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Cytotechnology

, Volume 15, Issue 1–3, pp 17–29 | Cite as

Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor

  • Liangzhi Xie
  • Daniel I. C. Wang
Article

Abstract

In our previous work (Xie and Wang, 1994a), a simplified stoichiometric model on energy metabolism for animal cell cultivation was developed. Fed-batch experiments were performed in T-flasks using this model in supplemental medium design (Xie and Wang, 1994b). In this work, the major pathways of glucose and glutamine metabolism were incorporated into the stoichiometric model. Fed-batch culture was conducted in a 2-liter bioreactor with appropriate process control strategies. Nutrient concentrations, especially glucose and glutamine, were maintained at constant but low levels through the automated feeding of a supplemental medium formulated using the improved stoichiometric model. The formation of toxic byproducts, such as ammonia and lactate (Hassellet al., 1991), was greatly reduced. The specific lactate production rate was decreased by 62-fold compared with batch culture in bioreactor and by 8-fold compared to fed-batch culture in T-flask using the previous stoichiometric model. Ammonia formation was also decreased compared with both the batch and fed-batch cultures. Most importantly, the monoclonal antibody concentration reached 900 mg l−1, an increase of 17- and 1.6-fold compared with the batch and fed-batch cultures respectively.

Key words

Ammonia animal cell bioreactor fed-batch culture feeding strategy lactate medium design 

Abbreviations

Camm, n, Camm, n−1

ammonia concentration in the n and (n−1)th samples respectively

mM; Camms

ammonia concentration in the supplemental medium, mM

CAb, n, CAb, n−1

antibody concentration in the n and (n−1)th samples respectively, mg l−1

Ci

concentration of the ith nutrient in the supplemental medium, mM

Clac, n, Clac, n−1

lactate concentration in the n and (n−1)th samples respectively, mM

Cks

concentration of glucose or glutamine in the supplemental medium, mM

Ck, n, Ck, n−1

concentration of glucose or glutamine in the n or(n−1)th sample respectively

mM; Ct

total concentration of glucose, amino acids, and vitamins in the supplemental medium, mM

F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12

fluxes described in Fig. 1, mmole cell−1

m

total number of samples

Nt

total cell number in the reactor at culture time t, number of cells

Nt, o

total cell number in the reactor at the beginning of culture, number of cells

Nt, n, Nt, n−1

total cells in the reactor when the n and (n−1)th samples were taken, respectively, number of cells

ΔNt, n, ΔNt, m

total cells produced since the initiation when the nth sample and the mth sample were taken, respectiveky, number of cells

Nv

viable cell number in the reactor at culture time t, number of cells

Nv, n, Nv, n−1

viable cells in the reactor when the n and (n−1)th samples were taken, respectively, number of cells

PAb, n

monoclonal antibody produced between the n and (n−1) samples, mg

Pamm, n

ammonia produced between the n and (n−1) samples, mmole

Pi, n

amount of lactate or ammonia produced between the n and (n−1)th samples, mmole

Plac, n

lactate produced between the n and (n−1) samples, mmole

P/O

number of ATP molecules generated per NADH molecule oxidized

qi

specific production rate of ammonia, lactate, or antibody respectively, mmole cell−1 h−1 (for ammonia and lactate), and mg cell−1 h−1 (for antibody)

t

culture time, h

tm

culture time when the mth sample (last sample) was taken, h

tn, tn−1

culture time when the n and (n−1)th samples were taken respectively, h

ΔVF

volume of supplemental medium fed to the reactor since the nth sample was taken, l

ΔVV, n

volume fed to the reactor between the (n−1)th and nth samples, l

Vn, Vn−1

total volume in the reactor after the nth or (n−1)th sample were taken respectively, l

Vs, j

volume of the jth sample, l

Vs, n, Vs, n−1

volume of the n and (n−1)th samples respectively, l

Xt, j

total cell density in the jth sample, cells l−1

Xt, n

total cell density of the nth sample, cells l−1

Xv, n, Xv, n−1

viable cell density in the n and (n−1)th samples respectively, cells l−1

Yamm/gln

ratio of ammonia production to glucose consumption, mmol mmol−1

Yi/cell

ratio of the total ammonia or lactate production to the total production of cells, mmole cell−1

Ylac/glc

ratio of lactate production to glucose consumption, mmol mmol−1

Greek letters

α

specific death rate, h−1

β

total stoichiometric coefficient of glucose, amino acids, and vitamins, mmole cell−1

δglc, n, δgln, n

Amount of glucose or glutamine crespectively, consumed between the n and the (n−1)th sample, mmole

δk, n

Amount of glucose or glutamine consumed between the n and the (n−1)th sample, mmole

μ

specific growth rate, h−1

θala, θasn, θasp, θglc, θglu, θgln, θgly, θpro, θser

stoichiometric coefficient of alanine, asparagine, aspartate, glucose, glutamate, glutamine, glycine, proline, and serine respectively including energy metabolism and biosynthesis of nonessential amino acids, mmole cell−1

θalacm, θasncm, θaspcm, θglccm, θglucm, θglncm, θglycm, θprocm, θsercm

stoichiometric coefficient of alanine, asparagine, aspartate, glucose, glutamate, glutamine, glycine, proline, and serine respectively in the biosyntheses of cell mass and product, excluding energy metabolism and synthesis from glutamine, mmole cell−1

θATP

stoichiometric coefficient for ATP in syntheses of cell mass and product, mmole cell−1

