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Francesc Godia - One of the best experts on this subject based on the ideXlab platform.
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Accelerating HIV-1 VLP production using stable insect cell pools
Biotechnology journal, 2020Co-Authors: Eduard Puente-massaguer, Reingard Grabherr, Florian Strobl, Gerald Striedner, Marti Lecina, Paula Grau-garcia, Francesc GodiaAbstract:Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve High product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed to produce a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with High producer Cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared to parental High Five Cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system. This article is protected by copyright. All rights reserved.
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pei mediated transient transfection of High Five Cells at bioreactor scale for hiv 1 vlp production
Nanomaterials, 2020Co-Authors: Eduard Puentemassaguer, Reingard Grabherr, Florian Strobl, Gerald Striedner, Marti Lecina, Francesc GodiaAbstract:High Five Cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of High titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five Cells is successfully demonstrated at bioreactor scale, resulting in a Higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five Cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100–200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins.
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Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells.
Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2020Co-Authors: Eduard Puente-massaguer, Marti Lecina, Paolo Saccardo, Neus Ferrer-miralles, Francesc GodiaAbstract:Advancements in the field of characterization techniques have broadened the opportunities to deepen into nanoparticle production bioprocesses. Gag-based virus-like particles (VLPs) have shown their potential as candidates for recombinant vaccine development. However, comprehensive characterization of the production process is still a requirement to meet the desired critical quality attributes. In this work, the production process of Gag VLPs by baculovirus (BV) infection in the reference High Five and Sf9 insect cell lines is characterized in detail. To this end, the Gag polyprotein was fused in frame to the enhanced green fluorescent protein (eGFP) to favor process evaluation with multiple analytical tools. Tracking of the infection process using confocal microscopy and flow cytometry revealed a pronounced increase in the complexity of High Five over Sf9 Cells. Cryogenic transmission electron microscopy (cryo-TEM) characterization determined that changes in cell complexity could be attributed to the presence of occlusion-derived BV in High Five Cells, whereas Sf9 Cells evidenced a larger proportion of the budded virus phenotype (23-fold). Initial evaluation of the VLP production process using spectrofluorometry showed that Higher levels of the Gag-eGFP polyprotein were obtained in High Five Cells (3.6-fold). However, comparative analysis based on nanoparticle quantification by flow virometry and nanoparticle tracking analysis (NTA) proved that Sf9 Cells were 1.7- and 1.5-fold more productive in terms of assembled VLPs, respectively. Finally, analytical ultracentrifugation coupled to flow virometry evidenced a larger sedimentation coefficient of High Five-derived VLPs, indicating a possible interaction with other cellular compounds. Taken together, these results Highlight the combined use of microscopy and flow cytometry techniques to improve vaccine development processes using the insect cell/BV expression vector system. © 2020 International Society for Advancement of Cytometry.
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Integrating nanoparticle quantification and statistical design of experiments for efficient HIV-1 virus-like particle production in High Five Cells
Applied Microbiology and Biotechnology, 2020Co-Authors: Eduard Puente-massaguer, Marti Lecina, Francesc GodiaAbstract:The nature of enveloped virus-like particles (VLPs) has triggered High interest in their application to different research fields, including vaccine development. The baculovirus expression vector system (BEVS) has been used as an efficient platform for obtaining large amounts of these complex nanoparticles. To date, most of the studies dealing with VLP production by recombinant baculovirus infection utilize indirect detection or quantification techniques that hinder the appropriate characterization of the process and product. Here, we propose the application of cutting-edge quantification methodologies in combination with advanced statistical designs to exploit the full potential of the High Five/BEVS as a platform to produce HIV-1 Gag VLPs. The synergies between CCI, MOI, and TOH were studied using a response surface methodology approach on four different response functions: baculovirus infection, VLP production, VLP assembly, and VLP productivity. TOH and MOI proved to be the major influencing factors in contrast with previous reported data. Interestingly, a remarkable competition between Gag VLP production and non-assembled Gag was detected. Also, the use of nanoparticle tracking analysis and flow virometry revealed the existence of remarkable quantities of extracellular vesicles. The different responses of the study were combined to determine two global optimum conditions, one aiming to maximize the VLP titer (quantity) and the second aiming to find a compromise between VLP yield and the ratio of assembled VLPs (quality). This study provides a valuable approach to optimize VLP production and demonstrates that the High Five/BEVS can support mass production of Gag VLPs and potentially other complex nanoparticles.
