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Specific Productivity (specific + productivity)
Selected AbstractsEffect of Simultaneous Application of Stressful Culture Conditions on Specific Productivity and Heterogeneity of Erythropoietin in Chinese Hamster Ovary CellsBIOTECHNOLOGY PROGRESS, Issue 4 2004Sung Kwan Yoon A single stressful culture condition induced by hypoosmotic stress (210 mOsm kg,1), low culture temperature (32 °C), or NaBu addition (1 mM) resulted in a 1.8- to 2.2-fold enhancement of specific erythropoietin (EPO) productivity (qEPO) of recombinant Chinese hamster ovary (rCHO) cells compared to normal culture condition (37 °C and 310 mOsm kg,1). Simultaneous application of these stressful conditions further enhanced qEPO up to approximately 5-fold. However, the quality of EPO was affected by stressful culture conditions. The proportion of acidic isoforms of EPO under a single stressful condition was 2.8,13.8% lower than that under normal culture condition. Simultaneous application of the stressful conditions further decreased the portion of acidic isoforms but not significantly. Despite 5-fold enhancement of qEPO, the portion of acidic isoforms under the simultaneous application of stressful culture conditions was 12.9,21.6% lower than that under normal culture condition. Taken together, these results suggest the potential of simultaneous application of different stressful culture conditions to the production phase of two-stage culture, where cell growth and production phases are separated, for improved EPO production. [source] The generation of stable, high MAb expressing CHO cell lines based on the artificial chromosome expression (ACE) technologyBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2009Malcolm L. Kennard Abstract The manufacture of recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high expressing cell lines rapidly, reproducibly and with relative ease. Commonly used methods incorporate transfection of mammalian cell lines with plasmid DNA containing the gene of interest. Identifying stable high expressing transfectants is normally laborious and time consuming. To improve this process, the ACE System has been developed based on pre-engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for the targeted transfection of single or multiple genes and eliminates the need for random integration into native host chromosomes. To illustrate the utility of the ACE System in generating stable, high expressing cell lines, CHO based candidate cell lines were generated to express a human monoclonal IgG1 antibody. Candidate cell lines were generated in under 6 months and expressed over 1,g/L and with specific productivities of up to 45,pg/cell/day under non-fed, non-optimized shake flask conditions. These candidate cell lines were shown to have stable expression of the monoclonal antibody for up to 70 days of continuous culture. The results of this study demonstrate that clonal, stable monoclonal antibody expressing CHO based cell lines can be generated by the ACE System rapidly and perform competitively with those cell lines generated by existing technologies. The ACE System, therefore, provides an attractive and practical alternative to conventional methods of cell line generation. Biotechnol. Bioeng. 2009; 104: 540,553 © 2009 Wiley Periodicals, Inc. [source] A study of monoclonal antibody-producing CHO cell lines: What makes a stable high producer?,BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Janet Chusainow Abstract Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg,cell,1,day,1 using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high- and low-producer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35,92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize that the decline in transgene mRNA levels upon long-term culturing without MTX was mainly caused by transgene silencing consequently leading to a loss in mAb productivity. The exact molecular mechanisms causing production instability are not yet fully understood. The herein described extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection. Biotechnol. Bioeng. 