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Amine Kamen - One of the best experts on this subject based on the ideXlab platform.
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Large-Scale Transient Transfection of Suspension Mammalian Cells for VLP Production.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Laura Cervera, Amine KamenAbstract:Large-scale Transient Transfection of mammalian cell suspension cultures enables the production of biological products in sufficient quantity and under stringent quality attributes to perform accelerated in vitro evaluations and has the potential to support preclinical or even clinical studies. Here we describe the methodology to produce VLPs in a 3L bioreactor, using suspension HEK 293 cells and PEIPro as a Transfection reagent. Cells are grown in the bioreactor to 1 × 106 cells/mL and transfected with a plasmid DNA-PEI complex at a ratio of 1:2. Dissolved oxygen and pH are controlled and are online monitored during the production phase and cell growth and viability can be measured off line taking samples from the bioreactor. If the product is labeled with a fluorescent marker, Transfection efficiency can be also assessed using flow cytometry analysis. Typically, the production phase lasts between 48 and 96 h until the product is harvested.
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292 towards large scale manufacturing of adeno associated virus by Transient Transfection of hek293 suspension cells in a stirred tank bioreactor using serum free medium
Molecular Therapy, 2016Co-Authors: Parminder Chahal, Amine Kamen, Erica Schulze, Alice Bernier, Stephane Lanthier, Nathalie Coulombe, Renald GilbertAbstract:Adeno-Associated Virus (AAV) vectors showing safety profile in phase I clinical trials and its ability to transduce gene expression in various tissues have made it a vector of choice for gene delivery. There are different modes of AAV vector production and each has advantages and disadvantages. Here we demonstrated that the production of AAV by Transient Transfection in a serum-free medium using NRC's patented cGMP compliant human embryonic kidney HEK293 cell line (clone HEK293SF-3F6) adapted for growth in suspension can be readily scaled-up in stirred tank bioreactors. We employed triple-plasmid / polyethylenimine (PEI) based Transient Transfection technique. As a proof of concept, we demonstrated that nine serotypes of AAV (AAV-1 to AAV-9) encoding GFP can be produced by our cell line HEK293SF with yields of about 1E+13 genome-containing particles per liter (Vg/L). Depending on the serotypes 4-30% of AAV is present in the supernatant of the cell culture at 48hpt. The presence of plasmids and plasmid polyplexes that were not taken up by the cells or were not brought into the cell nucleus were removed by Iodixanol-ultracentrifugation method and Benzonase treatment before analyzing by real-time PCR. About 25% loss in genome containing viral particle counts were observed by Iodixanol purification method based on infectivity assay. Productions of AAV2 and AAV6 encoding GFP were demonstrated in 3L stirred tank bioreactors. Purification scheme was based on column chromatography - a scalable process. Different chromatography media, such as cation exchanger, anion exchanger and hydrophobic interaction chromatography, were tested with each AAV serotypes for their ability to adsorb and elute efficiently. The purification scheme was then adopted by integrating best chromatography medium and sequence dependent upon the AAV serotype in use. We demonstrated the purification scheme for AAV2 based on ion-exchange and hydrophobic interaction chromatography steps. The SDS-PAGE showed the purity of the final product and the presence of three capsid proteins VP1, VP2 and VP3 on Western blot corresponding to the only three bands present in the final product on SDS-PAGE. To extend the storage life of AAV we explored lyophilization technique to study the stability of AAV2 and AAV6 under lyophilized conditions. The AAV2 and AAV6 were stable for over 40 weeks based on infectivity assay. We demonstrated the scalability of the process up to 45L. Productions tested in 20 and 500 mL cultures in shake flasks were scaled up in 2 and 45L cultures (in 3- and 60-L stirred tank bioreactors, respectively). The volumetric yields and purification recoveries were comparable at all of these production scale levels demonstrating scalability of Transient Transfection at even larger scale is possible to generate material necessary for dosages required for gene therapy application.
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Transient Transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII
BMC biotechnology, 2011Co-Authors: Kamilla Swiech, Amine Kamen, Sven Ansorge, Yves Durocher, Virgínia Picanço-castro, Elisa Maria De Sousa Russo-carbolante, Mario Soares De Abreu Neto, Dimas Tadeu CovasAbstract:Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of Transient Transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated Transfections done at 34° and 37°C. We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in Transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated Transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-Transfection. Serum-free suspension Transient Transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.