Paula M. Alves - One of the best experts on this subject based on the ideXlab platform.
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Integrating High cell density cultures with adapted laboratory evolution for improved Gag-HA virus-like particles production in stable insect cell lines.
Biotechnology and bioengineering, 2021Co-Authors: Bárbara Fernandes, Ricardo Correia, Paula M. Alves, Manuel J.t. Carrondo, Marcos Sousa, António RoldãoAbstract:Stable insect cell lines are emerging as an alternative to the insect cell-baculovirus expression vector system (IC-BEVS) for protein expression, benefiting from being a virus-free, non-lytic system. Still, the titers achieved are considerably lower. In this study, stable insect (Sf-9 and High Five) Cells producing Gag virus-like particles (VLPs) were first adapted to grow under hypothermic culture conditions (22 ºC instead of standard 27 ºC), and then pseudotyped with a model membrane protein (influenza hemagglutinin, HA) for expression of Gag-HA VLPs. Adaptation to lower temperature led to an increase in protein titers of up to 12-fold for p24 (as proxy for Gag-VLP) and 6-fold for HA, with adapted Sf-9 Cells outperforming High Five Cells. Resulting Gag-HA VLPs producer Sf-9 Cells were cultured to High cell densities (HCD), i.e. 100x106 cell/mL, using perfusion (ATF® 2) in 1 L stirred-tank bioreactors. Specific p24 and HA production rates were similar to those of batch culture, enabling to increase volumetric titers by 7-8 fold without compromising the assembly of Gag-HA VLPs. Importantly, the antigen (HA) quantity in VLPs generated using stable adapted Cells in perfusion was ≈ 5-fold Higher than that from IC-BEVS, with the added benefit of being a baculovirus-free system. This article is protected by copyright. All rights reserved.
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Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution.
Vaccines, 2020Co-Authors: Ricardo Correia, Bárbara Fernandes, Paula M. Alves, Manuel J.t. Carrondo, António RoldãoAbstract:The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of High-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect Cells. In this study, insect High Five Cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (ΔPh = 0.2–0.3), and Cells were maintained at each pH value for 2–3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted Cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted Cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted Cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five Cells.
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Adaptive laboratory evolution of stable insect cell lines for improved HIV-Gag VLPs production.
Journal of biotechnology, 2019Co-Authors: Bárbara Fernandes, Ricardo Correia, Paula M. Alves, Manuel J.t. Carrondo, Joao Vidigal, Ana P Teixeira, António RoldãoAbstract:Abstract Adaptive laboratory evolution (ALE) has been extensively used to modulate the phenotype of industrial model organisms (e.g. Escherichia. coli and Saccharomyces cerevisae) towards a specific trait. Nevertheless, its application to animal Cells, and in particular to insect cell lines, has been very limited. In this study, we describe employing an ALE method to improve the production of HIV-Gag virus-like particles (VLPs) in stable Sf-9 and High Five cell lines. Serial batch transfer was used for evolution experiments. During the ALE process, Cells were cultured under controlled hypothermic conditions (22 °C instead of standard 27 °C) for a prolonged period of time (over 3 months), which allowed the selection of a population of Cells with improved phenotype. Adapted Cells expressed up to 26-fold (Sf-9 Cells) and 10-fold (High Five Cells) more Gag-VLPs than non-adapted Cells cultured at standard conditions. The production of HIV Gag-VLPs in adapted, stable insect Sf-9 cell lines was successfully demonstrated at bioreactor scale. The Gag-VLPs produced at 22 °C and 27 °C were comparable, both in size and morphology, thus confirming the null impact of adaptation process and hypothermic culture conditions on VLP’s quality. This work demonstrates the suitability of ALE as a powerful method for improving yields in stable insect cell lines producing VLPs.
