Real-time monitoring and control of glucose and lactate concentrations in a mammalian cell perfusion reactor

On-line monitoring and control of cell culture fermentation is important for optimal and consistent production of biologicals. In this work, glucose and lactate concentrations are monitored on-line using a commercially available analyzer (Model 2700, Yellow Springs Instruments, Yellow Springs, OH) during batch and perfusion hybridoma cell fermentation. Cell free samples from the reactor are obtained using a 0.45 mum hollow fiber filtering system placed in a circulation loop. The samples were analyzed at specified times and the data are collected on a computer. A process control strategy was developed to control the concentrations of glucose and lactate in a perfusion reactor where the feed rate is adjusted to maintain their concentrations at desired set points. Hybridoma cells (A10G10) were cultivated in a high density perfusion culture where cell density increased from 2 to 14 million cells/mL. During this period the control algorithm successfully adjusted the perfusion rate while maintaining constant glucose and lactate concentrations. Glucose consumption and lactate accumulation rates as well as net lactate yield on glucose were monitored continuously during perfusion culture. These metabolic rates were observed to be independent of cell concentration and were used for the estimation of viable cell density in the reactor. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 372-378, 1997.     

流体动力对HEK293细胞的细胞团形成及细胞生长和代谢的影响

利用HEK293细胞在悬浮培养中具有聚集成团的体外培养特性,在250mL的Bellco的搅拌培养体系中,以HEK293细胞团的粒径、细胞数、细胞活力、葡萄糖比消耗率(qglc)、乳酸比产率(qlac)和乳酸转化率(Ylacglc)为观察指标,考察HEK293细胞在搅拌速度分别设置为25、50、75和100rmin的培养条件下的细胞团形成、粒径分布以及细胞生长和代谢。HEK293细胞在搅拌速度为50rmin和75rmin培养条件下所形成细胞团的粒径大小适中、离散度小。培养7d后,HEK293细胞团的平均粒径分别为201μm和175μm,其中粒径≥225μm的细胞团所占比例均低于10%;在整个培养过程中,细胞团中的HEK293细胞活力维持在90%以上,qglc、qlac和Ylacglc等反映HEK293细胞代谢的参数保持相对恒定。实验结果提示:合适的搅拌速度所产生的流体动力既可使细胞团的粒径控制在合适的范围内,也可为细胞团中的HEK293细胞提供基本满足其正常生长和代谢需要的物质传递效率。     

Acetate accumulation and regulation by process parameters control in Chinese hamster ovary cell culture

Chinese hamster ovary (CHO) cells represent a group of predominantly used mammalian hosts for producing recombinant therapeutic proteins. Known for their rapid proliferation rates, CHO cells undergo aerobic glycolysis that is characterized by fast glucose consumption, that ultimately gives rise to a group of small-molecule organic acids. However, only the function of lactate has been extensively studied in CHO cell culture. In this study, we observed the accumulation of acetate from the late exponential phase to harvest day, potentially contributing to the pH decline in late culture stage regardless of lactate consumption. In addition, we evaluated the acidification of the fresh media and the cell culture suspension, and the data revealed that acetate presented a lower acidification capacity compared to lactate and exhibited limited inhibitory effect on cells with less than 20 mM supplemented in the media. This study also explored the ways to control acetate accumulation in CHO cell culture by manipulating the process parameters such as temperature, glucose, and pH control. The positive correlation between the specific glucose consumption rate and acetate generation rate provides evidence of the endogenous acetate generation from overflow metabolism. Reducing these parameters (temperature, glucose consumption) and HCl-controlled low pH ultimately suppress acetate build-up. In addition, the specific acetate generation rate and relevant glucose consumption rate are found to be a metabolic trait associated with specific cell lines. Taken together, the results presented in these experiments provide a means to advance industrial CHO cell culture process control and development.     

