Glycolysis is governed by growth regime and simple enzyme regulation in adherent MDCK cells.

Due to its vital importance in the supply of cellular pathways with energy and precursors, glycolysis has been studied for several decades regarding its capacity and regulation. For a systems-level understanding of the Madin-Darby canine kidney (MDCK) cell metabolism, we couple a segregated cell growth model published earlier with a structured model of glycolysis, which is based on relatively simple kinetics for enzymatic reactions of glycolysis, to explain the pathway dynamics under various cultivation conditions. The structured model takes into account in vitro enzyme activities, and links glycolysis with pentose phosphate pathway and glycogenesis. Using a single parameterization, metabolite pool dynamics during cell cultivation, glucose limitation and glucose pulse experiments can be consistently reproduced by considering the cultivation history of the cells. Growth phase-dependent glucose uptake together with cell-specific volume changes generate high intracellular metabolite pools and flux rates to satisfy the cellular demand during growth. Under glucose limitation, the coordinated control of glycolytic enzymes re-adjusts the glycolytic flux to prevent the depletion of glycolytic intermediates. Finally, the model's predictive power supports the design of more efficient bioprocesses.

Cell separator operation within temperature ranges to minimize effects on Chinese hamster ovary cell perfusion culture.

A cell retention device that provides reliable high-separation efficiency with minimal negative effects on the cell culture is essential for robust perfusion culture processes. External separation devices generally expose cells to periodic variations in temperature, most commonly temperatures below 37 degrees C, while the cells are outside the bioreactor. To examine this phenomenon, aliquots of approximately 5% of a CHO cell culture were exposed to 60 s cyclic variations of temperature simulating an acoustic separator environment. It was found that, for average exposure temperatures between 31.5 and 38.5 degrees C, there were no significant impacts on the rates of growth, glucose consumption, or t-PA production, defining an acceptable range of operating temperatures. These results were subsequently confirmed in perfusion culture experiments for average exposure temperatures between 31.6 and 38.1 degrees C. A 2(5-1) central composite factorial design experiment was then performed to systematically evaluate the effects of different operating variables on the inlet and outlet temperatures of a 10L acoustic separator. The power input, ambient temperature, as well as the perfusion and recycle flow rates significantly influenced the temperature, while the cell concentration did not. An empirical model was developed that predicted the temperature changes between the inlet and the outlet of the acoustic separator within +/-0.5 degrees C. A series of perfusion experiments determined the ranges of the significant operational settings that maintained the acoustic separator inlet and outlet temperatures within the acceptable range. For example, these objectives were always met by using the manufacturer-recommended operational settings as long as the recirculation flow rate was maintained above 15 L day(-1) and the ambient temperature was near 22 degrees C.

High-performance anion-exchange chromatography using on-line electrolytic eluent generation for the determination of more than 25 intermediates from energy metabolism of mammalian cells in culture.

In this work, we present an improved method for the determination of a wide range of intracellular metabolites from mammalian cells by anion-exchange chromatography. The analysis includes the measurement of intermediates from glycolysis and tricarboxylic acid cycle as well as several additional nucleotides and sugar nucleotides. The use of an electrolytic on-line eluent generation device made the method highly convenient, reliable and prone to errors. Due to short delay times of the eluent generator, rapid KOH gradient changes could be applied to improve separation and to speed up elution. Suppressed conductivity and UV in series was used for detection. The detection wavelength of the UV detector was switched from 220 to 260 nm during the elution for a more selective signal depending on the absorption of analytes. Standards from more than 50 metabolites of major cellular pathways were chromatographically tested and compared to chromatograms from extraction samples of Madin-Darby canine kidney (MDCK) and BHK21 cells. A validation for most substances was performed. Detection limits were below the micromolar range and the coefficient of correlation (R(2)) was above 0.99 for most analytes. Working ranges were between 0.125-3.875 and 4.5-139.5 microM. Sample pH had a major impact on the quantification of several metabolites, but measurements were robust within a pH range of 6.5-9.0.

Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells.

In mammalian cell culture technology glutamine is required for biomass synthesis and as a major energy source together with glucose. Different pathways for glutamine metabolism are possible, resulting in different energy output and ammonia release. The accumulation of ammonia in the medium can limit cell growth and product formation. Therefore, numerous ideas to reduce ammonia concentration in cultivation broths have been developed. Here we present new aspects on the energy metabolism of mammalian cells. The replacement of glutamine (2 mM) by pyruvate (10 mM) supported cell growth without adaptation for at least 19 passages without reduction in growth rate of different adherent commercial cell lines (MDCK, BHK21, CHO-K1) in serum-containing and serum-free media. The changes in metabolism of MDCK cells due to pyruvate uptake instead of glutamine were investigated in detail (on the amino acid level) for an influenza vaccine production process in large-scale microcarrier culture. In addition, metabolite profiles from variations of this new medium formulation (1-10 mM pyruvate) were compared for MDCK cell growth in roller bottles. Even at very low levels of pyruvate (1 mM) MDCK cells grew to confluency without glutamine and accumulation of ammonia. Also glucose uptake was reduced, which resulted in lower lactate production. However, pyruvate and glutamine were both metabolized when present together. Amino acid profiles from the cell growth phase for pyruvate medium showed a reduced uptake of serine, cysteine, and methionine, an increased uptake of leucine and isoleucine and a higher release of glycine compared to glutamine medium. After virus infection completely different profiles were found for essential and nonessential amino acids.