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The role of host cell physiology in the productivity of the baculovirus-insect cell system: Fluxome analysis of Trichoplusia ni and Spodoptera frugiperda cell lines
Biotechnology and bioengineering, 2016Co-Authors: Francisca Monteiro, Vicente Bernal, Paula M. AlvesAbstract:The Insect Cell-Baculovirus Expression Vector System (IC-BEVS) is broadly used for the production of recombinant proteins and vaccine manufacture, yet the host physiological aspects that contribute to productivity are to be disclosed. This work provides the first quantitative analysis of the metabolic fluxes of High Five Cells. This analysis was conducted in comparison with Sf9 Cells, another major host for biologicals production via BEVS. Moreover, herein is presented, for the first time, quantitative data of the relative contribution of sugars and amino acids catabolism to the activity of the TCA cycle in Sf9 and High Five Cells. High Five Cells metabolic activity was markedly influenced by the amino acids concentration in culture medium, which determine the rates of amino acid catabolism, carbon overflow and by-product formation. This characteristic of High Five Cells was reflected in the activities of anaplerotic metabolism and the TCA cycle, which may not work as a true cycle as a function of medium composition. This was not the case for Sf9 Cells, in which the glucose carbon incorporation in the TCA cycle was significantly Higher and lactate production minor. Following infection, the decrease in by-product accumulation rates was accompanied by an increase in net ATP synthesis in Sf9 and High Five Cells, although through distinct mechanisms cell-line dependent. The impact of baculovirus infection on cellular metabolic status Highlights the capacity of this virus to re-direct the cellular fluxome toward ATP production to support replication and progeny generation. These results pave the way to deepen our knowledge on the relationship between a host cell and the virus, contributing to disclosing the metabolic determinants that contribute to productivity. Biotechnol. Bioeng. 2017;114: 674-684. © 2016 Wiley Periodicals, Inc.
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a cell sorting protocol for selecting High producing sub populations of sf9 and High Five Cells
Journal of Biotechnology, 2013Co-Authors: Joao Vidigal, Paula M. Alves, Manuel J.t. Carrondo, Mafalda M Dias, Fabiana Fernandes, Marco Patrone, Claudia Bispo, Claudia Andrade, Rui Gardner, Ana P TeixeiraAbstract:Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 Cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect Cells was developed here. The standard sorting conditions used for mammalian Cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ Cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold Higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent Cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect Cells.
Feng Yang - One of the best experts on this subject based on the ideXlab platform.
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Identification of three immediate-early genes of white spot syndrome virus.
Archives of virology, 2011Co-Authors: Fanyu Lin, He Huang, Feng YangAbstract:Viral immediate-early (IE) genes generally encode regulatory proteins that are critical for viral replication. Their transcription, which is independent of de novo viral protein synthesis, is driven directly by host transcription factors. In this study, we examined promoter activities of 12 predicted regulatory genes of white spot syndrome virus (WSSV) belonging to the zinc finger protein family by EGFP-reporter assays in High Five Cells. The results showed that the promoters of three genes (wsv056, wsv403 and wsv465) could drive reporter gene expression, and RT-PCR analysis revealed that their expression in WSSV-infected primary crayfish hemocytes was insensitive to the protein synthesis inhibitor cycloheximide (CHX). Therefore, they are IE genes of WSSV.
Reingard Grabherr - One of the best experts on this subject based on the ideXlab platform.
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Accelerating HIV-1 VLP production using stable insect cell pools
Biotechnology journal, 2020Co-Authors: Eduard Puente-massaguer, Reingard Grabherr, Florian Strobl, Gerald Striedner, Marti Lecina, Paula Grau-garcia, Francesc GodiaAbstract:Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve High product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed to produce a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with High producer Cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared to parental High Five Cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system. This article is protected by copyright. All rights reserved.
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pei mediated transient transfection of High Five Cells at bioreactor scale for hiv 1 vlp production
Nanomaterials, 2020Co-Authors: Eduard Puentemassaguer, Reingard Grabherr, Florian Strobl, Gerald Striedner, Marti Lecina, Francesc GodiaAbstract:High Five Cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of High titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five Cells is successfully demonstrated at bioreactor scale, resulting in a Higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five Cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100–200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins.
