Home About us Contact
|
Home About us Contact | ||
|
|
||
L Bioreactor (l + bioreactor)
Selected AbstractsIn situ magnetic separation for extracellular protein productionBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Tobias Käppler Abstract A new approach for in situ product removal from bioreactors is presented in which high-gradient magnetic separation is used. This separation process was used for the adsorptive removal of proteases secreted by Bacillus licheniformis. Small, non-porous bacitracin linked magnetic adsorbents were employed directly in the broth during the fermentation, followed by in situ magnetic separation. Proof of the concept was first demonstrated in shake flask culture, then scaled up and applied during a fed batch cultivation in a 3.7 L bioreactor. It could be demonstrated that growth of B. licheniformis was not influenced by the in situ product removal step. Protease production also remained the same after the separation step. Furthermore, degradation of the protease, which followed first order kinetics, was reduced by using the method. Using a theoretical modeling approach, we could show that protease yield in total was enhanced by using in situ magnetic separation. The process described here is a promising technique to improve overall yield in bio production processes which are often limited due to weak downstream operations. Potential limitations encountered during a bioprocess can be overcome such as product inhibition or degradation. We also discuss the key points where research is needed to implement in situ magnetic separation in industrial production. Biotechnol. Bioeng. 2009;102: 535,545. © 2008 Wiley Periodicals, Inc. [source] NS0 cell damage by high gas velocity sparging in protein-free and cholesterol-free culturesBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2008Ying Zhu Abstract Recent developments in high cell density and high productivity fed-batch animal cell cultures have placed a high demand on oxygenation and carbon dioxide removal in bioreactors. The high oxygen demand is often met by increasing agitation and sparging rates of air/O2 in the bioreactors. However, as we demonstrate in this study, an increase of gas sparging can result in cell damage at the sparger site due to high gas entrance velocities. Previous studies have showed that gas bubble breakup at the culture surface was primarily responsible for cell damage in sparged bioreactors. Such cell damage can be reduced by use of surfactants such as Pluronic F-68 in the culture. In our results, where NS0 cells were grown in a protein-free and cholesterol-free medium containing 0.5 g/L Pluronic F-68, high gas entrance velocity at the sparger site was observed as the second mechanism for cell damage. Experiments were performed in scaled-down spinners to model the effect of hydrodynamic force resulting from high gas velocities on antibody-producing NS0 cells. Cell growth and cell death were described by first-order kinetics. Cell death rate constant increased significantly from 0.04 to 0.18 day,1 with increasing gas entrance velocity from 2.3 to 82.9 m/s at the sparger site. The critical gas entrance velocity for the NS0 cell line studied was found to be ,30 m/s; velocities greater than 30 m/s caused cell damage which resulted in reduced viability and consequently reduced antibody production. Observations from a second cholesterol-independent NS0 cell line confirmed the occurrence of cell damage due to high gas velocities. Increasing the concentration of Pluronic F-68 from 0.5 to 2 g/L had no additional protective effect on cell damage associated with high gas velocity at the sparger. The results of gas velocity analysis for cell damage have been applied in two case studies of large-scale antibody manufacturing. The first is a troubleshooting study for antibody production carried out in a 600 L bioreactor, and the second is the development of a gas sparger design for a large bioreactor scale (e.g., 10,000 L) for antibody manufacturing. Biotechnol. Bioeng. 2008;101: 751,760. © 2008 Wiley Periodicals, Inc. [source] Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process controlBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2005Magnus Schroeder Abstract It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5,×,106 cells/mL after 9 days of differentiation. Approximately 30%,40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (,MHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28,×,109 cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications. © 2005 Wiley Periodicals, Inc. [source] Enhanced Production of Podophyllotoxin by Podophyllum hexandrum Using in Situ Cell Retention BioreactorBIOTECHNOLOGY PROGRESS, Issue 3 2003Saurabh Chattopadhyay The rhizomes of the rare plant Podophyllum hexandrum contain podophyllotoxin, which is a precursor of the anticancer drugs etoposide and teniposide. Batch cultivation of Podophyllumhexandrum was conducted using optimized medium in a 3 L bioreactor, which resulted in biomass and podophyllotoxin concentrations of 21.4 g/L and 13.8 mg/L in 24 and 26 days, respectively. The batch kinetics was used to identify the mathematical model. The model was extrapolated to identify the nutrient feeding rate (150 mL/d) and substrate concentration (105 g/L) in the incoming feed for nonlimiting and noninhibitory glucose concentration in the cell retention bioreactor. An improvement in cell growth to 53 g/L and intracellular podophyllotoxin accumulation of 48.8 mg/L was achieved in 60 days, when the bioreactor was operated in continuous cell retention cultivation mode. [source] A predictive high-throughput scale-down model of monoclonal antibody production in CHO cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009Rachel Legmann Abstract Multi-factorial experimentation is essential in understanding the link between mammalian cell culture conditions and the glycoprotein product of any biomanufacturing process. This understanding is increasingly demanded as bioprocess development is influenced by the Quality by Design paradigm. We have developed a system that allows hundreds of micro-bioreactors to be run in parallel under controlled conditions, enabling factorial experiments of much larger scope than is possible with traditional systems. A high-throughput