			Home About us Contact

Hybridoma Cells (hybridoma + cell)

Distribution by Scientific Domains

Life Sciences	85%
Medical Sciences	14%

Selected Abstracts

Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG2a Monoclonal Antibody in a Wave Bioreactor-Perfusion Culture System

BIOTECHNOLOGY PROGRESS, Issue 1 2007
Ya-Jie Tang
A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 ,m was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 × 105 and 200.5 × 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 × 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L·day) in perfusion culture were much higher than those (i.e., 22.3 × 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L·day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale. [source]

Hyperosmotic Stress in Murine Hybridoma Cells: Effects on Antibody Transcription, Translation, Posttranslational Processing, and the Cell Cycle

BIOTECHNOLOGY PROGRESS, Issue 2 2004
Zhe Sun
Mechanisms for increased antibody production in batch cultures of murine hybridoma cells in response to hyperosmotic stress were investigated. The rates of immunoglobulin transcription and protein translation and posttranslational processing were determined in control and hyperosmotic cultures. Changes in immunoglobulin transcription played a minor role in the increase in antibody production in response to hyperosmotic stress. In contrast, protein translation increased substantially in response to osmotic stress. However, the antibody translation rate remained relatively constant after correcting for the overall increase in protein translation. Cell size and intracellular antibody pool also increased in response to hyperosmolarity. The intracellular antibody pool increased proportionately with the increase in cell size, indicating that hyperosmotic cultures do not selectively increase their intracellular antibody population. Changes in cell cycle distribution in response to osmotic stress and the relationship between the cell cycle and antibody production were also evaluated. Hyperosmotic stress altered the cell cycle distribution, increasing the fraction of the cells in S-phase. However, this change was uncorrelated with the increase in antibody production rate. Immunoglobulin degradation was relatively low (,15%) and remained largely unchanged in response to hyperosmotic stress. There was no apparent increase in immunoglobulin stability as a result of osmotic stress. Antibody secretion rates increased approximately 50% in response to osmotic stress, with a commensurate increase in the antibody assembly rate. The rate of transit through the entire posttranslational processing apparatus increased, particularly for immunoglobulin light chains. The levels of endoplasmic reticulum chaperones did not increase as a fraction of the total cellular protein but were increased on a per cell basis as the result of an increase in total cellular protein. A difference in the interactions between the immunoglobulin heavy chains and BiP/GRP78 was observed in response to hyperosmotic conditions. This change in interaction may be correlated with the decrease in transit time through the posttranslational pathways. The increase in the posttranslational processing rate appears to be commensurate with the increase in antibody production in response to hyperosmotic stress. [source]

Protective Effect of Viral Homologues of bcl-2 on Hybridoma Cells under Apoptosis-Inducing Conditions

BIOTECHNOLOGY PROGRESS, Issue 1 2003
Joaquim Vives
Targets 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. [source]

Cybernetic Modeling and Regulation of Metabolic Pathways in Multiple Steady States of Hybridoma Cells

BIOTECHNOLOGY PROGRESS, Issue 5 2000
Maria Jesus Guardia
Hybridoma cells utilize a pair of complementary and partially substitutable substrates, glucose and glutamine, for growth. It has been shown that cellular metabolism shifts under different culture conditions. When those cultures at different metabolic states are switched to a continuous mode, they reach different steady states under the same operating conditions. A cybernetic model was constructed to describe the complementary and partial substitutable nature of substrate utilization. The model successfully predicted the metabolic shift and multiple steady-state behavior. The results are consistent with the experimental observation that the history of the culture affects the resulting steady state. [source]

