bluuebunny/crossref_metadata_2025 huggingface dataset:
The permeability of EUDRAGIT RL and HEMA–MMA microcapsules to glucose and inulin
Authors: Douglas J. A. , Sefton M. V.
Published: December 2004

Measurement of the rate of glucose diffusion from EUDGRAGIT RL and HEMA‐MMA microcapsules coupled with a Thiele modulus/Biot number analysis of the glucose utilization rate suggests that pancreatic islets and CHO (Chinese hamster ovary) cells (at moderate to high cell densities) should not be adversely affected by the diffusion restrictions associated with these capsule membranes. The mass transfer coefficients for glucose at 20 °C were of the same order of magnitude for both capsules, based on release measurements: ∼5 × 10−6 cm/s for EUDRAGIT RL and ∼2 × 10−6 for HEMA‐MMA. Inulin release from EUDRAGIT RL was slower than for glucose (mass transfer coefficient 14 ± 4 × 10−8 cm/s). The Thiele moduli were much less than 1, either for a single islet at the center of a capsule or CHO cells uniformly distributed throughout a capsule at 10−6 cells/ mL, so that diffusion restrictions within the cells in EUDRAGIT RL or 800 μm HEMA‐MMA capsules should be negligible. The ratio of external to internal diffusion resistance (Biot number) was less than 1, so that at most, only a small diffusion effect on glucose utilization should be expected (i.e., the overall effectiveness factors were greater than 0.8). These calculations were consistent with experimental observation of encapsulated islet behavior but not fully with CHO cell behavior. Permeability restricted cell viability and growth is potentially a major limitation of encapsulated cells; further analysis is warranted.
A structured kinetic model for Zymomonas mobilis ATCC10988
Authors: Veeramallu Uday , Agrawal Pramod
Published: December 2004

The inhibitory effects of glucose and ethanol on Zymomonas mobilis ATCC10988 were isolated through kinetic analysis of transient batch fermentation data. Growth of Z. mobilis was inhibited above a glucose concentration of 80 g/L. Growth was mildly inhibited by ethanol to 50 g/L, and severely inhibited above this concentration. Specific rates of ethanol production and glucose uptake were essentially invariant during batch fermentation. A structured kinetic model was developed, by way of augmentation of the Extended Bottleneck model, to quantify the kinetics of the growth and product formation processes. The model successfully describes the transient batch fermentation of Z. mobilis over a wide range of initial glucose concentration in a semidefined medium.
Balanced nutrient fortification enables high‐density hybridoma cell culture in batch culture
Authors: Jo Eui‐Cheol , Park Hae‐Joon , Park Jong‐Myun , Kim Kyong‐Ho
Published: December 2004

Cells of an in vitro culture system are not the same as for an in vivo system, metabolically and physiologically; ineffective utilization of nutrients occurs by cells in vitro. Therefore, a simpler approach is needed to examine closely and overcome differences between in vivo and in vitro cells.

Recognizing the ineffectiveness of nutrient utilization in vitro, we have constructed, a balanced, fortified high‐density medium based on RPMI 1640 medium previously optimized for relatively low‐density cell culture. The high‐density medium was used to cultivate a hybridoma line in a batch spinner flask culture. In this fortified medium, a hybridoma cell line 2c3.1 was cultivated to near 1 × 107 cells/mL in batch suspension culture. During the culture, glucose, glutamine, and 10 essential amino acids of concentrations five times richer than normal in the medium were almost thoroughly consumed. Combined analysis of these consumption profiles reveals that the balanced, fortified nutrient supply contributes much to cellular activity to overcome the limitations of in vitro cellular growth. Intermediate metabolites, such as ammonium ion and lactic acid, were produced over concentrations reported until now to be inhibitory. This observation suggests that the major conclusive factor against cellular growth over the critical cell density is not so‐called inhibitory metabolites. As a result of the high‐density culture, 5–8 times higher production of a monoclonal antibody for hepatitis B surface antigen (anti‐HBs) was obtained.

Active cellular consumption of all the essential nutrients and the corresponding production of MAb strongly support the potential of our approach to overcome the growth limitation of cells in vitro and to obtain high‐density hybridoma cell culture.
Complications encountered using cibacron blue F3G‐A as a ligand for affinity precipitation of lactate dehydrogenase
Authors: Morris John E. , Fisher Rod R.
Published: December 2004

The use of the affinity interaction between Cibacron Blue F3G‐A (CB) and NADH‐dependent enzymes to selectively precipitate these enzymes has been examined. An attempt was made to form crosslinked precipitates of lactate dehydrogenase (LDH) using bis‐ and poly‐CB conjugates. When precipitation was not observed, an examination of the interaction between the enzyme and the conjugated CB was made. Quasielastic light scattering indicated only a slight radius increase, the greatest being from 50 to 130 Å, when a CB‐dextran conjugate was added to a solution of LDH, and no increase when bis‐CB made with a 1, 6‐diaminohexane spacer was added to a similar solution. The results of enzyme inhibition studies showed that conjugated CB bound at the NAD+ site of LDH. Spectral measurements of the conjugated CB below 5 μM were similar to those reported for a stacking interaction that occurs in solutions with CB concentrations above 5 μM We conclude that the conjugated CB is binding to the LDH, but that a competing dye stacking interaction prevents extensive cross‐linking of the LDH, and thus inhibits precipitation.
A hollow‐fiber reactor design for NMR studies of microbial cells
Authors: Briasco Catherine A. , Ross Debra A. , Robertson Channing R.
Published: December 2004

