Monte Carlo simulation of the alpha-amylolysis of amylopectin potato starch. 2. alpha-amylolysis of amylopectin.

A model is presented that describes all the saccharides that are produced during the hydrolysis of starch by an alpha-amylase. Potato amylopectin, the substrate of the hydrolysis reaction, was modeled in a computer matrix. The four different subsite maps presented in literature for alpha-amylase originating from Bacillus amyloliquefaciens were used to describe the hydrolysis reaction in a Monte Carlo simulation. The saccharide composition predicted by the model was evaluated with experimental values. Overall, the model predictions were acceptable, but no single subsite map gave the best predictions for all saccharides produced. The influence of an alpha(1-->6) linkage on the rate of hydrolysis of nearby alpha(1-->4) linkages by the alpha-amylase was evaluated using various inhibition constants. For all the subsites considered the use of inhibition constants led to an improvement in the predictions (a decrease of residual sum of squares), indicating the validity of inhibition constants as such. As without inhibition constants, no single subsite map gave the best fit for all saccharides. The possibility of generating a hypothetical subsite map by fitting was therefore investigated. With a genetic algorithm it was possible to construct hypothetical subsite maps (with inhibition constants) that gave further improvements in the average prediction for all saccharides. The advantage of this type of modeling over a regular fit is the additional information about all the saccharides produced during hydrolysis, including the ones that are difficult to measure experimentally.

Optimization of a feed medium for fed-batch culture of insect cells using a genetic algorithm.

Insect cells have been cultured for over 30 years, but their application is still hampered by low cell densities in batch fermentations and expensive culture media. With respect to the culture method, the fed-batch culture mode is often found to give the best yields. However, optimization of the feed composition is usually a laborious task. In this report, the successful use of genetic algorithms (GAs) to optimize the growth of insect cells is described. A feed was developed from 11 different medium components, each used at a wide range of concentrations. The feed was optimized within four sets of 20 experiments. The optimized feed was tested in bioreactors and the addition scheme was further improved. The viable-cell density of HzAm1 (Helicoverpa zea) insect cells improved 550% to 19.5 x 10(6) cells/mL compared to a control fermentation in an optimized commercial medium. No accumulation of waste products was found, and none of the amino acids was depleted. Glucose was depleted, which suggests that even further improvement is possible. We show that GAs are a successful method to optimize a complex fermentation in a relatively short time frame and without the need of detailed information concerning the cellular physiology or metabolism.

Effect of low inoculation density in the scale-up of insect cell cultures.

The scale-up of insect cell cultures and the production of baculovirus with these cultures is dependent on the inoculation density applied. The effect of applying a low inoculation on the specific growth rate and on the duration of the lag phase was tested. Three different cell lines, HzAm1, Ha2302, and Sf21 were tested in a total of five cell line/medium combinations. Growth in suspension culture was examined, and data obtained were fitted with the Gompertz equation. A significant decline in specific growth rate with decreasing inoculation density was observed in all cell line/medium combinations, except for HzAm1. No critical inoculation density, below which no growth would occur, was found. In suspension culture in shake flasks, an inoculation density of 5 x 10(4) cells/mL is achievable, without severely influencing the overall growth rate. A lower inoculation density in suspension culture results in less steps in the scale-up process and might be a tool in bypassing the viral passage effect.

Metabolic-flux analysis of hybridoma cells under oxidative and reductive stress using mass balances.

Hybridoma cells were grown at steady state under both reductiveand oxidative stress and the intracellular fluxes weredetermined by mass-balancing techniques. By decreasing the dissolved oxygen pressure (pO(2)) in the bioreactor, the reduced formof nicotinamide adenine nucleotide (NADH) was enhanced relativeto the oxidized form (NAD(+)). Oxidative stress, as a resultof which the NAP(P)(+)/NAD(P)H-ratio increases, was generatedby both the enhancement of the pO(2) to 100% air saturationand by the addition of the artificial electron acceptorphenazine methosulphate (PMS) to the culture medium. It wasfound that fluxes of dehydrogenase reactions by which NAD(P)H isproduced decreased under hypoxic conditions. For example, thedegradation rates of arginine, isoleucine, lysine and theglutamate dehydrogenase flux were significantly lower at oxygenlimitation, and increased at higher pO(2) levels and when PMSwas added to the culture medium. In contrast, the prolinesynthesis reaction, which requires NADPH, decreased under PMSstress. The flux of the NADH-requiring lactate dehydrogenase reaction also strongly decreased from 19 to 3,4 pmol/cell/day,under oxygen limitation and under PMS stress, respectively. Thedata show that metabolic-flux balancing can be used to determinehow mammalian respond to oxidative and reduction stress.

