bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Metabolic 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

The 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

A 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

Rapid 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

In 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

The 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

The 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

Heterologous 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

A 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

Lipids 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

Arresting 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

Previously 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

Paucimannose 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.
Impact of Process Conditions on the Centrifugal Recovery of a Disabled Herpes Simplex Virus
Authors: Lotfian Pantea , Levy M. Susana , Coffin Robert. S. , Fearn T. , Ayazi‐Shamlou Parviz
Published: February 2003

Despite continuous improvements in culturing and recovery techniques, high‐titer stocks of purified disabled herpes simplex virus type‐1 (HSV‐1 DIS) vector for drug discovery and use in preclinical and clinical trials are currently difficult to achieve. Efforts to improve their centrifugal recovery have been addressed in this paper. The operation of a swing‐out centrifuge rotor was assessed, and its operational conditions were defined for the recovery of viable HSV‐1 DIS. 80% virus recovery was achieved after 90 min at 26000 g. The 20% loss of virus was attributed to damage to the viral envelope by overcompaction of the pellet and impaction with the base of the centrifuge tube. Virus recovery was increased by a further 10% by using a fixed‐angle centrifuge rotor operating at 26000 g. Plaque assays of recovered HSV‐1 DIS gave values on the order of 106 pfu/mL, compared to values typically above 109 pfu/mL obtained for the replication‐competent HSV‐1 viron.
Small Increases in pH Enhance Retroviral Vector Transduction Efficiency of NIH‐3T3 Cells
Authors: Jensen Tor W. , Chen Yong , Miller William M.
Published: February 2003

Increases in pH between 7.1 and 7.7 increase the efficiency of polybrene (Pb)‐ and protamine sulfate (PS)‐aided retroviral transduction of NIH‐3T3 cells in a serum‐lot‐dependent manner. The increase in Pb‐aided transduction efficiency at pH 7.7, relative to the value at pH 7.33, ranged from 13% to 49% for three serum lots. For a constant Moloney murine leukemia virus (MMLV) vector dilution at pH 7.33, three different serum lots resulted in absolute transduction efficiencies ranging from 29% to 53% using Pb. At the same vector dilution, PS‐aided transduction was less effective on an absolute basis than Pb‐aided transduction, but the benefit of elevated pH was more pronounced with PS. There was a similar enhancement with PS at elevated pH for a murine stem cell virus (MSCV) vector as for the MMLV vector. The benefit at pH 7.7 for PS‐aided transduction was partially due to greater PS stability at elevated pH. Heat inactivating the serum supplement or adding protease inhibitors helped to stabilize PS. This increased the absolute transduction efficiency but decreased the relative benefit of elevated pH to a level similar to that for Pb‐aided transduction. Incubating Pb with the vector at pH 7.1 for 10 min, prior to readjusting to pH 7.7 and transducing the cells, was sufficient to abrogate the beneficial effects of transduction at pH 7.7. In contrast, prior exposure of PS with vector at pH 7.1 did not affect subsequent transduction at pH 7.7. These results indicate that pH is an important variable in retroviral transduction and that the relative benefits of Pb or PS on retroviral vector transduction will vary with the pH, polymer addition method, and serum lot.
Improved Fermentation Processes for NS0 Cell Lines Expressing Human Antibodies and Glutamine Synthetase
Authors: Dempsey Jonathan , Ruddock Steve , Osborne Matthew , Ridley Alison , Sturt Simone , Field Ray
Published: February 2003