Microencapsulation of an anti-VE,cadherin antibody secreting 1B5 hybridoma cells

BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2001
G. Orive
Accumulating experimental evidence demonstrates that tumor growth and lethality are dependent on angiogenesis. Based on this concept, there is growing interest in the use of antiangiogenesis agents to inhibit tumor expansion. Compelling data implicate vascular endothelium (VE),cadherin (an endothelium specific protein) as a key factor in the last step of angiogenesis, where the endothelial cells join one to each other and form microtubules (future blood vessels). We propose a novel approach to the inhibition of angiogenesis by immobilizing VE,cadherin-secreting hybridoma cells in alginate,agarose microcapsules. Hybridoma cells can be protected with biocompatible and semipermeable membranes that permit exit of anti-VE,cadherin monoclonal antibodies but not entry of cellular immune mediators. Stability studies were performed to select the suitable microcapsule for cell immobilization. Alginate and agarose solid beads coated with poly- L -lysine and alginate were chosen according to their stability and diffusional properties. 1B5 hybridoma cells were grown within the microcapsules and secreted anti-VE,cadherin antibodies during the 9 days of culture, reaching a cumulative concentration of 1.7 ,g/mL. This antibody concentration inhibited microtubule formation (87%) in the in vitro angiogenesis Matrigel assay. Moreover, the antiangiogenic effect observed was antibody concentration related. These findings open a new alternative for the inhibition or prevention of angiogenesis and demonstrates the feasibility of using microencapsulated cells as a control-drug delivery system. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 285,294, 2001. [source]

Cybernetic Modeling and Regulation of Metabolic Pathways in Multiple Steady States of Hybridoma Cells

Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus species

CLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2000
W.-L. Lin
Background Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species. Objective The object of this study is to generate and characterize monoclonal antibodies against these serine proteinase allergens. Methods BALB/c mice were immunized individually with the Penicillium citrinum culture medium or the crude extract and culture medium preparations of Aspergillus fumigatus. Hybridoma cells that secrete monoclonal antibodies against serine proteinase allergens were selected by immunoblotting. Antigens in three different Penicillium (P. citrinum, P. notatum and P. oxalicum) and two different Aspergillus species (A. fumigatus, and A. flavus) recognized by these monoclonal antibodies were analysed by sodium dodecyl sulphate and two-dimensional polyacrylamide gel electrophoresis immunoblotting and N-terminal amino acid sequence analysis. Results Four (PCM8, PCM10, PCM16 and PCM39) and one (FUM20) monoclonal antibodies against serine proteinase allergens were generated after fusion of NS-1 cells with spleen cells obtained from BALB/c mice immunized with antigens from P. citrinum and A. fumigatus, respectively. Immunoblotting results showed that PCM8 reacted with an alkaline serine proteinase allergen in P. citrinum and P. notatum. PCM10 and PCM39 reacted with the alkaline serine proteinase in two Penicillium (P. citrinum, P. notatum) and two Aspergillus species (A. fumigatus, and A. flavus) tested. PCM16 reacted with the alkaline serine proteinase allergen in P. citrinum, A. fumigatus and A. flavus but not with that in P. notatum. MoAb FUM20 reacted with the alkaline serine proteinase allergen in two Aspergillus species (A. fumigatus and A. flavus) but not with that in two different Penicillium species (P. citrinum, P. notatum) tested. Among these five monoclonal antibodies generated, only PCM39 and FUM20 can react with the vacuolar serine proteinase allergen in P. notatum, P. oxalicum and in A. fumigatus. The 35 kDa P. citrinum component that reacted with FUM20 has an N-terminal amino acid sequence of DSPSVEKNAP. Conclusion Five monoclonal antibodies against different epitopes of the serine proteinase major allergens in prevalent Penicillium and Aspergillus species were generated in the present study. Antibodies obtained may be useful in the characterization and standardization of serine proteinase allergens in crude fungal extracts. [source]

Production of Monoclonal Antibodies to Sugarcane Yellow Leaf Virus Using Recombinant Readthrough Protein

