20 results on '"Perfusion Culture"'
Search Results
2. Optimization of HEK-293S cell cultures for the production of adenoviral vectors in bioreactors using on-line OUR measurements
- Author
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Gálvez, J., Lecina, M., Solà, C., Cairó, J.J., and Gòdia, F.
- Subjects
- *
CELL culture , *GENETIC vectors , *BIOREACTORS , *CELL lines , *GENE therapy , *CELL differentiation , *CULTURE media (Biology) - Abstract
Abstract: The culture of HEK-293S cells in a stirred tank bioreactor for adenoviral vectors production for gene therapy is studied. Process monitoring using oxygen uptake rate (OUR) was performed. The OUR was determined on-line by the dynamic method, providing good information of the process evolution. OUR enabled cell activity monitoring, facilitating as well the determination of the feeding rate in perfusion cultures and when to infect the culture. Batch cultures were used to validate the monitoring methodology. A cell density of 10×105 cell/mL was infected, producing 1.3×109 infectious viral particles/mL (IVP/mL). To increase cell density values maintaining cell specific productivity, perfusion cultures, based on tangential flow filtration, were studied. In this case, OUR measurements were used to optimize the dynamic culture medium feeding strategy, addressed to avoid any potential nutrient limitation. Furthermore, the infection protocol was defined in order to optimize the use of the viral inoculum, minimizing the uncontrolled release of particles through the filter unit mesh. All these developments enabled an infection at 78×105 cell/mL with the consequent production of 44×109 IVP/mL, representing a cell specific productivity 4.3 times higher than for the batch culture. [Copyright &y& Elsevier]
- Published
- 2012
- Full Text
- View/download PDF
3. Three-dimensional perfusion cultures of mouse and pig fetal liver cells in a packed-bed reactor: Effect of medium flow rate on cell numbers and hepatic functions
- Author
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Miyoshi, Hirotoshi, Ehashi, Tomo, Kawai, Hideki, Ohshima, Norio, and Suzuki, Satoshi
- Subjects
- *
FETAL liver cells , *TISSUE scaffolds , *CELL growth , *BIOREACTORS , *PERFUSION , *LIVER function tests , *ANIMAL models in research - Abstract
Abstract: To develop a tissue-engineered bioartificial liver (BAL), perfusion cultures of mouse and pig fetal liver cells (FLCs) immobilized within a three-dimensional (3D) porous scaffold were performed utilizing a packed-bed reactor system. These FLCs were cultured under different medium flow rate conditions, and the effects of the flow rates on cell growth and the hepatic functions of the FLCs were investigated. In the cultures of mouse FLCs, the medium flow suppressed cell growth and the albumin secretion activity of the FLCs, and considerably lower albumin secretion than that in the 3D stationary control cultures was obtained in the perfusion cultures. In the case of pig FLCs, cell growth was also inhibited by the medium flow, however, the cells exhibited higher tolerance to the medium flow compared with mouse FLCs. The albumin secretion activity of pig FLCs was well maintained under an extremely low flow rate condition (4.8mm/min in the reactor), and activity higher than the 3D stationary cultures was detected at a later stage (after 20 days in the perfusion cultures). These results revealed that FLCs are quite sensitive to medium flow and an extremely low shear stress is required for the perfusion cultures of FLCs. [Copyright &y& Elsevier]
- Published
- 2010
- Full Text
- View/download PDF
4. An on-line method for the reduction of fouling of spin-filters for animal cell perfusion cultures
- Author
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Vallez-Chetreanu, F., Fraisse Ferreira, L.G., Rabe, R., von Stockar, U., and Marison, I.W.