The effect of process conditions on the alpha-amylolytic hydrolysis of amylopectin potato starch: An experimental design approach.

The hydrolysis of amylopectin potato starch with Bacillus licheniformis alpha-amylase (Maxamyl) was studied under industrially relevant conditions (i.e. high dry-weight concentrations). The following ranges of process conditions were chosen and investigated by means of an experimental design: pH [5.6-7.6]; calcium addition [0-120 microg/g]; temperature [63-97 degrees C]; dry-weight concentration [3-37% [w/w]]; enzyme dosage [27.6-372.4 microL/kg] and stirring [0-200 rpm]. The rate of hydrolysis was followed as a function of the theoretical dextrose equivalent. The highest rate (at a dextrose equivalent of 10) was observed at high temperature (90 degrees C) and low pH (6). At a higher pH (7.2), the maximum temperature of hydrolysis shifted to a lower value. Also, high levels of calcium resulted in a decrease of the maximum temperature of hydrolysis. The pH, temperature, and the amount of enzyme added showed interactive effects on the observed rate of hydrolysis. No product or substrate inhibition was observed. Stirring did not effect the rate of hydrolysis. The oligosaccharide composition after hydrolysis (at a certain dextrose equivalent) did depend on the reaction temperature. The level of maltopentaose [15-24% [w/w]], a major product of starch hydrolysis by B. licheniformis alpha-amylase, was influenced mostly by temperature.

Error analysis of metabolic-rate measurements in mammalian-cell culture by carbon and nitrogen balances.

The analysis of metabolic fluxes of large stoichiometric systems is sensitive to measurement errors in metabolic uptake and production rates. It is therefore desirable to independently test the consistency of measurement data, which is possible if at least two elemental balances can be closed. For mammalian-cell culture, closing the C balance has been hampered by problems in measuring the carbon-dioxide production rate. Here, it is shown for various sets of measurement data that the C balance can be closed by applying a method to correct for the bicarbonate buffer in the culture medium. The measurement data are subsequently subject to measurement-error analysis on the basis of the C and N balances. It is shown at 90% reliability that no gross measurement errors are present, neither in the measured production- and consumption rates, nor in the estimated in- and outgoing metabolic rates of te subnetwork, that contains the glycolysis, the pentose-phosphate, and the glutaminolysis pathways.

IgG and hybridoma partitioning in aqueous two-phase systems containing a dye-ligand.

The effect of the important ATPS- and buffer parameters on IgG and hybridoma partitioning in ATPSs containing a PEG-dye-ligand was studied. Objective was to establish selection criteria for effective ligands for extractive fermentations with animal cells in ATPSs. In the presence of 1% PEG-dye-ligand the binding of IgG to the PEG-ligand was affected severely by the Na-chloride concentration. The tie-line length and pH affected IgG partitioning to a lesser extent. The desired partitioning of IgG into the top phase, was only obtained when, in addition to the 10 mmol/kg K-phosphate buffer, no Na-chloride was present. In an ATPS culture medium, with +/- 35 mmol/kg Na-bicarbonate and 60 mmol/kg Na-chloride, increasing the PEG-dye-ligand concentration up to 100% did increase the partition coefficient, but was not effective in concentrating the IgG in the top phase of ATPS culture medium at a pH of 7.8. Furthermore, addition of the PEG-dye-ligand to ATPS culture medium changed the hybridoma cell partitioning from the bottom phase to the interface.

Activity of glutamate dehydrogenase is increased in ammonia-stressed hybridoma cells.

The effect of added ammonia on the intracellular fluxes in hybridoma cells was investigated by metabolic-flux balancing techniques. It was found that, in ammonia-stressed hybridoma cells, the glutamate-dehydrogenase flux is in the reverse direction compared to control cells. This demonstrates that hybridoma cells are able to prevent the accumulation of ammonia by converting ammonia and alpha-ketoglutarate into glutamate. The additional glutamate that is produced by this flux, as compared to the control culture, is converted by the reactions catalyzed by alanine aminotransferase (45% of the extra glutamate) and aspartate aminotransferase (37%), and a small amount is used for the biosynthesis of proline (6%). The remaining 12% of the extra glutamate is secreted into the culture medium. The data suggest that glutamate dehydrogenase is a potential target for metabolic engineering to prevent ammonia accumulation in high-cell-density culture.