JOURNAL OF PHYTOPATHOLOGY, Issue 8-9 2002
J. Korimbocus
Abstract Yellow leaf syndrome (YLS) of sugarcane is associated with sugarcane yellow leaf virus (SCYLV), a member of the family Luteoviridae. A fragment of the coat protein and readthrough domain of SCYLV wasexpressed in a bacterial expression system. The resulting protein was purified and used to immunize mice for monoclonal antibody (MAb) production. Two MAbs, 3A2E3 and 2F7H5, were selected following the screening of hybridoma cells using both plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) and tissue blot immunoassay (TBIA). These MAbs can be incorporated into the TBIA assay currently used for the routine detection of SCYLV but could not be used in triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The two antibodies selected have slightly different specificities. Antibody 3A2E3 gave equivalent results to a polyclonal antiserum (raised to purified virus) in comparative testing using TBIA. The MAbs produced should provide a widely available, uniform reagent for SCYLV diagnosis with the potential to help manage YLS. [source]

Suppression of experimental lupus nephritis by aberrant expression of the soluble E-selectin gene

PATHOLOGY INTERNATIONAL, Issue 3 2002
Satoru Takahashi
Circulating leukocytes, particularly neutrophils and monocytes, are important effector cells in the induction of many forms of glomerulonephritis. Adhesion molecules, especially selectins, are also thought to be critical for the development of this disease. We examined the possible suppressive effect of soluble E-selectin on the development of experimental lupus nephritis induced by the injection of a hybridoma clone (2B11.3) derived from an MRL/MpJ- lpr/lpr lupus mouse. This clone produces IgG3 antibodies that induce severe proliferative glomerulonephritis resembling lupus nephritis when injected into normal mice. Transgenic mice with a soluble E-selectin gene were injected intraperitoneally with the hybridoma cells and histopathologically examined on day 15. As a result, the development of glomerulonephritis was significantly suppressed. This suppression was characterized by fewer inflammatory cell infiltrates, compared with non-transgenic litter mates, despite the fact that there were no remarkable differences in immunoglobulin deposits or expression of E-selectin between the two groups. These findings suggest that by controlling inflammatory cell infiltration, soluble E-selectin plays a preventative role in the development of a particular type of lupus nephritis. [source]

Reduced fragmentation of apoptotic chromatin is associated with nephritis in lupus-prone (NZB × NZW)F1 mice

ARTHRITIS & RHEUMATISM, Issue 3 2008
Svetlana N. Zykova
Objective Antinucleosome autoantibodies are pathogenic factors in lupus nephritis. Defects in apoptotic pathways may result in increased levels of apoptotic nucleosomes. The objectives of this study were 1) to determine whether low molecular weight oligonucleosomes are present in the kidneys of autoimmune (NZB × NZW)F1 mice, 2) to analyze whether the presence of glomerular membrane,associated TUNEL-positive electron-dense structures reflect the existence of low molecular weight oligonucleosomes, and 3) to determine an eventual temporal relationship between glomerular electron-dense structures, oligonucleosomes, and proteinuria in these mice. Methods DNA was isolated from mouse 111s34 hybridoma cells and from the kidneys of normal BALB/c mice in which apoptosis was induced by camptothecin and from the kidneys of (NZB × NZW)F1 mice at ages 4 weeks, 8 weeks, 20 weeks, and ,26 weeks (nephritic mice). The DNA fragmentation pattern was determined with an Agilent bioanalyzer. An electron microscopy,based TUNEL assay was performed to detect apoptotic chromatin in glomerular membranes, and immunoelectron microscopy was used to determine antibody binding. Transcription levels for nucleases associated with apoptosis and necrosis were determined by real-time polymerase chain reaction. Results DNA from camptothecin-treated cell lines and BALB/c mouse kidneys, but not that from untreated (NZB × NZW)F1 mouse kidneys, demonstrated DNA cleavage consistent with apoptotic fragmentation. DNA from (NZB × NZW)F1 mice was devoid of apoptotic fragmentation, irrespective of the age of the mice, whereas TUNEL-positive chromatin particles were detected in glomerular membranes in nephritic mice. Renal DNase I transcription was reduced in nephritic mice. Nucleosomal DNA fragmentation in response to camptothecin exposure was highly reduced in (NZB × NZW)F1 mouse kidneys compared with that in their normal counterparts. Conclusion The results of this study demonstrate that TUNEL-positive chromatin particles are deposited in the glomeruli of nephritic (NZB × NZW)F1 mice, due to reduced fragmentation and clearance of chromatin. [source]