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Tnao38, High Five and Sf9—evaluation of host–virus interactions in three different insect cell lines: baculovirus production and recombinant protein expression
Biotechnology Letters, 2014Co-Authors: Monika Wilde, Miriam Klausberger, Dieter Palmberger, Wolfgang Ernst, Reingard GrabherrAbstract:Purpose of work: A comparative analysis of new and established insect cell lines, in regard to process relevant parameters, provide data that can be exploited for designing more robust and effective protein production processes. The baculovirus-insect cell expression system has been efficiently used for the production of heterologous proteins. Three different insect cell lines Tnao 38, High Five and Sf 9 were compared in terms of virus susceptibility, baculovirus production and product yield of an intra-cellularly (YFP) and extra-cellularly (influenza A virus hemagglutinin)-expressed recombinant protein. The Tnao 38 and High Five cell lines exhibited Higher (tenfold) susceptibility to baculovirus infection than Sf 9 Cells, whereas Sf 9 Cells showed a Higher (100-fold) capacity for production of infectious virus particles. Analysis of recombinant protein expression revealed considerably Higher product yields in Tnao 38 and High Five Cells as compared to Sf 9 Cells, for both model proteins. Overall, the two Trichoplusia ni -derived cell lines, High Five and Tnao 38, were significantly more efficient in terms of secreting proteins such as the glycoprotein hemagglutinin of influenza A virus.
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Tnao38, High Five and Sf9--evaluation of host-virus interactions in three different insect cell lines: baculovirus production and recombinant protein expression.
Biotechnology letters, 2013Co-Authors: Monika Wilde, Miriam Klausberger, Dieter Palmberger, Wolfgang Ernst, Reingard GrabherrAbstract:Purpose of work: A comparative analysis of new and established insect cell lines, in regard to process relevant parameters, provide data that can be exploited for designing more robust and effective protein production processes. The baculovirus-insect cell expression system has been efficiently used for the production of heterologous proteins. Three different insect cell lines Tnao38, High Five and Sf9 were compared in terms of virus susceptibility, baculovirus production and product yield of an intra-cellularly (YFP) and extra-cellularly (influenza A virus hemagglutinin)-expressed recombinant protein. The Tnao38 and High Five cell lines exhibited Higher (tenfold) susceptibility to baculovirus infection than Sf9 Cells, whereas Sf9 Cells showed a Higher (100-fold) capacity for production of infectious virus particles. Analysis of recombinant protein expression revealed considerably Higher product yields in Tnao38 and High Five Cells as compared to Sf9 Cells, for both model proteins. Overall, the two Trichoplusia ni-derived cell lines, High Five and Tnao38, were significantly more efficient in terms of secreting proteins such as the glycoprotein hemagglutinin of influenza A virus.
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Insect Cells for antibody production: evaluation of an efficient alternative.
Journal of biotechnology, 2011Co-Authors: Dieter Palmberger, Dubravko Rendić, Philipp Tauber, Florian Krammer, Iain B H Wilson, Reingard GrabherrAbstract:In recent years there has been an increase in both availability and demand for therapeutic monoclonal antibodies. Currently, most of these antibodies are produced by stably transfected mammalian Cells. In this study we evaluated the use of different baculoviral insect cell systems as an alternative for commonly used production schemes. We expressed the human anti-gp41 antibody 3D6 in Spodoptera frugiperda Sf9, Trichoplusia ni BTI-TN5B1-4 "High Five", and Spodoptera frugiperda SfSWT-1 "Mimic™" insect Cells and compared product yield, specificity and glycosylation patterns with a 3D6 antibody expressed in Chinese hamster ovary Cells. Using "High Five" Cells we achieved amounts of secreted antibody comparable to those resulting from transient expression in mammalian Cells. We determined the N-linked oligosaccharide structures present on asparagine-297 in IgG₁ heavy chains and tested the functionality in terms of antigen binding and the ability to elicit effector functions. Antibodies expressed in all insect cell lines displayed Highly specific antigen binding. In general, the insect-produced antibodies carried, as the CHO-produced form, fucosylated N-glycans, including, in the case of "High Five" Cells, High levels of core α1,3-fucose. This indicates that in all systems glycoengineering may be required in order to produce optimal glycoforms of this antibody.
Marti Lecina - One of the best experts on this subject based on the ideXlab platform.