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Production of lentiviral vectors by large-scale Transient Transfection of suspension cultures and affinity chromatography purification
Biotechnology and bioengineering, 2007Co-Authors: María Mercedes Segura, Yves Durocher, Alain Garnier, Helene Coelho, Amine KamenAbstract:The use of lentiviral vectors as gene delivery vehicles has become increasingly popular in recent years. The growing interest in these vectors has created a strong demand for large volumes of vector stocks, which entails the need for scaleable vector manufacturing procedures. In this work, we present a simple and robust process for the production of lentiviral vectors using scaleable production and purification methodologies. Lentivirus particles were produced by Transient Transfection of serum-free suspension-growing 293 EBNA-1 cells with four plasmids encoding the vector components using linear polyethylenimine (PEI) as Transfection reagent. This process was successfully scaled-up from shake flasks to a 3-L bioreactor from which 10(10) IVP were recovered. In addition, an affinity chromatography protocol designed for purification of bioactive oncoretroviral vectors has been adapted in this work for the purification of VSV-G pseudotyped lentiviral vectors. Using heparin affinity chromatography, lentiviral particles were concentrated and purified directly from the clarified supernatants. During this step, a recovery of 53% of infective lentiviral particles was achieved while removing 94% of the impurities contained in the supernatant.
W Pear - One of the best experts on this subject based on the ideXlab platform.
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Transient Transfection methods for preparation of high-titer retroviral supernatants.
Current protocols in molecular biology, 2001Co-Authors: W PearAbstract:Generation of high-titer retrovirus by Transient production not only is less laborious than production of stable retroviral producer cell lines, but also has allowed the production of high-titer retroviral supernatants from cDNAs that cannot be achieved by stable producer cell lines. Transient Transfection has also increased the versatility of retrovirus-mediated gene transfer to include the rapid testing of different constructs, viral pseudotyping, and construction of retroviral cDNA libraries. Systems based on human 293 cells, an adenovirus-transformed human embryonic kidney cell line have produced the highest retroviral titers and are the most widely used. This unit describes methods for optimizing retroviral production from the 293-based systems and for growing and freezing 293 cells. Methods are included for pseudotyping the virus with VSV G protein by sequential Transfection or coTransfection. Virus produced by Transiently transfected cells can be used to infect cells. Protocols are provided for infection of adherent cells either directly with retroviral supernatant or by spin infection. In addition, procedures are included for infection of nonadherent cells by addition of retrovirus supernatant, cocultivation with producer cells, or spin infection. These infection methods are also applicable to retrovirus produced by any of the stable producer cell lines.
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Current Protocols in Molecular Biology - Transient Transfection methods for preparation of high-titer retroviral supernatants.
Current Protocols in Molecular Biology, 2001Co-Authors: W PearAbstract:Generation of high-titer retrovirus by Transient production not only is less laborious than production of stable retroviral producer cell lines, but also has allowed the production of high-titer retroviral supernatants from cDNAs that cannot be achieved by stable producer cell lines. Transient Transfection has also increased the versatility of retrovirus-mediated gene transfer to include the rapid testing of different constructs, viral pseudotyping, and construction of retroviral cDNA libraries. Systems based on human 293 cells, an adenovirus-transformed human embryonic kidney cell line have produced the highest retroviral titers and are the most widely used. This unit describes methods for optimizing retroviral production from the 293-based systems and for growing and freezing 293 cells. Methods are included for pseudotyping the virus with VSV G protein by sequential Transfection or coTransfection. Virus produced by Transiently transfected cells can be used to infect cells. Protocols are provided for infection of adherent cells either directly with retroviral supernatant or by spin infection. In addition, procedures are included for infection of nonadherent cells by addition of retrovirus supernatant, cocultivation with producer cells, or spin infection. These infection methods are also applicable to retrovirus produced by any of the stable producer cell lines.
Zhuyong Mei - One of the best experts on this subject based on the ideXlab platform.
