures: Evaluation of the hybridoma density and the medium composition from glucose concentration measurements. Cytotechnology. 1994;15:291–299. doi: 10.1007/BF00762404. [PubMed] [Cross Ref]
  • Sonderhoff SA, Kilburn DG, Piret JM. Analysis of mammalian viable cell biomass based on cellular ATP. Biotechnol Bioeng. 1992;39:859–864. doi: 10.1002/bit.260390807. [PubMed] [Cross Ref]
  • Van der Pol JJ, Joksch B, Gätgens J, Biselli M, De Gooijer CD, Tramper J, Wandrey C. On-line control of an immobilized hybridoma culture with multi-channel flow injection analysis. J Biotechnol. 1995;43:229–242. doi: 10.1016/0168-1656(95)00143-3. [PubMed] [Cross Ref]
  • Vits H, Hu W-S. Fluctuations in continuous mammalian cell bioreactors with retention. Biotechnol Prog. 1992;8:397–403. doi: 10.1021/bp00017a004. [PubMed] [Cross Ref]
  • Wu P, Ozturk S, Blackie JD, Thrift JC, Figueroa C, Naveh D. Evaluation and applications of optical cell density probes in mammalian cell bioreactors. Biotechnol Bioeng. 1995;45:495–502. doi: 10.1002/bit.260450606. [PubMed] [Cross Ref]
  • Zhou W, Hu WS. On line characterization of a hybridoma cell culture process. Biotechnol Bioeng. 1994;44:170–177. doi: 10.1002/bit.260440205. [PubMed] [Cross Ref]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.