- Subjects
- *
OLD age , *ADULTS , *AGE , *AGE groups , *HUMAN life cycle - Abstract
Abstract: The main limitation in the use of spin-filters during perfusion cultures of animal cells was revealed to be filter fouling. This phenomenon involves cell–sieve interactions as well as cell attachment to, and growth on, the filter surface. The cell attachment effect has been analysed in the present study during long-term perfusion simulations with CHO animal cells. It was demonstrated that at low filter acceleration, below 6.2m/s2, a high perfusion rate of 25cm/h induced rapid filter pore clogging within 3 days, whereas increasing the filter acceleration to 25m/s2 increased filter longevity from 3 to 25 days, for filters with a pore size of 8.5μm. Increasing the filter pore size to 14.5μm improved filter longevity by 84% with less viable and dead cell deposits on the filter surface. However, it was demonstrated that filter longevity was not necessarily dependent on the amount of cell deposit on the filter surface. In the second part of this study, ultrasonic technology was used to reduce filter fouling. Filter vibration, induced by a piezo actuator, improved filter longevity by 113% during CHO cells perfusion cultures. [Copyright &y& Elsevier]
- Published
- 2007
- Full Text
- View/download PDF
5. Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor
- Author
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Trabelsi, Khaled, Rourou, Samia, Loukil, Houssem, Majoul, Samy, and Kallel, Héla
- Subjects
- *
BLOOD plasma , *GLUCOSE , *AMINO acids , *GLUTAMINE - Abstract
Abstract: To improve rabies vaccine production by Vero cells, we have developed a strategy based on high cell density culture and optimization of virus yield. We have first optimized cell growth in spinner flask using a Taguchi''s L8 experimental design. We analyzed the effects of the following factors: initial glucose and glutamine concentrations, Cytodex 1 concentration and the regulation of glucose level at 1gl−1. We have also investigated the effect of the following factor interactions: Cytodex 1 concentration/glutamine concentration, Cytodex 1 concentration/glucose concentration and glucose concentration/glutamine concentration. Statistical analysis of the collected data pointed to the initial glucose concentration, the regulation of glucose level at 1gl−1 and the interactions between Cytodex 1 concentration/initial glucose concentration and Cytodex 1 concentration/initial glutamine concentration as the parameters that affected cell growth. Using the optimal conditions determined earlier, we have studied Vero cell growth in a 7-l bioreactor and in batch culture, and obtained a cell density level equal to 3.6±0.2×106 cellsml−1. Cell infection with rabies virus (LP 2061/Vero strain) at a multiplicity of infection (MOI) of 0.3 using M199 medium supplemented with 0.2% bovine serum albumin (BSA), yielded a maximal virus titer equal to 8±1.6×107 Fluorescent Focus Units (FFU) ml−1. We have also studied Vero cell growth in a 7-l bioreactor using recirculation as a perfusion culture mode during cell proliferation step and perfusion for virus multiplication phase. In comparison to batch culture, we reached a higher cell density level that was equal to 10.1±0.5×106 cellsml−1. Cell infection under the conditions previously indicated, yielded 14l of virus harvest that had a virus titer equal to 2.6±0.5×107 FFUml−1. The activity of the inactivated virus harvest showed a protective activity that meets WHO requirements. [Copyright &y& Elsevier]
- Published
- 2006
- Full Text
- View/download PDF
6. Bioprocess development for the production of a recombinant MUC1 fusion protein expressed by CHO-K1 cells in protein-free medium
- Author
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Link, T., Bäckström, M., Graham, R., Essers, R., Zörner, K., Gätgens, J., Burchell, J., Taylor-Papadimitriou, J., Hansson, G.C., and Noll, T.