Metabolite-balancing techniques vs. 13C tracer experiments to determine metabolic fluxes in hybridoma cells.

The estimation of intracellular fluxes of mammalian cells using only mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. In order to quantify fluxes in cyclic pathways the mass balance equations can be complemented with several constraints: (1) the mass balances of co-metabolites, such as ATP or NAD(P)H, (2) linear objective functions, (3) flux data obtained by isotopic-tracer experiments. Here, these three methods are compared for the analysis of fluxes in the primary metabolism of continuously cultured hybridoma cells. The significance of different theoretical constraints and different objective functions is discussed after comparing their resulting flux distributions to the fluxes determined using 13CO2 and 13C-lactate measurements of 1 - 13C-glucose-fed hybridoma cells. Metabolic fluxes estimated using the objective functions "maximize ATP" and "maximize NADH" are relatively similar to the experimentally determined fluxes. This is consistent with the observation that cancer cells, such as hybridomas, are metabolically hyperactive, and produce ATP and NADH regardless of the need for these cofactors.

Current good manufacturing practice in plant automation of biological production processes.

The production of biologicals is subject to strict governmental regulations. These are drawn up in current good manufacturing practices (cGMP), a.o. by the U.S. Food and Drug Administration. To implement cGMP in a production facility, plant automation becomes an essential tool. For this purpose Manufacturing Execution Systems (MES) have been developed that control all operations inside a production facility. The introduction of these recipe-driven control systems that follow ISA S88 standards for batch processes has made it possible to implement cGMP regulations in the control strategy of biological production processes. Next to this, an MES offers additional features such as stock management, planning and routing tools, process-dependent control, implementation of software sensors and predictive models, application of historical data and on-line statistical techniques for trend analysis and detection of instrumentation failures. This paper focuses on the development of new production strategies in which cGMP guidelines are an essential part.

On-line immunoanalysis of monoclonal antibodies during a continuous culture of hybridoma cells.

The monoclonal-antibody production of an immobilized hybridoma cell line cultivated in a fluidized-bed reactor was monitored on-line for nearly 900 h. The monoclonal antibody concentration was determined by an immuno affinity-chromatography method (ABICAP). Antibodies directed against the product, e.g. IgG, were immobilized on a micro-porous gel and packed in small columns. After all IgG present in the sample was bound to the immobilized antibodies, unbound proteins were removed by rinsing the column. Elution of the bound antibodies followed and the antibodies were determined by fluorescence. The analytical procedure was automated with a robotic device to enable on-line measurements. The correlation between the on-line determined data and antibody concentrations measured by HPLC was linear.A sampling system was constructed, which was based on a pneumatically actuated in-line membrane valve integrated into the circulation loop of the reactor. Separation of the cells from the sample stream was achieved by a depth filter made of glass-fibre, situated outside the reactor. Rapid obstruction of the filter by cells or cell debris and contamination of the sample system was avoided by intermittent rinsing of the sample system with a chemical solution. The intermittent rinsing of the filter, which had a surface of 4.8 cm(2), resulted in an operational capacity of up to 40 samples (1.0 l total sample volume). Both the sampling system and the analytical device functioned without failure during this long-term culture.The culture temperature was varied between 34 and 40 °C. Raising the temperature from 34 up to 37 °C resulted in a simultaneous increase of growth and specific antibody production rate. Specific metabolic rates of glucose, lactate, glutamine and ammonium stayed constant in this temperature range. A further enhancement of temperature up to 40 °C had a negative effect on the growth rate, whereas the specific monoclonal antibody production rate showed a small increase. The other specific metabolic rates also increased in the temperature range between 38 to 40 °C.

Automation of selective assays for on-line bioprocess monitoring by flow-injection analysis.

On-line analysis of one component in a complex media used for bioprocesses requires the application of selective tests such as enzymes assays. Because these assays are susceptible to interference by other medium components and have a limited detection range, automatic sample pretreatment is a prerequisite. The progress made with automatic sample pretreatment in flow-injection analysis makes this technique particularly suitable for on-line monitoring of bioprocesses. Moreover, newly developed software control systems may improve the necessary robustness of flow-infection analysis systems.

On-line estimation of the biomass activity during animal-cell cultivations.

