Integrating high cell density cultures with adapted laboratory evolution for improved Gag‐HA virus‐like particles production in stable insect cell lines

Abstract

Stable insect cell lines are emerging as an alternative to the insect cell-baculovirus expression vector system (IC-BEVS) for protein expression, benefiting from being a virus-free, nonlytic system. Still, the titers achieved are considerably lower. In this study, stable insect (Sf-9 and High Five) cells producing Gag virus-like particles (VLPs) were first adapted to grow under hypothermic culture conditions (22°C instead of standard 27°C), and then pseudotyped with a model membrane protein (influenza hemagglutinin [HA]) for expression of Gag-HA VLPs. Adaptation to lower temperature led to an increase in protein titers of up to 12-fold for p24 (as proxy for Gag-VLP) and sixfold for HA, with adapted Sf-9 cells outperforming High Five cells.Resulting Gag-HA VLPs producer Sf-9 cells were cultured to high cell densities, that is, 100 × 10 6 cell/ml, using perfusion (ATF® 2) in 1 L stirred-tank bioreactors. Specific p24 and HA production rates were similar to those of batch culture, enabling to increase volumetric titers by 7-8-fold without compromising the assembly of Gag-HA VLPs. Importantly, the antigen (HA) quantity in VLPs generated using stable adapted cells in perfusion was ≈5-fold higher than that from IC-BEVS, with the added benefit of being a baculovirus-free system. This study demonstrates the potential of combining stable expression in insect cells adapted to hypothermic culture conditions with perfusion for improving Gag-HA VLPs production.

Intensifying Continuous Production of Gag-HA VLPs at High Cell Density Using Stable Insect Cells Adapted to Low Culture Temperature

“…A similar approach has been successfully applied to antibody production using other cell lines (e.g., CHO and mouse hybridoma cells) (Konstantinov et al, 2006;Schulze et al, 2021). Indeed, by maintaining cell concentration at 20 × 10 6 cell/mL while decreasing the CSPR, the amount of HA and p24 produced per volume of culture medium consumed per unit of process time (STY) increased by up to 3-fold when compared to previous studies using batch and perfusion operation mode (Fernandes et al, 2021). The presence of particles resembling Gag VLPs (in size and morphology) with HA molecules (spikes) displayed on their surface could be confirmed, with their concentration increasing with reducing CSPR.…”

“…Insect Sf9 cells expressing Gag-HA VLPs (from now on named “Sf9 Gag-HA” cells), previously established in our lab ( Fernandes et al, 2021 ), were routinely sub-cultured as described elsewhere ( Fernandes et al, 2020 ). Briefly, cells were sub-cultured to 1 × 10 6 cell/mL every 3–4 days when cell density reached 2–3 × 10 6 cell/mL using Sf-900™ II SFM (Thermo Fisher Scientific) media.…”

“…A peristaltic pump Sartoflow ® Slice 200 benchtop crossflow system (Sartorius) was used in the TFF system. The cell-specific perfusion rate (CSPR) was set to 0.04 nL/d.cell when glucose (Glc) and glutamine (Gln) concentrations reached 48 and 6 mmol/L, respectively ( Fernandes et al, 2021 ). Culture volume in the bioreactor was kept constant by gravimetric feed control.…”

“…Despite yielding high-protein titers in short timeframes, IC-BEVS has several limitations impairing process performance and/or product quality (e.g., release of host and viral proteases due to the lytic infection and the complexity of continuous production) ( Fernandes et al, 2021 ). This has triggered interest in stable insect cells and the development of new bioprocess strategies for their culture aiming to improve yields ( Fernandes et al, 2020 ; Fernandes et al, 2021 ).…”

“…FIGURE 5 | Space-time yield of Gag-HA VLPs produced using stable adapted Sf9 cells at different bioreactor operation modes and cell-specific perfusion rates. Data from batch and perfusion mode have been reported elsewhere(Fernandes et al, 2021).…”