Abstract
Production of a TGFβ receptor with high density baculovirus infected Sf-9 cells (7×106cells ml-1) served as a test run for a retrofitted 150 L microbial fermentor. The entire 110 L batch run was performed in serum free medium, with an addition of a concentrated amino acid and yeastolate mixture at the time of infection. This addition strategy has been proven effective at a small scale by enabling cultures to maintain maximum product yield. In the bioreactor however, while cellular growth was comparable to that of the smaller scale control, TGFβ receptor production was three fold below the control. To minimize the mechanical stress, low flow rate of pure oxygen was used to control the dissolved oxygen at 40%. As a consequence, it seems that this aeration strategy involved an accumulation of dissolved carbon dioxide that in turn inhibited the protein production. A model has been developed that estimated the CO2 partial pressure in the culture to be in the vicinity of 0.15 atm. The effect of dissolved CO2 at this concentration has been assessed at smaller scale for TGFβ receptor and β-gal expression, in controlled atmosphere incubators.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- CST:
-
cumulative sparging time (s)
- dpi:
-
days post-infection (d)
- \(D_{CO_2 /H_2 O} /D_{O_2 ,H_2 O}\) :
-
CO2/O2 diffusivities ratio (=0.784 at 27 °C)
- DO:
-
% saturation of dissolved oxygen (%)
- hpi:
-
hours post-infection (h)
- He:
-
Henry coefficient (HeCO 2=32.1×10-3M atm-1, HeO 2=1.23×10-3 M atm-1 at 27 °C)
- kLa:
-
volumetric liquid-side mass transfer coefficient (h-1)
- LDH:
-
Lactate dehydrogenase
- MOI:
-
multiplicity of infection (pfu cell-1)
- N:
-
agitation speed (rpm)
- OTR:
-
oxygen transfer rate (mole O2 ml-1 h-1)
- OUR:
-
oxygen uptake rate (mole O2 ml-1 h-1)
- p:
-
partial pressure (atm)
- P:
-
total pressure (atm)
- pfu:
-
plaque forming units
- q:
-
specific consumption or production rate (mole cell-1 h-1)
- QH, OUT :
-
headspace outlet gas flow rate
- QS,NOM :
-
nominal volumetric sparged gas flow rate (92 ml s-1 at bioreactor conditions)
- R:
-
ideal gas constant (82.05 ml atm mole-1 K-1)
- SRV L :
-
molar sparging rate per unit liquid volume (mole ml-1 h-1)
- SSR:
-
specific sparging rate (mole cell-1 h-1)
- T:
-
temperature (C or K)
- VL :
-
culture volume (ml)
- VVD:
-
volume of feed per volume of culture per day (d-1)
- X:
-
cell concentration (cell ml-1)
- Y:
-
yield coefficient
- CO2, O2 :
-
related to CO2 or O2, respectively
- glc:
-
glucose
- H:
-
headspace gas phase or gas/liquid interface
- L:
-
liquid phase
- lact:
-
lactate
- S:
-
sparged phase or gas/liquid interface
- V:
-
viable
References
Bédard C, Kamen A, Tom R and Massie B (1994) Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch culture. Cytotechnology 15: 129–138.
Bédard C, Tom R and Kamen A (1993) Growth, nutrient consumption, and end-product accumulation in Sf-9 and BTI-EAA insect cell cultures: Insights into growth limitation and metabolims. Biotechnol. Prog. 9: 615–624.
Brown S, Rose PB and Marowitch J (1995) Scale up of production of a recombinant membran-bound protein in an insect cell-baculovirus system to 100L. Baculovirus and Insect Cell Gene Expression Conference, Pinehurst, N.C., March 26–30, 1995.
Caron AW, Archambault J and Massie B (1990) High-level recombinant protein production in bioreactors using the baculovirusinsect cell expression system. Biotechnol. Bioeng. 36: 1133–1140.
Caron AW, Tom RL, Kamen AA and Massie B (1994) Baculovirus expression system scale-up by perfusion of high-density Sf-9 cell cultures. Biotechnol. Bioeng. 43: 881–891.
Ho CS and Shanahan JF (1986) Carbon dioxide transfer in bioreactors. CRC Critical Reviews in Biotechnology 4(2): 185–252.
Jäger V, Kobold A, Köhne C, Deutschmann SM, Grabenhorst E, Karger C and Conradt HS (1994) High density insect cell culture for the production of recombinant proteins with the baculovirus expression system. In: Spier RE, Griffiths JB, Berthold W (eds.) Animal Cell Technology: Products of Today, Prospects of Tomorrow. Butterworth-Heinemann, Oxford.
Junker BH, Hunt G, Burgess B, Aunins J and Buckland BC (1994) Modified microbial fermenter performance in animal cell culture and its implications for flexible fermenter design. Bioprocess Engineering. 11: 57–63.
Kamen AA, Chavarie C, André G and Archambault J (1992) Design parameters and performance of a surface baffled helical ribbon impeller bioreactor for the culture of shear sensitive cells. Chem. Eng. Sci. 47: 2375–2380.
Kamen AA, Bédard C, Tom R, Perret S and Jardin B (1996) On-line monitoring of respiration in recombinant baculovirus-infected and uninfected insect cell bioreactor cultures. Biotechnol. Bioeng. 50: 36–48.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Lazarte JE, Tosi PF and Nicolau C (1992) Optimization of the production of full-length rCD4 in baculovirus-infected Sf-9 cells. Biotechnol. Bioeng. 40: 214–217.
Lindsay DA and Betenbaugh MJ (1992) Quantification of cell culture factors affecting recombinant protein yields in baculovirus-infected insect cells. Biotechnol. Bioeng. 39: 614–618.
Nguyen B, Jarnagin K, Williams S, Chan H and Bamett J (1993) Fed-batch culture of insect cells: a method to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirus expression system. J. Biotechnol. 31: 205–217.
Royce PNC and Thornhill NF (1991) Estimation of dissolved carbon dioxide concentrations in aerobic fermentations. AIChE J. 37: 1680–1686.
Summers MD and Smith GE (1987) A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station Bulletin No. 1555.
Tom RL, Debanne MT, Bédard C, Caron AW, Massie B and Kamen AA (1995) Improved yields of the extracellular domain of the epidermal growth factor receptor produced using the baculovirus expression system by medium replacement following infection. Appl. Microbiol. Biotechnol. 44: 53–58.
Towbin H, Staehelin T and Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350–4354.
Wang MY, Kwong S and Bentley WE (1993) Effects of oxygen/glucose/glutamine feeding on insect cell baculovirus protein expression: a study on epoxide hydrolase production. Biotechnol. Prog. 9: 355–361.
Wilke CR and Chang PC (1955) Correlation of diffusion coefficients in dilute solutions. AIChE J. 1: 264–273.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Garnier, A., Voyer, R., Tom, R. et al. Dissolved carbon dioxide accumulation in a large scale and high density production of TGFβ receptor with baculovirus infected Sf-9 cells. Cytotechnology 22, 53–63 (1996). https://doi.org/10.1007/BF00353924
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00353924