To meet the increasing requirement for therapeutic antibodies to conduct clinical trials, an enhanced culture medium and fed‐batch process was developed for GS‐NS0 cell lines. This process was shown to produce high concentrations of monoclonal antibodies for several cell lines expressing different antibodies. Cells were adapted to growth in a glutamine‐ and serum‐free medium containing bovine serum albumin (BSA), cholesterol, and transferrin. A number of amino acids were found to be depleted during cell culture. The concentrations of these amino acids were increased, and further cell culture analyses were performed. This process of cell growth and analysis was repeated over multiple cycles until no depletion was detected. This resulted in an amino acid supplement that was shown to be generic and enhanced antibody productivity up to 5‐fold for the three cell lines tested. Transferrin was replaced using tropolone, a lipophilic iron chelator and ferric ammonium citrate. Cell growth was equivalent to that in transferrin‐containing medium over the wide ranges tested. A concentrated feed solution, based on the amino acid supplement and the components of the serum‐and protein‐free supplements, was formulated. Addition of this feed in response to metabolic requirements resulted in a harvest titer a further 2‐fold higher than the enhanced culture medium. Harvest antibody titers of up to 600 mg/L were achieved for three cell lines expressing different antibodies, representing an increase of 10‐fold over the starting concentrations.
Metabolic Screening of Mammalian Cell Cultures Using Well‐Plates
Authors: Balcarcel R. Robert , Clark Lindsey M.
Published: February 2003

For use in a broad spectrum of cell culture applications, we have devised a novel method, termed High‐Throughput Metabolic Screening (HTMS), with which to more rapidly screen the overall activity of major metabolic pathways of mammalian cells. This current protocol uses adaptations of theoretical and experimental techniques from metabolic and cell culture engineering. First, HTMS makes use of a simplified metabolic network for metabolic flux analysis. Despite its simplicity, the network is capable of generating flux distributions and ATP production rates that are comparable to a more detailed network. Second, HTMS makes use of microtiter well‐plate technology and adaptations of well‐known enzymatic assays to increase precision and throughput for cell culture experiments. Multireplicate, multiparallel cultures in the sub‐milliliter scale yield very precise metabolic rates using common laboratory equipment and at a fraction of the cost and time of traditional experiments with T‐flasks, spinner flasks, or bioreactor systems. The simplicity of the network and the well‐plate assays synergistically comprise a new, extremely useful, broadly applicable, and relatively inexpensive way to probe cell cultures for metabolic effects, screen drugs and toxins, optimize media, and support the development of bioprocesses. The simplified network and cell culture and analytical assays are also useful for undergraduate, graduate, and professional training.
Optimization of an Acoustic Cell Filter with a Novel Air‐Backflush System
Authors: Gorenflo Volker M. , Angepat Sumitra , Bowen Bruce D. , Piret James M.
Published: February 2003

Increasing worldwide demand for mammalian cell production capacity will likely be partially satisfied by a greater use of higher volumetric productivity perfusion processes. An important additional component of any perfusion system is the cell retention device that can be based on filtration, sedimentation, and/or acoustic technologies. A common concern with these systems is that pumping and transient exposure to suboptimal medium conditions may damage the cells or influence the product quality. A novel air‐backflush mode of operating an acoustic cell separator was developed in which an injection of bioreactor air downstream of the separator periodically returned the captured cells to the reactor, allowing separation to resume within 20 s. This mode of operation eliminated the need to pump the cells and allows the selection of a residence time in the separator depending on the sensitivity of the cell line. The air‐backflush mode of operating a 10L acoustic separator was systematically tested at 107 cells/mL to define reliable ranges of operation. Consistent separation performance was obtained for wide ranges of cooling airflow rates from 0 to 15 L/min and for backflush frequencies between 10 and 40 h−1. The separator performance was optimized at a perfusion rate of 10 L/day to obtain a maximum separation efficiency of 92 ± 0.3%. This was achieved by increasing the power setting to 8 W and using duty cycle stop and run times of 4.5 and 45 s, respectively. Acoustic cell separation with air backflush was successfully applied over a 110 day CHO cell perfusion culture at 107 cells/mL and 95% viability.
Strategies for Improved dCO2 Removal in Large‐Scale Fed‐Batch Cultures
Authors: Mostafa Sigma S. , Gu Xuejun (Sherry)
Published: February 2003

