bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Critical Batch Operating Parameters in Fed‐Batch Recombinant E. coli Fermentations Using Decision Tree Analysis
Authors: Buck Kristan K. S. , Subramanian Venkatanarayanan , Block David E. 
Published: December 2002

AbstractTo develop a useful fermentation process model, it is first necessary to identify which batch operating parameters are critical in determining the process outcome. To identify critical processing inputs in large databases, we have explored the use of Decision Tree Analysis with the decision metrics of Gain (i.e., Shannon Entropy changes), Gain Ratio, and a multiple hypergeometric distribution. The usefulness of this approach lies in its ability to treat “categorical” variables, which are typical of archived fermentation databases, as well as “continuous” variables. In this work, we demonstrate the use of Decision Tree Analysis for the problem of optimizing recombinant green fluorescent protein production in E. coli. A database of 85 fermentations was generated to examine the effect of 15 process input parameters on final biomass yield, maximum recombinant protein concentration, and productivity. The use of Decision Tree Analysis led to a considerable reduction in the fermentation database through the identification of the significant as well as insignificant inputs. However, different decision metrics selected different inputs and different numbers of inputs to classify the data for each output.
Kinetics of the Alkaline Phosphatase Catalyzed Hydrolysis of Disodium p‐Nitrophenyl Phosphate in Frozen Model Systems
Authors: Terefe Netsanet Shiferaw , Mokwena Kereilemang Khanah , Van Loey Ann , Hendrickx Marc E. 
Published: December 2002

AbstractThe alkaline phosphatase catalyzed hydrolysis of disodium‐ p‐nitrophenyl phosphate was studied in four model systems comprising sucrose, maltodextrin, carboxymethylcellulose (CMC), and CMC‐lactose in a temperature range of –28 to 20 °C. In the maltodextrin and CMC‐lactose model systems, the reaction rate decreased to a very low value as the glass transition temperature was approached. In the CMC and CMC‐lactose systems with low initial solute concentration, as a consequence of freeze‐concentration, a rate maximum around the initial freezing temperature was observed. The Arrhenius equation described the temperature dependence of the reaction rate both in the liquid and the glassy states in all systems studied, while a slightly curved Arrhenius plot was observed in the “rubbery” state of the CMC and CMC‐lactose systems. The WLF equation with system‐dependent coefficients described the kinetics in the rubbery state of all the model systems except sucrose, excluding the short temperature range where reaction rate enhancement with decreasing temperature was observed.
Comparison of Techniques for Monitoring Antibody Fragment Production in E. coli Fermentation Cultures
Authors: Bowering Leigh C. , Bracewell Daniel G. , Kesharvarz‐Moore Eli , Hoare Mike , Weir A. Neil C. 
Published: December 2002

AbstractThe use of an optical biosensor for monitoring antibody fragment accumulation following induction in a batch fermentation of recombinant E. coli is compared to the more traditional method of ELISA quantification. Using the biosensor, concentration data can be obtained within minutes of sample addition to the device, compared to an average assay time of 3–4 h for the ELISA. We describe two biosensor assays developed as an alternative to ELISA and compare them with ELISA in the ability to provide quantitative product accumulation profiles during fermentation. Discrepancies in titers recorded by the assays are explained by a combination of differences in product variants detected by each assay and interference from sample contaminants. Method of sample preparation is also shown to be important if accurate concentration data is required. Both biosensor assays are shown to be capable of providing product accumulation profiles comparable to those obtained by ELISA. The use of a rapid extraction technique would allow such data to be obtained during process operation, enabling improved fermentation control and more rapid process development.
Secretion of Cellobiase Is Mediated via Vacuoles in Termitomyces clypeatus
Authors: Mukherjee Sumana , Khowala Suman 
Published: December 2002