analytics workflow was also developed using commercially available instruments to obtain product quality information for each cell culture condition. The micro-bioreactor system was tested by executing a factorial experiment varying four process parameters: pH, dissolved oxygen, feed supplement rate, and reduced glutathione level. A total of 180 micro-bioreactors were run for 2 weeks during this DOE experiment to assess this scaled down micro-bioreactor system as a high-throughput tool for process development. Online measurements of pH, dissolved oxygen, and optical density were complemented by offline measurements of glucose, viability, titer, and product quality. Model accuracy was assessed by regressing the micro-bioreactor results with those obtained in conventional 3,L bioreactors. Excellent agreement was observed between the micro-bioreactor and the bench-top bioreactor. The micro-bioreactor results were further analyzed to link parameter manipulations to process outcomes via leverage plots, and to examine the interactions between process parameters. The results show that feed supplement rate has a significant effect (P,<,0.05) on all performance metrics with higher feed rates resulting in greater cell mass and product titer. Culture pH impacted terminal integrated viable cell concentration, titer and intact immunoglobulin G titer, with better results obtained at the lower pH set point. The results demonstrate that a micro-scale system can be an excellent model of larger scale systems, while providing data sets broader and deeper than are available by traditional methods. Biotechnol. Bioeng. 2009; 104: 1107,1120. © 2009 Wiley Periodicals, Inc. [source] Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibody during fed-batch cultureBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2007Scott H. Stansfield Abstract In this study we have analyzed the dynamic covariation of the mammalian cell proteome with respect to functional phenotype during fed-batch culture of NS0 murine myeloma cells producing a recombinant IgG4 monoclonal antibody. GS-NS0 cells were cultured in duplicate 10 L bioreactors (36.5°C, 15% DOT, pH 7.0) for 335 h and supplemented with a continuous feed stream after 120 h. Cell-specific growth rate declined continuously after 72 h of culture. Cell-specific recombinant monoclonal antibody production rate (qP) varied sixfold through culture. Whilst qP correlated with relative recombinant heavy chain mRNA abundance up to 216 h, qP subsequently declined, independent of recombinant heavy chain or light chain mRNA abundance. GS-NS0 cultures were sampled at 48 h intervals between 24 and 264 h of culture for proteomic analyses. Total protein abundance and nascent polypeptide synthesis was determined by 2D PAGE of unlabeled proteins visualized by SYPRO® Ruby and autoradiography of 35S-labeled polypeptides, respectively. Covariation of nascent polypeptide synthesis and abundance with biomass-specific cell growth, glucose and glutamate consumption, lactate and Mab production rates were then examined using two partial least squares regression models. Most changes in polypeptide synthesis or abundance for proteins previously identified by mass spectrometry were positively correlated with biomass-specific growth rate. We conclude that the substantial transitions in cell physiology and qP that occur during culture utilize a relatively constant complement of the most abundant host cell machines that vary primarily with respect to induced changes in cell growth rate. Biotechnol. Bioeng. 2007;97: 410,424. © 2006 Wiley Periodicals, Inc. [source] Study of laccase production by Pleurotus ostreatus in a 5 l bioreactor and application of the enzyme to determine the antioxidant concentration of human plasmaLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2008S. Mazumder Abstract Aims:, To achieve high laccase production from Pleurotus ostreatus in a bench top bioreactor and to utilize the enzyme for determination of the total antioxidant concentration (TAC) of human plasma. Methods and Results:, Laccase production by P. ostreatus studied in a benchtop bioreactor was as high as, 874·0 U ml,1 in presence of copper sulfate. The enzyme was used to replace metmyoglobin and hydrogen peroxide for the estimation of TAC in human plasma. The trolox equivalent antioxidant concentrations determined by the laccase-based method and metmyoglobin method ranged from 1·63 ± 0·011 to 1·80 ± 0·006 mmol l,1 and from 1·41 ± 0·004 to 1·51 ± 0·008 mmol l,1 plasma, respectively. Conclusions:,Pleurotus ostreatus produced high amount of extracellular laccase in a benchtop bioreactor. The enzyme can be used to assay TAC of blood plasma without the interference encountered with the hydrogen peroxide and metmyoglobin mediated assay method. Significance and Impact of the Study:, Laccase production by P. ostreatus obtained in this study was the highest among all reported laccase producing white-rot fungi. Moreover, an accurate laccase-based assay method was developed for detection of TAC in human plasma. [source] Improvement for the production of clavulanic acid by mutant Streptomyces clavuligerusLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2002S.D. Lee Aims:,To improve the production of clavulanic acid through the development of strains, the selection of a production medium and a pH shift strategy in a bioreactor. Methods and Results:,Streptomyces clavuligerus mutant 15 was selected by antibacterial activities. As a result of pH control in a 2·5 l bioreactor, the highest productivity (3·37 ,g ml,1 h,1) was obtained at a controlled pH of 7·0. Conclusions:,The highest level of production obtained was an increase of about 36% compared with a non-controlled pH. When the production of clavulanic acid reached the maximum level, the pH was shifted from 7·0 to 6·0 for reduction in decomposition rate. The maximum concentration of clavulanic acid was maintained for 24 h as a result of the pH shift control, and a significant reduction in the decomposition of clavulanic acid was obtained. Significance and Impact of the Study:,Clavulanic acid decomposition was considerably reduced as a result of the pH shift control. The results of this study can be applied for the efficient production of ,-lactamase inhibitory antibiotics. [source] | ||||||||||