Metabolic effects of influenza virus infection in cultured animal cells: Intra- and extracellular metabolite profiling.

BACKGROUND: Many details in cell culture-derived influenza vaccine production are still poorly understood and approaches for process optimization mainly remain empirical. More insights on mammalian cell metabolism after a viral infection could give hints on limitations and cell-specific virus production capacities. A detailed metabolic characterization of an influenza infected adherent cell line (MDCK) was carried out based on extracellular and intracellular measurements of metabolite concentrations. RESULTS: For most metabolites the comparison of infected (human influenza A/PR/8/34) and mock-infected cells showed a very similar behavior during the first 10-12 h post infection (pi). Significant changes were observed after about 12 h pi: (1) uptake of extracellular glucose and lactate release into the cell culture supernatant were clearly increased in infected cells compared to mock-infected cells. At the same time (12 h pi) intracellular metabolite concentrations of the upper part of glycolysis were significantly increased. On the contrary, nucleoside triphosphate concentrations of infected cells dropped clearly after 12 h pi. This behaviour was observed for two different human influenza A/PR/8/34 strains at slightly different time points. CONCLUSIONS: Comparing these results with literature values for the time course of infection with same influenza strains, underline the hypothesis that influenza infection only represents a minor additional burden for host cell metabolism. The metabolic changes observed after 12 h pi are most probably caused by the onset of apoptosis in infected cells. The comparison of experimental data from two variants of the A/PR/8/34 virus strain (RKI versus NIBSC) with different productivities and infection dynamics showed comparable metabolic patterns but a clearly different timely behavior. Thus, infection dynamics are obviously reflected in host cell metabolism.

Simultaneous extraction of several metabolites of energy metabolism and related substances in mammalian cells: optimization using experimental design.

As a basis for the development of predictive mathematical models in systems biology and a quantitative understanding of cellular metabolism, reliable experimental data sets of intracellular metabolites are indispensable. A prerequisite for the acquisition of such data is the identification of a suitable sample preparation method. In this work, the extraction procedure for the simultaneous measurement of a wide range of intracellular metabolites from adherent mammalian cells in culture was optimized. A screening of several commonly used extraction protocols with Madin-Darby canine kidney (MDCK) cells found the methanol/chloroform (MeOH/CHCl(3)) and MeOH/Boil methods to be promising candidates for further analysis by anion-exchange chromatography. Both methods were optimized based on experimental design techniques with four response variables: Nucleotide Content, Energy Charge, Fructose 1,6-Bisphosphate content (F16bP), and Absorption at 280 nm. After data evaluation and with the help of desirability functions, an overall optimum for the extraction conditions was found. Using optimal settings, the extraction performances for MDCK and Vero cell cultivations of both methods were compared. Both methods extracted nearly the same absolute amounts of intracellular metabolites, suggesting that these methods are equal. However, recoveries for nucleotide diphosphates were significantly above 100% for both methods. This most likely was due to remaining nucleotide kinase activity during extraction. After combining individual steps of both methods, recoveries close to 100% for all metabolites could be reached. Absolute values of intracellular metabolites extracted with this modified method are comparable to the results of the two previously optimized methods, indicating a good extraction procedure according to the chosen response variables.

Characterization and optimization of acoustic filter performance by experimental design methodology.

Acoustic cell filters operate at high separation efficiencies with minimal fouling and have provided a practical alternative for up to 200 L/d perfusion cultures. However, the operation of cell retention systems depends on several settings that should be adjusted depending on the cell concentration and perfusion rate. The impact of operating variables on the separation efficiency performance of a 10-L acoustic separator was characterized using a factorial design of experiments. For the recirculation mode of separator operation, bioreactor cell concentration, perfusion rate, power input, stop time and recirculation ratio were studied using a fractional factorial 2(5-1) design, augmented with axial and center point runs. One complete replicate of the experiment was carried out, consisting of 32 more runs, at 8 runs per day. Separation efficiency was the primary response and it was fitted by a second-order model using restricted maximum likelihood estimation. By backward elimination, the model equation for both experiments was reduced to 14 significant terms. The response surface model for the separation efficiency was tested using additional independent data to check the accuracy of its predictions, to explore robust operation ranges and to optimize separator performance. A recirculation ratio of 1.5 and a stop time of 2 s improved the separator performance over a wide range of separator operation. At power input of 5 W the broad range of robust high SE performance (95% or higher) was raised to over 8 L/d. The reproducible model testing results over a total period of 3 months illustrate both the stable separator performance and the applicability of the model developed to long-term perfusion cultures.