A hollow‐fiber membrane reactor was designed and constructed to allow perfusion of entrapped, dense Escherichia coli cells with nutrient medium during examination of cell metabolism using nuclear magnetic resonance (NMR) spectroscopy. Phosphorus‐31 NMR spectra of the perfused cells included peaks for nucleoside di‐ and triphosphates, sugar phosphates, and pH‐sensitive peaks for inorganic phosphate. The observed intensity of the lumenal inorganic phosphate peak was found to depend on flow rate, ruling out the use of this peak as a concentration reference. Absolute intracellular pH values obtained from NMR measurements were found to be accurate to 0.2 pH units due to uncertainties in intracellular ionic concentrations. Relative pH values, however, were found to be sensitive to cell energetic status. The response of E. coli intracellular pH following a shift to carbon starvation medium was monitored with a resolution of 3 min. Use of a hollow‐fiber reactor for cell containment and perfusion during NMR spectroscopy enables metabolic experiments of longer duration and of greater variety than is possible using standard, nonperfused sample tubes.
The role of the plasma membrane fluidity on the shear sensitivity of hybridomas grown under hydrodynamic stress
Authors: Ramírez Octavio T. , Mutharasan R.
Published: December 2004

The role of the plasma membrane fluidity (PMF) on the shear sensitivity of HB‐32 hybridomas to laminar fluid shear was investigated. Steady‐state fluorescence anisotropy (rs) of the cationic fluorescent probe 1‐[4‐(trimethylamino) phenyl]‐6‐phenylhexa‐1,3,5‐triene, was used to evaluate the PMF of whole hybridoma cells. The PMF was manipulated by the addition of the anesthetic benzyl alcohol, by temperature changes and by cholesterol enrichment. The effect of these PMF modifying procedures on the shear sensitivity of HB‐32 was assessed by exposing the cells to defined levels of laminar shear stress in a Couette flow device. Conditions that resulted in lower rs values (indicating higher PMF) yielded a more fragile cell. Batch cultivations supplemented with the shear protective agent Pluronic® F‐68 showed higher values of rs compared to control experiments during various growth phases, suggesting that the protective mechanism of Pluronic F‐68 relies on its ability to decrease the PMF through direct interaction with the plasma membrane. The protective mechanism of serum against turbulent fluid shear is also discussed from analysis of growth and death kinetics of agitated and static cultures at increasing serum levels. The results of this study show that the fluid state of the plasma membrane is important in determining the integrity of hybridomas when exposed to lethal shear levels. It is concluded that increasing membrane fluidity correlates with increasing shear sensitivity.
Determination of growth and coupled nitrification/denitrification by immobilized Thiosphaera pantotropha using measurement and modeling of oxygen profiles
Authors: Hooijmans C. M. , Geraats S. G. M. , van Neil E. W. J. , Robertson L. A. , Heijnen J. J. , Luyben K. Ch. A. M.
Published: December 2004

An oxygen microsensor in combination with mathematical modeling was used to determine the behavior of immobilized Thiosphaera pantotropha. This organism can convert ammonia completely to nitrogen gas under aerobic conditions (coupled nitrification/denitrification) and denitrifies nitrate at highest rates under anaerobic conditions. Immobilization of T. pantotropha can result in aerobic and anaerobic zones inside the biocatalyst particle which will be advantageous for the conversion of ammonia and nitrate from wastewater. However, information of the effects of immobilization on the physiology of T. pantotropha is necessary for the development of such a system. This article gives the extension of a model developed to describe the behavior of chemostat cultures of T. pantotropha so that it can be used for immobilized cells. The original model was based on metabolic reaction equations. Kinetic and diffusion equations have now been added. Experimental verification was carried out using a stirred tank reactor and a Kluyver flask. After immobilization in agarose, the cells were grown in the particles under continuous culture conditions for 3 days. After 24 h the oxygen penetration depth showed a constant value of 100 μ, indicating that a steady state was reached. Scanning electron micrographs showed that large colonies of cells were present in this 100‐μm aerobic layer.