Development of a fed-batch cultivation for antibody-producing cells based on combined feeding strategy of glucose and galactose

In order to achieve enhanced cell mass and productivity with less lactate accumulation, a fed-batch culture based on a combined feeding strategy of glucose and galactose was developed. Cell performance was first examined with feeding of galactose alone. While cell growth was improved compared with glucose-feeding culture, cell maintenance was inefficient with rapid lactate depletion and considerable ammonium accumulation. Subsequently, to improve cell maintenance, a combined feeding strategy of glucose and galactose was proposed focusing on optimizing the ratio of glucose to galactose and feeding time. In addition, the compositions of amino acids and vitamins in feeding medium were refined for balanced supply of nutrients. With the combined feeding strategy, the metabolic shift of lactate from production to consumption occurred, but not accompanied by rapid lactate depletion and ammonium production. Furthermore, energy metabolism was more efficient and better utilization of carbon sources was achieved. Compared with the glucose-feeding culture in bioreactor, maximum lactate concentration was reduced by 55%; IVCC and the specific production rate of antibody were increased by 45% and 143%, respectively.     

Adaptive control at low glucose concentration of HEK-293 cell serum-free cultures.

Fed-batch cultures were implemented to study the metabolism of HEK-293 cells. Glucose, measured every 30 min by a FIA biosensor system, was maintained at 1 mM throughout the culture using an adaptive nonlinear controller based on minimal process modeling. The controller performed satisfactorily at both low and high cell concentrations without the need for retuning between different culture phases. Overall, lactate production was significantly reduced by maintaining a low glucose concentration, thus decreasing the rate of glycolysis. The rates of glucose and glutamine uptake as well as the lactate and ammonia production were compared to those obtained in batch mode with an initial glucose concentration of 21 mM. Basically, three phases were observed in both culture modes. The metabolic shift from the first to the second phase was characterized by a significant reduction in glucose consumption and lactate production while maximum growth rate was maintained. The specific respiration rate appeared unchanged during the first two phases, suggesting that no change occurred in the oxidative pathway capacity. In the third phase, cell growth became slower very likely due to glutamine limitation.     

Catabolic control of hybridoma cells by glucose and glutamine limited fed batch cultures

Substrate limited fed batch cultures were used to study growth and overflow metabolism in hybridoma cells. A glucose limited fed batch, a glutamine limited fed batch, and a combined glucose and glutamine limited red batch culture were compared with batch cultures. In all cultures mu reaches its maximum early during growth and decreases thereafter so that no exponential growth and decreases thereafter so that no exponential growth rate limiting, although the glutamine concentration (>0.085mM) was lower than reported K(s) vales and glucose was below 0.9mM; but some other nutrients (s) was the cause as verified by simulations. Slightly more cells and antibodies were produced in the combined fed batch compared with the batch culture. The specific rates for consumption of glucose and glutamine were dramatically influenced in fed batch cultures resulting in major metabolic changes. Glucose limitation decreased lactate formation, but increased glutamine consumption and ammonium formation. Glutamine limitation decreased ammonium and alanine formation of lactate, alanine, and ammonium was negligible in the dual-substrate limited fed batch culture. The efficiency of the energy metabolism increased, as judged by the increase in the cellular yield coefficient for glucose by 100% and for glutamine by 150% and by the change in the metabolic ratios lac/glc, ala/ln, and NH(x)/ln, in the combined fed culture. The data indicate that a larger proportion of consumed glutamine enters the TCA cycle through the glutamate dehydrogenase pathway, which releases more energy from glutamine than the transamination pathway. We suggest that the main reasons for these changes are decreased uptake rates of glucose and glutamine, which in turn lead to a reduction of the pyruvate pool and a restriction of the flux through glutaminase and lactate dehydrogenase. There appears to be potential for further cell growth in the dual-substrate-limited fed batch culture as judged by a comparison of mu in the different cultures. (c) 1994 John Wiley & Sons, Inc.     

