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Daniel I. C. Wang - One of the best experts on this subject based on the ideXlab platform.

  • material balance studies on Animal Cell metabolism using a stoichiometrically based reaction network
    Biotechnology and Bioengineering, 2000
    Co-Authors: Liangzhi Xie, Daniel I. C. Wang
    Abstract:

    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%.

  • energy metabolism and atp balance in Animal Cell cultivation using a stoichiometrically based reaction network
    Biotechnology and Bioengineering, 2000
    Co-Authors: Liangzhi Xie, Daniel I. C. Wang
    Abstract:

    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 FADH(2). 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 FADH(2) 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.

  • stoichiometric analysis of Animal Cell growth and its application in medium design
    Biotechnology and Bioengineering, 1994
    Co-Authors: Liangzhi Xie, Daniel I. C. Wang
    Abstract:

    Abstract 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. (c) 1994 John Wiley & Sons, Inc.

  • A fiber-bed bioreactor for anchorage-dependent Animal Cell cultures: part I. Bioreactor design and operations.
    Biotechnology and bioengineering, 1991
    Co-Authors: Tzyy-wen Chiou, Sei Murakami, Daniel I. C. Wang
    Abstract:

    A concentric-cylinder airlift reactor, in which the annulus is a packed bed of glass fibers, has been developed in order to facilitate the scaleup and enhance the volumetric productivity of anchorage-dependent Animal Cell cultures. In this bio-reactor, oxygen-containing gas is sparged through the inner draft tube, causing bubble-free medium to flow through the fiber bed in the outer cylinder and providing both oxygenation and convective nutrient transfer to the Cells. Several other desirable features for reactor operation are also provided by this design. Cell cultivations in this bioreactor have been successfully carried out and provide data for the feasibility of the large-scale Cell cultivation.

Liangzhi Xie - One of the best experts on this subject based on the ideXlab platform.

  • energy metabolism and atp balance in Animal Cell cultivation using a stoichiometrically based reaction network
    Biotechnology and Bioengineering, 2000
    Co-Authors: Liangzhi Xie, Daniel I. C. Wang
    Abstract:

    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 FADH(2). 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 FADH(2) 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.

  • material balance studies on Animal Cell metabolism using a stoichiometrically based reaction network
    Biotechnology and Bioengineering, 2000
    Co-Authors: Liangzhi Xie, Daniel I. C. Wang
    Abstract:

    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%.

  • stoichiometric analysis of Animal Cell growth and its application in medium design
    Biotechnology and Bioengineering, 1994
    Co-Authors: Liangzhi Xie, Daniel I. C. Wang
    Abstract:

    Abstract 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. (c) 1994 John Wiley & Sons, Inc.

Henrik Garoff - One of the best experts on this subject based on the ideXlab platform.

  • a new generation of Animal Cell expression vectors based on the semliki forest virus replicon
    Nature Biotechnology, 1991
    Co-Authors: Peter Liljeström, Henrik Garoff
    Abstract:

    We have developed a novel DNA expression system, based on the Semliki Forest virus (SFV) replicon, which combines a wide choice of Animal Cell hosts, high efficiency and ease of use. DNA of interest is cloned into SFV plasmid vectors that serve as templates for in vitro synthesis of recombinant RNA. The RNA is transfected with virtually 100% efficiency into Animal tissue culture Cells by means of electroporation. Within the Cell, the recombinant RNA drives its own replication and capping and leads to massive production of the heterologous protein while competing out the host protein synthesis. The expression system also includes an in vivo packaging procedure whereby recombinant RNA is packaged into infectious virus particles using cotransfection with packaging–deficient helper RNA molecules. The resulting high titer recombinant virus stock can be used to infect a wide range of Animal Cells with subsequent high expression of the heterologous gene product, but without expression of any structural proteins of the helper. The infected Cells produce protein for up to 75 hours post infection after which the heterologous product can constitute as much as 25% of the total Cell protein. The general utility of the system is demonstrated through the expression of human transferrin receptor, mouse dihydrofolate reductase, chick lysozyme and Escherichia coli β–galactosidase.

  • a new generation of Animal Cell expression vectors based on the semliki forest virus replicon
    Nature Biotechnology, 1991
    Co-Authors: Peter Liljeström, Henrik Garoff
    Abstract:

    We have developed a novel DNA expression system, based on the Semliki Forest virus (SFV) replicon, which combines a wide choice of Animal Cell hosts, high efficiency and ease of use. DNA of interest is cloned into SFV plasmid vectors that serve as templates for in vitro synthesis of recombinant RNA. The RNA is transfected with virtually 100% efficiency into Animal tissue culture Cells by means of electroporation. Within the Cell, the recombinant RNA drives its own replication and capping and leads to massive production of the heterologous protein while competing out the host protein synthesis. The expression system also includes an in vivo packaging procedure whereby recombinant RNA is packaged into infectious virus particles using cotransfection with packaging-deficient helper RNA molecules. The resulting high titer recombinant virus stock can be used to infect a wide range of Animal Cells with subsequent high expression of the heterologous gene product, but without expression of any structural proteins of the helper. The infected Cells produce protein for up to 75 hours post infection after which the heterologous product can constitute as much as 25% of the total Cell protein. The general utility of the system is demonstrated through the expression of human transferrin receptor, mouse dihydrofolate reductase, chick lysozyme and Escherichia coli beta-galactosidase.

