Bioprocess characterization of virus‐like particle production with the insect cell baculovirus expression system at nanoparticle level

Abstract

Background. Virus-like particles (VLPs) are a multivalent platform showing great promise for the development of vaccines, gene therapy, diagnostic, and drug delivery approaches. Particularly, HIV-1 Gag VLPs provide a robust and flexible scaffold for the presentation of a variety of antigens. The insect cell baculovirus expression vector system (BEVS) is nowadays one of the reference systems to produce these complex nanoparticles, but information about VLP quality, quantity, stability, as well as cell performance is scarce, especially at bioreactor scale. Results. VLPs produced in the reference High Five and Sf9 insect cell lines share similar physicochemical properties, with VLPs produced in Sf9 cells showing lower levels of double stranded DNA and protein contaminants, and a higher degree of VLP assembly. Besides VLPs, other nanoparticle populations are divergently encountered in each cell line. Hi5 supernatants contain a higher load of extracellular vesicles, while Sf9 supernatants exhibit higher concentrations of baculovirus particles. Similar titers are achieved when comparing Gag to Gag-eGFP VLP production, with the size of most of the nanoparticles produced comprised at the 150-250 nm range. Eventually, Gag VLP production in a 2 L stirred tank bioreactor is successfully demonstrated, validating bioprocess transference to the final product candidate. Conclusions. This work provides two potentially valuable strategies for the production of HIV-1 Gag VLPs and a detailed analysis of the different nanoparticle populations produced.

Characterisation of extracellular vesicles in baculovirus infection of Spodoptera frugiperda cells

“…While EV secretion by S. frugiperda cells had been described previously, no characterisation of these EVs has yet been reported (Hashimoto et al, 2017;Ishikawa et al, 2020;Puente-Massaguer et al, 2022). Small EVs released by Trichoplusia ni cells during baculovirus infection were recently analysed by mass spectrometry, yet viral proteins were not analysed due to possible contamination by virions (Hausjell et al, 2023).…”

“…Most studies on EVs in viral infection have focused either on human cells or on insect vectors of human viruses such as ticks and mosquitos. Though the presence of EVs has been described in S. frugiperda cells, little research has been performed on the role they may play in baculovirus infection (Hashimoto et al., 2017 ; Hodgson et al., 2022 ; Ishikawa et al., 2020 ; Puente‐Massaguer et al., 2022 ). Recently, sEVs were characterised by mass spectrometry in AcMNPV‐infected T. ni cells, although the presence of viral proteins was not evaluated since no full separation of sEVs from BV was achieved (Hausjell et al., 2023 ).…”