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

Abstract

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.

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

“…The titers shown here are in the range of those obtained with stable High Five cell pools, 27 but lower than those reported in insect cells with the baculovirus expression vector system (BEVS) 19,39 and TGE, 21,22 probably reflecting the lower amount of Gag-eGFP produced in the stable cell pool. However, the possibility of having a continuous platform to produce VLPs devoid of baculoviruses and baculovirus-derived proteins makes the use of this system appealing, especially in combination with production techniques enabling increased cell concentrations, such as continuous perfusion, and subsequently the final VLP titers.…”

“…Similar results have recently been described with stable High Five cell pools. 27 Continuous Gag VLP production in Sf9 cells has not been shown to be cytotoxic, 28 but a slight effect on cell viability was observed here. Differences between both recombinant products were also detected after achieving expression stability in the unsorted stable cell pools.…”

“…45 No lactate formation was observed, suggesting that there was no oxygen limitation in any of the cell cultures. 32 Of note, lactate concentrations of ≤28 mmol L −1 were measured in High Five cell pools in the same culture conditions, 27 which highlights a more efficient metabolism in Sf9 cells. Serine, despite being consumed at lower rates than glucose and glutamine, was completely exhausted by the end of the exponential phase; this amino acid has been shown to be very important for Sf9 cell culture and involved in the generation of biomass and energy production.…”

“…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.…”

“…These results differ from studies with High Five cells cultured in the same cell culture medium showing a higher dependency on asparagine. 21,27 Glucose is mainly consumed via the glycolysis pathway, being converted to pyruvate before entrance into the Krebs cycle (KC), whereas glutamine is incorporated into the KC after conversion to ⊍-ketoglutarate. 43 Previous studies with Sf9 cells indicate that glucose consumption is around 3-fold higher than that of glutamine.…”