13 results on '"Durocher Y"'
Search Results
2. Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells.
- Author
-
Stuible M, Gervais C, Lord-Dufour S, Perret S, L'Abbé D, Schrag J, St-Laurent G, and Durocher Y
- Subjects
- Animals, CHO Cells, Cricetulus, Gene Expression, HEK293 Cells, Humans, SARS-CoV-2, Transfection, Protein Domains, Recombinant Proteins, Spike Glycoprotein, Coronavirus genetics
- Abstract
Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 spike ectodomain. A high-cell-density protocol using DXB11-derived CHO
BRI/55E1 cells gave substantially better yields than the other methods. Different forms of the spike ectodomain were expressed, including the wild-type SARS-CoV-2 sequence and a mutated form (to favor expression of the full-length spike ectodomain stabilized in pre-fusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L in the harvested medium. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)- Published
- 2021
- Full Text
- View/download PDF
3. Production of α2,6-sialylated and non-fucosylated recombinant alpha-1-antitrypsin in CHO cells.
- Author
-
Lalonde ME, Koyuturk I, Brochu D, Jabbour J, Gilbert M, and Durocher Y
- Subjects
- Animals, Biosimilar Pharmaceuticals metabolism, CHO Cells, Cricetulus, Humans, Recombinant Proteins, Sialyltransferases genetics, alpha 1-Antitrypsin genetics, beta-D-Galactoside alpha 2-6-Sialyltransferase, Leukocyte Elastase antagonists & inhibitors, Sialyltransferases metabolism, alpha 1-Antitrypsin metabolism
- Abstract
Alpha-1-antitrypsin (A1AT) is an abundant serum inhibitor of serine proteases. A1AT deficiency is a common genetic disorder which is currently treated with augmentation therapies. These treatments involve weekly injections of patients with purified plasma-derived A1AT. Such therapies can be extremely expensive and rely on plasma donors. Hence, large-scale production of recombinant A1AT (rA1AT) could greatly benefit these patients, as it could decrease the cost of treatments, reduce biosafety concerns and ensure quantitative and qualitative controls of the protein. In this report, we sought to produce α2,6-sialylated rA1AT with our cumate-inducible stable CHO pool expression system. Our different CHO pools could reach volumetric productivities of 1,2 g/L. The human α2,6-sialyltransferase was stably expressed in these cells in order to mimic elevated α2,6-sialylation levels of native A1AT protein. Sialylation of the recombinant protein was stable over the duration of the fed-batch production phase and was higher in a pool where cells were sorted and enriched by FACS based on cell-surface α2,6-sialylation. Addition of ManNAc to the cell culture media during production enhanced both α2,3 and α2,6 A1AT sialylation levels whereas addition of 2F-peracetylfucose potently inhibited fucosylation of the protein. Finally, we demonstrated that rA1AT proteins exhibited human neutrophil elastase inhibitory activities similar to the commercial human plasma-derived A1AT., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
