bluuebunny/crossref_metadata_2025_split huggingface dataset:
Influence of Carboxymethyl Dextran and Ferric Citrate on the Adhesion of CHO Cells on Microcarriers
Authors: Landauer Karlheinz , Wiederkum Susanne , Dürrschmid Markus , Klug Helga , Simic Gordana , Blüml Gerald , Doblhoff‐Dier Otto 
Published: February 2003

AbstractDue to the inherent risks of animal‐derived raw materials, the biopharmaceutical industry has an increasing demand for serum‐free and protein‐free media for industrial cell culture bioprocesses. The absence of serum often changes the characteristics of mammalian cells, especially growth, productivity, and adherence properties. This study is mainly focused on the influence of media additives on cell adherence characteristics. An array of different carboxymethyl dextrans and different ferric citrate concentrations was tested with a number of CHO clones, using standard cell culture Roux‐flasks and Cytoline 1 macroporous microcarriers. A prototype mixing system with controlled shear force input was developed as a screening system for adherence characteristics. The results of this evaluation revealed a negatively correlated dose‐dependent influence on adhesion for ferric citrate. It was also found that certain carboxymethyl dextrans are capable of increasing the adherence on Roux‐flasks and microcarriers.
Protective Effect of Viral Homologues of bcl‐2 on Hybridoma Cells under Apoptosis‐Inducing Conditions
Authors: Vives Joaquim , Juanola Sandra , Cairó Jordi J. , Prats Eva , Cornudella Lluís , Gòdia Francesc 
Published: February 2003

AbstractTargets for metabolic engineering have been identified in a hybridoma cell line to make it more robust in culture toward potential limitations inducing apoptosis. The cells were genetically modified with plasmids harboring endogenous bcl‐2 gene and also with viral Bcl‐2 homologues, particularly ksbcl‐2 and bhrf‐1 genes. When cells were exposed to apoptosis‐inducing conditions (i.e., glutamine‐free medium), the control cells exhibited a decrease in viable cell number within the first 12 h, whereas, for the bcl‐2 and ksbcl‐2 transfected cell cultures, the viable cell number did not exhibit any clear decrease until after 60 h. Furthermore, hybridoma cells expressing the viral homologue bhrf‐1 were even more resistant to cell death, showing a decrease in viability of only 50% at 72 h of culture in glutamine‐deprived medium, substantially lower than the 90% viability decrease observed for the control culture. In addition, and most relevant for further bioprocess applications, the cells genetically modified could be brought back to growth conditions even after being exposed to glutamine‐deprived conditions during a significant time window, up to 72 h.
Improving Glucose and Glutamine Metabolism of Human HEK 293 and Trichoplusiani Insect Cells Engineered To Express a Cytosolic Pyruvate Carboxylase Enzyme
Authors: Elias Cynthia B. , Carpentier Eric , Durocher Yves , Bisson Louis , Wagner Roland , Kamen Amine 
Published: February 2003

AbstractMetabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect ( Trichoplusia ni High‐Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed.
High‐Level Expression of Recombinant IgG in the Human Cell Line PER.C6
Authors: Jones David , Kroos Nathalie , Anema Regina , Van Montfort Bart , Vooys Andre , Kraats Sven van der , Helm Esmeralda van der , Smits Shirley , Schouten Jan , Brouwer Kirsten , Lagerwerf Fija , Van Berkel Patrick , Opstelten Dirk‐Jan , Logtenberg Ton , Bout Abraham 
Published: February 2003