Carbon dioxide buildup in large‐scale reactors can be detrimental to cell growth and productivity. In case of protein X, a therapeutic glycoprotein, when cultures were scaled up from bench scale to the pilot plant, there was a 40% loss of specific productivity. The dissolved CO2 (dCO2) level was 179 ± 9 mmHg at the pilot plant scale and 68 ± 13 mmHg at bench scale. The authors proposed a comprehensive approach to maintain dCO2 levels between 40 and 120 mmHg throughout the 14‐day fed‐batch process. A cell‐free experiment was used to investigate the impact of the following parameters on dCO2 removal: (1) sparge rate, (2) agitator speed, (3) bubble size, (4) bicarbonate concentration, (5) impeller position, and (6) aeration rate at the headspace of bioreactor. dCO2 was measured using a fiber optic based probe. dCO2 removal rate was a strong function of sparge rate and a weak function of agitator speed. Bubble size was modulated by the presence or absence of a sparge stone (10 μm pore size, 1 cm pipe i.d.). Open pipe provided 3‐ to 4‐fold better dCO2 removal for the same mass transfer coefficient ( kLa) value. A mathematical model and a bench‐scale experiment indicated that the benefit of a lower level of sodium bicarbonate in the culture medium was transient for batch and fed‐batch cultures. Thus, this strategy was not used at pilot scale. Decreasing top impeller position improved kLa of dCO2 by 2‐fold. Changing headspace aeration rate from 0.02 to 0.04 vvm had no impact on dCO2 removal. Two pilot runs were conducted using (A) open pipe and (B) antifoam in the presence of sparge stone, both in conjunction with lower impeller position. The presence of antifoam may interfere in product purification; however, demonstration of antifoam removal can be difficult. Open pipe allowed an alternative to using antifoam, as foam level with open pipe was significantly less. Both strategies successfully reduced dCO2 level by 2.5‐fold (179 ± 9 vs 72 ± 9 mmHg). Titer at day 10 of culture improved by 1.5‐fold. Specific productivity improved by 41%. Historically, cultures were harvested around day 9–11 because of the high amount of foam; both strategies allowed the cultures to be extended up to day 14, resulting in 2‐fold higher titer compared to that of the historical control without compromising protein quality.
Heat Inactivation of Mammalian Cell Cultures for Biowaste Kill System Design
Authors: Gregoriades Niki , Luzardo Melissa , Lucquet Beatrice , Ryll Thomas
Published: February 2003

A biowaste kill system was implemented to treat biological waste generated from a clinical manufacturing and R&D antibody facility. To confirm that design parameters of this continuous decontamination system are sufficient to inactivate mammalian cell culture waste, bench‐scale experiments were conducted. The biowaste kill system heat inactivates mammalian cell cultures before they are piped to a neutralization tank and subsequently released to the sewage system. Heat inactivation of cells is accomplished by exposing cells to 80 °C for 1 min. Small‐scale heat inactivation studies were performed on CHO, 293‐HEK, and hybridoma cells. Cells at 1 × 106 cells/mL or 1 × 107 cells/mL were exposed to 37, 60, 70, or 80 °C for 0, 30, 60, and 120 s. Viability based on trypan blue exclusion method and ability to proliferate was assessed after exposure to heat. Data suggest that exposure of cells to 80 °C for 60 s is sufficient to inactivate these cultures before they are released to the sewage system.
Fluid Mechanics, Cell Distribution, and Environment in Cell Cube Bioreactors
Authors: Auniņš John G. , Bader Brett , Caola Anthony , Griffiths Janet , Katz Maayan , Licari Peter , Ram Kripa , Ranucci Colette S. , Zhou Weichang
Published: February 2003

Cultivation of MRC‐5 cells and attenuated hepatitis A virus (HAV) for the production of VAQTA, an inactivated HAV vaccine ( 1), is performed in the Cell Cube reactor, a laminar flow fixed‐bed bioreactor with an unusual diamond‐shaped, diverging‐converging flow geometry. These disposable bioreactors have found some popularity for the production of cells and gene therapy vectors at intermediate scales of operation ( 2, 3). Early testing of the Cell Cube revealed that the fluid mechanical environment played a significant role in nonuniform cell distribution patterns generated during the cell growth phase. Specifically, the reactor geometry and manufacturing artifacts, in combination with certain inoculum practices and circulation flow rates, can create cell growth behavior that is not simply explained. Via experimentation and computational fluid dynamics simulations we can account for practically all of the observed cell growth behavior, which appears to be due to a complex mixture of flow distribution, particle deposition under gravity, fluid shear, and possibly nutritional microenvironment.
Production of a Sialylated N‐Linked Glycoprotein in Insect Cells: Role of Glycosidases and Effect of Harvest Time on Glycosylation
Authors: Joosten Christoph E. , Shuler Michael L.
Published: February 2003

