Abstract
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 6.8·1010 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.
Highlights
Since the human immunodeficiency virus 1 (HIV-1) was first reported in 1959, 74.9 million people have been infected (Whiteside and Wilson, 2018)
The energetic demand is solved with the constant addition of fresh media but the cell density effect (CDE) comprises molecular causes that cannot be solved only by perfusion (Petiot et al, 2015). We propose overcoming this problem by changing the approach of perfusion using cell retention devices, like alternating tangential flow (ATF), not to achieve high cell densities, but to ensure the constant media replacement in order to maintain the cell culture in a re-transfectable state for a longer period of time in the bioreactor and prolong production
This suggests that 1RV/day is enough to meet the nutrient demand of the culture and further cell growth could be hindered by other factors such as oxygen diffusion in the flask
Summary
Since the human immunodeficiency virus 1 (HIV-1) was first reported in 1959, 74.9 million people have been infected (Whiteside and Wilson, 2018). We propose overcoming this problem by changing the approach of perfusion using cell retention devices, like alternating tangential flow (ATF), not to achieve high cell densities, but to ensure the constant media replacement in order to maintain the cell culture in a re-transfectable state for a longer period of time in the bioreactor and prolong production This perfusion approach is not intended to operate at steady state and cell bleeding is not necessary, as continuous perfusion is used to constantly renew media while cells are retransfected to keep a high proportion of cells producing VLPs, up to a certain limited process span. The overall significance of the model was determined by analysis of variance (ANOVA) F test, whereas the significance of each coefficient was determined by the corresponding t test
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