4. Assessment of fed-batch cultivation strategies for an inducible CHO cell line.
- Author
-
Mellahi K, Brochu D, Gilbert M, Perrier M, Ansorge S, Durocher Y, and Henry O
- Subjects
- Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal genetics, CHO Cells, Cell Survival genetics, Cricetulus, Glucose metabolism, Humans, Rituximab chemistry, Rituximab genetics, Antibodies, Monoclonal biosynthesis, Batch Cell Culture Techniques methods, Cell Proliferation genetics, Rituximab biosynthesis
- Abstract
In order to maximize cell growth and productivity for an inducible CHO cell line expressing rituximab, various fed-batch culture strategies were investigated. In each case, the performance was evaluated for cultures induced at moderate and high cell density conditions (4 × 10
6 and 10 × 106 cells/mL) to assess the impact of the timing of induction. We first demonstrate the importance of starting the feeding process during the growth phase, as this translated into significantly improved integral of viable cells and antibody concentration, when compared to post-induction feeding only. Secondly, we investigated the impact of the feed rate by maintaining different levels of glucose (25, 35 and 50 mM) via a dynamic feeding strategy. The highest antibody concentrations were achieved under a moderate feeding regime for both cell densities at induction, highlighting the risks of under- or over-feeding the cultures. We then evaluated the impact of performing a temperature shift at induction by testing different mild hypothermia conditions. At small-scale, the highest production yields (1.2 g/L) were achieved when the temperature was reduced from 37 to 30 °C during the production phase of a culture induced at high cell density. When the strategy was applied in bioreactor, the better controlled conditions led to even greater product concentrations (1.8 g/L). Furthermore, this production protocol was shown to promote a more galactosylated glycan profile than a bioreactor culture initiated at 34 °C during growth and downshifted to 30 °C during the production phase., (Copyright © 2019. Published by Elsevier B.V.)- Published
- 2019
- Full Text
- View/download PDF
5. Reducing recombinant protein expression during CHO pool selection enhances frequency of high-producing cells.
- Author
-
Poulain A, Mullick A, Massie B, and Durocher Y
- Subjects
- Animals, Antibodies, Monoclonal genetics, Antibodies, Monoclonal immunology, CHO Cells, Cricetinae, Cricetulus, Gene Expression Regulation immunology, Immunoglobulin Fc Fragments genetics, Immunoglobulin Fc Fragments immunology, Recombinant Proteins genetics, Recombinant Proteins immunology, Stress, Physiological genetics, Transfection, Antibodies, Monoclonal biosynthesis, Batch Cell Culture Techniques methods, Immunoglobulin Fc Fragments biosynthesis, Recombinant Proteins biosynthesis
- Abstract
Chinese hamster ovary (CHO) cells are the most widely used mammalian host for industrial-scale production of monoclonal antibodies (mAbs) and other protein biologics. Isolation of rare high-producing CHO cell lines from heterogeneous populations of stable transfectants is a daunting task and delays the process of manufacturing of novel biologics. A variety of factors that contribute to the low frequency of high-producing clones have been described; however, the impact of metabolic burden and other stresses (eg. ER stress) associated with sustained high-level expression of recombinant protein (r-protein) during selection of stable transfectants has not been fully appreciated. CHO cell line development has not traditionally received much optimization in this area because the vast majority of platforms use constitutive expression systems to produce biologics. Previously, we developed a cell line (CHO
BRI/rcTA ) containing a robust inducible expression system, based on the cumate gene switch, that allows r-protein expression to be down-regulated during selection. Using this switch, we generated inducible CHOBRI/rcTA pools expressing an Fc-fusion protein within two weeks of transfection with volumetric productivity of up to 1.1 g/L at 17 days post-induction in a fed-batch culture process. Herein, we show that the ability to regulate r-protein expression during pool generation confers a substantial advantage for selecting high-producing stable clones. Reducing expression levels ("off-state") during pool selection dramatically enhances high-producer frequency compared to a pool in which expression was maintained at a high level during selection ("on-state", mimicking a constitutive expression system). Overexpression of the r-protein during the pool selection process negatively affects pool recovery and is associated with subtle but significant increases in BiP expression and cell death compared to pool selection in the "off-state". Our data shows that the cumate gene switch is a valuable platform for stable clone generation and supports the wider application of inducible systems for scalable production of biologics in CHO cells., (Copyright © 2019. Published by Elsevier B.V.)- Published
- 2019
- Full Text
- View/download PDF
6. Optimization of a high-cell-density polyethylenimine transfection method for rapid protein production in CHO-EBNA1 cells.
