Abstract
Virus-like particles (VLPs) are excellent platforms for the development of influenza vaccine candidates. Nonetheless, their characterization is challenging due to VLPs’ unique biophysical and biochemical properties. To cope with such complexity, multiple analytical techniques have been developed to date (e.g., single-particle analysis, thermal stability, or quantification assays), most of which are rarely used or have been successfully demonstrated for being applicable for virus particle characterization. In this study, several biophysical and biochemical methods have been evaluated for thorough characterization of monovalent and pentavalent influenza VLPs from diverse groups (A and B) and subtypes (H1 and H3) produced in insect cells using the baculovirus expression vector system (IC-BEVS). Particle size distribution and purity profiles were monitored during the purification process using two complementary technologies — nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS). VLP surface charge at the selected process pH was also assessed by this last technique. The morphology of the VLP (size, shape, and presence of hemagglutinin spikes) was evaluated using transmission electron microscopy. Circular dichroism was used to assess VLPs’ thermal stability. Total protein, DNA, and baculovirus content were also assessed. All VLPs analyzed exhibited similar size ranges (90–115 nm for NTA and 129–141 nm for TRPS), surface charges (average of −20.4 mV), and morphology (pleomorphic particles resembling influenza virus) exhibiting the presence of HA molecules (spikes) uniformly displayed on M1 protein scaffold. Our data shows that HA titers and purification efficiency in terms of impurity removal and thermal stability were observed to be particle dependent. This study shows robustness and generic applicability of the tools and methods evaluated, independent of VLP valency and group/subtype. Thus, they are most valuable to assist process development and enhance product characterization.
Highlights
Influenza Virus-like particles (VLPs) are traditionally composed of hemaglutinin (HA) protein displayed on the surface of M1 protein
While the H1 subtype monovalent culture shows higher HA titers compared to pentavalent culture, the opposite occurs for H3 and B
The highest HA titer was observed for B pentavalent culture, with peak production reached at 72 hpi
Summary
Influenza VLPs are traditionally composed of hemaglutinin (HA) protein displayed on the surface of M1 protein (the scaffold). They can be composed by HA of only one subtype/strain (i.e., monovalent) or multiple subtypes/strains (i.e., multivalent). These VLPs are enveloped particles, they contain host cell proteins (HCP) acquired during the budding process (Vicente et al, 2011). These particles can be produced using several expression systems, namely, Escherichia coli, eukaryotic mammalian, insect, and plant cells (Pillet et al, 2016; Tretyakova et al, 2016; VenereoSanchez et al, 2016; Huang et al, 2017). The envelope composition affects particle complexity impacting VLPs’ structure and stability (Deng, 2018; GonzálezDomínguez et al, 2020)
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