θglcen

stoichiometric coefficient of glucose in energy metabolism, mmol cell−1

θi

stoichiometric coefficient of nutrient, mmol cell−1

θicm

stoichiometric coefficient of nutrient in cell mass and product formation without consideration of consumption in energy formation and the production of nonessential amino acids from glutamine, mmol cell−1

τ

integration of viable cells over culture time, cells h

Superscripts

cm

cell mass (include product) syntheses

en

energy metabolism

s

supplemental medium

Subscripts

Ab

antibody

ala

alanine

amm

ammonia

asn

asparagine

asp

aspartate

F

feeding

glc

glucose

gln

glutamine

glu

glutamate

gly

glycine

i

index for nutrients, index for ammonia or lactate

j

index of sample

k

stands for glucose or glutamine

lac

lactate

m

total number of sample

n

index of sample

p

product

pro

proline

ser

serine

t

time or total cells

v

vitamin or viable cells

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References

  1. Adamson SR, Fitzpatrick SL and Behie LA (1983) In vitro production of high titer monoclonal antibody by hybridoma cells in dialysis culture. Biotechnol. Lett. 5: 573–578.Google Scholar
  2. Bligh EG and Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911–917.Google Scholar
  3. Fike R, Kubiak J, Price P and Jayme D (1993) Feeding strategies for enhanced hybridoma productivity: automated concentrate supplementation. BioPharm. 6: 49–54.Google Scholar
  4. Folch J, Lees M and Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497–509.Google Scholar
  5. Gerschenson LE, Mead JF, Harary I and Haggerty Jr. DF (1967) Studies on the essential fatty acids on growth rate, fatty acid composition, oxidative phosphorylation and respiratory control of HeLa cells in culture. Biochim. Biophys. Acta 131: 42–49.Google Scholar
  6. Glasken MW, Huang C and Sinskey AJ (1989) Mathematical descriptions of hybridoma culture kinetics. III. simulation of fedbatch bioreactors. J. Biotechnol. 10: 39–66.Google Scholar
  7. Glacken MW (1987) Development of mathematical descriptions of mammalian cell culture kinetics for the optimization of fed-batch bioreactors. Ph. D. thesis, MIT, Cambridge, MA, USAGoogle Scholar
  8. Hanson RS and Philips JA (1981) Chemical Composition. In: Gerhardt P et al. (eds.). Manual of Methods for General Bacteriology (pp. 329–364) American Society for Microbiology, Washington, D.C.Google Scholar
  9. Hassell T, Gleave S and Butler M (1991) Growth inhibition in animal cell culture: The effect of lactate and ammonia. Applied Biochem. Biotechnol. 30: 29–41.Google Scholar
  10. Hu WS, Dodge TC, Frame KK and Himes VB (1987) Effect of glucose on the cultivation of mammalian cells. Develop. Biol. Stand. 66: 279–290.Google Scholar
  11. Linardos TI, Kalogerakis N and Behie LA (1991) The effect of specific growth rate and death rate on monoclonal antibody production in hybridoma chemostat cultures. Can. J. Chem. Eng. 69: 429–438.Google Scholar
  12. Linardos TI, Kalogerakis N and Behie LA (1992) Monoclonal antibody production in dialyzed continuous suspension culture. Biotechnol. Bioeng. 39: 504–510.Google Scholar
  13. Lindell PI (1992) Dynamic operation of mammalian cell fed-batch bioreactors. Ph.D. thesis, MIT, Cambridge, MA, USA.Google Scholar
  14. Luan YT, Mutharasan R and Magee WE (1987) Strategies to extend the longevity of hybridomas in culture and promote yield of monoclonal antibodies. Biotechnol. Lett. 9: 691–696.Google Scholar
  15. Nelson GJ (1975) Isolation and purification of lipids from animal tissues. In: Perkins EG (ed.) Analysis of lipids and lipoproteins (pp. 1–22) American Oil Chemists Society, Champaign, Illinois.Google Scholar
  16. Oh SKW, Vig P, Chua F, Teo WK Yap MGS (1993) Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate. Biotechnol. Bioeng. 42: 601–610.Google Scholar
  17. Ozturk S.S and Palsson BO (1991) Effect of medium osmolarity on hybridoma growth, metabolism, and antibody production. Biotechnol. Bioeng. 37: 989–993.Google Scholar
  18. Packer L (1967) Experiments in cell physiology. Academic press, New York.Google Scholar
  19. Pendse GJ and Bailey JE (1990) Effects of growth factors on cell proliferation and monoclonal antibody production of batch hybridoma cultures. Biotechnol. Lett. 12: 487–492.Google Scholar
  20. Read SM (1984) Techniques in proteins and enzyme biochemistry, part I supplement: techniques for determining protein concentration. In: Tipton KF (ed.) Techniques in the life sciences. (pp. 1–34) Elsevier Scientific, New York.Google Scholar
  21. Stein J and Smith G (1982) Techniques in lipid and membrane biochemistry, part I: extraction methods. In: Hesketh TR et al. (eds.) Techniques in the life sciences. (pp. 1–10) Elsevier/North-Holland Scientific, New York.Google Scholar
  22. Xie L and Wang DIC (1994a) Stoichiometric analysis of animal cell growth and its application of medium design. Biotechnol. Bioeng. 43: 1164–1174.Google Scholar
  23. Xie L and Wang DIC (1994b) Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies. Biotechnol. Bioeng. 43: 1175–1189.Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Liangzhi Xie
    • 1
  • Daniel I. C. Wang
    • 1
  1. 1.Department of Chemical Engineering, Biotechnology Process Engineering CenterMassachusetts Institute of TechnologyCambridgeUSA

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