Real-time method for determining the colony-forming cell content of human hematopoietic cell cultures

Glucose and lactate metabolic rates were evaluated for cultures of cord blood (CB) mononuclear cell (MNC), peripheral blood (PB) MNC, and PB CD34(+) cell cultures carried out in spinner flasks and in T-flasks in both serum-containing and serum-free media. Specific glucose uptake rates (q(gluc), in micromoles per cell per hour) and lactate generation rates (q(lac)) correlated with the percentage of colony-forming cells (CFC) present in the culture for a broad range of culture conditions. Specifically, the time of maximum CFC percentage in each culture coincided with the time of maximum q(gluc) and q(lac) in cultures with different seeding densities and cytokine combinations. A two-population model (Q(lac) = alpha[CFC] + beta([TC] - [CFC ]), where [TC] is total cell concentration; Q(lac) is volumetric lactate production rate in micromoles per milliliter per hour; alpha is q(lac) for an average CFC; and beta is q(lac) for an average non-CFC) was developed to describe lactate production. The model described lactate production well for cultures carried out in both T-flasks and spinner flasks and inoculated with either PB or CB MNC or PB CD34(+) cells. The values for alpha and beta that were derived from the model varied with both the inoculum density and the cytokine combination. However, preliminary results indicate that cultures carried out under the same conditions from different samples with similar initial CD34(+) cell content have similar values for beta and beta. These findings suggest that it should be possible to use lactate production data to predict the harvest time that corresponds to the maximum number of CFC in culture. The ability to harvest ex vivo hematopoietic cultures for transplantation when CFC are at a maximum has the potential to speed the rate at which immunocompromised patients recover. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 693-700, 1997.     

Understanding factors that limit the productivity of suspension-based perfusion cultures operated at high medium renewal rates

One of the key parameters in perfusion culture is the rate of medium replacement (D). Intensifying D results in enhanced provision of nutrients, which can lead to an increase in the viable cell density (X(v)). The daily MAb production of hybridoma cells can thus be increased proportionally without modifying the bioreactor scale, provided that both viable cell yield per perfusion rate (Y(Xv/D)) and specific MAb productivity (q(MAb)) remain constant at higher D. To identify factors prone to limit productivity in perfusion, a detailed kinetic analysis was carried out on a series of cultures operated within a D range of 0.48/4.34 vvd (volumes of medium/reactor volume/day) in two different suspension-based systems. In the Celligen/vortex-flow filter system, significant reductions in Y(Xv/D) and q(MAb) resulting from the use of gas sparging were observed at D > 1.57 vvd (X(v) > 15 x 10(6) cells/mL). Through glucose supplementation, we have shown that the decrease in Y(Xv/D) encountered in presence of sparging was not resulting from increased cellular destruction or reduced cell growth, but rather from glucose limitation. Thus, increases in hydrodynamic shear stress imparted to the culture via intensification of gas sparging resulted in a gradual increase in specific glucose consumption (q(glc)) and lactate production rates (q(lac)), while no variations were observed in glutamine-consumption rates. As a result, while glutamine was the sole limiting-nutrient under non-sparging conditions, both glutamine and glucose became limiting under sparging conditions. Although a reduction in q(MAb) was observed at high-sparging rates, inhibition of MAb synthesis did not result from direct impact of bubbles, but was rather associated with elevated lactate levels (25-30 mM), resulting from shear stress-induced increases in q(lac), q(glc), and Y(lac/glc). Deleterious effects of sparging on Y(Xv/D) and q(MAb) encountered in the Celligen/vortex-flow filter system were eliminated in the sparging-free low-shear environment of the Chemap-HRI/ultrasonic filter system, allowing for the maintenance of up to 37 x 10(6) viable cells/mL. A strategy aimed at reducing requirements for sparging in large-scale perfusion cultures by way of a reduction in the oxygen demand using cellular engineering is discussed.     

