Integrating high cell density cultures with adapted laboratory evolution for improved Gag‐HA virus‐like particles production in stable insect cell lines
Bárbara Fernandes, Ricardo Correia, Marcos F. Q. Sousa, Manuel J.T. Carrondo, Paula M. Alves, António Roldão
Abstract
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.