From the dynamics of the start‐up phase, several parameters were determined from measurements of the oxygen concentration profiles made every few hours. The profiles simulated by the model were fitted to the measured data. The average value for the maximum specific growth rate was 0.52 h−1, and the maximum oxygen conversion rate was 1.0 mol Cmol−1 h−1. The maximum specific acetate uptake rate was 2.0 mol Cmol−1 h−1, and the Monod constant for acetate was 2.9 × 10−2 mol m−3. The maximum specific nitrification rate was 0.58 × 10−1 mol Cmol−1 h−1, and the amount of oxygen necessary for nitrification was 11% of the total oxygen uptake rate. Most of the kinetic parameters determined for the immobilized cells were in good agreement with those for the suspended cells. Only the maximum specific growth rate was significantly higher, and the maximum specific nitrification rate was some what lower than for suspended cells. The experimental results clearly show that an oxygen microsensor, in combination with mathematical modeling, can successfully be used to elucidate the kinetic behavior of immobilized, oxygen‐consuming, cells.
Moisture‐induced aggregation of lyophilized proteins in the solid state
Authors: Liu W. Robert , Langer Robert , Klibanov Alexander M.
Published: December 2004

A critical problem in the storage and delivery of pharmaceutical proteins is their aggregation induced by moisture. A model system has been elaborated and investigated to elucidate the mechanism of this phenomenon. When 10 mg of bovine serum albumin lyophilized from an aqueous solution of pH 7.3 are wetted with just 3 μL of a buffered physiological saline solution and incubated in the solid state at 37°C, the protein progressively loses its solubility in water; e.g., after a 24 h incubation 97% of the protein becomes insoluble. This moisture‐induced aggregation of albumin has been discovered to be due to an intermolecular S—S bond formation via the thiol‐disulfide interchange reaction. The dependence of the extent of the solid‐state aggregation on the amount and mode of addition of moisture and the atmosphere, additives, temperature, and history of the protein powder have been investigated. The moisture‐induced solid‐state aggregation has also been established and studied for three other lyophilized proteins: ovalbumin, glucose oxidase, and β‐lactoglobulin. In all cases, the loss of solubility is caused by thiol‐disulfide interchange either alone or in combination with a conformational (noncovalent) process. The aggregation can be minimized by lyophilizing the proteins from acidic aqueous solutions, by adding inorganic salts, by co‐lyophilizing the proteins with water‐soluble polymers, or by controlling the moisture content at optimal levels.
A structured model for monoclonal antibody synthesis in exponentially growing and stationary phase hybridoma cells
Authors: Bibila T. , Flickinger M. C.
Published: December 2004

An initial structured unsegregated kinetic model describing monoclonal antibody synthesis by a murine hybridoma cell line (9.2.27) grown in 1 liter batch cultures is described. The model is based on the intracellular balances of the heavy and light chain coding mRNAs, the intracellular balances of heavy and light chains and the description of the kinetics of heavy and light chain assembly. Model parameters were varied with specific growth rate in order to account for changes in the rates of antibody synthesis and secretion with entrance of the cells from the exponential into the stationary phase of growth. The parameters were varied based. on experimental data obtained in our laboratory on the variation of total cellular RNA content and the half‐lives of heavy (H) and light (L) chain mRNAs with specific growth rate, and data from other investigators on immunoglobulin synthesis and secretion. The model successfully predicts the experimentally observed decrease in the intracellular heavy and light chain mRNA levels with entrance of 9.2.27 cells from the exponential into the stationary phase of growth, as well as the extracellular accumulation of antibody (IgG2a) during batch culture.
Protein adsorption and leakage in carrier–enzyme systems
Authors: Ulbrich Renate , Golbik Ralph , Schellenberger Alfred
Published: December 2004

The capability of binding enzymes adsorptively to unmodified and silanized silica and glass as well as modified polystyrene carriers was studied for α‐amylase, β‐amylase, and α‐chymotrypsin. In most cases a high percentage of protein was bound very firmly under considerable loss of activity. The leakage of protein from the carriers was studied by measuring the intrinsic protein fluorescence on β‐amylase adsorptively bound to aminopropyl silica, aminomethyl, and hexadecylaminomethyl polystyrene. It was compared with the leakage of β‐amylase covalently bound to the same carriers via glutaraldehyde, trichloro‐triazine, or benzoquinone. In the absence and in the presence of substrate, at 25 and at 60°C, the leakage rates of the adsorptively bound enzymes were not higher than in the covalently bound systems. The poorest binding stability was found in benzoquinone‐coupled β‐amylase derivatives. It is even reduced at higher temperatures, whereas the temperature did not show any remarkable influence on the leakage of the other derivatives. In adsorptively as well as in all the covalently bound systems, the presence of substrate did not promote the protein leakage.
Determination of kinetic parameters related to the piasmid instability: For the recombinant fermentation under repressed condition
Authors: Park Sung Hoon , Ryu Dewey D. Y. , Lee Sun Bok
Published: December 2004

The plasmid stability under the repressed state of cloned gene was studied theoretically as well as experimentally using recombinant E. coli K12ΔH1Δtrp/pPLc23trpA1 as a “host‐vector” model system. The important kinetic parameters studied were the plasmid loss rate (θ) describing the rate at which the plasrnid‐harboring cells lose plas‐mids and the plasmid‐free cells are generated per unit time and the difference in growth rates (Δ) between the two genotypes. These parameters were carefully defined, studied, and compared with other key kinetic parameters involved in the recombinant fermentation to further our understanding of metabolism of recombinants. The ratio of the concentration of plasmid‐free cells to plasmid‐harboring cells (Ω) was introduced, and the mathematical model was derived and used for the determination of the kinetic parameters associated with plasmid instability. These methods developed based on the theoretical considerations were tested experimentally. The results of these methods were compared, and the best method was selected and recommended. The effect of temperature and dilution rate on kinetic parameters θ and Δ were also studied in continuous culture, in order to provide some practical information related to the operation and control of recombinant fermentation processes.
Papain in organic solvents: Determination of conditions suitable for biocatalysis and the effect on substrate specificity and inhibition
Authors: Stevenson David E. , Storer Andrew C.
Published: December 2004