2009;102: 1182,1196. © 2008 Wiley Periodicals, Inc. [source] Virus-like particle production at low multiplicities of infection with the baculovirus insect cell systemBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003Luis Maranga Abstract The baculovirus insect cell expression system (BEVS) was used for the production of self-forming Porcine parvovirus -like particles (VLPs) in serum-free medium. A low multiplicity of infection (MOI) strategy was used to overcome an extra virus amplification step, undesirable in industrial production, and to minimize the virus passage effect. It was confirmed that the time of infection (TOI) and MOI are dependent variables. Higher cell densities were obtained at low MOIs, keeping a constant TOI; however, both volumetric and specific productivities were lower. In synchronous infection, at high MOI, the specific productivity decreased when the cells were infected in the late phase of growth. Product degradation due to cell lysis strongly influenced the optimal time of harvest (TOH). Time of harvest was found to be highly dependent on the MOI, and a direct relationship with the cell yield was obtained. Analysis of the culture medium reveals that glutamine depletion occurs in the late phase of the growth. Supplementation of glutamine to uninfected cell cultures resulted in an increased cell yield. Its addition to cultures infected in the middle phase of the growth curve was also able to restore the productivity levels, but addition to cells in their stationary phase caused no observable effect on product expression. The study clearly shows that for a specific TOI it is not obvious what the correct MOI should be to obtain the best volumetric productivity. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 245,253, 2003. [source] 293 cell cycle synchronisation adenovirus vector productionBIOTECHNOLOGY PROGRESS, Issue 1 2009Tiago B. Ferreira Abstract As the market requirements for adenovirus vectors (AdV) increase, the maximisation of the virus titer per culture volume per unit time is a key requirement. However, despite the fact that 293 cells can grow up to 8 × 106 cell/mL in simple batch mode operations, for optimal AdV infection a maximum cell density of 1 × 106 cell/mL at infection time has usually been utilized due to the so called "cell density effect". In addition, AdV titer appears to be dependent upon cell cycle phase at the time of infection. To evaluate the dependence of AdV production upon cell cycle phase, 293 cells were chemically synchronised at each phase of the cell cycle; a 2.6-fold increase on AdV cell specific titer was obtained when the percentage of cells at the S phase of the cell cycle was increased from 36 to 47%; a mathematical equation was used to relate AdV cell specific productivities with cell synchronisation at the S phase using this data. To avoid the use of chemical inhibitors, a temperature shift strategy was also used for synchronisation at the S phase. S phase synchronisation was obtained by decreasing the culture temperature to 31°C during 67 h and restoring it to 37°C during 72 h. By using this strategy we were able to synchronise 57% of the population in the S phase of the cell cycle obtaining an increase of 7.3-fold on AdV cell specific titer after infection. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Bioreactor Production of Human ,1 -Antitrypsin Using Metabolically Regulated Plant Cell CulturesBIOTECHNOLOGY PROGRESS, Issue 3 2002Melody M. Trexler Transgenic rice cell cultures, capable of producing recombinant human ,1 -antitrypsin (rAAT), were scaled up from shake flasks to a 5-L bioreactor. The maximum specific growth rates (,max) observed from two bioreactor runs were 0.40 day,1 (doubling time of 1.7 days) and 0.47 day,1 (doubling time of 1.5 days), and the maximum specific oxygen uptake rates were 0.78 and 0.84 mmol O2/(g dw h). Using a metabolically regulated rice ,-amylase (RAmy3D) promoter, signal peptide, and terminator, sugar deprivation turned on rAAT expression, and rAAT was secreted into the culture medium. After 1 day of culture in sugar-free medium, there was still continued biomass growth, oxygen consumption, and viability. Extracellular concentrations of 51 and 40 mg active rAAT/L were reached 1.7 and 2.5 days, respectively, after induction in a sugar-free medium. Volumetric productivities for two batch cultures were 7.3 and 4.6 mg rAAT/(L day), and specific productivities were 3.2 and 1.6 mg rAAT/(g dw day). Several different molecular weight bands of immunoreactive rAAT were observed on immunoblots. [source] Detailed understanding of enhanced specific antibody productivity in NS0 myeloma cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Soo Hean Gary Khoo Abstract The understanding of how cellular productivity is modulated in cell lines is of significant importance in the biopharmaceutical industry. Often, single molecular mechanisms fail to fully explain how specific antibody productivity is enhanced during proliferation arrest. Previously, we reported that certain physiological changes occur when proliferation is arrested by p21CIP1 over expression. In this work, we correlate physiological and molecular factors to enhance antibody productivity. Using biomass, cell volume and total cellular protein content as a basis for determining specific productivity, it was found that total cellular protein correlated best with cellular productivity. This meant that there was no preferential increase in antibody production relative to cellular proteins in arrested cultures. However, molecular