I W Marison - One of the best experts on this subject based on the ideXlab platform.

  • monitoring of temperature effects on Animal Cell metabolism in a packed bed process
    Biotechnology and Bioengineering, 2002
    Co-Authors: P Ducommun, P A Ruffieux, A Kadouri, U Von Stockar, I W Marison
    Abstract:

    Animal Cell (Chinese Hamster Ovary) concn. was detd. online in a packed bed process using dielec. spectroscopy. This enabled the evaluation of the effect of temp. on specific metabolic rates during 3 mo of continuous culture. The effect of low cultivation temp. on Cell growth and metab. was monitored, and the data were used for process development. At 37 DegC Cells grew exponentially with a specific growth rate of 0.038 d-1 and specific glucose uptake and lactate prodn. rates increased continually. Redn. of the temp. to 33.5 DegC resulted in a lowering of these metabolic rates while having no effect on Cell proliferation. Subsequent redn. of the temp. to 32 DegC resulted in stabilization of the Cell concn. at a high d. (3.6 * 107 Cell per mL of packed bed). In addn., the specific prodn. rate of the protein of interest increased by a factor of 6 compared to the value at 37 DegC. During the stationary phase at 32 DegC, all other specific metabolic rates could be controlled to low and const. levels. [on SciFinder (R)]

  • a new method for on line measurement of the volumetric oxygen uptake rate in membrane aerated Animal Cell cultures
    Journal of Biotechnology, 2000
    Co-Authors: P Ducommun, P A Ruffieux, I W Marison, Mariapilar Furter, Urs Von Stockar
    Abstract:

    Oxygen is a key substrate in Animal Cell metab. and its consumption is thus a parameter of great interest for bioprocess monitoring and control. A system for measuring it based on an oxygen balance on the liq. phase was developed. The use of a gas-permeable membrane offered the possibility to provide the required quantity of oxygen into the culture, while avoiding problems of foaming or shear stress generally linked to sparging. This aeration system allowed moreover to keep a known and const. kLa value through cultures up to 400 h. Oxygen uptake rate (OUR) was measured online with a very good accuracy of +-5%, and the specific OUR for a CHO Cell line was detd. during batch (growth phase) and continuous culture as, resp., equal to 2.85*10-13 and 2.54*10-13 mol O2 Cell-1 h-1. It was also shown that OUR continuous monitoring gives actually more information about the metabolic state of the culture than the Cell concn. itself, esp. during transition phases like the end of the growth phase in a batch culture. [on SciFinder (R)]

  • measurement of volumetric our and determination of specific qo2 oxygen uptake rates in Animal Cell cultures
    Journal of Biotechnology, 1998
    Co-Authors: P A Ruffieux, Urs Von Stockar, I W Marison
    Abstract:

    Oxygen is a key substrate in Animal Cell metabolism. It has been reported that the oxygen uptake rate (OUR) is a good indicator of Cellular activity, and even under some conditions, a good indicator of the number of viable Cells. The measurement of OUR is difficult due to many different reasons. In particular, the very low specific consumption rate (0.2 x 10(-12) mol Cell h-1), the sensitivity of the Cells to variations in dissolved oxygen concentration and the difficulty to provide oxygen without damaging the Cells are problems which must be taken into account for the development of OUR measurement methods. Different solutions based on an oxygen balance on either the liquid phase or around the entire reactor, and with a variable or stable concentration of dissolved oxygen have been reported. The accuracy of the OUR measurements and the required analytical devices are very different from method to method.

G W Barton - One of the best experts on this subject based on the ideXlab platform.

  • a structured dynamic model for Animal Cell culture systems application to murine hybridoma
    Biochemical Engineering Journal, 1999
    Co-Authors: C S Sanderson, Jae Deog Jang, J P Barford, G W Barton
    Abstract:

    Abstract A detailed, structured Animal Cell model has a number of interesting potential applications. In this paper, a structured model will be `tuned' to data for two different murine hybridoma Cell lines, 2HG11 and AFP27. With an appropriate set of parameters, the model was able to describe the behaviour of both these cultures accurately. In order to demonstrate that the model could predict the behaviour of AFP27 Cells under different conditions, a further AFP27 experiment was performed and its results compared to model predictions. The model was capable of matching the behaviour of this culture using the parameters derived from the earlier data set. Differences between the predictions and the experimental data suggest regions in which either the model needs to be strengthened or in which the experimental data is questionable.

  • a structured dynamic model for Animal Cell culture application to baculovirus insect Cell systems
    Biochemical Engineering Journal, 1999
    Co-Authors: C S Sanderson, J P Barford, G W Barton, T K K Wong, Steven Reid
    Abstract:

    Viral infection can have significant effects on the behaviour of Cell cultures and the interaction of baculoviruses with insect Cells is currently of particular interest. This paper examines a number of existing mathematical models for this phenomenon and describes the development of an extension to a structured model for Cellular metabolism. This extension is capable of describing the effects of multiple and secondary infection, the actions of defective interfering particles and the release of viral products through Cell lysis, as well as considering the metabolic effects of infection. The basic model is applied to uninfected SF9 cultures, demonstrating that, with a suitable adjustment of its parameters, a model that successfully described hybridoma Cells can also be used for insect Cells. The extended model is applied to an infected culture and describes the infection process well.