Fractionation of cell mixtures using acoustic and laminar flow fields

BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2005
Manoj Kumar
Abstract A fractionation method applicable to different populations of cells in a suspension is reported. The separation was accomplished by subjecting the suspension to a resonant ultrasonic field and a laminar flow field propagating in orthogonal directions within a thin, rectangular chamber. Steady, laminar flow transports the cell suspension along the chamber, while the ultrasonic field causes the suspended cells to migrate to the mid-plane of the chamber at rates related to their size and physical properties. A thin flow splitter positioned near the outlet divides the effluent cell suspension into two product streams, thereby allowing cells that respond faster to the acoustic field to be separated from those cells that respond more slowly. Modeling of the trajectories of individual cells through the chamber shows that by altering the strength of the flow relative to that of the acoustic field, the desired fractionation can be controlled. Proof-of-concept experiments were performed using hybridoma cells and Lactobacillus rhamnosus cells. The two populations of cells could be effectively separated using this technique, resulting in hybridoma/Lactobacillus ratios in the left and right product streams, normalized to the feed ratio, of 6.9 ± 1.8 and 0.39 ± 0.01 (vol/vol), respectively. The acoustic method is fast, efficient, and could be operated continuously with a high degree of selectivity and yield and with low power consumption. © 2004 Wiley Periodicals, Inc. [source]

Microencapsulation of an anti-VE,cadherin antibody secreting 1B5 hybridoma cells

Robust parameter estimation during logistic modeling of batch and fed-batch culture kinetics

BIOTECHNOLOGY PROGRESS, Issue 3 2009
Chetan T. Goudar
Abstract Methods for robust logistic modeling of batch and fed-batch mammalian cell cultures are presented in this study. Linearized forms of the logistic growth, logistic decline, and generalized logistic equation were derived to obtain initial estimates of the parameters by linear least squares. These initial estimates facilitated subsequent determination of refined values by nonlinear optimization using three different algorithms. Data from BHK, CHO, and hybridoma cells in batch or fed-batch cultures at volumes ranging from 100 mL,300 L were tested with the above approach and solution convergence was obtained for all three nonlinear optimization approaches for all data sets. This result, despite the sensitivity of logistic equations to parameter variation because of their exponential nature, demonstrated that robust estimation of logistic parameters was possible by this combination of linearization followed by nonlinear optimization. The approach is relatively simple and can be implemented in a spreadsheet to robustly model mammalian cell culture batch or fed-batch data. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]

Dynamic Metabolic Modeling for a MAB Bioprocess

BIOTECHNOLOGY PROGRESS, Issue 1 2007
Jianying Gao
Production of monoclonal antibodies (MAb) for diagnostic or therapeutic applications has become an important task in the pharmaceutical industry. The efficiency of high-density reactor systems can be potentially increased by model-based design and control strategies. Therefore, a reliable kinetic model for cell metabolism is required. A systematic procedure based on metabolic modeling is used to model nutrient uptake and key product formation in a MAb bioprocess during both the growth and post-growth phases. The approach combines the key advantages of stoichiometric and kinetic models into a complete metabolic network while integrating the regulation and control of cellular activity. This modeling procedure can be easily applied to any cell line during both the cell growth and post-growth phases. Quadratic programming (QP) has been identified as a suitable method to solve the underdetermined constrained problem related to model parameter identification. The approach is illustrated for the case of murine hybridoma cells cultivated in stirred spinners. [source]

Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG2a Monoclonal Antibody in a Wave Bioreactor-Perfusion Culture System

Logistic Equations Effectively Model Mammalian Cell Batch and Fed-Batch Kinetics by Logically Constraining the Fit

