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1

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Material balance studies on animal cell metabolism using a stoichiometrically based reaction network (1996)

Xie, Liangzhi ; Wang, Daniel I. C.

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 52 (1996), S. 579-590

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ISSN: 0006-3592

Keywords: fed batch ; hybridoma ; material balance ; reaction network ; stoichiometric analysis ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: A detailed reaction network of mammalian cell metabolism contains hundreds of enzymatic reactions. By grouping serial reactions into single overall reactions and separating overlapped pathways into independent reactions, the total number of reactions of the network is significantly reduced. This strategy of manipulating the reaction network avoids the manipulations of a large number of reactions otherwise needed to determine the reaction extents. A stoichiometric material balance model is developed based on the stoichiometry of the simplified reaction network. Closures of material balances on glucose and each of the 20 amino acids are achieved using experimental data from three controlled fed-batch and one-batch hybridoma cultures. Results show that the critical role of essential amino acids, except glutamine, is to provide precursors for protein synthesis. The catabolism of some of the essential amino acids, particularly isoleucine and leucine, is observed when an excess amount of these amino acids is available in the culture medium. It was found that the reduction of glutamine utilization (for reducing ammonia production) is accompanied by an increase in the uptake of nonessential amino acids (NAAs) from the culture medium. This suggests that NAAs are necessary even though they are not essential for cell growth. A glutamine balance shows that less than 20% of the glutamine nitrogen is utilized for essential roles, such as protein and nucleotide syntheses. A relatively constant percentage (about 45%) of the glutamine nitrogen is utilized for NAA biosynthesis, despite the fact that the absolute amount varies among the four experiments. As to the carbon skeleton of glutamine, a significant portion enters the tricarboxylic acid (TCA) cycle. A material balance on glucose shows that most of the glucose (81%) is converted into lactate when glucose is in excess. On the other hand, when glucose is limited, lactate production is considerably reduced, while a major portion of glucose (48%) enters the TCA cycle. The fraction of glucose used for the synthesis of cellular components ranges from 9 to 28%. © 1996 John Wiley & Sons, Inc.

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2

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Energy metabolism and ATP balance in animal cell cultivation using a stoichiometrically based reaction network (1996)

Xie, Liangzhi ; Wang, Daniel I. C.

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 52 (1996), S. 591-601

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ISSN: 0006-3592

Keywords: animal cell metabolism ; ATP balance ; energy metabolism ; reaction network ; stoichiometric analysis ; hybridoma ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: A metabolic reaction network is developed for the estimation of the stoichiometric production of adenosine triphosphate (ATP) in animal cell culture. By using the material balance data from fed-batch and batch cultures of hybridoma cells, the stoichiometric ATP productions are determined with estimated effective P/O ratios of 2 for NADH and 1.2 for FADH2. A significant percentage of the ATP requirement (16-41%) in hybridoma cells is generated directly from free energy release without the participation of oxygen. The oxidative phosphorylation of NADH accounts for about 60% of the total ATP production in the fed-batch cultures and about 47% in the batch culture. The oxidative phosphorylation of FADH2 accounts for less then 20% of the total ATP production in all cases.A fractional model is devised to analyze the contribution of each nutrient to the ATP production. Results show that a majority of the ATP is produced from glucose metabolism (60-76%). Less than 30% of the ATP is derived from glutamine, and less than 11% is derived from other essential amino acids. The analysis also shows that the glycolytic pathway generates more ATP in the batch (41%) than in the fed-batch (〈27%) cultures. The TCA cycle provides 51-68% of the total ATP production. The calculated stoichiometric oxygen consumption differs among the batch and fed-batch cultures, depending on the glucose concentration. This result suggests that the relationship between the oxygen uptake rate (OUR) and cell growth may change with the culture conditions. However, the calculated respiratory quotient (RQ) is relatively constant in all cases.A linear relationship is obtained between the specific ATP production rate and the specific cell growth rate. The maximum ATP yield and the maintenance ATP requirement are determined based on this linear relationship. The biosynthetic ATP demand estimated from the dry cell weight and cell composition is significantly lower than that calculated from the maximum ATP yield, indicating that the non-growth-associated ATP demand may contain other factors than what is considered in the estimation of the biosynthetic ATP demand. © 1996 John Wiley & Sons, Inc.