On-line monitoring of respiration in recombinant-baculovirus infected and uninfected insect cell bioreactor cultures

Respiration rates in Spodoptera frugiperda (Sf-9) cell bioreactor cultures were successfully measured on-line using two methods: The O(2) uptake rate (OUR) was determined using gas phase pO(2) values imposed by a dissolved oxygen controller and the CO(2) evolution rate (CER) was measured using an infrared detector. The measurement methods were accurate, reliable, and relatively inexpensive. The CER was routinely determined in bioreactor cultures used for the production of several recombinant proteins. Simple linear relationships between viable cell densities and both OUR and CER in exponentially growing cultures were used to predict viable cell density. Respiration measurements were also used to follow the progress of baculoviral infections in Sf-9 cultures. Infection led to increases in volumetric and per-cell respiration rates. The relationships between respiration and several other culture parameters, including viable cell density, cell protein, cell volume, glucose consumption, lactate production, viral titer, and recombinant beta-galactosidase accumulation, were examined. The extent of the increase in CER following infection and the time postinfection at which maximum CER was attained were negatively correlated with the multiplicity of infection (MOI) at multiplicities below the level required to infect all the cells in a culture. Delays in the respiration peak related to the MOI employed were correlated with delays in the peak in recombinant protein accumulation. DO levels in the range 5-100% did not exert any major effects on viable cell densities, CER, or product titer in cultures infected with a baculovirus expressing recombinant beta-galactosidase. (c) 1996 John Wiley & Sons, Inc.     

High density culture of HeLa cells in a CelliGen perfusion system

A hollow fiber cartridge may be used in an extraneous recycle loop to facilitate perfusion operation of a stirred tank bioreactor. Retention of cells while removing waste products and replenishment with fresh nutrients allows higher than normal cell densities obtained in batch or continuous culture systems. This system successfully propagated HeLa cells to over 11 million viable cells per milliliter. Much higher perfusion rates (up to 4 vessel volumes per day) were necessary for high density culture of HeLa cells compared to BHK or a hybridoma cell line because of a much higher specific cellular metabolic rate. Cell specific glucose consumption rate, lactate production and ammonia production rates are several times higher for HeLa cells. Reproducible high cell densities and viabilities can be repeatedly obtained after harvest and dilution of a HeLa cell culture by partial drainage and reconstitution in the bioreactor.     

Characteristics of CHO-K1 cell culture producing two types of recombinant soluble thrombomodulin

Recombinant CHO-K1 cells, expressing human soluble thrombomodulin, were cultured in a serum-free medium and characteristics of the culture associated with glucose and lactate were investigated. In 3 L fermentor (3LFM) cultures, the cell density was found to have a proportional relationship with the volumetric glucose consumption rate, and the specific glucose consumption rates were constant at about 0.2 mg/(10⁶ cells·d) despite many differences in the culture conditions. Thus, it was concluded that the glucose consumption rate is little influenced by the condition of the cells or the culture conditions, and that the cell density can be estimated by the glucose consumption rate calculated from glucose measurement. Two types of thrombomodulin (rsTMα and rsTMβ) were produced, in which rsTMβ possesses chondroitin-4-sulfate and has greater anticoagulant activities than rsTMα. Therefore, it is important to investigate the rsTMα and rsTMβ production properties, and to determine the optimal culture conditions for high rsTMβ production. The most important factor to increase the production of rsTMβ relative to rsTMα (the β/α ratio) was effective aeration. Moreover, a lower ratio of lactate production/glucose consumption (the L/G ratio) with sufficient oxygen, high glucose concentration, and a longer medium exchange interval contributed to a higher specific rsTMβ production rate. Since there was a linear relationship between the production rate of each type of rsTM and the overall rsTM production rate per liter, it is expected that the rsTMα and rsTMβ production rates may be able to be estimated from the overall rate and the rsTMβ production increased by increasing the overall rsTM production with a lower L/G ratio.     

Hybridoma growth and antibody production as a function of cell density and specific growth rate in perfusion culture

Steady state metabolic parameters for hybridoma cell line H22 were determined over a wide range of cell densities and specific growth rates in a filtration based homogeneous perfusion reactor. Operating the reactor at perfusion rates of 0.75, 2.0, and 2.9 day(-1)(each at four different specific growth rates), viable cell densities as high as 2 x 10(7) cells/mL were obtained. For the cell line under investigation, the specific monoclonal antibody production rate was found to be a strong function of the viable cell density, increasing with increasing cell density. In contrast, most of the substrate consumption and product formation rates were strong functions of the specific growth rate. Substrate metabolism became more efficient at high cell densities and low specific growth rates. The Specific rates of metabolite formation and the apparent yields of lactate from glucose and ammonia from glutamine decreased at low specific growth rates and high cell densities. While the specific oxygen consumption rate was independent of the specific growth rate and cell density, ATP production was more oxidative at lower specific growth rate and higher cell density. These observed shifts are strong indications of the production potential of high-density perfusion culture. (c) 1995 John Wiley & Sons, Inc.     