Synthesis of N‐CBZ‐(N‐Carbobenzoxy)‐1‐amino‐acid methyl esters from N‐CBZ‐amino acids and methanol has been used as an assay to examine the properties of papain in organic solvents containing small amounts of water. Papain is active in solvents ranging in polarity from acetonitrile to tetrachloromethane. The optimal activity in each solvent varied only about three to four fold, but the amount of added water required to achieve it varied from 4% (v/v) in acetonitrile to 0.05% (v/v) in tetrachloromethane. The enzyme was generally more stable in hydrophobic solvents and at lower water contents. The apparent Km value of CBZ‐glycine was 26 times higher in acetonitrile than in toluene due to differential partitioning of the substrate between aqueous and organic phases. The substrate specificity of the enzyme was qualitatively little different from that in aqueous solution, with amino acid derivatives still the best substrates. Nitrile analogs of substrates inhibited the enzyme, as they do in aqueous solution, and inhibition by a variety of substituted aromatic hydrocarbons showed that the main specificity of papain for hydrophobic side chains at its S2 subsite, was little affected. The results show that papain can catalyze reactions under a variety of conditions in organic solvents but its substrate specificity is little changed from that in aqueous media.
Kinetics and modeling of temperature effects on batch xanthan gum fermentation
Authors: Shu Chin‐Hang , Yang Shang‐Tian
Published: December 2004

Batch fermentation kinetics of xanthan gum production from glucose by Xanthomonas campestris at temperatures between 22°C and 35°C were studied to evaluate temperature effects on cell growth and xanthan formation. These batch xanthan fermentations were modeled by the logistic equation for cell growth, the Luedeking–Piret equation for xanthan production, and a modified Luedeking–Piret equation for glucose consumption. Temperature dependence of the parameters in this model was evaluated. Growth‐associated rate constants increased to a maximum at ∼30°C and then decreased to zero at ∼35°C. This temperature effect can be modeled using a square‐root model. On the contrary, non‐growth‐associated rate constants increased with increasing temperature, following the Arrhenius relationship, in the entire temperature range studied. The model developed in this work fits the experimental data very well and can be used in a simulation study. However, due to the empirical nature of the model, the parameter values need to be reevaluated if the model is to be applied to different growth conditions.
Hydrolysis of inulin: A kinetic study of the reaction catalyzed by an inulinase from Aspergillus ficuum
Authors: Carniti Paolo , Beltrame Pier Luigi , Guardione Donatella , Focher Bonaventura , Marzetti Annamaria
Published: December 2004

A kinetic study of the hydrolysis of inulin was performed by using as catalyst a commercial inulinase from Aspergillus ficuum. The reaction was studied carrying out initial rate as well as time course measurements. Both inulinase and invertase activities of the enzyme were taken into account, and the corresponding kinetic parameters were determined in the temperature range 30‐50°C. The activation energies of the turnover constant for inulinase and invertase activities were found to be similar (56–57 kJ · mol−1). The ratio S/I of invertase to inulinase activity was 1.6 regardless of temperature. The thermal degradation of the enzyme was also investigated up to 70°C, and an activation energy of 350‐370 kJ · mol−1 was evaluated.
Transient response method for evaluating the rate constants of reactions over immobilized enzymes
Authors: Wu Jau‐Yann , Weng Hung‐Shan
Published: December 2004

The transient response method was utilized to evaluate the rate constants of reaction over immobilized enzyme. Glucose oxidation catalyzed by the immobilized glucose oxidase in a fixed‐bed reactor was selected as an example. A theoretical model including the effects of axial dispersion, film diffusion, and intraparticle diffusion was established for the reactor. The individual rate constant of each elementary step of this enzymatic reaction was determined through direct fitting of the experimental response data to the model.
A novel technique for the measurement of disruption in high‐pressure homogenization: Studies on E. coli containing recombinant inclusion bodies
Authors: Middelberg Anton P. J. , O'Neill Brian K. , L. Bogle I. David , Snoswell Mark A.
Published: December 2004

The high‐pressure homogenization of Escherichia coli, strain JM101, containing inclusion bodies of recombinant porcine somatotropin was investigated. A novel technique employing an analytical disc centrifuge was used to monitor the disruption. This a direct technique which measures cell disintegration rather than soluble protein release. The technique is particularly suited to measurements where the disruption approaches 100%. The disk centrifuge provides a size distribution of the homogenate, and furnishes evidence for the preferential disruption of larger cells. For E. coli containing inclusion bodies, and increase in the cell feed concentration from 145 g/L (wet weight) to 330 g/L resulted is poorer homogenization. Poorer disruption was also obtained by lowering the feed temperature from 20°C to 5°C. Only slight variations in performance were obtained by increasing the feed pH from 7.5 to 9.0 or by storing the feed at 4°C for 24 h prior to disruption. Comparison with uninduced E. coli strain JM101, showed that the disruption obtained is higher for bacteria containing a recombinant inclusion body.
Routine determination of enzyme kinetics using Plate reader
Authors: Galgani F. , Cadiou Y. , Bocquene G.
Published: December 2004