- Subjects
- *
PROTEINS , *IMMUNOTHERAPY , *BREAST cancer , *BIOCHEMICAL engineering - Abstract
The mucin MUC1 is a candidate for use in specific immunotherapy against breast cancer, but this requires the large-scale production of a MUC1 antigen. In this study, a bioprocess for the expression of a recombinant MUC1 fusion protein with a cancer associated glycosylation in CHO-K1 cells has been developed. Cells permanently expressing parts of the extracellular portion of MUC1 fused to IgG Fc were directly transferred from adherent growth in serum-containing medium to suspension culture in the protein-free ProCHO4-CDM culture medium. Using the Cellferm-pro® system, optimal culture parameter as pH and pO2 were determined in parallel spinner flask batch cultures. A pH of 6.8–7.0 and a pO2 of 40% of air saturation was found to give best cell growth and productivity of secreted recombinant protein. Specific productivity strongly depended the pO2 and correlated with the online monitored oxygen uptake rate (OUR) of the cells, which indicates a positive influence of the rate of oxidative phosphorylation on productivity. The optimised conditions were applied to continuous perfusion culture which gave very high cell densities and space time yields of the recombinant MUC1 fusion protein, allowing production at gram scale. The product degradation was much lower in supernatants from continuous perfusion culture compared to batch mode. Antibodies reacting with cancer associated MUC1 glycoforms strongly bound to the fusion protein, indicating that the desired glycoforms were obtained and suggesting that the recombinant MUC1 protein could be tested for use in immunotherapy. [Copyright &y& Elsevier]
- Published
- 2004
- Full Text
- View/download PDF
7. Fabrication of engineered tubular tissue for small blood vessels via three-dimensional cellular assembly and organization ex vivo
- Author
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Yuka Yamagishi, Taisuke Masuda, Mitsuhiro Ukiki, Fumihito Arai, Michiya Matsusaki, Utako Yokoyama, and Mitsuru Akashi
- Subjects
0301 basic medicine ,Scaffold ,Materials science ,Biocompatibility ,Myocytes, Smooth Muscle ,Pulsatile flow ,Cell Culture Techniques ,Bioengineering ,02 engineering and technology ,Applied Microbiology and Biotechnology ,03 medical and health sciences ,Perfusion Culture ,Myosin ,Animals ,Aorta ,Cells, Cultured ,biology ,Tissue Engineering ,General Medicine ,Equipment Design ,Microfluidic Analytical Techniques ,021001 nanoscience & nanotechnology ,Rats ,Perfusion ,030104 developmental biology ,biology.protein ,Blood Vessels ,0210 nano-technology ,Elastin ,Ex vivo ,Biotechnology ,Biomedical engineering - Abstract
Although there is a great need for suitable vascular replacements in clinical practice, much progress needs to be made toward the development of a fully functional tissue-engineered construct. We propose a fabrication method of engineered tubular tissue for small blood vessels via a layer-by-layer cellular assembly technique using mouse smooth muscle cells, the construction of a poly-(l-lactide-co-e-caprolactone) (PLCL) scaffold, and integration in a microfluidic perfusion culture system. The cylindrical PLCL scaffold is incised, expanded, and its surface is laminated with the cell layers. The construct confirms into tubular structures due to residual stress imposed by the cylindrical PLCL scaffold. The perfusion culture system allows simulation of static, perfusion (laminar flow), and perfusion with pulsatile pressure (Pulsatile flow) conditions in which mimicking the in vivo environments. The aim of this evaluation was to determine whether fabricated tubular tissue models developed their mechanical properties. The cellular response to hemodynamic stimulus imposed by the dynamic culture system is monitored through expression analysis of fibrillin-1 and fibrillin-2, elastin and smooth muscle myosin heavy chains isoforms transcription factors, which play an important role in tissue elastogenesis. Among the available materials for small blood vessel construction, these cellular hybrid vascular scaffolds hold much potential due to controllability of the mechanical properties of synthetic polymers and biocompatibility of integrated cellular components.
- Published
- 2018
8. Robust on-line sampling and analysis during long-term perfusion cultivation of mammalian cells
- Author
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Mehdi Saghafi, Christoph Knappe, Stefan Steigmiller, Cary Matanguihan, Chetan T. Goudar, and LeeWey Chong
- Subjects
Sample (material) ,Cell Culture Techniques ,Analytical chemistry ,Sampling (statistics) ,Bioengineering ,General Medicine ,Applied Microbiology and Biotechnology ,Bioreactors ,Perfusion Culture ,Robustness (computer science) ,Bioreactor ,Animals ,Environmental science ,Process control ,Sample preparation ,Perfusion ,Biotechnology ,Biomedical engineering - Abstract
In an attempt to support robust automated sampling and analysis of mammalian cell bioreactors, an integrated platform, BaychroMAT®, was developed which includes an innovative sterile sampling device, automated sample transport, a sample preparation module, online analyzers, and communication interfaces to process automation systems. The robustness of this platform was verified by applying it to a laboratory-scale perfusion bioreactor that was operated for over 100 days. Both manual and automated samples were collected over the course of the run and a comparison was made for cell density, viability, glucose, and lactate concentrations. The highest variability (14.4%) was seen for cell density estimates while those for viability, glucose, and lactate were 0.7, 12.9, and 8.2%, respectively. In addition, cell density set-point changes were made towards the end of the perfusion culture and the high frequency automated samples provided a higher resolution description of the dynamics of cell density change compared to less frequent manual sampling. Overall, our results indicate stable and robust operation of the BaychroMAT® platform in a long-term perfusion culture. This success should readily translate to shorter duration fed-batch cultures thereby enabling feed-back control based on real-time nutrient measurements.