- Author
-
Stuible M, Burlacu A, Perret S, Brochu D, Paul-Roc B, Baardsnes J, Loignon M, Grazzini E, and Durocher Y
- Subjects
- Animals, CHO Cells, Cell Count, Cricetulus, Gene Expression, Epstein-Barr Virus Nuclear Antigens genetics, Polyethyleneimine, Transfection methods, Trastuzumab biosynthesis
- Abstract
For pre-clinical evaluation of biotherapeutic candidates, protein production by transient gene expression (TGE) in Chinese Hamster Ovary (CHO) cells offers important advantages, including the capability of rapidly and cost-effectively generating recombinant proteins that are highly similar to those produced in stable CHO clones. We have established a novel CHO clone (CHO-3E7) expressing a form of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) with improved TGE productivity relative to parental CHO cells. Taking advantage of a new transfection-compatible media formulation that permits prolonged, high-density culture, we optimized transfection parameters (cell density, plasmid vector and polyethylenimine concentrations) and post-transfection culture conditions to establish a new, high-performing process for rapid protein production. The growth media is chemically defined, and a single hydrolysate feed is added post-transfection, followed by periodic glucose supplementation. This method gave significantly higher yields than our standard low-cell density, F17-based CHO-3E7 TGE method, averaging several hundred mg/l for a panel of recombinant proteins and antibodies. Purified antibodies produced using the two methods had distinct glycosylation profiles but showed identical target binding kinetics by SPR. Key advantages of this new protein production platform include the cost-effectiveness of the transfection reagent, the commercial availability of the culture media and the ability to perform high-cell-density transfection without media change., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
7. Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch.
- Author
-
Poulain A, Perret S, Malenfant F, Mullick A, Massie B, and Durocher Y
- Subjects
- Animals, Batch Cell Culture Techniques, CHO Cells, Cricetinae, Cricetulus, Gene Expression, Genetic Vectors, Methionine Sulfoximine pharmacology, Plasmids metabolism, Promoter Regions, Genetic, Recombinant Proteins genetics, Plasmids genetics, Protein Engineering methods, Recombinant Proteins metabolism
- Abstract
To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHO
BRI/rcTA ) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHOBRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHOBRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
8. Therapeutic glycoprotein production in mammalian cells.
- Author
-
Lalonde ME and Durocher Y
- Subjects
- Animals, Cell Engineering, Cell Line, Genomics, Humans, Recombinant Proteins biosynthesis, Glycoproteins biosynthesis
- Abstract
Over the last years, the biopharmaceutical industry has significantly turned its biologics production towards mammalian cell expression systems. The presence of glycosylation machineries within these systems, and the fact that monoclonal antibodies represent today the vast majority of new therapeutic candidates, has largely influenced this new direction. Recombinant glycoproteins, including monoclonal antibodies, have shown different biological properties based on their glycan profiles. Thus, the industry has developed cell engineering strategies not only to improve cell's specific productivity, but also to adapt their glycosylation profiles for increased therapeutic activity. Additionally, the advance of "omics" technologies has recently given rise to new possibilities in improving these expression platforms and will significantly help developing new strategies, in particular for CHO (Chinese Hamster Ovary) cells., (Copyright © 2017. Published by Elsevier B.V.)