AbstractThe number of therapeutic monoclonal antibodies in production is expected to rise rapidly in the next few years. As a result, there is much focus on the optimization of antibody expression platforms. Several issues are important including the speed of transition from bench to manufacturing, yield of IgG, and quality (particularly of the glycan structures present on immunoglobulins). We have characterized the human cell line PER.C6 for its ability to produce recombinant IgG. Production yields are still being optimized, but in nonfed batch culture, PER.C6 is able to grow to a cell density of 5 × 106 cells/mL and produce 300–500 mg/L IgG; this is likely to increase significantly in fed batch cultures. The generation of antibody‐producing cell lines is fast, as rounds of amplification of inserted genes are not required for high production yields. The gene copy number of inserted genes is in the region of 1–10 copies per genome. In addition, PER.C6 is a human cell line, and so does not add glycans, which are immunogenic in humans. A core fucose molecule is essentially always present, and galactose residues are present at a physiological level (0, 1, and 2 galactose residues per glycan are present at a ratio of 1:2:1). No hybrid or high‐mannose structures are seen.
Exploring Vaccinia Virus as a Tool for Large‐Scale Recombinant Protein Expression
Authors: Bleckwenn Nicole A. , Bentley William E. , Shiloach Joseph 
Published: February 2003

AbstractA recombinant vaccinia virus was engineered to express enhanced green fluorescent protein (EGFP) under control of the T7 promoter using the VOTE expression system in HeLa cells. Infection of HeLa cells with this virus and induction with IPTG demonstrated the utility of this construct for easily measuring protein expression. This construct was used to evaluate several production parameters, specifically, multiplicity of infection (MOI), volume during infection, and serum concentration during the infection phase. In static culture, increasing multiplicity of infection was found to increase expression of EGFP up to a plateau around MOI of 1.0. Expression was also shown to increase with decreasing volume during the infection phase. Serum concentration during the infection phase was only marginally significant from 0 to 7.5%. Cytodex 3 microcarriers were found to have the best characteristics for HeLa cell growth. These cells were grown and infected in microcarrier spinner flask culture, and the maximum expression was 2.2 μg EGFP/(million cells at the time of infection), demonstrating the ability of this system to successfully express recombinant proteins at larger scale.
Effect of Time of Harvest of Budded Virus on the Selection of Baculovirus FP Mutants in Cell Culture
Authors: Lua Linda H. L. , Reid Steven 
Published: February 2003

AbstractRapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large‐scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of time of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can be reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the BV formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.
Effect of Doxycycline‐Regulated ERp57 Expression on Specific Thrombopoietin Productivity of Recombinant CHO Cells
Authors: Hwang Sun Ok , Chung Joo Young , Lee Gyun Min 
Published: February 2003

AbstractIn an attempt to increase the specific thrombopoietin (TPO) productivity ( qTPO) of recombinant Chinese hamster ovary (rCHO) cells (TPO‐33), the effect of expression level of ERp57, an isoform of protein disulfide isomerase, on qTPO was investigated. To regulate ERp57 expression level, the Tet‐Off system was first introduced in TPO‐33 cells and stable Tet‐Off cells (TPO‐33‐Tet‐Off) were screened by the luciferase assay. The rCHO cells with a doxycycline‐regulated ERp57 expression system (TPO‐33‐ERp57) were obtained by cotransfection of pTRE‐ERp57 and pTK‐Hyg expression vectors into TPO‐33‐Tet‐Off cells and subsequent screening by Western blot analysis of ERp57 and an enzyme‐linked immunosorbent assay of secreted TPO. Western blot analysis showed that ERp57 expression level in TPO‐33‐ERp57 cells could be regulated tightly by the addition of different concentrations of doxycycline to a culture medium. A doxycycline concentration of 1 μg/mL, which did not influence cell growth and TPO production of TPO‐33‐Tet‐Off cells, was high enough to suppress the ERp57 expression to a basal level. Compared with the basal level, a 1.7‐fold increase in ERp57 expression level was obtained in the absence of doxycycline. This increased expression level of ERp57 resulted in a 2.1‐fold increase in qTPO without growth inhibition, probably as a result of the chaperone‐like activity of ERp57 in CHO cells. Taken together, the results obtained here demonstrate that qTPO of rCHO cells can be increased by elevating the expression level of ERp57.
Enhancement of Sf‐9 Cell Growth and Longevity through Supplementation of Culture Medium with Hemolymph
Authors: Maranga Luis , Mendonça Ronaldo Z. , Bengala André , Peixoto Cristina C. , Moraes Roberto H. P. , Pereira Carlos A. , Carrondo Manuel J. T. 
Published: February 2003