A method is described for the routine determination of kinetics parameters from colorimetric enzyme reactions using a 96‐well microtiter plate reader commonly used in immunoassay. A program in Turbo Pascal have been developed for the analysis of results. The system performs the description of plate format, the definition of conditions of assays, the measurement of enzyme reactions and collection of data, analysis, visualization of results and modification of data. Measurement of Km and Vmax is made with different methods, including iterative determination, Lineweaver‐Burk transformation, and Hofster transformation, to give the final values of kinetic parameters with their associated standards errors. This approach should be applicable to routine determination of kinetic parameters.
Diffusion in gels containing immobilized cells: A critical review
Authors: Westrin Bengt A. , Axelsson Anders
Published: December 2004

Eleven experimental investigation of diffusion in gels containing immobilized cells are reviewed. The experimental data, which quantitatively express the diffusion coefficient as a function of the cell concentration, are compared with a number of well‐known equations developed for mass transfer in heterogeneous media. Based on this comparison, a procedure for the theoretical prediction of effective diffusion coefficients in cell‐containing gels is recommended.
Affinity partitioning of vancomycin
Authors: Godbole Pradyumna P. , Tsai Ruey‐S. , Clark William M.
Published: December 2004

The use of the Flanagan and Barondes model14 describing affinity partitioning as an aid in designing separation systems is discussed. Experimental studies are described for affinity partitioning of vancomycin, a glycopeptide antibiotic, in a water–methoxypolyethylene glycol‐dextran system using methoxypolyethylene glycol‐dextran system using methoxypolyethylene glycol bound D‐alanyl‐Dalanyl‐D‐alanine or D‐alanyl‐D‐alanine as the reversible affinity ligand. Even for this ideal case of 1:1 binding interaction, the model only qualitatively predicts the affinity effect when all model parameters are measured independently. The discrepancy between measured and predicted values can be attributed to a difference in exposed surface of the free antibiotic and ligand compared to that in the bound state.

The effect of experimentally varying model parameters is also described. It was determined that a polymers‐ligand which partitions more strongly to the top phase would provide the most significant enhancement to this affinity partitioning system. Such an improvement can be made by increasing the molecular weight of the hydrophobicity of the polymer‐ligand. A process for vancomycin recovery from fermentation broth using D‐‐alanyl‐D‐alanine sepharose as affinity ligand is described.
Factors controlling cell proliferation and antibody production in mouse hybridoma cells: I. Influence of the amino acid supply
Authors: Duval D. , Demangel C. , Munier‐Jolain K. , Miossec S. , Geahel I.
Published: December 2004

To investigate the influence of medium amino acid resources on hybridoma cell proliferation and monoclonal antibody secretion, we first determined, in two different cell lines, the pattern of amino acid consumption. We then showed that complementation of each cell line by an amino acid solution prepared to cover its needs led in both cases to a marked increase in cell density and monoclonal antibody production. This observation suggests that the amino acid supply is one of the factors limiting cell growth and productivity in typical batch processes, and we demonstrated that a similar limitation occurs in a semicontinuous culture. Finally, we showed that the cell decline observed after a period of amino acid deficiency is not irreversible and that proliferation could be resumed if the required amino acids were added.
Cell‐cycle‐dependent protein accumulation by producer and nonproducer murine hybridoma cell lines: A population analysis
Authors: Kromenaker Sandra J. , Srienc Friedrich
Published: December 2004

Single‐cell rates of accumulation of cellular protein have been determined as a function of total protein content using flow cytometry and population balance equations for exponentially growing murine hybridoma cells in the individual G1, S1 and G2 + M cell cycle phases. A novel flow cytometric technique for the identification of hybridoma cells in mitosis was developed and implemented. The data were obtained from a producer cell line which synthesizes and secretes high levels of monoclonal antibodies, and from a nonproducer clone which does not synthesize and secrete substantial amounts of antibody. The results indicate that the kinetics of single‐cell protein accumulation in these two cell lines are considerably different. In particular, low protein content G1 phase producer cells were characterized by a rate of protein accumulation which was approximately five times higher than the mean rate observed for higher protein content producer cells cycle phase. In contrast, the rate of accumulation of protein increased continuously with totalprotein content for the G1 phase nonproducer cells. S phase hybridoma cells were characterized by a considerably lower rate of protein accumulation which did not vary much with protein content for either cell line. Finally, G2 + M phase producer cells demonstrated a negative rate of protein accumulation which indicates that the rates of protein synthesis. It was hypothesized that these differences in total protein accumulation are caused by differences in monoclonal antibody accumulation. The distribution of rates suggests the need for a segregated approach to the modeling of the kinetics of antibody production in hybridomas.
Molecular design of expression systems: Comparison of different control configurations using molecular mechanism models
Authors: Chen Wilfred , Bailey James E. , Lee Sun Bok
Published: December 2004

