Evaluation of screening platforms for virus-like particle production with the baculovirus expression vector system in insect cells

Strobl, Florian; Ghorbanpour, Sahar Masoumeh; Palmberger, Dieter; Striedner, Gerald

doi:10.1038/s41598-020-57761-w

Cited by 8 publications

(9 citation statements)

References 27 publications

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“…These results are in agreement with the VLP productivity increase observed in HEK 293 cells when cultured in bioreactor [ 49 ]. An increase of 1.7-fold in VLP production by baculovirus infection of Tnms 42 insect cells in bioreactor culture conditions has also been reported [ 50 ].…”

Section: Resultsmentioning

confidence: 99%

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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: Resultsmentioning

confidence: 99%

“…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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“…Variations in insect cell performance with the BEVS have been recently reported and attributed to the different cell culture strategies employed. [ 43 ]…”

Section: Resultsmentioning

confidence: 99%

“…[42] In the culture strategies employed. [43] The number of Gag-eGFP expressing cells in the stable cell pool cultured in bioreactor was kept constant and at the same level observed in shake flasks ( Figure 3E). In terms of Gag-eGFP production, higher yields were measured throughout the experiment in the bioreactor condition, with intracellular Gag-eGFP concentration peaking between 72 and 96 h ( Figure 3F).…”

Section: Vlp Production In Bioreactormentioning

confidence: 99%

Accelerating HIV‐1 VLP production using stable High Five insect cell pools

Puente‐Massaguer

Grau-Garcia

Strobl

et al. 2020

Biotechnology Journal

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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 for the production of 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 1.5fold, 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 with 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.

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“…The maintenance of DO and pH setpoints in the cell culture might also explain that cell viabilities remained at >95% for a longer period of time. 31 pH fluctuations in shake flasks did not exceed the 5.9-6.4 range, showing that Sf9 cells maintain a more stable pH in culture than other insect cell lines such as High Five cells. 27,32 In terms of production, VLP titers achieved in bioreactor were 1.4-fold higher than those obtained in shake flasks, illustrating the scalability of stable cell pools and also reinforcing the necessity of controlling culture parameters to improve the production of recombinant proteins.…”

Section: Discussionmentioning

confidence: 87%

Stable Sf9 cell pools as a system for rapid HIV‐1 virus‐like particle production

Puente‐Massaguer

Grau-Garcia

Strobl

et al. 2021

J of Chemical Tech & Biotech

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BACKGROUND: The emergence of infectious diseases is accelerating the intensification of bioprocess strategies to support the increasing demand for the manufacture of higher quantities of vaccines in short timeframes. Here, the development of stable Sf9 cell pools producing human immunodeficiency virus serotype 1 (HIV-1) Gag-eGFP virus-like particles (VLPs) is assessed.RESULTS: Fluorescence-activated cell sorting (FACS) was employed to select high producing cells, achieving an 8.1-fold increase in fluorescence intensity compared to unsorted cell pools after three rounds of cell sorting. The transferability of this system to bioreactor scale was also successfully achieved, attaining a 1.4-fold increase in VLP production and maintaining a higher cell viability than shake flask controls. Analysis of the metabolism of stable cell pools and parental Sf9 cells did not show significant differences regarding metabolite consumption and production, even though a better performance and more efficient metabolism were observed in bioreactor compared with shake flask cultures, highlighting the flexibility of these cells to adapt to different culture conditions and heterologous recombinant protein production.CONCLUSIONS: Stable Sf9 cell pools represent a suitable system for shortening bioprocess development times and accelerating vaccine production.

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Evaluation of screening platforms for virus-like particle production with the baculovirus expression vector system in insect cells

Cited by 8 publications

References 27 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

Accelerating HIV‐1 VLP production using stable High Five insect cell pools

Stable Sf9 cell pools as a system for rapid HIV‐1 virus‐like particle production

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