A software sensor was developed to determine the volumetric biomass activity of animal cell cultivations on-line. It was based on the on-line estimation of the ATP-production rate from the oxygen uptake and the lactic-acid production rate. The sensor was verified for a batch culture of Vero cells, and a batch and a continuous culture of hybridoma cells. For the hybridoma cells, the sensor showed a good correlation with the biomass concentration. However, this was not the case for the Vero cells. As soon as glutamine was exhausted, the biomass activity stabilized, whereas the amount of biomass almost doubled. Because the sensor developed responds to nutrient limitations much faster than becomes visible through cell density measurements, and because the volumetric biomass activity can be related to the volumetric consumption rates and production rates of important metabolites, it shows excellent possibilities for control purposes.

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

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, O(2), CO(2), NH(4), 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 CO(2) or the NAD(P)H mass balance into account is shown to be in agreement with the measured O(2) and CO(2) 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 (i)The majority of the consumed glucose (>90%) is channeled through the pentose-phosphate pathway in rapidly proliferating cells.(ii)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 CO(2).(iii)The flux from glutamate to alpha-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.(iv)Transhydrogenation plays a significant role in hybridoma cells under our experimental conditions. NADPH is produced at relatively high rates (11 x 10(-12) to 13 x 10(-12) mol . cell(-1) . day(-1)) compared to other fluxes in both culture media.

Hybridoma and CHO cell partitioning in aqueous two-phase systems.

The partitioning of mouse/mouse hybridoma cell line BIF6A7, mouse/rat hybridoma PFU-83, and CHO DUKX B11-derived cell line BIC-2 in aqueous two-phase systems (ATPSs) of poly(ethylene glycol) (PEG) and dextran was studied. The partitioning of BIF6A7 was investigated systematically by using a statistical experimental design. The aims were to identify the key factors governing cell partitioning and to select ATPSs with suitable cell partitioning for extractive bioconversions with animal cells. The influence of five factors, i.e., the poly(ethylene glycol) molecular weight (PEG MW), dextran molecular weight (Dx MW), tie-line length (TLL), pH, and the ratio of potassium phosphate to potassium chloride, defined as the fraction KPi/(KPi + KCl), on BIF6A7 cell partitioning was characterized by using a full factorial experimental design. The cell partitioning ranged from complete partitioning into the interface to an almost complete partitioning to the lower phase. In all cases less than 1% of the cells partitioned to the top phase. The potassium phosphate fraction had the largest effect on cell partitioning. Low potassium phosphate fractions increased the proportion of cells in the lower phase. To a lesser extent the other factors also played a role in the cell partitioning. The best partitioning for the BIF6A7 cell line was obtained in ATPSs with PEG MW = 35,000, Dx MW = 40,000, TLL = 0.10 g/g, pH 7.4, and KPi/(KPi + KCl) = 0.1, where 93% of the cells were present in the lower phase. The previously reported partitioning of BIF6A7 cells in ATPS culture medium, corresponded well with the current findings. The partitioning of mouse/rat hybridoma cell line PFU-83 and CHO cell line BIC-2 was studied in an ATPS culture medium with PEG 35,000, dextran 40,000, TLL = 0.12 g/g, and hybridoma culture medium. Both cell lines partitioned almost completely into the lower phase. Moreover, the PFU-83 cell line was able to grow in the ATPS hybridoma culture medium. This ATPS hybridoma culture medium therefore seems to be suitable for extractive bioconversions with a wide range of hybridoma cell lines, provided that their product can be partitioned into the upper PEG-rich phase.

Separation of hybridoma cells from their IgG product using aqueous two-phase systems.