AbstractThe majority of cellobiase activity in Termitomyces clypeatus was localized in vacuolar fractions of the fungus under secretory and nonsecretory conditions of growth. Activities of marker proteins for subcellular organelles, e.g., vacuoles, cytosol, ER, and mitochondria, in mycelial extracts from the secreting conditions increased by approximately 20, 12, 5, and 2.5 times, respectively, as compared to those obtained from mycelium grown in nonsecreting conditions. The average size and concentration of vacuoles visualized by electron microscopy were also increased in secreting conditions in the fungus. The specific activity of cellobiase in vacuoles isolated in Ficoll‐sucrose gradient, as obtained from mycelial growth in secretory medium, was more than 40 times higher in comparison to that found from nonsecretory medium. The results indicated that subcellular localization of cellobiase in vacuoles is regulated by the cellular signaling prevailing in the fungus. Mycelial extraction of intracellular proteins by hand grinding and by bead‐beater from cells frozen in the presence or absence of liquid nitrogen was also compared. Maximum recovery of intracellular protein was obtained with the bead‐beater under aerobic conditions in the absence of nitrogen. Highest recovery of vacuoles up to 85% was obtained by single‐step ultracentrifugation of the mycelial extract of the fungus in Ficoll‐sucrose gradient. The method appeared to be useful for separation of other subcellular organelles in filamentous fungi.
Aqueous Extraction of β‐Glucuronidase from Transgenic Canola: Kinetics and Microstructure
Authors: Bai Yun , Nikolov Zivko L. , Glatz Charles E. 
Published: December 2002

AbstractAqueous extraction kinetics of recombinant β‐glucuronidase (rGUS) from transgenic canola ( Brassica napus) was investigated in terms of the particle size and microstructural characteristics resulting from canola seed processing. The canola had been transformed to express recombinant GUS intracellulary in the seed, and electron microscopy showed that the cells are distributed among (1) disrupted cells in a thin layer at or adjacent to the particle surface, (2) disrupted cells within the interior, and (3) intact cells within the interior. A simple compartmental model containing two extractable pools and a third nonextractable pool fitted the batch extraction results very well. Comparing the rate constants from the model to estimates of expected transport rates from the observed cell fractions showed that the two extractable pools roughly correspond to the two disrupted cell fractions. Both flaking, causing more extensive cell wall damage throughout the seed, and grinding, increasing the total surface area, increase the size of the first pool and, therefore, the extraction yield. Mass transfer from the same type of pool from two types of processed seed behaved similarly. GUS extraction from the first extractable pool is 10–20 times faster (<1 min) than from the second extractable pool.
On‐Line Determination of Biomass in a Microalga Bioreactor Using a Novel Computerized Flow Injection Analysis System
Authors: Meireles Luís A. , Azevedo José L. , Cunha João P. , Malcata F. Xavier 
Published: December 2002

AbstractA flow injection analysis (FIA) device has been developed, which is able to assay successfully for biomass in a microalga bioreactor. The device is fully computerized and is operated via diluting small aliquots of the culture followed by measuring optical density (OD); this figure is then accurately correlated with biomass, in terms of both cell number and ash‐free dry weight, during the entire culture time. Furthermore, the device is not expensive, is highly versatile, and is easy to operate owing to specifically developed, user‐friendly software. The growth rate and biomass productivity of Pavlova lutheri, cultivated under batch and semicontinuous modes, were monitored as experimental testing model.
Microcarrier Culture of Lepidopteran Cell Lines: Implications for Growth and Recombinant Protein Production
Authors: Ikonomou Laertis , Drugmand Jean‐Christophe , Bastin Georges , Schneider Yves‐Jacques , Agathos Spiros N. 
Published: December 2002

