Extended gene expression for Gag VLP production achieved at bioreactor scale

Fuenmayor, Javier; Cervera, Laura; Gòdia, Francesc; Kamen, Amine

doi:10.1002/jctb.5777

Cited by 17 publications

(19 citation statements)

References 29 publications

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“…Nanomaterials 2020, 10, x; doi: FOR PEER REVIEW www.mdpi.com/journal/nanomaterials 293 cells when cultured in bioreactor [49]. An increase of 1.7-fold in VLP production by baculovirus infection of Tnms42 insect cells in bioreactor culture conditions has also been reported [50].…”

Section: Transferability Of Vlp Production To Bioreactormentioning

confidence: 93%

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

et al. 2020

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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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Section: Transferability Of Vlp Production To Bioreactormentioning

confidence: 93%

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

et al. 2020

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“…Cumulative VLP production ( Figure 3G) achieved was 6.8•10 10 VLPs/mL, improving in 86.7% or 7.54 fold the previous reported results (Cervera et al, 2015) using the original EGE protocol in shake flasks. Comparing the EGE protocol carried out in bioreactor prior to the developed optimization (Fuenmayor et al, 2019), the presented work achieved a reactor and media volumetric productivity of 7.1•10 12 and 8.7•10 11 VLP•L −1 •day −1 , respectively, improving 26.8% or 1.36 fold and 67.8% or 3.1 fold, respectively, the reported results. This improvement led to the achievement of an average VLP specific productivity of 3,000 VLP•cell −1 •day −1 (Figure 3C).…”

Section: Intensification Of the Optimized Protocol In Bioreactor Usinmentioning

confidence: 54%

“…However, perfusion cultures are fed with a continuous addition of fresh media and removal of metabolic waste, preventing energy depletion. Encountering the CDE in continuous cultures has been already reported (Genzel et al, 2014;Fuenmayor et al, 2019) and different molecular and metabolic factors have been described to influence the CDE (Lavado-García et al, 2020). Therefore, using perfusion to achieve very high cell densities, as implemented when working with stable gene expression (SGE) cell-based platforms, is no longer useful for bioprocesses using TGE.…”

Section: Optimization Of the Second Retransfection Parameters Bioreacmentioning

confidence: 99%

“…One of the main advantages of continuous bioprocessing is that this mode of operation allows the support of high cell density cultures, dramatically increasing volumetric productivity as the product is being constantly harvested and operation times extended. The fact of achieving higher cell densities in shorter times was one of the issues encountered in the first attempt of transferring the EGE protocol to a bioreactor operated in continuous mode without further optimization (Fuenmayor et al, 2019). Indeed, cells need to be maintained around 2•10 6 cells/mL for an efficient transfection.…”

Section: Introductionmentioning

confidence: 99%

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An Alternative Perfusion Approach for the Intensification of Virus-Like Particle Production in HEK293 Cultures

Lavado‐García

Cervera

Gòdia

2020

Front. Bioeng. Biotechnol.

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Virus-like particles (VLPs) have gained interest over the last years as recombinant vaccine formats, as they generate a strong immune response and present storage and distribution advantages compared to conventional vaccines. Therefore, VLPs are being regarded as potential vaccine candidates for several diseases. One requirement for their further clinical testing is the development of scalable processes and production platforms for cell-based viral particles. In this work, the extended gene expression (EGE) method, which consists in consecutive media replacements combined with cell retransfections, was successfully optimized and transferred to a bioreactor operating in perfusion. A process optimization using design of experiments (DoE) was carried out to obtain optimal values for the time of retransfection, the cell specific perfusion rate (CSPR) and transfected DNA concentration, improving 86.7% the previously reported EGE protocol in HEK293. Moreover, it was successfully implemented at 1.5L bioreactor using an ATF as cell retention system achieving concentrations of 6.8•10 10 VLP/mL. VLP interaction with the ATF hollow fibers was studied via confocal microscopy, field emission scanning electron microscopy, and nanoparticle tracking analysis to design a bioprocess capable of separating unassembled Gag monomers and concentrate VLPs in one step.

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“…The preferred strategies to produce enveloped VLPs are transient gene expression (TGE) in mammalian cell lines and the baculovirus expression vector system (BEVS) in insect cells (Table 1; Genzel, 2015). These production platforms are widely used in industry, and have been brought to the production scale, achieving competitive production yields for preclinical studies (Cruz et al, 1998; Fuenmayor, Cervera, Gòdia, & Kamen, 2019; Venereo‐Sanchez et al, 2017). Although transiently produced VLPs in mammalian cells show lower productivities, the BEVS present a major bottleneck in product purity (Cervera et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Quantification of the HIV‐1 virus‐like particle production process by super‐resolution imaging: From VLP budding to nanoparticle analysis

Puente‐Massaguer

Cervera

Gòdia

2020

Biotech & Bioengineering

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Virus‐like particles (VLPs) offer great promise in the field of nanomedicine. Enveloped VLPs are a class of these nanoparticles and their production process occurs by a budding process, which is known to be the most critical step at intracellular level. In this study, we developed a novel imaging method based on super‐resolution fluorescence microscopy (SRFM) to assess the generation of VLPs in living cells. This methodology was applied to study the production of Gag VLPs in three animal cell platforms of reference: HEK 293‐transient gene expression (TGE), High Five‐baculovirus expression vector system (BEVS) and Sf9‐BEVS. Quantification of the number of VLP assembly sites per cell ranged from 500 to 3,000 in the different systems evaluated. Although the BEVS was superior in terms of Gag polyprotein expression, the HEK 293‐TGE platform was more efficient regarding the assembly of Gag as VLPs. This was translated into higher levels of non‐assembled Gag monomer in BEVS harvested supernatants. Furthermore, the presence of contaminating nanoparticles was evidenced in all three systems, specifically in High Five cells. The SRFM‐based method here developed was also successfully applied to measure the concentration of VLPs in crude supernatants. The lipid membrane of VLPs and the presence of nucleic acids alongside these nanoparticles could also be detected using common staining procedures. Overall, a complete picture of the VLP production process was achieved in these three production platforms. The robustness and sensitivity of this new approach broaden the applicability of SRFM toward the development of new detection, diagnosis and quantification methods based on confocal microscopy in living systems.

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Extended gene expression for Gag VLP production achieved at bioreactor scale

Cited by 17 publications

References 29 publications

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

An Alternative Perfusion Approach for the Intensification of Virus-Like Particle Production in HEK293 Cultures

Quantification of the HIV‐1 virus‐like particle production process by super‐resolution imaging: From VLP budding to nanoparticle analysis

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