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Retroviral vector production by Transient Transfection of 293VEC-GALV cells
Cytotherapy, 2019Co-Authors: Adrian P. Gee, Zhuyong MeiAbstract:Background & Aim Introduction Retroviral vectors can permanently integrate their genetic material into chromosomes in the target cells for long-term, stable expression of the introduced genetic elements. It has been widely used since the 1980s and is the second most commonly employed system for gene transfer. Traditionally, retroviral vector production involves the preparation and characterization of a stable producer cell line from which the vector is produced as supernatant. It usually takes about 12 to 20 months from generation the stable producer cell line clone to the final release of the retroviral vector for clinical use. Due to the recent rapid growth of CAR-T cell therapies, a faster, more efficient method for retroviral vector production is required. We have, therefore, employed a novel manufacturing system using Transient Transfection for rapid GMP preparation of retroviral vectors. Methods, Results & Conclusion Methods Clinical retroviral vectors are produced from a master cell bank of 293Vec-Galv (BioVec Pharma, Quebec, Canada), a human embryonic kidney HEK293-based packaging cell line that stably expresses the Moloney Murine Leukemia gag-pol and Gibbon Ape Leukemia Virus envelope viral proteins. The cells are transfected with plasmid DNA that carries the gene(s) of interest (purified plasmid DNAs are produced by Puresyn Inc. Malvern PA, USA or Aldevron Inc. Fargo ND, USA). The Transfection is carried out at 37°C and is mediated by PEIpro®-HQ (Polyplus Transfection, Illkirch, France), which is a highly qualified grade of polyethylenimine developed for large-scale bioproduction processes. About 24 hours post-Transfection the medium is replaced by fresh complete medium. Supernatant containing the retrovirus is harvested at 48, 72 and 96 hours post-Transfection and filtered through 0.45µm filter. The filtered supernatant is aliquoted, snap frozen and immediately transferred to storage in -80°C freezers. Results and Conclusion Eight retroviral vectors have been manufactured using this novel system at the CAGT GMP Vector Production Facility. Vector production from a single master cell bank of 293Vec-Galv takes about 4 weeks. Four vectors have been released for clinical trials. The transduction rate, phenotype and cytotoxic effect of T cells transduced by vectors produced by this novel system met release requirements, including absence of replication-competent retrovirus. Retroviral vector production by Transient Transfection of 293Vec-Galv cells saves time, is cost-efficient and safe ( Table 1 ).
María Mercedes Segura - One of the best experts on this subject based on the ideXlab platform.
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Intracellular characterization of Gag VLP production by Transient Transfection of HEK 293 cells
Biotechnology and bioengineering, 2017Co-Authors: Laura Cervera, María Mercedes Segura, Irene González-domínguez, Francesc GòdiaAbstract:Transient Transfection is a fast, flexible, and cost-effective approach to produce biological products. Despite the continued interest in Transient Transfection, little is known regarding the Transfection process at the intracellular level, particularly for complex products, such as virus-like particles (VLPs). The kinetics of PEI-mediated Transfection following an established in-house protocol is reported in this work with the aim of characterizing and understanding the complete process leading to VLP generation and identifying important events driving process improvement. For this purpose, DNA/PEI polyplexes' internalization in cells was tracked using Cy3 DNA staining. The production of a fluorescently labeled Gag polyprotein (a Gag-GFP fusion construct that forms fluorescent Gag-VLPs) was monitored by flow cytometry and confocal microscopy, and the VLP concentration in supernatants was measured by fluorometry. DNA/PEI polyplexes have been shown to interact with the cell membrane immediately after polyplex addition to the cell culture. A linear increase in the number of cells expressing the protein is observed during the first 60 minutes of contact between the cells and polyplexes. No additional improvement in the number of cells expressing the protein (up to 60%) or VLP production (up to 1 × 1010 VLPs/mL) is observed with additional contact time between the cells and polyplexes. Polyplexes can be detected in the cytoplasm of transfected cells as early as 1.5 hours post-Transfection (hpt) and reach the nucleus approximately 4 hpt. GFP fluorescence is observed homogeneously in the cytoplasm of transfected cells 24 hpt, but generalized VLP budding is not observed by microscopy until 48 hpt. Although all cells have internalized a polyplex soon after Transfection, only a fraction of cells (60%) express the fluorescent Gag protein. VLP production kinetics was also studied. Fluorescence in the supernatant (enveloped VLPs) is 40% less than total fluorescence, supernatant plus pellet (free Gag-GFP), indicating that there is a fraction of Gag that remains inside the cells. The maximum VLP concentration in the cell culture supernatant with cell viability >89% was observed at 72 hpt, which was determined to be the optimal harvest time. This article is protected by copyright. All rights reserved
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Optimized production of HIV-1 virus-like particles by Transient Transfection in CAP-T cells.