AbstractSeveral microcarrier systems were screened with Sf‐9 and High‐Five cell lines as to their ability to support cell growth and recombinant (β‐galactosidase) protein production. Growth of both cell lines on compact microcarriers, such as Cytodex‐1 and glass beads, was minimal, as cells detached easily from the microcarrier surface and grew as single cells in the medium. Cell growth was also problematic on Cytopore‐1 and ‐2 porous microcarriers. Cells remained attached for several days inside the microcarrier pores, but no cell division and proliferation were observed. On the contrary, insect cells grew well in the interior of Fibra‐Cel disks mainly as aggregates at points of fiber intersection, reaching final (plateau) densities of about 4 × 106 (Sf‐9) and 2.7 × 106 (High‐Five) cells mL‐1 (8 × 106 and 5.5 × 106 cells per cm2 of projected disk area, respectively). Their growth was described well by the logistic equation, which takes into account possible inhibition effects. β‐Galactosidase (β‐gal) production of Sf‐9 cells on Fibra‐Cel disks (infected at 3.3 × 106 cells mL‐1) was prolonged (192 h), and specific protein production was similar to that of high‐density free cell infection. Cultispher‐S microcarriers were found to be a very efficient system for the growth of High‐Five cells, whereas no growth of Sf‐9 cells took place for the same system. Concentrations of about 9 × 106 cells mL‐1 were reached within 120 h, with cell growth in both microcarriers and aggregates, appearance of cellular bridges between microcarriers and aggregates, and eventual formation of macroaggregates incorporating several microcarriers. Specific protein productions after β‐gal baculovirus infection at increasing cell concentrations were almost constant, thus leading to elevated volumetric protein production: final β‐gal titers of 946, 1728, and 1484 U mL‐1 were obtained for infection densities of 3.4, 7.2, and 8.9 × 106 cells mL‐1, respectively.
Design of Methanol Feed Control in Pichia pastoris Fermentations Based upon a Growth Model
Authors: Zhang Wenhui , Smith Leonard A. , Plantz Bradley A. , Schlegel Vicki L. , Meagher Michael M. 
Published: December 2002

AbstractThe methylotrophic yeast Pichia pastoris is an effective system for recombinant protein productions that utilizes methanol as an inducer, and also as carbon and energy source for a Mut+ (methanol utilization plus) strain. Pichia fermentation is conducted in a fed‐batch mode to obtain a high cell density for a high productivity. An accurate methanol control is required in the methanol fed‐batch phase (induction phase) in the fermentation. A simple “on‐off” control strategy is inadequate for precise control of methanol concentrations in the fermentor. In this paper we employed a PID (proportional, integral and derivative) control system for the methanol concentration control and designed the PID controller settings on the basis of a Pichia growth model. The closed‐loop system was built with four components: PID controller, methanol feed pump, fermentation process, and methanol sensor. First, modeling and transfer functions for all components were derived, followed by frequency response analysis, a powerful method for calculating the optimal PID parameters Kc (controller gain), θI (controller integral time constant), and θD (controller derivative time constant). Bode stability criteria were used to develop the stability diagram for evaluating the designed settings during the entire methanol fed‐batch phase. Fermentations were conducted using four Pichia strains , each expressing a different protein, to verify the control performance with optimal PID settings. The results showed that the methanol concentration matched the set point very well with only small overshoot when the set point was switched, which indicated that a very good control performance was achieved. The method developed in this paper is robust and can serve as a framework for the design of other PID feedback control systems in biological processes.
Preservation of Chitinolytic Pantoae agglomerans in a Viable Form by Cellular Dried Alginate‐Based Carriers
Authors: Zohar‐Perez C. , Ritte E. , Chernin L. , Chet I. , Nussinovitch A. 
Published: December 2002

AbstractImproved viability of Gram‐negative bacteria during freeze‐dehydration, storage, and soil inoculation is of crucial importance to their efficient application. The chitinolytic Pantoae ( Enterobacter) agglomeransstrain IC1270, a potential biocontrol agent of soil‐borne plant‐pathogenic fungi, was used as a model organism to study the efficacy of freeze‐dried alginate‐based beads (macrocapsules) as possible carriers for immobilized Gram‐negative bacterial cells. These macrocapsules were produced by freeze‐dehydration of alginate gel spherical beads, in which different amounts of bacteria, glycerol, and colloidal chitin were entrapped. Subsequent drying produced different unexpected structures, pore‐size distributions, and changes in the outer and inner appearance of the resultant dried cellular solid. With increasing glycerol content, the proportion of larger pores increased. These structures can be related to changes in the slow‐release properties of the dried beads. The amount of glycerol in the beads differed from that in the alginate solution as a result of leakage during the beads' preparation and dehydration. Entrapping 109 cells per bead produced from alginate solution containing 30% glycerol and 1% chitin resulted in improved (in comparison to other studies) survival prospects (95%) during freeze‐drying. Moreover, immobilization of the bacterium sharply improved its survival in nonsterile irrigated and dry soils compared to bacteria in a water suspension. The results suggest that optimized conservation of Gram‐negative bacteria in dry glycerol‐containing alginate‐based cellular solids is not only possible but applicable for a variety of uses.
Facile and Statistical Optimization of Transfection Conditions for Secretion of Foreign Proteins from Insect Drosophila S2 Cells using Green Fluorescent Protein Reporter
Authors: Shin Hwa Sung , Cha Hyung Joon 
Published: December 2002

