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

Abstract

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.

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

“…This indicates that this amino acid might play an important role in High Five cells cultured in low hydrolysate media, and its consumption could be associated to cell growth since leucine metabolization decreases when the cell growth rate of High Five cells is reduced, as recently observed in the transient gene expression of these cells. [47] In parallel to lactate formation, the production of increasing amounts of alanine was also detected, achieving a maximum yield of 28 mM in the stable cell pool cultured in shake flasks ( Figure 4C and D). In this condition, the highest level of lactate generation was also observed (23 mM).…”

“…This indicates that this amino acid might play an important role in High Five cells cultured in low hydrolysate media, and its consumption could be associated to cell growth since leucine metabolization decreases when the cell growth rate of High Five cells is reduced, as recently observed in the transient gene expression of these cells. [ 47 ]…”

Nonviral Locally Injected Magnetic Vectors for In Vivo Gene Delivery: A Review of Studies on Magnetofection

“…As for the magnetic vector itself, these were transMAG PEI (Chemicell) particles—superparamagnetic iron oxide nanoparticles with an average size of 200 nm (by dynamic light scattering), coated with polyethylenimine (PEI, 800 kDa). PEI is the most effective nonviral vector due to its favorable characteristics of DNA protection, cell binding and uptake, and endosomal escape and release from the carrier [ 106 , 107 , 108 ]; an example of the structure of a PEI-coated magnetic particle is shown in Figure 2 . Therefore, in most studies, magnetic particles coated with PEI are used.…”

High shear resistance of insect cells: the basis for substantial improvements in cell culture process design

“…However, this type of process operation leads to a number of problems. High gas flow rates create more gas bubbles in the system, which in turn lead to increased foaming and shear due to the bursting of air bubbles on the liquid surface 20 , 21 . Low stirring speeds, in turn, can lead to mixing problems and thus increase the formation of zones and gradients—a problem that is particularly relevant in scale-up 22 .…”