- Published
- 2013
9. Three-dimensional perfusion cultures of mouse and pig fetal liver cells in a packed-bed reactor: Effect of medium flow rate on cell numbers and hepatic functions
- Author
-
Satoshi Suzuki, Norio Ohshima, Hirotoshi Miyoshi, Hideki Kawai, and Tomo Ehashi
- Subjects
medicine.medical_specialty ,Liver cytology ,Bioengineering ,Biology ,Prosthesis Design ,Applied Microbiology and Biotechnology ,law.invention ,Mice ,Bioreactors ,Perfusion Culture ,Tissue engineering ,law ,Internal medicine ,medicine ,Animals ,Secretion ,Cells, Cultured ,Tissue Engineering ,Tissue Scaffolds ,Cell growth ,Bioartificial liver device ,Albumin ,General Medicine ,Liver, Artificial ,Molecular biology ,Equipment Failure Analysis ,Mice, Inbred C57BL ,Perfusion ,Endocrinology ,Liver ,Hepatocytes ,Biotechnology - Abstract
To develop a tissue-engineered bioartificial liver (BAL), perfusion cultures of mouse and pig fetal liver cells (FLCs) immobilized within a three-dimensional (3D) porous scaffold were performed utilizing a packed-bed reactor system. These FLCs were cultured under different medium flow rate conditions, and the effects of the flow rates on cell growth and the hepatic functions of the FLCs were investigated. In the cultures of mouse FLCs, the medium flow suppressed cell growth and the albumin secretion activity of the FLCs, and considerably lower albumin secretion than that in the 3D stationary control cultures was obtained in the perfusion cultures. In the case of pig FLCs, cell growth was also inhibited by the medium flow, however, the cells exhibited higher tolerance to the medium flow compared with mouse FLCs. The albumin secretion activity of pig FLCs was well maintained under an extremely low flow rate condition (4.8 mm/min in the reactor), and activity higher than the 3D stationary cultures was detected at a later stage (after 20 days in the perfusion cultures). These results revealed that FLCs are quite sensitive to medium flow and an extremely low shear stress is required for the perfusion cultures of FLCs.
- Published
- 2010
10. An on-line method for the reduction of fouling of spin-filters for animal cell perfusion cultures
- Author
-
R. Rabe, U. von Stockar, F. Vallez-Chetreanu, Ian W. Marison, and L.G. Fraisse Ferreira
- Subjects
Pore size ,Time Factors ,Materials science ,Transducers ,Cell ,Cell Culture Techniques ,Analytical chemistry ,Bioengineering ,CHO Cells ,Online Systems ,Vibration ,Applied Microbiology and Biotechnology ,law.invention ,Cricetulus ,Perfusion Culture ,law ,Cricetinae ,medicine ,Animals ,Ultrasonics ,Porosity ,Filtration ,Fouling ,General Medicine ,Perfusion ,medicine.anatomical_structure ,Filter (video) ,Biophysics ,Biotechnology - Abstract
The main limitation in the use of spin-filters during perfusion cultures of animal cells was revealed to be filter fouling. This phenomenon involves cell-sieve interactions as well as cell attachment to, and growth on, the filter surface. The cell attachment effect has been analysed in the present study during long-term perfusion simulations with CHO animal cells. It was demonstrated that at low filter acceleration, below 6.2 m/s2, a high perfusion rate of 25 cm/h induced rapid filter pore clogging within 3 days, whereas increasing the filter acceleration to 25 m/s2 increased filter longevity from 3 to 25 days, for filters with a pore size of 8.5 microm. Increasing the filter pore size to 14.5 microm improved filter longevity by 84% with less viable and dead cell deposits on the filter surface. However, it was demonstrated that filter longevity was not necessarily dependent on the amount of cell deposit on the filter surface. In the second part of this study, ultrasonic technology was used to reduce filter fouling. Filter vibration, induced by a piezo actuator, improved filter longevity by 113% during CHO cells perfusion cultures.