analysis of mRNA transcription and stability indicated that both processes were altered in arrested cultures resulting in up to threefold increased heavy chain mRNA levels. While flow cytometric analysis revealed that arrested cells had elevated translational capacity for both heavy and light chains, the heavy to light chain polypeptide ratio was 10,50% higher than in the control. This resulted in a lower extracellular accumulation of light chains and a better utilization of cellular resources for the formation of complete antibodies. Active transcriptional regulation of heavy and light chain mRNA and the modulation of translational activities play a vital role in the modulation of overall antibody productivity of these cells. The combined effect of heavy chain mRNA enhancement and the increased cellular assembly capacity was determined to effectively increase specific productivity. Biotechnol. Bioeng. 2009;102: 188,199. © 2008 Wiley Periodicals, Inc. [source] Comparative transcriptome analysis to unveil genes affecting recombinant protein productivity in mammalian cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Joon Chong Yee Abstract Low temperature culture (33°C) has been shown to enhance the specific productivity of recombinant antibodies in Chinese hamster ovary (CHO) cells but did not affect antibody productivity in hybridoma (MAK) cells. We probed the transcriptional response of both cells undergoing temperature shift using cDNA microarrays. Among the orthologous gene probes, common trends in the expression changes between CHO and MAK are not prominent. Instead, many transcriptional changes were specific to only one cell line. Notably, oxidative phosphorylation and ribosomal genes were downregulated in MAK but not in CHO. Conversely, several protein trafficking genes and cytoskeleton elements were upregulated in CHO but remained unchanged in MAK. Interestingly, at 33°C, immunoglobulin heavy and light chain showed no significant changes in CHO, but the immunoglobulin light chain was downregulated in MAK. Overall, a clear distinction in the transcriptional response to low temperature was seen in the two cell lines. To further elucidate the set of genes responsible for increased antibody productivity, the expression data of low temperature cultures was compared to that of butyrate treatment which increased specific antibody productivity in both cell lines. Genes which are commonly differentially expressed under conditions that increased productivity are likely to reflect functional classes that are important in the productivity changes. This comparative transcriptome analysis suggests that vesicle trafficking, endocytosis and cytoskeletal elements are involved in increased specific antibody productivity. Biotechnol. Bioeng. 2009;102: 246,263. © 2008 Wiley Periodicals, Inc. [source] Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfectionBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2008Sebastien Chenuet Abstract Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines. Biotechnol. Bioeng. © 2008 Wiley Periodicals, Inc. [source] Gene transcription acceleration: main cause of hepatitis B surface antigen production improvement by dimethyl sulfoxide in the culture of Chinese hamster ovary cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007Wenying Wang Abstract The production and specific productivity of hepatitis B surface antigen (HBsAg) in recombinant Chinese hamster ovary (CHO) cells were increased by 81% and threefold, respectively, when supplemented with 1.5% dimethyl sulfoxide (DMSO) in the culture medium. To investigate the mechanism of DMSO effect on HBsAg production improvement, HBsAg mRNA level was measured by real-time PCR. HBsAg mRNA was increased by about 1.5-fold at 1.5% DMSO. The increase could derive from the increase of HBsAg gene copy number, the improvement of HBsAg mRNA stability, or the acceleration of HBsAg gene transcription. It was found that HBsAg gene copy number was not significantly changed in the cells stimulated with DMSO. HBsAg mRNA stability of cells with DMSO treatment was also not obviously different from control, and the mRNA half-life of 5.58 h in the cells at 1.5% DMSO was comparable to that of 5.36 h in the control culture. DMSO resulted in 80% increase in HBsAg gene transcription activity assessed using a nuclear run-on transcription assay. It could be deduced that the acceleration of HBsAg gene transcription is the main cause of HBsAg production improvement. Biotechnol. Bioeng. 