Molecular‐level mathematical models have been used to evaluate the effectiveness of eight different configurations of repressor synthesis control on the regulation of cloned gene expression initiated from a promoter‐operator sequence. Both single and dual‐repressor situations were considered, employing genetically structured models for the lac and λPR promoter‐operators in example calculations. Simulation results suggest that the most effective mode of cloned gene expression control is a cross‐regulation configuration carried on a multicopy plasmid. This system was able to control cloned gene transcription in the uninduced state over a broad range of plasmid copy number and also provided the highest overall transcription rate in the induced state. The general strategies suggested by these simulations should be applicable for other repressor‐operator‐promoter systems in diverse hosts.
Kinetic study of hybridoma cell growth in continuous culture: II. Behavior of producers and comparison to nonproducers
Authors: Frame Kelly K. , Hu Wei‐Shou
Published: December 2004

A hybridoma cell line, AFP‐27‐P, was cultivated in continuous culture under glucose‐limited conditions. The viable cell concentration, dead‐cell concentration, and cell volume all varied with the dilution rate. A model previously developed for a nonproducing clone of the same cell line, AFP‐27‐NP, was extended to describe the behavior of the cells. The relationship between the specific growth rate and glucose concentration is described by a function similar to the Monod model. A threshold glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentration and a minimum specific growth rate are incorporated; the model is meaningful only at glucose concentrations and specific growth rates above these levels. The relationship between the death rate and the glucose concentration is described by an inverted Monod‐type function. Furthermore, the yield coefficient based on glucose is constant in the lower range of specific growth rates and changes to a new constant value in the upper range of specific growth rates. No maintenance term for glucose consumption is used; in the plot of specific glucose consumption rate vs. specific growth rate, the line intercepts the specific growth rate at a value close to the minimum growth rate. The productivity of antibody as a function of the specific growth rate is described by a mixed type model with a noon‐growth‐associated term and a negative‐growth‐associated term. The values for the model parameters were determined from regression analysis of the steady state data.
Simple structured model for α‐amylase synthesis by Bacillus amyloliquefaciens
Authors: Ponzo John H. , Weigand William A.
Published: December 2004

A predictive, simple, structured model describing the synthesis of α‐amylase by Bacillus amyloliquefaciens was formulated. Three key intracellular processes were identified (i.e, translation, and excretion) along with two key intracellular components (i.e., mRNA and the intracellular form of the α‐amylase enzyme). Nearly all the model parameters were estimated by means of performing independent experiments, primarily fed‐batch experiments. The model was shown to predict transient system behavior in batch and in fed‐batch operation with some limitation and minor model parameter revisions. Since a principal objective was to demonstrate that independent experimental parameter determination can be used to construct the predictive model, further fine‐tuning of the parameters may be necessary before application for optimization and control purposes.
Assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum‐free and serum‐supplemented media using a temperature‐sensitive baculovirus
Authors: King G. , Kuzio J. , Daugulis A. , Faulkner P. , Allen B. , Wu J. , Goosen M.
Published: December 2004

Spodoptera frugiperda insect cells were grown in Sf‐900 serum‐free medium and two kinds of serum‐supplemented media (IPL −41 and Grace's). The specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h−1 respectively, at 33°C. The IPL −41 medium supported to highest maximum cell density (10.6 × 106 cells/mL) compared to 3.5 × 106 and 8.7 × 106 cells/mL with the Grace's and serum‐free media, respectively. In temperature shifdown experiments with a temperature‐sensitive baculo‐virus (acts10YM1CAT), virus titer and chloramphenicol acetyl transferase (CAT) expression were highest in the IPL −41 (5.1 × 107 PFU/mL and 20000 U/mL). Use of Grace's medium gave higher virus titers than the serum‐free medium (4.4 × 106 vs 4.1 × 105 PFU/mL) as well as higher CAT titers (7050 vs 1980 U/mL). Interestingly, in the three media used, the highest virus and CAT titers were obtained at MOI (multiplicity of infection) of 0.02 At MOI of 2.0 virtually no increase in virus of CAT titer was observed. This result is contrary to those obtained at constant‐temperature (27°C) infection and cell culture, in which higher virus titers and recombinant protein expression and obtained at higher MOI.
Effects of Glutamate, Glucose, Phosphate, and Alkali metal ions on cephamycin C production by Nocardia lactamdurans in defined media
Authors: Kirpekar A. C. , Kirwan D. J. , Stieber R. W.
Published: December 2004

A High cephamycin C producing strain of Nocardia lactam‐durans was used to study cell growth and antibiotics production in defined media. Batch fermentations in shake flasks and stirred tanks showed that antibiotic production occurred during cell growth and the production rate rapidly decline as the growth slowed. Glutamate served as a primary substrate during this phase. Later, ammonia was utilized along with a remainder of the glucose. Rapid antibiotic production occurred in this phase. Increased glutamate promoted higher growth, a rise in ammonium ion concentration, and a marked reduction in antibiotic titers. An increase of the glucose concentration along with the glutamate concentration balanced to the medium; no ammonium ion rise occurred and a peak specific antibiotic titer comparable to the control medium was obtained. In a phosphate‐limited medium, cell growth equivalent to the control medium and increased antibiotic titers were obtained. In these experiments, adjustment of Na+ and K+ ion concentration equal to that in the control medium was found to be important. Based on carbon and nitrogen balances, the activity of the key nitrogen metabolism enzymes, and the published literature, a two‐stage model of regulation is suggested.
Electrically promoted protein production by mammalian cells cultured on the electrode surface
Authors: Kojima Junichiro , Shinohara Hiroaki , Ikariyama Yohihito , Aizawa Masuo , Nagaike Kazuhiro , Morioka Satoshi
Published: December 2004