Applied microbiology and biotechnology, 2015Co-Authors: Sonia Gutiérrez-granados, Laura Cervera, María Mercedes Segura, Jens Wölfel, Francesc GòdiaAbstract:HIV-1 virus-like particles (VLPs) have great potential as new-generation vaccines. The novel CAP-T cell line is used for the first time to produce Gag-GFP HIV-1 VLPs by means of polyethylenimine (PEI)-mediated Transient Transfection. CAP-T cells are adapted to grow to high cell densities in serum-free medium, and are able to express complex recombinant proteins with human post-translational modifications. Furthermore, this cell line is easily transfected with PEI, which offers the flexibility to rapidly generate and screen a number of candidates in preclinical studies. Transient Transfection optimization of CAP-T cells has been performed systematically in this work. It is determined that for optimal production, cells need to be growing at mid-exponential phase, Protein Expression Medium (PEM) medium has to be added post-Transfection, and cells can be transfected by independent addition of DNA and PEI with no prior complexation. A Box-Behnken experimental design is used to optimize cell density at time of Transfection, DNA/cell and PEI/cell ratios. The optimal conditions determined are Transfection at a density of 3.3E + 06 cells/mL with 0.5 pg of DNA/cell and 3 pg of PEI/cell. Using the optimized protocol, 6 × 1010 VLP/mL are obtained, demonstrating that CAP-T is a highly efficient cell line for the production of HIV-1 VLPs and potentially other complex viral-based biotherapeutics.
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Production of lentiviral vectors by large-scale Transient Transfection of suspension cultures and affinity chromatography purification
Biotechnology and bioengineering, 2007Co-Authors: María Mercedes Segura, Yves Durocher, Alain Garnier, Helene Coelho, Amine KamenAbstract:The use of lentiviral vectors as gene delivery vehicles has become increasingly popular in recent years. The growing interest in these vectors has created a strong demand for large volumes of vector stocks, which entails the need for scaleable vector manufacturing procedures. In this work, we present a simple and robust process for the production of lentiviral vectors using scaleable production and purification methodologies. Lentivirus particles were produced by Transient Transfection of serum-free suspension-growing 293 EBNA-1 cells with four plasmids encoding the vector components using linear polyethylenimine (PEI) as Transfection reagent. This process was successfully scaled-up from shake flasks to a 3-L bioreactor from which 10(10) IVP were recovered. In addition, an affinity chromatography protocol designed for purification of bioactive oncoretroviral vectors has been adapted in this work for the purification of VSV-G pseudotyped lentiviral vectors. Using heparin affinity chromatography, lentiviral particles were concentrated and purified directly from the clarified supernatants. During this step, a recovery of 53% of infective lentiviral particles was achieved while removing 94% of the impurities contained in the supernatant.
Francesc Gòdia - One of the best experts on this subject based on the ideXlab platform.
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Production of HIV virus-like particles by Transient Transfection of CAP-T cells at bioreactor scale avoiding medium replacement.
Journal of biotechnology, 2017Co-Authors: Sonia Gutiérrez-granados, Queralt Farràs, Kerstin Hein, Javier Fuenmayor, Pablo Félez, Mercedes Segura, Francesc GòdiaAbstract:Human-derived CAP-T cell line has been demonstrated to be a powerful platform for high-titer production of HIV virus-like particles (VLPs) by PEI-mediated Transient Transfection. Scale-up of Transfection processes is key to ensure the necessary quantities for pre-clinical and clinical testing. One of the major operational challenges of large-scale Transient Transfection is the medium replacement step that is often required before Transfection. In this work, CAP-T cells were cultured in 1L bioreactor with addition of sodium bicarbonate and surface aeration, which were observed to improve cell state for Transfection. Remarkably, the medium replacement step was avoided by culturing the cells in a combination of media (FreeStyleF17+1% of PEM) compatible with cell growth and PEI-mediated Transient Transfection. In the conditions developed in this work, 0.5×106cells/mL were seeded in 1L bioreactor. Two days later, ∼2×106cells/mL were transfected without medium exchange, using 0.5pg of DNA/cell and 3pg of PEI/cell. Transfection efficiency and VLP production comparable to shake flasks were obtained with a production of 4×1010VLPs/mL. This novel strategy significantly simplifies large-scale Transient Transfection, while suitable cell growth, Transfection efficiency, and high quality VLP production are achieved.