Using a nonengineered Trichoplusia ni insect cell line, Tn‐4s, infected with an Autographa californica recombinant baculovirus, 20% sialylation of human secreted placental alkaline phosphatase (SEAP) was observed. In contrast to this level of sialylation, intermediate complex forms with terminal galactose or N‐acetylglucosamine were found in low proportions (<3% and <1%, respectively). We tested whether time of harvest or degradation of intermediate complex forms is responsible for this distribution of glycoforms. Spinner‐flask cultures were infected with the SEAP baculovirus expression vector, and the cultures were harvested 48, 72, and 96 h post‐infection. Structural analysis revealed that the glycoform distribution of SEAP was very similar at the different times of harvest, indicating that the cellular machinery was not significantly affected by the progress of infection and that the glycoforms obtained were stable. High levels of β‐galactosidase and N‐acetylglucosaminidase activity were detected throughout infection. In contrast, sialidase activity was below detection level both in cell extracts and in supernatants. These levels of glycosidases activities raise the possibility that intermediate complex glycoforms may be degraded while sialylated forms should not experience significant degradation in this cell line. However, culture in the presence of extracellular β‐galactosidase and N‐acetylglucosaminidase inhibitors did not significantly improve glycosylation, suggesting that extracellular degradation processes are not taking place. Instead, results suggest that the intracellular machinery of the Tn‐4s cells tends to either shunt the glycans to paucimannosidic forms or drive them completely to sialylation.
Greater inotropic and cyclic AMP responses evoked by noradrenaline through Arg389 β1‐adrenoceptors versus Gly389 β1‐adrenoceptors in isolated human atrial myocardium
Authors: Sandilands A J , O'Shaughnessy K M , Brown M J
Published: February 2003

We studied the biochemical and contractile responses of isolated human myocardial tissue expressing native receptor variants of the 389G>R β1‐adrenoceptor polymorphism.

Right atrial appendage was obtained from homozygous RR patients (n=37) and homozygous GG patients (n=17) undergoing elective cardiac surgery. The positive inotropic effect of noradrenaline in these tissues, mediated through β1‐adrenoceptors, was studied using electrically stimulated (1 Hz) atrial strips, as well as the effects of noradrenaline on cyclic AMP levels and cyclic AMP‐dependent protein kinase.

Tissue from RR homozygotes (n=14) showed significantly increased inotropic potency to noradrenaline (−log EC50, M=6.92±0.12) compared to GG homozygotes (n=8, −log EC50, M=6.36±0.11, P<0.005). This difference was not dependent on tissue basal force.

Tissue cyclic AMP levels (pmol mg−1) were also greater in RR homozygotes (basal 34.8±3.7 n=12, 300 nM noradrenaline 41.4±7.6 n=9, 30 μM noradrenaline 45.2±3.2 n=22, 0.2 mM isoprenaline 48.3±4.2 n=16) compared to GG homozygotes (basal 30.7±4.4 n=5, 300 nM noradrenaline 32.6±6.92 n=5, 30 μM noradrenaline 38.1±3.1 n=8, 0.2 mM isoprenaline 42.6±5.2 n=6, P=0.007). There were no differences between the variants in terms of cyclic AMP‐dependent protein kinase activity.

These data provide the first evidence that enhanced G‐protein coupling of the R389 β1‐adrenoceptor variant reported in rodent fibroblast expression systems is also present in native human receptors. The functional consequence of this is to significantly alter the inotropic potency of β1‐adrenoceptor activation depending on its genotype at the 389 position.