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3

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High cell density and high monoclonal antibody production through medium design and rational control in a bioreactor (1996)

Xie, Liangzhi ; Wang, Daniel I. C.

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 51 (1996), S. 725-729

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ISSN: 0006-3592

Keywords: bioreactor control ; fed batch ; high antibody ; high density ; medium design ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: A simple feeding strategy was developed and successfully employed for nutritional control in a 2-L fed-batch culture of hybridoma cells. A previously developed stoichiometric model for animal cell growth was used to design a supplemental medium for feeding. Undialyzed fetal bovine serum and trace metals (Fe2+, SeO32-, Li+, Zn2+, and Cu2+) were fed to the cells periodically in addition to the automatic feeding of other nutrients in the supplemental medium. In this study, the maximum viable cell density was increased from 6.3 × 106 to 1.7 × 107 cells/mL, and the culture span was extended from 340 to 550 hours. The final monoclonal antibody titer achieved was 2400 mg/L. The specific production rates for ammonia and lactate were further reduced from 0.0045 and 0.0048 in our previous fed-batch experiments to 0.0028 and 0.0036 mmol/109 cell h, respectively. Only 3.4% of the total glucose consumption was converted into lactate, compared to 67% in a conventional batch culture.

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4

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Influence of Primatone RL supplementation on sialylation of recombinant human interferon-γ produced by Chinese hamster ovary cell culture using serum-free media (1997)

Gu, Xuejun ; Xie, Liangzhi ; Harmon, Bryan J. ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 56 (1997), S. 353-360

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ISSN: 0006-3592

Keywords: Primatone RL ; sialylation ; interferon-γ ; serum substitutes ; cell ; CHO cell culture ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Although serum-free media have been widely used in mammalian cell culture for therapeutic protein production, the effects of serum-substitutes on product quality have not been extensively examined. This study observed an adverse effect of Primatone RL, an animal tissue hydrolysate commonly used as a serum-substitute to promote cell growth, on sialylation of interferon-γ (IFN-γ) derived from Chinese hamster ovary (CHO) cell culture in both batch and fed-batch modes. In batch cultures, decreased sialylation was observed at each of the glycosylation sites (i.e., Asn25 and Asn97) of IFN-γ with the use of elevated concentrations of the peptone. Although poorest sialylation was obtained with the use of a growth-inhibiting concentration of Primatone RL, diminished sialylation was observed at the optimal peptone concentration for cell growth and product yield. Since incubation of the product in Primatone RL-supplemented acellular medium did not result in decreased sialylation, the negative effect of Primatone RL could not be attributed to extracellular desialylation of IFN-γ by components of the peptone. In the fed-batch mode, a culture utilizing a serum-free feeding medium supplemented with Primatone RL demonstrated poorer sialylation than a similar culture not fed the peptone. The results of both the batch and fed-batch experiments indicate that the adverse effect of the peptone was not due solely to ammonia accumulation. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 353-360, 1997.

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5

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Gamma-interferon production and quality in stoichiometric fed-batch cultures of Chinese hamster ovary (CHO) cells under serum-free conditions (1997)

Xie, Liangzhi ; Nyberg, Gregg ; Gu, Xuejun ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 56 (1997), S. 577-582

