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 Sousa, Manuel J.T. Carrondo, Paula M. Alves, António Roldão

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

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 × 106 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.

Original languageEnglish
Pages (from-to)2536-2547
Number of pages12
JournalBiotechnology and Bioengineering
Volume118
Issue number7
DOIs
Publication statusPublished - Jul 2021

Keywords

  • adaptive laboratory evolution
  • high cell density
  • influenza virus-like particles
  • insect cells
  • perfusion

Fingerprint

Dive into the research topics of 'Integrating high cell density cultures with adapted laboratory evolution for improved Gag-HA virus-like particles production in stable insect cell lines'. Together they form a unique fingerprint.

Cite this