British Journal of Pharmacology (2003) 138, 386–392. doi:10.1038/sj.bjp.0705030
Receptor gene polymorphisms: lessons on functional relevance from the β1‐adrenoceptor
Authors: Michel Martin C , Insel Paul A
Published: February 2003

British Journal of Pharmacology (2003) 138, 279–282. doi:10.1038/sj.bjp.0705031
Effects of the AT1‐receptor antagonist eprosartan on the progression of left ventricular dysfunction in dogs with heart failure
Authors: Suzuki George , Mishima Takayuki , Tanhehco Elaine J , Sharov Victor G , Todor Anastassia , Rostogi Sharad , Gupta Ramesh C , Chaudhry Pervaiz A , Anagnostopoulos Petros V , Nass Omar , Goldstein Sidney , Sabbah Hani N
Published: February 2003

We examined the effects of eprosartan, an AT1 receptor antagonist, on the progression of left ventricular (LV) dysfunction and remodelling in dogs with heart failure (HF) produced by intracoronary microembolizations (LV ejection fraction, EF 30 to 40%).

Dogs were randomized to 3 months of oral therapy with low‐dose eprosartan (600 mg once daily, n=8), high‐dose eprosartan (1200 mg once daily, n=8), or placebo (n=8).

In the placebo group, LV end‐diastolic (EDV) and end‐systolic (ESV) volumes increased after 3 months (68±7 vs 82±9 ml, P<0.004, 43±1 vs 58±7 ml, P<0.003, respectively), and EF decreased (37±1 vs 29±1%, P<0.001). In dogs treated with low‐dose eprosartan, EF, EDV, and ESV remained unchanged over the course of therapy, whereas in dogs treated with high‐dose eprosartan, EF increased (38±1 vs 42±1%, P<0.004) and ESV decreased (41±1 vs 37±1 ml, P<0.006), Eprosartan also decreased interstitial fibrosis and cardiomyocyte hypertrophy.

We conclude that eprosartan prevents progressive LV dysfunction and attenuates progressive LV remodelling in dogs with moderate HF and may be useful in treating patients with chronic HF.

British Journal of Pharmacology (2003) 138, 301–309. doi:10.1038/sj.bjp.0705032
Functional characterization of α1‐adrenoceptor subtypes in vascular tissues using different experimental approaches:a comparative study
Authors: Gisbert Regina , Madrero Yolanda , Sabino Valentina , Noguera M Antonia , Ivorra M Dolores , D'Ocon Pilar
Published: February 2003

The α1‐adrenergic responses of rat aorta and tail artery have been analysed measuring the contractility and the inositol phosphate (IP) formation induced by noradrenaline. Three antagonists, prazosin, 5‐methylurapidil (α1A selective) and BMY 7378 (α1D selective) have been used in different experimental procedures.

Noradrenaline possesses a greater potency inducing contraction and IP accumulation in aorta (pEC50‐contraction=7.32±0.04; pEC50‐IPs=6.03±0.08) than in the tail artery (pEC50‐contraction=5.71±0.07; pEC50‐IPs=5.51±0.10). Although the maximum contraction was similar in both tissues (Emax‐tail=619.1±55.6 mg; Emax‐aorta‐698.2±40.8 mg), there were marked differences in the ability of these tissues to generate intracellular second messengers the tail artery being more efficient (Emax‐tail=1060±147%; Emax‐aorta=108.1±16.9%).

Concentration response curves of noradrenaline in presence of antagonist together with concentration inhibition curves for antagonists added before (CICb) or after (CICa) noradrenaline‐induced maximal response in Ca2+‐containing or Ca2+‐free medium have been performed. A comparative analysis of the different procedures as well as the mathematical approaches used in each case to calculate the antagonist potencies, were completed.

The CICa was the simplest method to characterize the predominant α1‐adrenoceptor subtype involved in the functional response of a tissue.

In aorta, where constitutively active α1D‐adrenoeptors are present, the use of different experimental procedures evidenced a complex equilibrium between α1D‐ and α1A‐adrenoceptor subtypes.

The appropriate management of LiCl in IP accumulation studies allowed us to reproduce the different experimental procedures performed in contractile experiments giving more technical possibilities to this methodology.

British Journal of Pharmacology (2003) 138, 359–368. doi:10.1038/sj.bjp.0705033
Characterization of opioid receptors that modulate nociceptive neurotransmission in the trigeminocervical complex
Authors: Storer R J , Akerman S , Goadsby P J
Published: February 2003

Opioid agonists have been used for many years to treat all forms of headache, including migraine. We sought to characterize opioid receptors involved in craniovascular nociceptive pathways by in vivo microiontophoresis of μ‐receptor agonists and antagonists onto neurons in the trigeminocervical complex of the cat.