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Intracellular characterization of Gag VLP production by Transient Transfection of HEK 293 cells
Biotechnology and bioengineering, 2017Co-Authors: Laura Cervera, María Mercedes Segura, Irene González-domínguez, Francesc GòdiaAbstract:Transient Transfection is a fast, flexible, and cost-effective approach to produce biological products. Despite the continued interest in Transient Transfection, little is known regarding the Transfection process at the intracellular level, particularly for complex products, such as virus-like particles (VLPs). The kinetics of PEI-mediated Transfection following an established in-house protocol is reported in this work with the aim of characterizing and understanding the complete process leading to VLP generation and identifying important events driving process improvement. For this purpose, DNA/PEI polyplexes' internalization in cells was tracked using Cy3 DNA staining. The production of a fluorescently labeled Gag polyprotein (a Gag-GFP fusion construct that forms fluorescent Gag-VLPs) was monitored by flow cytometry and confocal microscopy, and the VLP concentration in supernatants was measured by fluorometry. DNA/PEI polyplexes have been shown to interact with the cell membrane immediately after polyplex addition to the cell culture. A linear increase in the number of cells expressing the protein is observed during the first 60 minutes of contact between the cells and polyplexes. No additional improvement in the number of cells expressing the protein (up to 60%) or VLP production (up to 1 × 1010 VLPs/mL) is observed with additional contact time between the cells and polyplexes. Polyplexes can be detected in the cytoplasm of transfected cells as early as 1.5 hours post-Transfection (hpt) and reach the nucleus approximately 4 hpt. GFP fluorescence is observed homogeneously in the cytoplasm of transfected cells 24 hpt, but generalized VLP budding is not observed by microscopy until 48 hpt. Although all cells have internalized a polyplex soon after Transfection, only a fraction of cells (60%) express the fluorescent Gag protein. VLP production kinetics was also studied. Fluorescence in the supernatant (enveloped VLPs) is 40% less than total fluorescence, supernatant plus pellet (free Gag-GFP), indicating that there is a fraction of Gag that remains inside the cells. The maximum VLP concentration in the cell culture supernatant with cell viability >89% was observed at 72 hpt, which was determined to be the optimal harvest time. This article is protected by copyright. All rights reserved
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Optimized production of HIV-1 virus-like particles by Transient Transfection in CAP-T cells.
Applied microbiology and biotechnology, 2015Co-Authors: Sonia Gutiérrez-granados, Laura Cervera, María Mercedes Segura, Jens Wölfel, Francesc GòdiaAbstract:HIV-1 virus-like particles (VLPs) have great potential as new-generation vaccines. The novel CAP-T cell line is used for the first time to produce Gag-GFP HIV-1 VLPs by means of polyethylenimine (PEI)-mediated Transient Transfection. CAP-T cells are adapted to grow to high cell densities in serum-free medium, and are able to express complex recombinant proteins with human post-translational modifications. Furthermore, this cell line is easily transfected with PEI, which offers the flexibility to rapidly generate and screen a number of candidates in preclinical studies. Transient Transfection optimization of CAP-T cells has been performed systematically in this work. It is determined that for optimal production, cells need to be growing at mid-exponential phase, Protein Expression Medium (PEM) medium has to be added post-Transfection, and cells can be transfected by independent addition of DNA and PEI with no prior complexation. A Box-Behnken experimental design is used to optimize cell density at time of Transfection, DNA/cell and PEI/cell ratios. The optimal conditions determined are Transfection at a density of 3.3E + 06 cells/mL with 0.5 pg of DNA/cell and 3 pg of PEI/cell. Using the optimized protocol, 6 × 1010 VLP/mL are obtained, demonstrating that CAP-T is a highly efficient cell line for the production of HIV-1 VLPs and potentially other complex viral-based biotherapeutics.