AbstractInsect Drosophila melanogaster S2 cells were developed as a plasmid‐based and therefore nonlytic expression system for functional foreign proteins. Transfection is an important step to introduce foreign target DNA into cells and should be properly optimized to obtain maximum production yield. Single factor search (SFS) methodology is still generally used to determine optimal condition in a biological system. Although this method is relatively simple to perform, it has many disadvantages such as not considering interactions between several factors and not covering the entire region of the solution pool. Therefore, we approached this optimization problem statistically with response surface (RSM) and evolutionary operation (EVOP) methodologies and compared the transfection efficiencies with the traditional SFS method. We employed secreted green fluorescent protein (GFP) as a reporter for determination of optimal transfection condition and secreted human erythropoietin (hEPO) as a confirming foreign model protein. Consequently, we arrived at the best optimal transient transfection condition (1 μg of plasmid DNA, 5 μg of lipofectin, 2 × 106 cells of initial cell number, and 18 h of transfection duration time) through a systematic access in a series of SFS, RSM, and EVOP. The secreted hEPO yield using optimal transient transfection condition by EVOP methodology was enhanced by about 1.8‐fold compared to that of traditional SFS. This optimized transient transfection condition can be used as a basis for optimal stable transfections. A linear relationship between secreted GFP fluorescence intensity and secreted hEPO concentration indicated that facile and noninvasive determination of optimal transfection conditions for expression and secretion of foreign proteins in S2 cell cultures was made possible by simple measurement of GFP fluorescence.
On‐Line Purification of Monoclonal Antibodies Using an Integrated Stirred‐Tank Reactor/Expanded‐Bed Adsorption System
Authors: Ohashi Ryo , Otero Jose M. , Chwistek Adam , Yamato Ichiro , Hamel Jean‐François P. 
Published: December 2002

AbstractWhile expanded‐bed adsorption (EBA) units have been used to recover proteins from whole cell cultures, the development of a more efficient, on‐line process could streamline the traditional multistep process. This study implements a bench‐scale on‐line purification system in which whole cell cultures are loaded directly into a chromatography column to capture a monoclonal antibody (mAb) in a single step. The on‐line purification system used here integrates a stirred‐tank reactor (STR) and an EBA unit into a new hybrid (STR‐EBA) system. To conduct this work, first, column and buffer conditions were optimized to capture immunoglobulin G from a hybridoma cell culture. A high cell removal (>95%) was achieved in part by removing the top flow distributor and mesh. Then, the 95% extent of removal was sustained for four successive cycles, each using PBS. With 20 mM phosphate buffer, however, the removal decreased from 95% to 75% stepwise. Next, the operational constraints of the EBA system were determined for the hybridoma cell culture, focusing on the effects of cell viability and density on cell removal. This study shows that the cell removal was not significantly different in the range of 80% to 0% viability. Cell density was also varied between 1 × 106 and 1 × 108 cells/mL. From 0.1 to 6 × 107 cells/mL, cell retention in the column was less than 5% and product recovery remained high, approximately 95%. After characterizing the working conditions of the EBA system, on‐line purification was performed. With 1.1 L of culture containing 3 × 106 cells/mL and 100 mg/L of IgG, repeated‐batch cultures were implemented. Half of the culture volume (550 mL) was directly sent to the EBA system every day, for 11 days, and the same amount of fresh medium was fed into the STR. During on‐line purification, productivity was 58 mg of IgG/cycle (day) and purity was greater than 95%. Simple batch culture alone produced 17 mg of IgG/day. This result suggests that the on‐line STR‐EBA system can achieve higher and faster production compared with STR batch and off‐line EBA purification. Overall, the STR‐EBA system with repeated‐batch mode was an effective and flexible system for bench‐scale mAb production.
Standing Wave Design and Experimental Validation of a Tandem Simulated Moving Bed Process for Insulin Purification
Authors: Xie Yi , Mun Sungyong , Kim Jinhyun , Wang Nien‐Hwa Linda 
Published: December 2002