- Published
- 2007
11. Bioprocess development for the production of a recombinant MUC1 fusion protein expressed by CHO-K1 cells in protein-free medium
- Author
-
Ros Graham, Ruth Essers, K Zörner, J. Burchell, Thomas Noll, Gunnar C. Hansson, Jochem Gätgens, Joyce Taylor-Papadimitriou, Thomas Link, and Malin Bäckström
- Subjects
Glycosylation ,Time Factors ,Glutamine ,Recombinant Fusion Proteins ,Cell Culture Techniques ,Gene Expression ,Cell Count ,Bioengineering ,CHO Cells ,Biology ,Applied Microbiology and Biotechnology ,Culture Media, Serum-Free ,Cell Line ,law.invention ,chemistry.chemical_compound ,Bioreactors ,Perfusion Culture ,law ,Cricetinae ,Animals ,Humans ,Bioprocess ,MUC1 ,Chinese hamster ovary cell ,Mucin-1 ,General Medicine ,Hydrogen-Ion Concentration ,Fusion protein ,Glucose ,chemistry ,Biochemistry ,Cell culture ,Recombinant DNA ,Biotechnology - Abstract
The mucin MUC1 is a candidate for use in specific immunotherapy against breast cancer, but this requires the large-scale production of a MUC1 antigen. In this study, a bioprocess for the expression of a recombinant MUC1 fusion protein with a cancer associated glycosylation in CHO-K1 cells has been developed. Cells permanently expressing parts of the extracellular portion of MUC1 fused to IgG Fc were directly transferred from adherent growth in serum-containing medium to suspension culture in the protein-free ProCHO4-CDM culture medium. Using the Cellferm-pro system, optimal culture parameter as pH and pO(2) were determined in parallel spinner flask batch cultures. A pH of 6.8-7.0 and a pO(2) of 40% of air saturation was found to give best cell growth and productivity of secreted recombinant protein. Specific productivity strongly depended the pO(2) and correlated with the online monitored oxygen uptake rate (OUR) of the cells, which indicates a positive influence of the rate of oxidative phosphorylation on productivity. The optimised conditions were applied to continuous perfusion culture which gave very high cell densities and space time yields of the recombinant MUC1 fusion protein, allowing production at gram scale. The product degradation was much lower in supernatants from continuous perfusion culture compared to batch mode. Antibodies reacting with cancer associated MUC1 glycoforms strongly bound to the fusion protein, indicating that the desired glycoforms were obtained and suggesting that the recombinant MUC1 protein could be tested for use in immunotherapy.
- Published
- 2004
12. Secreted enzyme production by fungal pellets in a perfusion bioreactor
- Author
-
Wei Wen Su and Bing Jun He
- Subjects
Chromatography ,biology ,Phosphatase ,Acid phosphatase ,Pellets ,Bioengineering ,General Medicine ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Neurospora crassa ,Perfusion Culture ,Biochemistry ,biology.protein ,Extracellular ,Bioreactor ,Perfusion ,Biotechnology - Abstract
In this study, extracellular enzyme production by fungal pellets cultured in a novel continuous perfusion bioreactor is investigated. Cell retention during perfusion culture is achieved by incorporating an internal settling zone into an external-loop air-lift bioreactor. Production of an extracellular enzyme, acid phosphatase, by the filamentous fungus Neurospora crassa was chosen as a model system. In order to control culture morphology to allow effective long-term perfusion culture, an anionic polymer Carbopol (carboxypolymethylene) at 0.1% was added to the culture medium to promote growth in a more dispersed form. The bioreactor has shown a high pellet retention efficiency over a wide range of medium perfusion rates. The fungal pellets were successfully cultivated in the bioreactor for over 30 days. By operating the bioreactor under phosphate limitation, and by a step-wise increase of the perfusion rate from 0.5 to 1 d−1, a steady state phosphatase volumetric productivity of ca. 900 U l−1 d−1 was reached while cell dry weight was maintained at over 4 g l−1.