Protein production of mammalian cells has been promoted by applying a small constant potential to the surface of an electrode on which cells are cultured. Human carcinoma line of MKN45 cells were cultured on the surface of a platinum‐coated plastic plate electrode. Low d.c. voltage of constant potential was applied to the electrode during 4‐day culture to modulate the production of carcinoembryonic antigen (CEA). The amounts of both secreted and membrane‐bound CEA were dependent on the applied potential during culture. Secreted CEA was more than twice in amount in the potential range from 0.2 V to 0.6 V vs. Ag/Agcl as compared with that of normal culture. In the potential range, CEA was also increased in membrane‐bound form. The potential‐controlled cell culture may have an enhanced effect on protein production.
A study on enzymatic reaction using a liquid emulsion membrane technique
Authors: Ha Heung Yong , Hong Seong‐Ahn
Published: December 2004

An enzymatic reaction using a liquid emulsion membrane technique was studied to investigate the effects of some experimental variables on the stability of liquid membrane, enzyme deactivation, and transport of substrates and products. The hydrolysis of L‐phenylalanine methyl ester by α‐chymotrypsin was selected as a model reaction system. First, a transport mechanism for the substrates and products across the membrane was qualitatively identified. Second, it was found that the pH of the internal phase was one of the most important variables to determine the enzyme activity in a liquid membrane. Third, the effect of membrane phase which consists of surfactant, carrier, and organic solvent on the emulsion stability was investigated. It was found that the properties of the organic solvents greatly affect the emulsion stability. For an optimum condition, it was possible to reuse the emulsion which consists of membrane phase and internal phase without further separation. It was finally concluded that the enzyme in a liquid membrane retained 60% of its native activity in spite of vigorous mixing during the emulsification step.
A two‐step enzymatic synthesis of dipeptides
Authors: Schwarz A. , Wandrey C. , Steinke D. , Kula M. R.
Published: December 2004

A simple system is introduced to produce dipeptides continuously by enzyme catalyzed condensation of amino acid esters and amino acid amides. Synthesis of N‐terminal free dipeptide‐amides is achieved by means of carboxypeptidase Y. The peptide‐amide is deamidated utilizing a newly isolated peptide‐amide is deamidated utilizing a newly isolated peptide‐amidase. Separation of substrates and products is accomplished by anion‐exchange chromatography. Modeling of the reactions shows that the two reactions have to be carried out in a cascade of two reactors in order to prevent hydrolysis of the peptide by the carboxypeptidase. Continuous production of Kyotorphin (H‐TyrArg‐OH) with a space–time yield of 257 g/L · d shows the feasibility of this concept.
Stabilization of substilisin E in organic solvents by site‐directed mutagenesis
Authors: Martinez Pascal , Van Dam Mariana E. , Robinson Amy C. , Chen Keqin , Arnold Frances H.
Published: December 2004

Subtilisin E was rationally engineered to improve its stability in polar organic solvents such as dimethylformamide (DMF). A charged surface residue, Asp248, was substituted by three amino acids of increasing hydrophobicity, Asn, Ala, and Leu; all three variants were stabilized with respect to wild type in 80% DMF. This stabilization was only observed in the presence of high concentrations of the organic solvent: no stability enhancements were observed in 40% DMF. In contrast, the mutation Asn218 → Ser alters internal hydrogen bonding interactions and stabilizes subtilisin E in both 40% and 80% DMF. This study provides additional evidence that substitution of surface‐charged residues is a generally useful mechanism for stabilizing enzymes in organic media and that the stabilizing effects of such substitutions are unique to highly altered solvent environments. The effects of the single amino acid substitutions on free energies of stabilization are additive in the Asp248 → Asn + Asn218 → Ser combination variant, yielding an enzyme that is 3.4 times more stable than wild type in 80% DMF.
Molecular imprinting of proteins and other macromolecules resulting in new adsorbents
Authors: Dabulis Katherine , Klibanov Alexander M.
Published: December 2004