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ISSN: 0006-3592

Keywords: fed-batch ; gamma-Interferon ; CHO cells ; glycosylation ; glucose starvation ; protein quality ; nutritional control ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: The application of a stoichiometric medium design approach was studied in fed-batch cultivation of Chinese hamster ovary (CHO) cells. A serum-free medium containing a very low protein concentration (2 mg/L insulin) was developed. A supplemental medium was formulated according to the stoichiometric equation governing cell growth using cell composition obtained from hybridoma cells. Fed-batch culture was conducted in spinner flasks using the supplemental medium for feeding. Significant improvement in cell growth, by-product reduction, and Gamma-Interferon (IFN-γ) production was achieved as compared to a typical batch culture. Results indicate that the stoichiometric approach, originally developed for hybridoma cultures, is a fast and effective method for cell culture process design and improvement. The glycosylation of IFN-γ was monitored off-line during the culture process. The accumulative IFN-γ glycosylation efficiency was slightly improved as compared to that of the batch culture, due to the nutritional control through the stoichiometric feeding. Periodic glucose starvation was observed during the fed-batch culture as a result of the manual feeding. Pulse-chase radiolabeling assay shows that glucose starvation leads to a deteriorated IFN-γ glycosylation efficiency. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 577-582, 1997.

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6

Electronic Resource

Fed-batch cultivation of animal cells using different medium design concepts and feeding strategies (1994)

Xie, Liangzhi ; Wang, Daniel I. C.

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 43 (1994), S. 1175-1189

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ISSN: 0006-3592

Keywords: fed-batch ; medium design ; animal cell culture ; ammonia ; lactate ; hybridoma ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: In animal cell cultivation, cell density and product concentration are often low due to the accumulation of toxic end-products such as ammonia and lactate and/or the depletion of essential nutrients. A hybridoma cell line (CRL-1606) was cultivated in T-flasks using a newly devised medium feeding strategy. The goals were to decrease ammonia and lactate formation by the design of an initial medium which would provide a starting environment to achieve optimal cell growth. This was followed by using a stoichiometric equation governing animal cell growth and then designing a supplemental medium for feeding strategy used to control the nutritional environment. The relationship between the stoichiometric demands for glutamine and nonessential amino acids was also studied. Through stoichiometric feeding, nutrient concentrations were controlled reasonably well. Consequently, the specific production rate of lactate was decreased by fourfold compared with conventional fed-batch culture and by 26-fold compared with conventional batch culture. The specific production rate of ammonia was decreased by tenfold compared with conventional fed-batch culture and by 50-fold compared with conventional batch culture. Most importantly, total cell density and monoclonal antibody concentration were increased by five- and tenfold respectively, compared with conventional batch culture. © 1994 John Wiley & Sons, Inc.

Additional Material: 10 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/bit.260431123

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7

Electronic Resource

Stoichiometric analysis of animal cell growth and its application in medium design (1994)

Xie, Liangzhi ; Wang, Daniel I. C.

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 43 (1994), S. 1164-1174

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ISSN: 0006-3592

Keywords: stoichiometric analysis ; medium design ; animal cell culture ; feeding strategy ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Animal cell cultivation in vitro has been studied for more than 40 years. However, the culture medium composition has not been designed on the basis of the stoichiometric nutritional demands for animal cell growth. In this article, a model was developed to study the stoichiometric demands for nutrients (including glucose, 20 amino acids, and 10 vitamins)for the synthesis of cell mass and product. The coefficients for these nutrients in the stoichiometric equation governing animal cell growth were determined based on cell composition. In addition, a detailed analysis of the nutrients′ roles in the synthesis of cell mass and product was also performed. Applications of the stoichiometric analysis in animal cell cultivation, such as culture medium design, supplemental medium formulation, and feeding strategy will also be discussed. The stoichiometric analysis can be potentially employed to analyze results from animal cell cultures, to improve the performance of culture processes, and to design new process rationally. It can also help to provide a better understanding of animal cell metabolism. Simplifications on the cellular energy metabolism were made in order to simplify the model and to provide the preliminary bases to test the process performance. However, this could introduce inaccuracies for the model and results in errors in the calculations of glucose and glutamine concentrations when employed in medium design. © 1994 John Wiley & Sons, Inc.

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URL: http://dx.doi.org/10.1002/bit.260431122

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8

Electronic Resource

Applications of improved stoichiometric model in medium design and fed-batch cultivation of animal cells in bioreactor (1994)

Xie, Liangzhi ; Wang, Daniel I. C.

Springer

Cytotechnology 15 (1994), S. 17-29

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ISSN: 1573-0778

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

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology

Notes: 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.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00762376

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