- Published
- 2017
- Full Text
- View/download PDF
9. Altering the central carbon metabolism of HEK293 cells: Impact on recombinant glycoprotein quality.
- Author
-
Karengera E, Robotham A, Kelly J, Durocher Y, De Crescenzo G, and Henry O
- Subjects
- Ammonia metabolism, Bioreactors, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Glucose metabolism, Glutamine metabolism, Glycoproteins genetics, Glycosylation, Humans, Interferon alpha-2, Interferon-alpha chemistry, Interferon-alpha metabolism, Lactic Acid metabolism, Metabolic Engineering methods, Pyruvate Carboxylase genetics, Pyruvate Carboxylase metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Yeasts genetics, Carbon metabolism, Glycoproteins metabolism, HEK293 Cells metabolism, Recombinant Proteins metabolism
- Abstract
The accumulation of metabolic by-products remains a critical challenge in the development of mammalian cells culture processes as it impacts cellular growth, productivity and product quality. Although the overexpression of the PYC2 gene was shown to significantly improve the nutrient metabolism efficiency of mammalian cells, its impact on recombinant protein quality has not been investigated yet. In this study, we assess the effect of this metabolic engineering strategy on the quality of a recombinant therapeutic glycoprotein, the human interferon α2b (IFNα2b). As inferred from densitometry analysis of SDS-PAGE gels, PYC2-overexpressing cells sustained a higher percentage of intact glycosylated IFNα2b at the late stage of batch cultures, which was correlated with prolonged viability and reduced accumulation of waste metabolites. Contrarily to the IFNα2b produced by the PYC2 cells, LC-MS analysis confirmed the presence of less glycosylated IFNα2b as well as the occurrence of proteolytic cleavage in the IFNα2b produced in the parental cells. Taken together, these results indicate that PYC2-overexpression in mammalian cells leads to extended favorable conditions for glycosylation and offer an attractive approach to mass-produce high-quality recombinant proteins., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
10. An omics approach to rational feed: Enhancing growth in CHO cultures with NMR metabolomics and 2D-DIGE proteomics.
- Author
-
Blondeel EJM, Ho R, Schulze S, Sokolenko S, Guillemette SR, Slivac I, Durocher Y, Guillemette JG, McConkey BJ, Chang D, and Aucoin MG
- Subjects
- Animals, CHO Cells, Cell Culture Techniques, Cricetinae, Cricetulus, Culture Media, Humans, Isoelectric Focusing, Magnetic Resonance Spectroscopy, Metabolic Networks and Pathways, Tandem Mass Spectrometry, Two-Dimensional Difference Gel Electrophoresis, Metabolomics methods, Proteomics methods, Recombinant Proteins analysis, Recombinant Proteins biosynthesis, Recombinant Proteins metabolism
- Abstract
Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression of endogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabolites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteins following stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identified and cross-referenced with cellular functions and metabolic pathways to identify points of cell stress. Subsequently, 1D-(1)H NMR metabolomics experiments analyzed cultures to observe nutrient depletion and waste metabolite accumulations to further examine these cell stresses and pathways. From among fifty metabolites tracked in time-course, eight were observed to be completely depleted from the production media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypoxanthine, while twenty-three excreted metabolites were also observed to accumulate. The differentially expressed proteins, as well as the nutrient depletion and accumulation of these metabolites corresponded with upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPH replenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redox modulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cell stresses to achieve a ∼75% improvement to peak cell densities., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
11. Comparative study of polyethylenimines for transient gene expression in mammalian HEK293 and CHO cells.
- Author
-
Delafosse L, Xu P, and Durocher Y
- Subjects
- Acylation, Animals, Antibodies metabolism, CHO Cells, Cell Death, Cricetinae, Cricetulus, DNA metabolism, Endocytosis, Genetic Vectors metabolism, HEK293 Cells, Humans, Kinetics, Luminescent Proteins metabolism, Mammals, Proton Magnetic Resonance Spectroscopy, Transfection, Gene Expression, Polyethyleneimine metabolism
- Abstract
Three commercially available linear polyethylenimines (25kDa LPEI, 40kDa PEI"Max" and PEIpro™) were compared regarding their potency to transfect serum-free growing and suspension-adapted HEK293 and CHO cells. We determined the optimal DNA:PEI ratios for maximal expression of the reporter gene SEAP while monitoring cytotoxicity following transfection. PEIs acylation was determined by (1)H NMR and their apparent size and polydispersity assessed by size-exclusion chromatography. The propensity of PEIs to condense plasmid DNA was evaluated by agarose-gel electrophoresis. The zeta potentials and particle sizes at optimal DNA:PEI ratio were analyzed. Polyplex attachment to the cells and internalization kinetics were monitored. The quantity of PEIpro™ needed to efficiently transfect the cells was significantly lower than with LPEI and PEI"Max" and, interestingly, the maximal amount of internalized PEIpro™-based polyplexes was approximately half of that observed with its counterparts. PEIpro™ was the largest and least polydisperse polymer, but also the most cytotoxic. The optimal transfection conditions were subsequently used to express three monoclonal antibodies at larger-scale. The use of the deacylated PEI"Max" and PEIpro™ resulted in a significant increase of recombinant protein expression compared to LPEI. These findings demonstrate the importance of properly choosing the most suitable polymers to obtain optimal recombinant protein transient expression., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