- Published
- 1997
13. Optimization of HEK-293S cell cultures for the production of adenoviral vectors in bioreactors using on-line OUR measurements
- Author
-
J. Gálvez, Jordi J. Cairó, Martí Lecina, Carles Solà, and Francesc Gòdia
- Subjects
Virus Cultivation ,Cell Survival ,Cell ,Genetic Vectors ,Bioengineering ,Cell Count ,Biology ,Applied Microbiology and Biotechnology ,Cross-flow filtration ,Adenoviridae ,Perfusion Culture ,Bioreactors ,Cell density ,Bioreactor ,medicine ,Humans ,Chromatography ,business.industry ,HEK 293 cells ,Virion ,General Medicine ,Biotechnology ,Culture Media ,Oxygen ,Perfusion ,medicine.anatomical_structure ,Glucose ,HEK293 Cells ,Oxygen uptake rate ,Cell culture ,business - Abstract
The culture of HEK-293S cells in a stirred tank bioreactor for adenoviral vectors production for gene therapy is studied. Process monitoring using oxygen uptake rate (OUR) was performed. The OUR was determined on-line by the dynamic method, providing good information of the process evolution. OUR enabled cell activity monitoring, facilitating as well the determination of the feeding rate in perfusion cultures and when to infect the culture. Batch cultures were used to validate the monitoring methodology. A cell density of 10×10(5)cell/mL was infected, producing 1.3×10(9) infectious viral particles/mL (IVP/mL). To increase cell density values maintaining cell specific productivity, perfusion cultures, based on tangential flow filtration, were studied. In this case, OUR measurements were used to optimize the dynamic culture medium feeding strategy, addressed to avoid any potential nutrient limitation. Furthermore, the infection protocol was defined in order to optimize the use of the viral inoculum, minimizing the uncontrolled release of particles through the filter unit mesh. All these developments enabled an infection at 78×10(5)cell/mL with the consequent production of 44×10(9)IVP/mL, representing a cell specific productivity 4.3 times higher than for the batch culture.
- Published
- 2011
14. High productivity of human recombinant beta-interferon from a low-temperature perfusion culture
- Author
-
N. Huzel, Kevin Sunley, Michael Butler, Jose Rodriguez, C. Lodewyks, Tharmala Tharmalingam, and Maureen Spearman
- Subjects
Protein Denaturation ,Glycosylation ,Cell Culture Techniques ,Bioengineering ,CHO Cells ,Biology ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,law.invention ,chemistry.chemical_compound ,Perfusion Culture ,Bioreactors ,Cricetulus ,law ,Cricetinae ,medicine ,Bioreactor ,Animals ,Humans ,Denaturation (biochemistry) ,Escherichia coli ,Chinese hamster ovary cell ,Temperature ,General Medicine ,Interferon-beta ,Recombinant Proteins ,Culture Media ,chemistry ,Biochemistry ,Recombinant DNA ,Perfusion ,Biotechnology - Abstract
Recombinant human interferon-beta (β-IFN), used in the therapeutic treatment of multiple sclerosis (MS), can be produced on a large-scale from genetically engineered Chinese hamster ovary (CHO) cells. However, its hydrophobicity causes non-reversible, molecular aggregation in culture. The parameters affecting aggregation were determined to be concentration, culture residence time, temperature and glycosylation. Although the protein can be produced in Escherichia coli in a non-glycosylated form, the addition of glycans confers a reduced rate of aggregation as well as a 10-fold higher bioactivity. We report on the application of a low temperature perfusion culture designed to control the parameters that cause aggregation. In this three-phase culture system there is a transition to a low temperature (32°C) in a batch mode prior to implementing perfusion at 1 volume/day using an acoustic cell separator. Perfusion at the low temperature resulted in a 3.5-fold increase in specific productivity and a 7-fold increase in volumetric productivity compared to the batch culture at 37°C. The percentage aggregation of β-IFN was reduced from a maximum of 43% in batch culture to a minimum of 5% toward the end of the perfusion phase. The glycosylation profile of all samples showed predominantly sialylated biantennary fucosylated structures. The extent of sialylation, which is important for bioactivity, was enhanced significantly in the perfusion culture, compared to the batch culture.