AbstractA tandem simulated moving bed (SMB) process for insulin purification has been proposed and validated experimentally. The mixture to be separated consists of insulin, high molecular weight proteins, and zinc chloride. A systematic approach based on the standing wave design, rate model simulations, and experiments was used to develop this multicomponent separation process. The standing wave design was applied to specify the SMB operating conditions of a lab‐scale unit with 10 columns. The design was validated with rate model simulations prior to experiments. The experimental results show 99.9% purity and 99% yield, which closely agree with the model predictions and the standing wave design targets. The agreement proves that the standing wave design can ensure high purity and high yield for the tandem SMB process. Compared to a conventional batch SEC process, the tandem SMB has 10% higher yield, 400% higher throughput, and 72% lower eluant consumption. In contrast, a design that ignores the effects of mass transfer and nonideal flow cannot meet the purity requirement and gives less than 96% yield.
Evaluation of Factors Promoting Astaxanthin Production by a Unicellular Green Alga, Haematococcus pluvialis, with Fractional Factorial Design
Authors: Choi Y. E. , Yun Y.‐S. , Park J. M. 
Published: December 2002

AbstractFactors affecting the astaxanthin production by a unicellular green alga, Haematococcus pluvialis UTEX 16, were evaluated with sequential fractional factorial design. To simulate an actual production mode, a two‐stage process was adapted for astaxanthin production: the alga was first cultivated under vegetative growth conditions, and then astaxanthin production was induced by applying various induction methods. A high dose of irradiation was most effective for the production of astaxanthin both in weight (mg/g) and in cellular (pg/cell) contents. A combination of nitrogen deficiency and acetate addition also significantly increased the astaxanthin content of cells on a dry weight basis. Meanwhile, the acetate addition alone increased only the cellular content of astaxanthin. Although the addition of ferrous ion alone had a negative effect on the weight content of astaxanthin, simultaneous addition of ferrous ion and acetate was effective for increasing the cellular content of astaxanthin.
On‐Line Monitoring of Adhesion and Proliferation of Cultured Hepatoma Cells Using Optical Waveguide Lightmode Spectroscopy (OWLS)
Authors: Hug T. S. , Prenosil J. E. , Maier P. , Morbidelli M. 
Published: December 2002

AbstractMonitoring of cell adhesion, cell spreading, and cell proliferation opens attractive perspectives in the on‐line control of monolayer cell cultures in toxicity tests, in bioreactors as used for the serial production of skin grafts, or in extracorporeal liver devices. In this study the hepatoma Hep G2 cell adhesion and proliferation was monitored using an integrated optical method, optical waveguide lightmode spectroscopy (OWLS). This method is based upon refractive index measurements within a 100‐nm thin layer above a Si(Ti)O2 surface on which the cells were cultured and exposed to cytotoxic and cytostatic agents. The OWLS signal was proportional to cell density during the spreading period (4 h), and in long‐term experiments (46 h) the OWLS signal correlated on a logarithmic scale with cell density. After administration of the protein synthesis inhibitor cycloheximide (4 μg/mL) to fully spread hepatoma cells, cell growth was arrested and change of the OWLS signal became noticeable within 6 h after drug administration. For exposure to increasing concentrations of the anticancer drug cyclophosphamide (2.5–20 mM) a concentration‐dependent reduction of the OWLS signal was found. For cycloheximide and cyclophospamide the OWLS signal was also confirmed by cell viability measurements using the neutral red assay, the thiazolylblue tetrazoliumbromide assay, total protein measurements, and cell morphology. It was demonstrated that the OWLS signal detects minor changes in cell adhesion, which serve as indicators of metabolic state and growth behavior. OWLS is thus a quantitative tool to characterize impaired cell growth mediated by culture medium, by extracellular matrix, or after exposure to a toxin.