Cats were anaesthetized with α‐chloralose 60 mg kg−1, i.p. and 20 mg kg−1, i.v. supplements after induction and surgical preparation using halothane. Units were identified in the trigeminocervical complex responding to supramaximal electrical stimulation of the superior sagittal sinus, and extracellular recordings of activity made.

Seven‐ or nine‐barrelled glass micropipettes incorporating tungsten recording electrodes in their centre barrels were used for microiontophoresis of test substances onto cell bodies.

Superior sagittal sinus (SSS)‐linked cells whose firing was evoked by microiontophoretic application of L‐glutamate (n=8 cells) were reversibly inhibited by microiontophoresis of H2N‐Tyr‐D‐Ala‐Gly‐N‐Me‐Phe‐Gly‐ol (DAMGO) (n=12), a selective μ‐receptor agonist, in a dose dependent manner, but not by control ejection of sodium or chloride ions from a barrel containing saline.

The inhibition by DAMGO of SSS‐linked neurons activated with L‐glutamate could be antagonized by microiontophoresis of selective μ‐receptor antagonists D‐Phe‐Cys‐Tyr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP) or D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2 (CTAP), or both, in all cells tested (n=4 and 6, respectively).

Local iontophoresis of DAMGO during stimulation of the superior sagittal sinus resulted in a reduction in SSS‐evoked activity. This effect was substantially reversed 10 min after cessation of iontophoresis. The effect of DAMGO was markedly inhibited by co‐iontophoresis of CTAP.

Thus, we found that μ‐receptors modulate nociceptive input to the trigeminocervical complex. Characterizing the sub‐types of opioid receptors that influence trigeminovascular nociceptive transmission is an important component to understanding the pharmacology of this synapse, which is pivotal in primary neurovascular headache.

British Journal of Pharmacology (2003) 138, 317–324. doi:10.1038/sj.bjp.0705034
Inhibition of kinin breakdown prolongs retention and action of bradykinin in a myocardial B2 receptor compartment
Authors: Dendorfer Andreas , Folkers Verena , Klinger Matthias , Wolfrum Sebastian , Dominiak Peter
Published: February 2003

The high efficacy of ACE inhibitors to potentiate the actions of kinins might be explained by a hypothetical compartment in which B2‐receptors are colocalized with kinin degrading enzymes. To demonstrate the functional consequence of such a compartment we compared the myocardial uptake and the persistence of action of bradykinin under the influence of kininase inhibitors.

Bradykinin‐induced vasodilation and uptake of tritiated bradykinin were studied in perfused rat hearts during inhibition of ACE and aminopeptidase P. B2‐receptors were localized by immuno‐gold labelling and electron‐microscopy.

The EC50 of bradykinin‐induced vasodilation (5.1±0.8 nM) was shifted to 14 fold lower concentrations during inhibition of both kininases. The maximum persistence of vasodilation after termination of bradykinin application (half‐life 112±20 s) was increased by kininase inhibitors to 398±130 s. This prolongation was reversed when B2‐receptors were blocked simultaneously with the termination of bradykinin infusion.

Tritiated bradykinin (perfused for 1 min) was partially (1.7±0.24%) retained by the myocardium and consecutively released with a half‐life of 70±9 s. Kinin uptake was increased during kininase inhibition (7.7±2.6%), and was normalized by HOE 140 (2.0±0.34%), or when a tritiated B2‐receptor antagonist (NPC 17731) was used as label.

B2‐receptors were localized in plasmalemmal and cytosolic vesicles of capillary endothelium.

Bradykinin is locally incorporated and can associate with B2‐receptors repeatedly when kinin breakdown is inhibited. This is the kinetic and functional consequence of a colocalization of kininases and B2‐receptors in a compartment constituted by endothelial membrane vesicles.

British Journal of Pharmacology (2003) 138, 310–316. doi:10.1038/sj.bjp.0705036