The partitioning of IgG in aqueous two-phase systems (ATPSs) of PEG and Dextran was studied systematically using a statistical experimental design. Aim was to improve the separation of hybridoma cells and their IgG product, by identifying the key variables governing IgG partitioning, and by comparing the IgG partitioning data with the hybridoma cell partitioning data obtained in previous work. The influence of five factors, i.e. the poly(ethylene glycol) molecular weight (PEG Mw), dextran molecular weight (Dx Mw), tie-line length (TLL), pH and potassium phosphate fraction (KPi/(KPi+KCl)), on IgG partitioning was characterized using a full-factorial experimental design. In all of the ATPS's the IgG partitioned predominantly into the lower phase. The partition coefficient varied between 0.78 (Variable settings: PEG Mw = 6000, Dx Mw = 500000, TLL = 0.10 g g-1, KPi/(KPi+KCl) = 1.0 and pH = 7.4) and 0.0002 (Variable settings: PEG Mw = 35000, Dx Mw = 40000, TLL = 0.20 g g-1 KPi/(KPi+KCl) = 1.0 and pH 6.6). The tie-line length, the dextran molecular weight and the PEG molecular weight had the most pronounced effect on IgG partitioning. Matching the partitioning data of the IgG product with previously obtained data of the hybridoma cell partitioning, showed that within the experimental design no ATPS could be found giving a good separation of the hybridoma cells and their IgG product. There are, however, ATPS's available in which the cells partition to, and grow in the lower dextran-rich phase. To achieve a good separation of the hybridoma cells and their IgG product in these ATPSs, the IgG product has to be specifically extracted into the PEG-rich top phase. For this purpose the use affinity ligands coupled to PEG may offer a solution. Therefore, a number of commercially available dye-resins was screened for their ability to bind the BIF6A7 IgG antibody. The mimetic green 1 A6XL dye-resin was found to bind BIF6A7 IgG. The dye-ligand coupled to PEG was used to manipulate the IgG partitioning in an ATPS. In the presence of the PEG-ligand, the IgG partitioned almost completely to the top phase. The IgG-partition coefficient increased three orders of magnitude, resulting in a 25-fold higher IgG concentration in the top phase than in the bottom phase.

Scale up aspects of sparged insect-cell bioreactors.

CONCLUSION: In this chapter we have attempted to evaluate the most important parameters which can be useful for the pur-pose of design and scale up. Insect cells and animal cells in general can be grown well in large vessels. However, none of the theories and parameters discussed in this chapter have been validated on a larger scale than laboratory and small pilot reactors. Selection of the most suitable design and scale-up method there-fore needs in particular studies in larger vessels. The Kolmogorov theory and the killing-volume model are in this respect the most promising approaches for the optimal design of large-scale animal-cell bioreactors.

Oxygen gradients in small and big sparged insect-cell bioreactors.

CONCLUSIONS: It should be clear from the above that the calculations described here are at best rough estimations yielding order-of-magnitude values. Even though, the following general conclusions can be drawn. The gradients in stagnant layers surrounding the particles which are characteristic for animal-cell bioreactors are relatively small as compared to the gradients which can be expected in the bulk-liquid phases of the three bioreactors considered, in particular to the gradients in the stagnant layer surrounding the air bubbles. It can be concluded that under almost all circumstances gradients are likely to exist and can be very steep in larger vessels and in particular at high cell densities. The effects of gradients, however, are largely unknown; therefore research on the effects of gradients on specific and volumetric productivities and product quality seems to be an interesting area.

Death rate in a small air-lift loop reactor of vero cells grown on solid microcarriers and in macroporous microcarriers.

The death rate of Vero cells grown on Cytodex-3 microcarriers was studied as a function of the gas flow rate in a small air-lift loop reactor. The death rate may be described by first-order death-rate kinetics. The first-order death-rate constant as calculated from the decrease in viable cells, the increase in dead cells and the increase in LDH activity is linear proportional to the gas flow rate, with a specific hypothetical killing volume in which all cells are killed of about 2·10(-3) m(3) liquid per m(3) of air bubbles. In addition, an experiment was conducted in the same air-lift reactor with Vero cells grown inside porous Asahi microcarriers. The specific hypothetical killing volume calculated from this experiment has a value of 3·10(-4) m(3) liquid per m(3) of air bubbles, which shows that the porous microcarriers were at least in part able to protect the cells against the detrimental hydrodynamic forces generated by the bubbles.

Hybridomas in a bioreactor cascade: modeling and determination of growth and death kinetics.

Hybridomas were cultured under steady-state conditions in a series of two continuous stirred-tank reactors (CSTRs), using a serum-free medium. The substrate not completely converted in the first CSTR, was transported with the cells to the second one and very low growth rates, high death rates, and lysis of viable cells were observed in this second CSTR. These conditions are hardly accessible in a single vessel, because such experiments would be extremely time-consuming and unstable due to a low viability. In contrast to what is often observed in literature, kinetic parameters could thus be derived without the neccessity for extrapolation to lower growth rates. Good agreement with literature averages for other hybridomas was found. Furthermore, showing that the reactor series is a valuable research tool for kinetic studies under extreme conditions, the possibility to observe cell death under stable and defined steady-state conditions offers interesting opportunities to investigate apoptosis and necrosis. Additionally, a model was developed that describes hybridoma growth and monoclonal antibody production in the bioreactor cascade on the basis of glutamine metabolism. Good agreement between the model and the experiments was found.