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

Xie, Liangzhi; Wang, Daniel I. C.

doi:10.1007/bf00762376

Cited by 111 publications

(94 citation statements)

References 14 publications

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“…The total concentrations of arginine, lysine and cysteine present in the cell were assumed 70.5 lmol ml-packedcell À1 , 105 lmol ml-packed-cell À1 and 39.74 lmol ml-packed-cell À1 , respectively. The concentration of each amino acid was determined from the overall amino acid composition of proteins in animal cells (Xie and Wang 1994). To determine the cysteine residue concentration, the ratio of cysteine to cystine residues (11.5:1) was taken to be the average of the reported ratios for 85 enzymes and other proteins (Jocelyn 1977).…”

Section: Model 2: Methylglyoxal Interactions With Proteins In Vivomentioning

confidence: 99%

Analysis of Methylglyoxal Metabolism in CHO Cells Grown in Culture

Kingkeohoi

Chaplen

2005

Cytotechnology

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Recent evidence suggests that several unknown or ill-characterized factors strongly influence cell growth and function in culture. Isolating these factors is necessary in order to maximize culture productivities. Methylglyoxal (MG), a potent protein and nucleic acid modifying agent, has been identified as a player in the signaling pathways associated with cell death and is known to be detrimental to cultured cells. This compound is produced in all mammalian systems by spontaneous phosphate elimination from glycolytic pathway intermediates. A kinetic model that qualitatively describes the cellular distribution of proteinassociated MG in the absence of enzymatic adduct formation predicted far lower levels of reversibly bound MG than measured in cultured CHO cells. This suggests that the targeted modification of proteins through enzymatically mediated mechanisms is a significant sink for cellular methylglyoxal. The model was validated with measurements of carbon flux through the glyoxalase pathway to D-lactic acid, a unique end product of MG metabolism in mammalian systems. Fluxes to D-lactic acid of up to 16.8 mmol ml-packed cells À1 day À1 were measured with CHO cells grown in batch culture or 100-fold more than found in normal tissues.

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Section: Model 2: Methylglyoxal Interactions With Proteins In Vivomentioning

confidence: 99%

Analysis of Methylglyoxal Metabolism in CHO Cells Grown in Culture

Kingkeohoi

Chaplen

2005

Cytotechnology

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“…We conducted fed-batch and batch cultivations in 1-L bioreactors under appropriate conditions. Fed batch cultivations were conducted with initial medium containing Substitute (standard concentration) or Substitute (high concentration) instead of serum and feeding medium which was designed by Xie and Wang (1994). In batch cultivations, Iscove's Modified Dulbecco's medium (IMDM) containing 5% fetal bovine serum was used as a conventional cultivation, while IBL Media III was used in order to compare with serum-free fed-batch cultivation.…”

Section: Resultsmentioning

confidence: 99%

“…In the serum-free fed-batch cultivation we prepared the culture medium according to the method of Xie and Wang (1994). The culture medium consisted of an initial medium and a feeding medium.…”

Section: Cell Line and Culture Mediummentioning

confidence: 99%

“…Fed-batch cultivations with Substitute (standard concentration) and Substitute (high concentration) were done with IMDM based initial medium containing Substitute (standard concentration) or Substitute (high concentration) instead of serum and feeding medium. The feeding medium was designed by Xie and Wang (1994) Cytotechnology (2008) 57:187-197 193 The specific formation rate of ammonia (the slope of the line) varied depending on the stage of a culture but significant differences were not seen between fedbatch cultivations with Substitute (high concentration) and Substitute (standard concentration) nor between batch cultivation with serum and batch cultivation with Substitute (standard concentration). The slope of the line for fed-batch cultivations was lower than that of batch cultivations at every IVC value.…”

Section: Cell Growthmentioning

confidence: 99%

Section: Comparison Of Culture Performancementioning

confidence: 99%

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A serum substitute for fed-batch culturing of hybridoma cells

2008

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We developed a substitute for serum to produce fed-batch cultures of hybridoma cells in serum-free medium and confirmed that the cells could be successfully cultivated this way. Our substitute consisted of 12 components. The specific production rates of lactate and ammonia, which are harmful byproducts from the cells, were significantly reduced compared with a conventional serum-containing batch culture. This reduction led to a higher cell concentration and a longer production lifetime. As a result, the final concentration of monoclonal antibody was 400 mg/L, or five times greater than that in the conventional serum-containing batch culture. The developed substitute is expected to enable fed-batch cultivation in a serum-free condition.

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Metabolic flux analysis of hybridoma cells in different culture media using mass balances

Bonarius

Hatzimanikatis

Meesters

et al. 1996

Biotechnol. Bioeng.

242

174

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The estimation of the intracellular fluxes of mammalian cells using only the mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. Either additional experimental flux data or additional theoretical constraints are required to find one unique flux distribution out of the solution space that is bound by the mass balances. Here, a method is developed using the latter approach. The uptake and production rates of amino acids, glucose, lactate, O2, CO2, NH4, MAB, and the intracellular amino acid pools have been determined for two different steady‐states. The cellular composition {total protein and protein composition, total lipids and fatty acid distribution, total carbohydrates, DNA and RNA} has been measured to calculate the requirements for biosynthesis. It is shown to be essential to determine the uptake/production rates of ammonia and either carbon dioxide or oxygen. In mammalian cells these are cometabolites of cyclic metabolic pathways. The flux distribution that is found using the Euclidean minimum norm as the additional theoretical constraint and taking either the CO2 or the NAD(P)H mass balance into account is shown to be in agreement with the measured O2 and CO2 metabolic rates. The metabolic fluxes in hybridoma cells in continuous culture at a specific growth rate of 0.83 day−1 are estimated for a medium with (optimal medium) and without (suboptimal medium) Primatone RL, an enzymatic hydrolysate of animal tissue that causes a more than twofold increase in cell density. It is concluded that The majority of the consumed glucose (>90%) is channeled through the pentose‐phosphate pathway in rapidly proliferating cells. Pyruvate oxidation and tricarboxylic acid (TCA) cycle activity are relatively low, i.e., 8% of the glucose uptake in suboptimal and 14% in optimal medium, respectively. Under both conditions, only a small fraction of pyruvate is further oxidized to CO2. The flux from glutamate to α‐ketoglutarate (catalyzed by glutamate dehydrogenase) is almost zero in medium with and even slightly reversed in medium without Primatone RL. Almost all glutamate enters the TCA cycle due to the action of transaminases. Transhydrogenation plays a significant role in hybridoma cells under our experimental conditions. NADPH is produced at relatively high rates (11 × 10−12 to 13 × 10−12 mol · cell−1 · day−1) compared to other fluxes in both culture media. © 1996 John Wiley & Sons, Inc.

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

Cited by 111 publications

References 14 publications

Analysis of Methylglyoxal Metabolism in CHO Cells Grown in Culture

Analysis of Methylglyoxal Metabolism in CHO Cells Grown in Culture

A serum substitute for fed-batch culturing of hybridoma cells

Metabolic flux analysis of hybridoma cells in different culture media using mass balances

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