12. Metabolic engineering of CHO cells to alter lactate metabolism during fed-batch cultures.
- Author
-
Toussaint C, Henry O, and Durocher Y
- Subjects
- Animals, Antibodies, Monoclonal biosynthesis, Bioreactors, Cricetinae, Cricetulus, Glucose metabolism, Glycosylation, Pyruvate Carboxylase biosynthesis, Pyruvate Carboxylase genetics, Pyruvate Carboxylase metabolism, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Recombinant Proteins metabolism, Batch Cell Culture Techniques methods, CHO Cells metabolism, Lactic Acid metabolism, Metabolic Engineering methods
- Abstract
Recombinant yeast pyruvate carboxylase (PYC2) expression was previously shown to be an effective metabolic engineering strategy for reducing lactate formation in a number of relevant mammalian cell lines, but, in the case of CHO cells, did not consistently lead to significant improvement in terms of cell growth, product titer and energy metabolism efficiency. In the present study, we report on the establishment of a PYC2-expressing CHO cell line producing a monoclonal antibody and displaying a significantly altered lactate metabolism compared to its parental line. All clones exhibiting strong PYC2 expression were shown to experience a significant and systematic metabolic shift toward lactate consumption, as well as a prolonged exponential growth phase leading to an increased maximum cell concentration and volumetric product titer. Of salient interest, PYC2-expressing CHO cells were shown to maintain a highly efficient metabolism in fed-batch cultures, even when exposed to high glucose levels, thereby alleviating the need of controlling nutrient at low levels and the potential negative impact of such strategy on product glycosylation. In bioreactor operated in fed-batch mode, the higher maximum cell density achieved with the PYC2 clone led to a net gain (20%) in final volumetric productivity., (Copyright © 2015. Published by Elsevier B.V.)
- Published
- 2016
- Full Text
- View/download PDF
13. Exploiting the metabolism of PYC expressing HEK293 cells in fed-batch cultures.
- Author
-
Vallée C, Durocher Y, and Henry O
- Subjects
- Bioreactors, HEK293 Cells, Humans, Pyruvate Carboxylase genetics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Batch Cell Culture Techniques, Pyruvate Carboxylase metabolism
- Abstract
The expression of recombinant yeast pyruvate carboxylase (PYC) in animal cell lines was shown in previous studies to reduce significantly the formation of waste metabolites, although it has translated into mixed results in terms of improved cellular growth and productivity. In this work, we demonstrate that the unique phenotype of PYC expressing cells can be exploited through the application of a dynamic fed-batch strategy and lead to significant process enhancements. Metabolically engineered HEK293 cells stably producing human recombinant IFNα2b and expressing the PYC enzyme were cultured in batch and fed-batch modes. Compared to parental cells, the maximum cell density in batch was increased 1.5-fold and the culture duration was extended by 2.5 days, but the product yield was only marginally increased. Further improvements were achieved by developing and implementing a dynamic fed-batch strategy using a concentrated feed solution. The feeding was based on an automatic control-loop to maintain a constant glucose concentration. This strategy led to a further 2-fold increase in maximum cell density (up to 10.7×10(6)cells/ml) and a final product titer of 160mg/l, representing nearly a 3-fold yield increase compared to the batch process with the parental cell clone., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.