AbstractThe benefits of insect cell culture medium supplementation with hemolymph ofLonomia obliquawere investigated. The addition of hemolymph to the medium induced high levels of cell growth, and the viability was maintained for longer periods. The maximum cell yield increased almost 3‐fold after hemolymph supplementation. Cultures in their stationary phase were rescued through hemolymph supplementation, also reaching high cell concentrations. These actions were much dependent on the concentration of hemolymph; low hemolymph concentration had a positive effect in cell growth, whereas high hemolymph concentration showed a deleterious effect. Fractionation of hemolymph by gel filtration chromatography showed the presence of three factors with different activity in insect cell culture: an potential anti‐apoptotic factor, a growth‐promoting factor, and an enzyme that hydrolyzes sucrose. Addition of hemolymph to the medium induced high levels of glucose production. The sucrose to glucose conversion was also linearly dependent upon the hemolymph concentration. Therefore, we conclude that cell growth and longevity can be increased by supplementation of the culture medium with hemolymph.
pH, pCO2, and Temperature Effect on R‐Adenovirus Production
Authors: Jardon Mario , Garnier Alain 
Published: February 2003

AbstractThe effects of pH, carbon dioxide vapor pressure, pCO2, and temperature on E1 and E3 deleted recombinant adenovirus vector (rAV) production with HEK293S cells have been studied in the ranges of pH = 6.7–7.7, pCO2 = 0.05–0.20 atm, and T = 32–39 °C, respectively. The experiments were performed in four 500‐mL bioreactors in parallel, which make possible the reduction of inter‐run variability. Cell concentration and viability, relative oxygen uptake rate (OUR), fluorescence, and viral titer were measured. It was found that, although pH and pCO2 did not affect significantly cell viability in the range studied, they had an important effect on virus titer. pCO2 allowed the maximum production of rAV at 0.05 atm, and pH showed a very sharp optimum at 7.2. Temperature had an effect on both cell metabolism and virus titer. Low temperature prolonged cell viability and high OUR. Most of all, a 3‐fold increase in virus yield was found at 35 °C compared to that at 37 °C, while 32 °C was not as beneficial (1.5‐fold increase). This finding could have an important impact on large‐scale production. This phenomenon was modeled using a simple 3‐parameter synthesis‐decay model. This model shows how the optimum gain in virus production at 35 °C is due to a balance between the production and decay processes at that temperature.
Toward Higher Order Control Modalities in Mammalian Cells‐Independent Adjustment of Two Different Gene Activities
Authors: Fux Cornelia , Fussenegger Martin 
Published: February 2003

AbstractHeterologous higher order control modalities will be important tools for targeted multigene interventions in next‐generation gene therapy, tissue engineering, and sophisticated gene‐function studies. In this study, we present the design and rigorous quantitative analysis of a variety of different dual‐regulated gene transcription control configurations combining streptogramin‐ and tetracycline‐responsive expression systems in a one‐vector format. Quantitative assessment of dual‐regulated expression performance in various mammalian and human cell lines is based on two compatible secreted reporter genes, SEAP, the human placental secreted alkaline phosphatase, and the recently developed SAMY, the secreted α‐amylase. Assembly of streptogramin‐and tetracycline‐responsive transgene control units in consecutive (→ →), divergent (← →), and convergent (→ ←) orientation showed excellent regulation characteristics in most genetic arrangements exemplified by neglectable interference and high transgene induction ratios in all four control settings (ON/ON, OFF/ON, ON/OFF, OFF/OFF). The overall regulation performance of divergent dual‐regulated expression configurations could be substantially increased when placing noncoding stuffer fragments or insulator modules between the divergently oriented antibiotic‐responsive promoters. Dual‐regulated expression technology pioneers artificial higher order gene control networks that will likely enable new opportunities in multigene metabolic engineering and generate significant therapeutic impact.
Modulation of Cell Cycle for Enhancement of Antibody Productivity in Perfusion Culture of NS0 Cells
Authors: Ibarra Neysi , Watanabe Shikiko , Bi Jin‐Xiu , Shuttleworth John , Al‐Rubeai Mohamed 
Published: February 2003

