BACKGROUND: Transient gene expression has been widely used for virus-like particles (VLP) production in both small- and large-scale systems. The extended gene expression (EGE) technique is based on repeated medium exchanges and retransfections of cell culture to prolong the production phase. The aim of this study was to demonstrate scalability of the approach by operating EGE continuously in a controlled bioreactor to obtain similar results to those achieved with shake flask EGE for VLP production. A standard batch cultivation was also carried out in shake flask. RESULTS: For EGE in shake flasks and in a bioreactor, cell viability was comparable; however, the bioreactor enabled much higher cell densities and specific growth rates than the shake flasks. Owing to this increased cell growth in the bioreactor, the percentage of GFP-positive cells was considerably lower at the end than in the shake flasks. GagGFP VLP titers were similar in both shake flasks and bioreactor. Nanoparticle tracking analysis revealed that the ratio of VLPs/total particles (VLPs and cell vesicles) was higher in the shake flasks than in the bioreactor, possibly due to higher cell densities achieved in the bioreactor. CONCLUSIONS: In this study, EGE methodology was carried out in a bioreactor system for the first time while maintaining GagGFP production titers. Overall, our findings call for further optimization and implementation of common downstream processing steps to improve yield and VLP quality. (c) 2018 Society of Chemical Industry