Unique stabilizing mechanism provided by biocompatible choline-based ionic liquids for inhibiting dissociation of inactivated foot-and-mouth disease virus particles

Inactivated virus and virus-like particles (VLPs) are important classes of biopharmaceuticals for vaccines, immunotherapy and oncotherapy. Their complex particle structures are easily denatured during processing and storage, leading to loss in their biofunctionality. Ionic liquids (ILs) as stabilizing excipients have garnered interest in protein-based pharmaceutical research, but their stabilizing capacity for inactivated virus antigens remains unknown. Here, three biocompatible choline-based ILs, including [Cho][H2PO4], [Cho][Cl], and [Cho][SO4], were tested as potential stabilizers for the inactivated foot-and-mouth disease virus (iFMDV), which are extremely unstable virus particles easily dissociating into smaller pentamers named 12S. Based on differential scanning fluorimetry technology for thermal stability analysis, together with high-performance size-exclusion chromatography for quantitative determination of 146S, it was found that [Cho][Cl] and [cho][SO4] can improve the thermo- and long-term storage stability of iFMDV particles, while [Cho][H2PO4] showed a destabilizing effect. Animal experiments indicated that the immunogenicity of iFMDV antigens was not attenuated in all three ILs. By monitoring the microenvironmental pH of the virus particles in different ILs, a relatively lower proton intensity was observed in [Cho][Cl] and [Cho][SO4] than in buffers and [Cho][H2PO4]. Therefore, the stabilizing mechanism was supposed to be mainly due to suppression of protonation of histidine residues in the inter-pentamer interface of virus particles in [Cho][Cl] and [Cho][SO4], which is distinct from the mechanism reported for other proteins with relatively simple structures. The results suggest that the choline-based ILs with appropriate anions are promising stabilizing excipients for iFMDV or other vaccine antigens.