BIOTECHNOLOGY PROGRESS, Issue 4 2005
Chetan T. Goudar
A four-parameter logistic equation was used to fit batch and fed-batch time profiles of viable cell density in order to estimate net growth rates from the inoculation through the cell death phase. Reduced three-parameter forms were used for nutrient uptake and metabolite/product formation rate calculations. These logistic equations constrained the fits to expected general concentration trends, either increasing followed by decreasing (four-parameter) or monotonic (three-parameter). The applicability of this approach was first verified for Chinese hamster ovary (CHO) cells cultivated in 15-L batch bioreactors. Cell density, metabolite, and nutrient concentrations were monitored over time and used to estimate the logistic parameters by nonlinear least squares. The logistic models fit the experimental data well, supporting the validity of this approach. Further evidence to this effect was obtained by applying the technique to three previously published batch studies for baby hamster kidney (BHK) and hybridoma cells in bioreactors ranging from 100 mL to 300 L. In 27 of the 30 batch data sets examined, the logistic models provided a statistically superior description of the experimental data than polynomial fitting. Two fed-batch experiments with hybridoma and CHO cells in benchtop bioreactors were also examined, and the logistic fits provided good representations of the experimental data in all 25 data sets. From a computational standpoint, this approach was simpler than classical approaches involving Monod-type kinetics. Since the logistic equations were analytically differentiable, specific rates could be readily estimated. Overall, the advantages of the logistic modeling approach should make it an attractive option for effectively estimating specific rates from batch and fed-batch cultures. [source]

Hyperosmotic Stress in Murine Hybridoma Cells: Effects on Antibody Transcription, Translation, Posttranslational Processing, and the Cell Cycle

Protective Effect of Viral Homologues of bcl-2 on Hybridoma Cells under Apoptosis-Inducing Conditions

Production and Molecular Characterization of Clinical Phase I Anti-Melanoma Mouse IgG3 Monoclonal Antibody R24

BIOTECHNOLOGY PROGRESS, Issue 5 2001
Sven E. Kemminer
R24 is a mouse IgG3 monoclonal antibody (mab) that reacts with the ganglioside GD3 expressed by cells of neuroectodermal origin. The anti-tumor activity of R24 has been demonstrated in initial phase I and pilot trials in patients suffering from metastatic melanoma. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important antibody. Growth, metabolism, and IgG production of R24 secreting hybridoma cells were analyzed on 1 L bioreactor bench scale using repeated-batch mode. The amount of 57 mg of pure mab was obtained from 1.6 L crude supernatant by protein A chromatography. Western blot binding assays with sugar-specific lectins revealed glycosylation of the heavy chains, whereas no carbohydrates were detectable on the light chains. Because glycosylation is essential for antibody effector functions in vivo (such as complement fixation or binding to macrophage Fc receptors), mab R24 was subjected to both enzymatic deglycosylation using PNGase F and chemical deglycosylation by hydrazinolysis. Released glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Six major biantennary chains of the complex glycosylation phenotype were found with variations in galactosylation and core fucosylation. The predominant N-linked structure, indicating the high degree of agalactosyl glycoforms, was the agalacto biantennary chain with a relative percentage of 57% (51% core-fucosylated, 6% nonfucosylated). The second most abundant oligosaccharide was the monogalacto biantennary chain amounting to 30% (26% core- and 4% nonfucosylated). The antibody contained 0.46 ,g sialic acid per mg protein, which splits into 0.243 ,g Neu5Gc and 0.217 ,g Neu5Ac, corresponding to a Neu5Ac:Neu5Gc ratio of 1:1.06. Furthermore, the antigen specificity of R24 was determined by immunodetection of GD3 on thin-layer chromatograms, and real time GD3-antibody binding interactions were measured with an optical biosensor (BIAcore). From the structural data obtained in this study it is concluded that glycosylation of the antibody may be important in the clinical outcome of targeted anti-cancer immunotherapy. [source]