Comparative metabolic analysis of lactate for CHO cells in glucose and galactose

t-PA producing CHO cells have been shown to undergo a metabolic shift when the culture medium is supplemented with a mixture of glucose and galactose. This metabolic change is characterized by the reincorporation of lactate and its use as an additional carbon source. The aim of this work is to understand lactate metabolism. To do so, Chinese hamster ovary cells were grown in batch cultures in four different conditions consisting in different combinations of glucose and galactose. In experiments supplemented with glucose, only lactate production was observed. Cultures with glucose and galactose consumed glucose first and produced lactate at the same time, after glucose depletion galactose consumption began and lactate uptake was observed. Comparison of the metabolic state of cells with and without the shift by metabolic flux analysis show that the metabolic fluxes distribution changes mostly in the reactions involving pyruvate metabolism. When not enough pyruvate is being produced for cells to support their energy requirements, lactate dehydrogenase complex changes the direction of the reaction yielding pyruvate to feed the TCA cycle. The slow change from high fluxes during glucose consumption to low fluxes in galactose consumption generates intracellular conditions that allow the influx of lactate. Lactate consumption is possible in cell cultures supplemented with glucose and galactose due to the low rates at which galactose is consumed. Evidence suggests that an excessive production and accumulation of pyruvate during glucose consumption leads to lactate production and accumulation inside the cell. Other internal conditions such as a decrease in internal pH, forces the flow of lactate outside the cell. After metabolic shift the intracellular pool of pyruvate, lactate and H⁺ drops permitting the reversal of the monocarboxylate transporter direction, therefore leading to lactate uptake. Metabolic analysis comparing glucose and galactose consumption indicates that after metabolic shift not enough pyruvate is produced to supply energy metabolism and lactate is used for pyruvate synthesis. In addition, MFA indicates that most carbon consumed during low carbon flux is directed towards maintaining energy metabolism.     

杂交瘤细胞培养中摄氧速率(OUR)的在线检测及其与细胞生长及代谢的关系

在批式及灌流培养条件下研究了杂交瘤细胞在无血清培养基中的生长、代谢情况与氧消耗的关系。应用动力学方法在线进行OUR的检测,同时离线取样检测其他参数。结果发现OUR与谷氨酰胺的消耗、抗体的生成及活细胞密度间有明显的相关关系,进一步的分析还发现在对数生长期,OUR与活细胞密度间具有良好的线性关系,qOUR(0.103±0.028)×10^-12mol/cell/h,可以通过它来进行细胞密度的在线检测。并通过以ΔOUR=0时刻作为灌流调整点进行连续灌流培养的初步实验验证了OUR作为培养过程反馈控制参数的可能性。     

Glucose and glutamine utilization by rat lymphocytes, monocytes and neutrophils in culture: a comparative study