- Published
- 2010
15. Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells
- Author
-
R. Dunker, M. Thomaz, C. Burger, A. E. Cunha, José L. Moreira, Manuel J.T. Carrondo, João J. Clemente, E. Rieke, Cristina Peixoto, Helder J. Cruz, H.S. Conradt, and Elsa M. Dias
- Subjects
Glycosylation ,Recombinant Fusion Proteins ,Cell Culture Techniques ,Mice, Nude ,Oligosaccharides ,Bioengineering ,Chemostat ,Biology ,Kidney ,Applied Microbiology and Biotechnology ,Cell-free system ,law.invention ,Cell Line ,chemistry.chemical_compound ,Mice ,Perfusion Culture ,Bioreactors ,law ,Cricetinae ,Bioreactor ,Tumor Cells, Cultured ,Animals ,Humans ,Mice, Inbred BALB C ,Cell-Free System ,Antibodies, Monoclonal ,General Medicine ,Chromatography, Ion Exchange ,Fusion protein ,Perfusion ,Biochemistry ,chemistry ,Cell culture ,Immunoglobulin G ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Recombinant DNA ,Interleukin-2 ,Immunoglobulin Heavy Chains ,Cell Division ,Biotechnology - Abstract
The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.
- Published
- 2002
16. A novel perfusion system for animal cell cultures by two step sequential sedimentation
- Author
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Xiao-Wei Teng, Zhiyou Wen, and Feng Chen
- Subjects
Countercurrent exchange ,Sedimentation (water treatment) ,Cell Survival ,Cell Culture Techniques ,Bioengineering ,Cell Count ,Cell Separation ,Biology ,Applied Microbiology and Biotechnology ,Cell Line ,Immunoglobulin kappa-Chains ,Mice ,Perfusion Culture ,Bioreactors ,Settling ,Bioreactor ,Animals ,Chromatography ,Hybridomas ,Airlift ,Antibodies, Monoclonal ,General Medicine ,Rats ,Perfusion ,Volume (thermodynamics) ,Cell culture ,Immunoglobulin G ,Biotechnology ,Gravitation - Abstract
A novel perfusion system was developed for high density culture of animal cells. The system consists of an airlift bioreactor, a setting tank and a flat settler. Both the settling tank and flat settler have two connecting pipes for transporting the cells from and back to the reactor, respectively. Thus, the cell flow in the settlers can be controlled in uni-direction, avoiding the countercurrent flow of the cells. During perfusion cultures, the cells firstly settled in the settling tank, then, unsettled cells in the tank were transferred to the flat settler for re-settling. With the application of the system to hybridoma cell cultures, it was found that the maximum viable cell density, monoclonal antibody concentration and average productivity were 1.31×10 7 cells ml −1 , 400 mg l −1 and 461 mg l −1 d −1 , respectively, which were much higher than those of a batch culture. Both theoretical analysis and experimental results showed a much higher separation efficiency in such a two-step sedimentation system than that in a conventional one-step sedimentation system. In addition, the volumetric ratio of the sedimentation devices to the culture volume in our developed system is much lower, which may be potentially useful on an industrial-scale.
