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.
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Abbreviations
- Camm, n, Camm, n−1 :
-
ammonia concentration in the n and (n−1)th samples respectively
- mM; C samm :
-
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
- C sk :
-
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
- 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
- θ cmala , θ cmasn , θ cmasp , θ cmglc , θ cmglu , θ cmgln , θ cmgly , θ cmpro , θ cmser :
-
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
- θ englc :
-
stoichiometric coefficient of glucose in energy metabolism, mmol cell−1
- θi :
-
stoichiometric coefficient of nutrient, mmol cell−1
- θ cmi :
-
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
- cm:
-
cell mass (include product) syntheses
- en:
-
energy metabolism
- s:
-
supplemental medium
- 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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Xie, L., Wang, D.I.C. Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor. Cytotechnology 15, 17–29 (1994). https://doi.org/10.1007/BF00762376
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DOI: https://doi.org/10.1007/BF00762376