AbstractA prolonged period of high productivity at high cell density is desirable for industrial production of biopharmaceuticals. Previous efforts have shown that cessation of cell proliferation in low cell density culture results in increased productivity. We report here further results on multigenic manipulation of cell cycle and apoptosis to enhance productivity at high cell density. The NS0 6A1/4–9F myeloma cell line, which constitutively expresses a chimeric IgG4 antibody and inducibly expresses the p21CIP1 cyclin‐dependent kinase inhibitor has been further engineered to constitutively overexpress the Y28 mutant Bcl‐2 anti‐apoptotic protein. The effects of overexpression of p21CIP1 and Bcl‐2 on cell proliferation, cell viability, and antibody production has been investigated in batch and continuous perfusion cultures. In both cultures the p21CIP1 protein arrested cell proliferation, confirming the previous results in low‐density culture of 4‐fold increase in antibody production, whereas mutant Bcl‐2 expression has not resulted in any significant improvement in cell viability of arrested cells. This study demonstrates that it is possible to enhance the productivity of relatively high‐density continuous mammalian cell cultures by arresting the cell cycle in G1 phase.
Efficient Lipid Delivery to Hybridoma Culture by Use of Cyclodextrin in a Novel Granulated Dry‐Form Medium Technology
Authors: Walowitz Jennifer L. , Fike Richard M. , Jayme David W. 
Published: February 2003

AbstractLipids are critical nutrients for high density eukaryotic cell cultivation, but inclusion of lipid components into dry‐form media has been technically challenging. Lipid supplements are usually supplied for separate addition after powder reconstitution and filtration, which increases manipulation and risk in a biopharmaceutical manufacturing facility. Advanced Granulation Technology (AGT) is a novel dry‐form media format designed for large‐scale biopharmaceutical manufacture. All components of a complex formulation are homogeneously incorporated into a single granulated medium that simply requires water addition to yield a complete reconstituted 1X medium. We investigated whether cyclodextrin technology could be combined with the AGT process to deliver usable lipid in a dry medium format. The test lipids were cholesterol and several fatty acids supplied either as an aseptic supplement to liquid media or as part of a complete AGT formulation. The test system utilized a cholesterol auxotroph, NS0, and a protein‐free basal formulation (CD Hybridoma Medium), with or without supplemental lipid. Post‐filtration recovery of cholesterol from AGT‐processed materials compared favorably with media supplemented with liquid lipid concentrates. Cell growth and viability and expression of recombinant protein were equivalent in all test media.
Cell Growth Arrest by Nucleotides, Nucleosides and Bases as a Tool for Improved Production of Recombinant Proteins
Authors: Carvalhal Ana V. , Santos Sónia Sá , Calado José , Haury Matthias , Carrondo Manuel J. T. 
Published: February 2003