- Published
- 2000
17. A comparison of intensive cell culture bioreactors operating with hybridomas modified for inhibited apoptotic response
- Author
-
H Bierau, A Perani, A. N. Emery, and Mohamed Al-Rubeai
- Subjects
medicine.drug_class ,Cell ,Genetic Vectors ,Cell Culture Techniques ,Bioengineering ,Apoptosis ,Biology ,Monoclonal antibody ,Applied Microbiology and Biotechnology ,Perfusion Culture ,Bioreactors ,medicine ,Bioreactor ,Humans ,Ultrasonics ,Hybridomas ,Cell growth ,Antibodies, Monoclonal ,General Medicine ,Transfection ,Cell biology ,medicine.anatomical_structure ,Proto-Oncogene Proteins c-bcl-2 ,Cell culture ,Immunology ,Cell Division ,Filtration ,Biotechnology - Abstract
It is demonstrated, using two different perfusion reaction systems, that hybridoma modified by inhibiting their apoptotic response can give improved process performance in terms of cell number and viability in intensive cell culture. Two cell perfusion systems, one using a spin filter and the other an ultrasonic filter, are compared using two cell lines. One cell line is transfected with the bcl-2 gene (TB:C3 bcl-2) which encodes the ‘anti-apoptotic’ human bcl-2 protein and the other cell line (TB:C3 pEF) with a negative transfection vector. Both reactor systems give similar retention performance for both cell lines. Bcl-2 transfected cells reach higher cell densities than the control cell line, and the percentage of apoptotic cells is clearly lower than with pEF cells. The maximum cell numbers of the bcl-2 cell line are 1.2110 7 ml 1 in the ultrasonic filter culture and 1.58 10 7 ml 1 in the spin filter culture, respectively. Using the pEF cell line the maximum cell number reaches 6.0 10 6 ml 1 with ultrasonic retention and 5.9 10 6 ml 1 in the spin filter. The use of ultrasound in this cell retention system has no apparent influence on cell growth, productivity or viability. Selective retention of viable cells is detectable but the effect of removing non-viable cells is negligible. © 1998 Elsevier Science B.V. All rights reserved.
- Published
- 1998
18. HBs-MAb production in perfusion culture with selective ammonia removal system
- Author
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Fidel Rey P. Nayve, Tomoya Misato, Hiroshi Kataoka, and Masatoshi Matsumura
- Subjects
Ratón ,medicine.drug_class ,medicine.medical_treatment ,Cytological Techniques ,Bioengineering ,Biology ,Monoclonal antibody ,Applied Microbiology and Biotechnology ,Ammonia ,chemistry.chemical_compound ,Mice ,Perfusion Culture ,medicine ,Animals ,Hepatitis B Antibodies ,Cells, Cultured ,Chromatography ,Hepatitis B Surface Antigens ,Hybridomas ,Growth factor ,Antibodies, Monoclonal ,Membranes, Artificial ,General Medicine ,Culture Media ,Glutamine ,Perfusion ,chemistry ,Cell culture ,Biotechnology - Abstract
Conventional serum-free perfusion cultures of hybridoma TO-405 cells using medium supplemented with additional amounts of glucose, glutamine, β-mercaptoethanol and growth factors failed to yield cell densities and monoclonal antibody (MAb) concentrations which were significantly different from the results of unsupplemented perfusion cultures. Ammonia building-up to inhibitory concentrations in all the cultures was regarded as one of the primary reasons. When perfusion cultures and medium supplementation were done coupled to the ammonia removing system, the viable cell density grew to a maximum of 2.5 × 107 cells per ml at high percentage viability. This value is more than a 300% increase from that of conventional perfusion cultures and better compared to serum-supplemented cultures. The monoclonal antibody accumulated to a concentration as high as 26.3 × 105 mIU per ml. This is about 10-times when compared to that which can be achieved in ordinary perfusion cultures.
- Published
- 1994
19. Optimization of flow field in inclined gravitational settler for animal cells perfusion culture process
- Author
-
Wen-Song Tan, Lu Zhang, and Xu Zhang
- Subjects
Gravitation ,Perfusion Culture ,Chemistry ,Scientific method ,Bioengineering ,General Medicine ,Mechanics ,Applied Microbiology and Biotechnology ,Flow field ,Biotechnology - Published
- 2008
20. [Untitled]
- Subjects
Chromatography ,Cell growth ,Chinese hamster ovary cell ,Bioengineering ,General Medicine ,Biology ,Applied Microbiology and Biotechnology ,law.invention ,Matrix (chemical analysis) ,Erlenmeyer flask ,Perfusion Culture ,Volume (thermodynamics) ,law ,Bioreactor ,Perfusion ,Biotechnology - Abstract
Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 × 10(6)viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 °C to 29 °C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 °C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.
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