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Accelerating HIV-1 VLP production using stable insect cell pools
Biotechnology journal, 2020Co-Authors: Eduard Puente-massaguer, Reingard Grabherr, Florian Strobl, Gerald Striedner, Marti Lecina, Paula Grau-garcia, Francesc GodiaAbstract:Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve High product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed to produce a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with High producer Cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared to parental High Five Cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system. This article is protected by copyright. All rights reserved.
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pei mediated transient transfection of High Five Cells at bioreactor scale for hiv 1 vlp production
Nanomaterials, 2020Co-Authors: Eduard Puentemassaguer, Reingard Grabherr, Florian Strobl, Gerald Striedner, Marti Lecina, Francesc GodiaAbstract:High Five Cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of High titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five Cells is successfully demonstrated at bioreactor scale, resulting in a Higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five Cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100–200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins.
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Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells.
Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2020Co-Authors: Eduard Puente-massaguer, Marti Lecina, Paolo Saccardo, Neus Ferrer-miralles, Francesc GodiaAbstract:Advancements in the field of characterization techniques have broadened the opportunities to deepen into nanoparticle production bioprocesses. Gag-based virus-like particles (VLPs) have shown their potential as candidates for recombinant vaccine development. However, comprehensive characterization of the production process is still a requirement to meet the desired critical quality attributes. In this work, the production process of Gag VLPs by baculovirus (BV) infection in the reference High Five and Sf9 insect cell lines is characterized in detail. To this end, the Gag polyprotein was fused in frame to the enhanced green fluorescent protein (eGFP) to favor process evaluation with multiple analytical tools. Tracking of the infection process using confocal microscopy and flow cytometry revealed a pronounced increase in the complexity of High Five over Sf9 Cells. Cryogenic transmission electron microscopy (cryo-TEM) characterization determined that changes in cell complexity could be attributed to the presence of occlusion-derived BV in High Five Cells, whereas Sf9 Cells evidenced a larger proportion of the budded virus phenotype (23-fold). Initial evaluation of the VLP production process using spectrofluorometry showed that Higher levels of the Gag-eGFP polyprotein were obtained in High Five Cells (3.6-fold). However, comparative analysis based on nanoparticle quantification by flow virometry and nanoparticle tracking analysis (NTA) proved that Sf9 Cells were 1.7- and 1.5-fold more productive in terms of assembled VLPs, respectively. Finally, analytical ultracentrifugation coupled to flow virometry evidenced a larger sedimentation coefficient of High Five-derived VLPs, indicating a possible interaction with other cellular compounds. Taken together, these results Highlight the combined use of microscopy and flow cytometry techniques to improve vaccine development processes using the insect cell/BV expression vector system. © 2020 International Society for Advancement of Cytometry.
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Integrating nanoparticle quantification and statistical design of experiments for efficient HIV-1 virus-like particle production in High Five Cells
Applied Microbiology and Biotechnology, 2020Co-Authors: Eduard Puente-massaguer, Marti Lecina, Francesc GodiaAbstract:The nature of enveloped virus-like particles (VLPs) has triggered High interest in their application to different research fields, including vaccine development. The baculovirus expression vector system (BEVS) has been used as an efficient platform for obtaining large amounts of these complex nanoparticles. To date, most of the studies dealing with VLP production by recombinant baculovirus infection utilize indirect detection or quantification techniques that hinder the appropriate characterization of the process and product. Here, we propose the application of cutting-edge quantification methodologies in combination with advanced statistical designs to exploit the full potential of the High Five/BEVS as a platform to produce HIV-1 Gag VLPs. The synergies between CCI, MOI, and TOH were studied using a response surface methodology approach on four different response functions: baculovirus infection, VLP production, VLP assembly, and VLP productivity. TOH and MOI proved to be the major influencing factors in contrast with previous reported data. Interestingly, a remarkable competition between Gag VLP production and non-assembled Gag was detected. Also, the use of nanoparticle tracking analysis and flow virometry revealed the existence of remarkable quantities of extracellular vesicles. The different responses of the study were combined to determine two global optimum conditions, one aiming to maximize the VLP titer (quantity) and the second aiming to find a compromise between VLP yield and the ratio of assembled VLPs (quality). This study provides a valuable approach to optimize VLP production and demonstrates that the High Five/BEVS can support mass production of Gag VLPs and potentially other complex nanoparticles.