Purification of virus-like particles using aqueous biphasic systems composed of natural deep eutectic solvents

Abstract

Virus-like particles (VLPs) mimic the structure of viral particles, presenting antigens in a repetitive and ordered structure, which makes them excellent vaccine immunogens. However, a major bottleneck in the VLPs industrial production process is the lack of robust purification methods, which can account for up to 80% of total manufacturing cost.Aqueous biphasic systems (ABS) are portrayed as viable and efficient platform in purification of wide variety of biomolecules, since they can be designed to be versatile, biocompatible and easy to scale-up, depending on the phase forming components. In the last years, natural deep eutectic solvents (NADES) have emerged and gained a lot of attention in several fields and different applications, including the ABS implementation, where they have been proposed as versatile, cost-effective and biocompatible phase forming compounds.In this work, a study of the liquid-liquid equilibria (LLE) of ABS composed of carbohydrates and carbohydrates-based NADES is reported and the mechanisms associated with phase equilibria discussed. The carbohydrates used in the NADES preparation are osmolytes, which can be regarded both as extractants and excipients that stabilize the final formulation of biopharmaceuticals, avoiding the tricky back extraction of VLPs. It is here shown that the use of NADES leads to an increase of two-phase region, when compared to the use of only one of the NADES components, thus offering liquid-liquid demixing at low concentrations of phase forming compounds. Furthermore, the cytotoxicity studies of these NADES and their individual components using two different human cell lines, HEK 293 and Huh-7, was evaluated, in order to conclude about the biocompatibility and suitability of proposed systems in vaccine development. Finally, the potential of the proposed ABS in purification of VLPs from bovine serum albumin (BSA) is demonstrated, while their structural properties are also assessed.