Glucose and glutamine utilization and production of glutamate and lactate were determined for up to 48 h in lymphocytes, monocytes and neutrophils cultured in medium rich in metabolites and vitamins. Glucose was utilized by the three cell types in culture in the following order: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the order: monocytes > neutrophils > lymphocytes. The consumption of glucose followed the activity of glucose-6-phosphate dehydrogenase but it was not related to hexokinase activity. Glutamine was consumed by the three leukocyte types in culture as follows: neutrophils > lymphocytes > or = monocytes. The consumption of glutamine was not fully related to the activity of phosphate-dependent glutaminase. The production of glutamate was not remarkably different among the three cell types. For comparison, glutamine and glucose utilization and glutamate and lactate production were also evaluated using 1-h incubated leukocytes. Under this condition, only glucose or glutamine was added to the medium. Glucose was utilized as follows: neutrophils > monocytes > lymphocytes, whereas lactate was produced in the following order: monocytes > or = neutrophils > lymphocytes. Glutamine was consumed as follows: neutrophils > lymphocytes > monocytes, whereas glutamate was produced as follows: neutrophils > or = monocytes = lymphocytes. The ratio of the amount of glucose/glutamine consumed by 1-h incubated cells was 0.5 for neutrophils, 1.5 for monocytes, and 0.3 for lymphocytes. However, the three cell types cultured for 48 h utilized glucose to a much higher degree than glutamine. The ratio of the amount of glucose/glutamine utilized by the cultured cells was 8.9 for neutrophils, 16.4 for monocytes, and 6.7 for lymphocytes. These observations support the proposition that glutamine is required in much higher amounts than glucose to accomplish the total metabolic requirement of leukocytes. Under conditions closer to physiological when the availability of a variety of metabolites and vitamins is not restricted, glucose is the preferred substrate for lymphocytes, monocytes and neutrophils.     

Feed development for fed‐batch CHO production process by semisteady state analysis

Semisteady state cultures are useful for studying cell physiology and facilitating media development. Two semisteady states with a viable cell density of 5.5 million cells/mL were obtained in CHO cell cultures and compared with a fed‐batch mode control. In the first semisteady state, the culture was maintained at 5 mM glucose and 0.5 mM glutamine. The second condition had threefold higher concentrations of both nutrients, which led to a 10% increase in lactate production, a 78% increase in ammonia production, and a 30% reduction in cell growth rate. The differences between the two semisteady states indicate that maintaining relatively low levels of glucose and glutamine can reduce the production of lactate and ammonia. Specific amino acid production and consumption indicated further metabolic differences between the two semisteady states and fed‐batch mode. The results from this experiment shed light in the feeding strategy for a fed‐batch process and feed medium enhancement. The fed‐batch process utilizes a feeding strategy whereby the feed added was based on glucose levels in the bioreactor. To evaluate if a fixed feed strategy would improve robustness and process consistency, two alternative feeding strategies were implemented. A constant volume feed of 30% or 40% of the initial culture volume fed over the course of cell culture was evaluated. The results indicate that a constant volumetric‐based feed can be more beneficial than a glucose‐based feeding strategy. This study demonstrated the applicability of analyzing CHO cultures in semisteady state for feed enhancement and continuous process improvement. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Continuous culture of CHO-K1 cells producing thrombomodulin and estimation of culture conditions

Recombinant Chinese hamster ovary (CHO-K1) cells expressing human soluble thrombomodulin (rsTM) were cultured in a continuous culture system with a fluidized-bed reactor. Cells were grown in a medium containing 1% serum for 10 d, and then cultured in a serum-free medium. The protein production rate increased remarkably in the serum-free culture, with a decrease in the lactate production rate. This suggests that CHO-K1 cells exhibit different physiological characteristics in response to serum removal from the medium, which resulted in a higher rsTM concentration (about 60 mg/l). A procedure for estimating protein productivity was developed using experimental glucose and lactate measurements. In this procedure, cell density was estimated from the glucose consumption rate, and the specific protein (rsTM) production rate was obtained from the ratio of lactate production/glucose consumption (ΔL/ΔG). Since the cell density and protein productivity in repeated batch culture were well estimated, the procedure was applied to continuous culture in a fluidized-bed bioreactor culture. The estimation procedure was also found to be effective in this continuous culture using the models derived from the repeated batch culture.     