When the model protein bovine serum albumin (BSA) was dissolved in a concentrated aqueous solution of the multifunctional ligand L‐malic acid, the solution was lyophilized, and the solid residue thoroughly washed with tetrahydrofuran to extract malic acid, then the resultant (“imprinted”) protein was capable of binding 26.4 ±0.9 mol equivalents of the ligand in anhydrous ethyl acetate. The nonimprinted BSA (i.e., that prepared in the same manner apart from the absence of malic acid) bound less then one‐tenth of that amount under identical conditions. Furthermore, both imprinted and nonimprinted BSA exhibited little binding of L‐malic acid in water. The imprinted BSA retained its “memory” for the ligand in ethyl acetate even after a prolonged incubation under vacuum; dissolution in water, however, eliminated the imprinted protein's binding capacity. The BSA imprinted with L‐malic acid displayed affinity for this ligand not only in ethyl acetate but also in many other anhydrous solvents. It was found that the higher the solvent's propensity to form hydrogen bonds, the lower the protein–ligand binding in it, thus pointing to hydrogen bonds as the driving force of this binding. Studies with completely or partially cleaved BSA, with other globular proteins, glutathione, and poly(L‐aspartic acid) revealed that the critical requirement for the imprintability is the presence of a sufficiently long polymeric chain. Moreover, many hydrogen‐bond‐forming macromolecules other than proteins, such as dextrans and their derivatives, partially hydrolyzed starch, and poly(methacrylic acid), also could be imprinted for subsequent binding in ethyl acetate. The mechanism of imprinting and binding inferred from these experiments involves a multipoint hydrogen bonding in water of each ligand molecule with two or more sites on the polymeric chain, thereby folding a segment of the latter into a cavity around the ligand; following lyophilization and extraction of the ligand, the cavities remain in organic solvents (but not in water) and give rise to ligand binding. This conclusion is supported by the results of binding of numerous malic acid analogs and related ligands to BSA imprinted with L‐malic acid. Finally, BSA imprinted with malic acid was used as a selective adsorbent for a chromatographic separation of an equimolar mixture of maleic and acrylic acids in ethyl acetate.
Use of a structured kinetic model of antibody synthesis and secretion for optimization of antibody production systems: II. Transient analysis
Authors: Bibila Theodora A. , Flickinger Michael C.
Published: December 2004

The dynamic behavior of the monoclonal antibody (MAb) secretory pathway is studied by transient simulations using our previously developed structured kinetic model for antibody synthesis and secretion by hybridoma cells. The response of the secretory pathway to blocks in specific pathway steps and step changes in characteristic pathway parameters is presented in order to gain a better understanding of pathway dynamics and identify possible ratelimiting steps in the pathway. Model simulations suggest that the step of antibody assembly in the endoplasmic reticulum (ER) is a very good candidate for a rate‐limiting step in the antibody secretory pathway in fast‐growing hybridoma cells, whereas translation of the heavy and light chains is most likely rate–limiting in slowly growing or stationary phase cells. Transient simulation results are compared with experimentally observed transient changes in specific antibody secretion rates and used to suggest strategies for optimizing antibody secretion in large‐scale production systems.
Production of soluble sugar from cellulosic materials by repeated use of a reversibly soluble‐autoprecipitating cellulase
Authors: Taniguchi Masayuki , Hoshino Kazuhiro , Watanabe Kouichi , Sugai Kazuhito , Fujii Michihiro
Published: December 2004

A reversibly soluble‐autoprecipitating cellulase was prepared by covalently immobilizing Meicelase on an enteric coating polymer (AS‐L, hydroxypropyl methylcellulose acetate succinate). The immobilized enzyme (M‐AS) was reversibly soluble‐insoluble depending on the pH of the reaction medium. Moreover, the enzymatic activity of M‐AS, in a soluble state, for microcrystalline cellulose was higher than that of the conventional solid immobilized enzyme. On the other hand, M‐AS, in an insoluble state, and good properties of self‐sedimentation, and a large portion of M‐AS spontaneously precipitated for about 15 min at pH 3.8. In the repeated hydrolysis of rice straw, delignified with sodium hydroxide using a bioreactor with a conical bottom, M‐AS was separated batchwise from a production solution by self‐sedimentation in a conical bottom of the bioreactor. When delignified, rice straw was hydrolyzed repeatedly by reuse of M‐AS; the total amount of soluble sugar produced from 5 g of delignified rice straw was 2.7 g. Further, by the hydrolysis method with repeated removal of reaction inhibitors (soluble sugar), the time required for converting the same amount of delignified rice straw to soluble sugar was significantly reduced, as compared with that by the standard reaction method.
Glucose‐limited chemostat culture of chinese hamster ovary cells producing recombinant human interferon‐γ
Authors: Hayter Paul M. , Curling Elisabeth M. A. , Baines Anthony J. , Jenkins Nigel , Salmon Ian , Strange Phillip G. , Tong Jeremy M. , Bull Alan T.
Published: December 2004

A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon‐γ (IFN‐γ) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h−1 with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose‐limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon‐γ production was maintained throughout the course of this culture, indicating that IFN‐γ expression was stable under these conditions. However, the specific rate of IFN‐γ production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN‐γ produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN‐γ increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN‐γ.
Compact automated displacement gas metering system for measurement of low gas rates from laboratory fermentors
Authors: Angelidaki I. , Ellegaard L. , Ahring B. K.
Published: December 2004

An automated metering system was developed for measuring biogas production from laboratory scale biogas digestors. The gas metering system is based on the principle of liquid displacement with a 100‐mL reversible cycle and registration. The gas meter is made entirely of plastic and rubber materials resistant to the corrosive components of biogas (e.g., H2S) and requires a 12 to 15 VDC power supply.