Bioanalytics for Influenza Virus-Like Particle Characterization and Process Monitoring

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.

Virus-mimicking nanosystems: from design to biomedical applications

“…In addition, the charge state of the viral surface can influence the immune system's response to the virus and the binding of antibodies. 96,97 Nanoparticles can often expand the application of nanotechnology in medicine and biology by drawing on the functional and design principles of viral surface charge, which can optimize the properties of nanoparticles for cellular uptake, drug delivery and immune interactions. 98,99 Cui et al proposed a charge-reversible virus-mimicking nanocapsule for the effective systemic treatment of retractable gliomas.…”

Genetic diversity of murine norovirus populations less susceptible to chlorine

“…Another study also observed that a mutation in the capsid protein of the Sindbis virus can affect aggregation ( Ferreira et al, 2003 ). Detailed study of viral aggregates, for instance, using electron microscopy, nanoparticle tracking analysis, flow virometry, or tunable resistive pulse sensing, can be beneficial for characterizing viral aggregation in terms of size distribution, morphology, surface charge, composition, structure, and stability ( Heider and Metzner, 2014 ; Zamora and Aguilar, 2018 ; Carvalho et al, 2022 ).…”

Influenza Virus-like Particle-Based Hybrid Vaccine Containing RBD Induces Immunity against Influenza and SARS-CoV-2 Viruses

“…LNPs not only enhance the stability of the viral mRNA but also act as adjuvants [55,56]. Virus-like particles (VLPs) are also considered nanomaterials because of their size (10-200 nm) [57]. These are successfully used to deliver multiple antigens as vaccines [58,59].…”