Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

Abstract

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

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

“…EVs were recently observed in VLP production studies with the BEVS in insect cells [33,34], showing that they are not an exclusive matter of mammalian cell lines [41,42]. Analysis of the average VLP size by NTA resulted in 157.2 ± 8.5 nm ( Figure 2D), in agreement with Gag-eGFP VLPs produced in insect cells with the BEVS [43]. EVs displayed a similar mean size of 152.4 ± 15.9 nm than VLPs (p-value > 0.05), which raises the need to develop methodologies enabling their separation.…”

Preparation and Characterization of Microemulsions Based on Antarctic Krill Oil

“…Cryo-TEM has become a more advantageous tool for evaluating the structure and morphology of nanoscale samples compared to traditional TEM. The advantage of cryo-TEM over other related techniques is that it enables sample visualization in native conditions at a lower temperature without staining, makes water directly into vitrification ice, and reduces the damage of the electron beam to the sample so that it keeps the original shape of the sample as much as possible, especially for a liquid sample [ 50 , 51 ].…”

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

“…37 The implementation of flow virometry for VLP quantification enabled the detection of the co-expression of EVs. Recent studies indicate that these nanoparticles not only impact mammalian cell cultures, but are also important to consider in insect cells, 24 particularly in continuous operation processes aiming to achieve reproducible and constant culture conditions. EVs facilitate the intercellular communication by transporting functional molecules 42 ; however, the influence of EVs in insect cell bioprocesses is still not well understood.…”

“…Intracellular mCherry and Gag-eGFP fluorescence intensities were analyzed by disrupting cell pellets with three freeze-thaw cycles (2.5 h at −20 °C and 0.5 h at 37 °C), vortexed for 5 s three times between cycles, and resuspended in tris/magnesium/saline (TMS) buffer. 24 mCherry fluorescence intensities were measured with a ⊗ ex = 587 nm (slit = 5), ⊗ em = 600-630 nm (slit = 10), whereas for Gag-eGFP the equipment settings were adjusted to ⊗ ex = 488 nm (slit = 5), ⊗ em = 500-530 nm (slit = 10). For the calculation of mCherry protein concentration, a standard curve based on the linear correlation between known mCherry concentrations (BioVision, Milpitas, CA, USA) and their corresponding fluorescence values in relative fluorescence units (R.F.U.)…”