乳酸脱氢酶基因调控对HEK-293细胞代谢和腺病毒生产的影响

减少乳酸积累一直是哺乳动物细胞生物技术产业的一个目标。体外培养动物细胞时,乳酸积累主要是2种代谢途径作用的综合结果:一方面,葡萄糖在乳酸脱氢酶A(lactate dehydrogenase A,LDHA)的作用下生成乳酸;另一方面,乳酸可通过乳酸脱氢酶B(LDHB)或乳酸脱氢酶C(LDHC)氧化为丙酮酸重新进入三羧酸循环。本研究综合评估了乳酸代谢关键基因调控对人胚胎肾细胞(human embryonic kidney 293 cells,HEK-293)细胞生长、代谢和人腺病毒(human adenovirus,HAdV)生产的影响,有效提高了HEK-293细胞的HAdV生产能力,并为哺乳动物细胞的乳酸代谢工程调控提供了理论基础。通过改造乳酸代谢关键调控基因(敲除ldha基因以及过表达ldhb和ldhc基因),有效改善了HEK-293细胞的物质和能量代谢效率,显著提高了HAdV的生产。与对照细胞相比,3个基因改造均能促进细胞生长,降低乳酸和氨的积累,明显增强细胞的物质和能量代谢效率,显著提高了HEK-293细胞的HAdV生产能力。ldhc基因过表达对HEK-293细胞的生长、代谢和HAdV生产调控最显著,最大细胞密度提高了约38.7%,乳酸对葡萄糖得率和氨对谷氨酰胺得率分别下降了33.8%和63.3%,HAdV滴度提高了至少16倍。此外,相比于对照细胞株,改造细胞株的腺苷三磷酸(adenosine triphosphate,ATP)生成速率、ATP/O_(2)比率、ATP与腺苷二磷酸(adenosine diphosphate,ADP)的比值以及还原型辅酶Ⅰ(nicotinamide adenine dinucleotide,NADH)含量均有不同程度的提高,能量代谢效率明显改善。     

The effect of oscillating dissolved oxygen concentrations on the metabolism of a Spodoptera frugiperda IPLB-Sf21-AE clonal isolate

The effect of oscillating dissolved oxygen (DO) concentration on the metabolism of a clonal isolate of the Spodoptera frugiperda IPLB-Sf21-AE insect cell line was investigated. Specifically, the effect on cell growth, re- combinant protein synthesis, glucose and glutamine consumption, and lactate accumulation was determined. Prior to conducting the oscillating DO experiments, it was found that the DO concentration could be reduced to 15% air saturation without adversely affecting the growth rate. Under these conditions, glucose and glutamine became depleted as the maximum cell density was reached. The introduction of DO oscillations, that is, cycles consisting of 30 min at 15% DO followed by 30 min of anoxia, significantly altered cell metabolism, including inhibition of cell growth and recombinant protein synthesis. The effect of DO oscillations on glucose consumption was dependent on the experimental conditions. Glucose exhaustion occurred when the DO oscillations contained either an "apparent" anoxia period (nitrogen sparging discontinued upon reaching 0% DO) without pH control or a "true" anoxia period (nitrogen sparging continued throughout anoxia period) with pH control. Glucose consumption was significantly decreased, however, when the cells were exposed to a "true" anoxia period without pH control, that is, low pH inhibited glucose utilization. Glutamine uptake was not significantly affected by DO oscillations. Lactate only accumulated in the oscillating DO runs, a finding consistent with previous results demonstrating that significant lactate accumulation only occurs under DO-limited conditions. (c) 1995 John Wiley & Sons, Inc.     

Modification of glucose and glutamine metabolism in hybridoma cells through metabolic engineering

The present work describes the genetic modification of a hybridoma cell line with the aim to change its metabolic behaviour, particularly reducing the amounts of ammonia and lactate produced by the cells. The cellular excretion of ammonia was eliminated by transfection of a cloned glutamine synthetase gene. The metabolic characterisation of the transformed cell line includes the analysis of the changes introduced in its intracellular metabolic fluxes by means of a stoichiometric model. Furthermore, the reduction of lactate accumulation was attempted through an antisense mRNA approach, aiming to generate a rate limiting step in the glycolytic pathway, thus lowering the glucose consumption rate. The physiological results obtained with the transformed cells are discussed. A maximum reduction of about 47% in the glucose consumption rate was obtained for one of the transformations. However a main drawback was the lack of stability of the transformed cells This revised version was published online in July 2006 with corrections to the Cover Date.