2007;97: 526,535. © 2006 Wiley Periodicals, Inc. [source] Rapamycin reduces hybridoma cell death and enhances monoclonal antibody productionBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2001R. Robert Balcarcel Abstract Rapamycin was used as a medium additive to slow the progression of CRL 1606 hybridomas through the cell cycle, under the hypothesis that such a modulation might reduce cell death. Cell cycle distributions for CRL hybridomas in the G1 phase of the cell cycle ranged from 20% to 35% during batch, fed-batch, and continuous culture experiments, independent of culture time, dilution rate, growth rates, or death rates. Rapamycin, an mTOR signaling inhibitor, immunosuppressant, and G1-phase arresting agent, was identified and tested for efficacy in restraining cell cycle progression in CRL 1606 hybridoma cultures. However, in the presence of 100 nM rapamycin, the percentage of cells in the G1 phase of the cell cycle during fed-batch cultures was only increased from 28% to 31% in control cultures to 37% to 48% for those with rapamycin. Accordingly, rapamycin only slightly reduced culture growth rate. Instead, the use of rapamycin more notably kept viability higher than that of control cultures by delaying cell death for 48 h, thereby enabling viable proliferation to higher maximum viable cell densities. Furthermore, rapamycin enhanced specific monoclonal antibody production by up to 100% during high-viability growth. Thus, over the course of 6-day fed-batch cultivations, the beneficial effects of rapamycin on viable cell density and specific productivity resulted in an increase in final monoclonal antibody titer from 0.25 to 0.56 g/L (124%). As rapamycin is reported to influence a much broader range of cellular functions than cell cycle alone, these findings are more illustrative of the influence that signal transduction pathways related to mTOR can have on overall cell physiology and culture productivity. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 76: 1,10, 2001. [source] Enhanced IFN, production in adenosine-treated CHOCells: A mechanistic studyBIOTECHNOLOGY PROGRESS, Issue 3 2009William P. K. Chong Abstract Adenosine causes growth arrest in recombinant mammalian cell cultures, which results in enhanced productivity of the recombinant protein. Adenosine is also known to increase intracellular ATP level when added to mammalian cells. As a cell's energy level affects its protein expression capacity, we investigated the factors that contribute to the increase in recombinant protein productivity. Chinese hamster ovary (CHO) cells expressing human interferon-gamma (IFN,) were treated with 1 mM adenosine on Day 2 of culture. The growth arrest resulted in 60% reduction in integral viable cell density when compared with control. However, IFN, titer improved 1.4-fold alongside a 2.5-fold increase in average specific productivity. The adenosine-treated cells also experienced a two-fold increase in ATP level that sustained for 3 days. Western blot studies revealed a relatively short-lived but strong activation of the energy sensor AMP-activated protein kinase (AMPK) in adenosine-treated cells. Activation of AMPK was probably due to adenosine being temporarily converted to AMP. Activated AMPK should have down-regulated protein translation by preventing mammalian target of rapamycin (mTOR) from phosphorylating and inactivating 4E-binding protein 1 (4E-BP1), a key repressor of protein translation initiation. However, Western blots showed increased phosphorylation of 4E-BP1 on Day 2 that lasted 3 days. This implied that a high concentration of ATP could keep 4E-BP1 inhibited, probably by directly modulating mTOR. This corroborated with an earlier in vitro observation (Dennis et al., Science. 2001;294:1102-1105). Inhibition of translation initiation repression is thus likely to contribute in part to the improvement in IFN,-specific productivity and titer. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Combined Effect of Agitation/Aeration and Fed-Batch Strategy on Ubiquin- one-10 Production by Pseudomonas diminutaCHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 6 2010Mahesh V. Bule Abstract The effects of aeration rate and agitation speed on ubiquinone-10 (CoQ10) submerged fermentation in a stirred-tank reactor using Pseudomonas diminuta NCIM 2865 were investigated. CoQ10 production, biomass formation, glycerol utilization, and volumetric mass transfer coefficient (kLa) were affected by both aeration and agitation. An agitation speed of 400,rpm and aeration rate of 0.5,vvm supported the maximum production (38.56,mg,L,1) of CoQ10 during batch fermentation. The fermentation run supporting maximum production had an kLa of 27.07,h,1 with the highest specific productivity and CoQ10 yield of 0.064,mg,g,1h,1 and 0.96,mg,g,1 glycerol, respectively. Fermentation kinetics performed under optimum aeration and agitation showed the growth-associated constant (a,=,5.067,mg,g,1) to be higher than the nongrowth-associated constant (,,=,0.0242,mg,g,1h,1). These results were successfully utilized for the development of fed-batch fermentation, which increased the CoQ10 production from 38.56,mg,L,1 to 42.85,mg,L,1. [source] | ||||||||||