AbstractArresting cell growth and thus decreasing cell division potentially lessens the chance for genetic drift in the cell population; this would be of utmost importance for the consistent production of biopharmaceuticals during long periods. The drawback of the addition of well‐known synchronizing agents, such as chemotherapeutics, is that they cause a disproportionate accumulation of cellular constituents, leading to cell death. The use of compounds that are naturally synthesized by the cell, as is the case of nucleotides, nucleosides, and bases (Nt/Ns/B), is shown in this work to be a promising tool. The addition of purines and pyrimidines was tested using a CHO cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP). From the chemical alternatives tested, AMP was the most promising compound for protein production improvement; it reduced cell growth and maintained the culture with high cell viability for long periods, while increasing SEAP specific productivity 3‐fold. The use of CHO and BHK mammalian cells producing Factor VII and the use of a insect cell line (Sf9) showed that the effect of AMP addition seems to be independent of the r‐protein and cell line. With the addition of AMP, accumulation of cells at the S phase was accompanied by an increase of the protein specific productivity. Addition of known synchronizing drugs (aphidicolin and doxorubicin) and application of environmental cell growth arrest strategies (depletion of nutrients and byproduct accumulation) showed also to effectively arrest CHO cell growth. A careful look onto cell cycle distribution in the different scenarios created, shows whether it is important to consider r‐protein expression dependency upon cell cycle in process optimization and operation strategies.
Versatile Expression System for Rapid and Stable Production of Recombinant Proteins
Authors: Cho M.‐S. , Yee H. , Brown C. , Mei B. , Mirenda C. , Chan S. 
Published: February 2003

AbstractPreviously we reported the development of a novel expression system with Tat/TAR‐oriP vectors and HKB11 cell line, which supports high level protein expression (Cho et al. Cytotechnology 2001, 37, 23–30). In the present study, we further demonstrated that HKB11 cells are suitable for high throughput expression (microgram scale) of genomic candidates in transient transfection system for in vitro evaluation of biological functions. HKB11 cells were also shown to support the production of milligram to gram quantities of protein drug candidates for in vivo evaluation of efficacy in various disease models. Stable HKB11 clones secreting high levels of a tissue factor (TF; 40–50 pg/c/d) and B‐domain deleted recombinant factor VIII (BDDrFVIII; 5–10 μU/c/d) were derived under serum‐free conditions. The specific productivity for these two proteins from the HKB11 cells was 10‐fold greater than those from CHO cells derived under the similar conditions. In conclusion, we have demonstrated that the HKB11 cell line is well‐suited for transient and long‐term production of recombinant proteins.
Novel Insect Cell Line Capable of Complex N‐Glycosylation and Sialylation of Recombinant Proteins
Authors: Palomares Laura A. , Joosten Christoph E. , Hughes Patrick R. , Granados Robert R. , Shuler Michael L. 
Published: February 2003

AbstractPaucimannose or oligomannose structures are usually attached to glycoproteins produced by insect cells, while mammalian glycoproteins usually have complex glycans. The lack of complex glycosylation has limited the use of the insect cell baculovirus expression vector system (BEVS), despite its high productivity and versatility. The availability of cell lines capable of complex glycosylation can overcome such a problem and potentially increase the utility of BEVS. In this work the capability of two novel cell lines, one from Pseudaletia unipuncta (A7S) and one from Danaus plexippus (DpN1), to produce and glycosylate a recombinant protein (secreted human placental alkaline phosphatase, SeAP) was assessed. SeAP produced by Tn5B1–4 cells at a low passage number (<200) was utilized for comparison. The optimal conditions for the production of SeAP by DpN1 cells were defined, and the glycosylation profiles of SeAP produced by the cell lines were quantitatively determined. Both the A7S and the DpN1 cells produced lower concentrations of SeAP than the Tn5B1–4 cells. Less than 5% of the glycans attached to SeAP produced by the Tn5B1–4 cells had complex forms. Glycans attached to SeAP from A7S cells contained 4% hybrid and 8% complex forms. Galactosylated biantennary structures were identified. Glycans attached to SeAP produced by the DpN1 cell line had 6% hybrid and 26% complex forms. Of the complex forms in SeAP from DpN1, 13% were identified as sialylated glycans. The galactosyltransferase activity of the three cell lines was measured and correlated to their ability to produce complex forms. Even though neither novel cell line produced as much recombinant protein as the Tn5B1–4 cells, the glycosylation of SeAP expressed by both cell lines was more complete. These novel cell lines represent interesting alternatives for the production of complex glycosylated proteins utilizing the BEVS.