Jorge F. B. PEREIRA1, Nathalia V. VERÍSSIMO2, Carolina F. SAPONI2, Filipa A. A. VICENTE3, Tamar L. GREAVES4
1CIEPQPF, Department of Chemical Engineering, FCTUC, University of Coimbra, Coimbra, Portugal
2Department of Engineering of Bioprocesses and Biotechnology, FCFAr, São Paulo State University, Araraquara, Brazil
3National Institute of Chemistry, Ljubljana, Slovenia
4RMIT University, Melbourne, Australia
The instability of biopharmaceuticals and other protein products still limits their applications and hinders their transport and storage. Therefore, the low stability and high costs of proteins is the main bottleneck limiting access to life-saving protein-based bioproducts in countries and communities of low income. To improve the instability of proteins, ionic liquids (ILs) have been employed as stabilizers of proteins considering their ability to enhance the solubility and stability of a wide range of biomolecules and unique properties for industrial and medical applications. However, several variables can impact the effect of ILs on proteins, including the nature, biocompatibility, and concentration of ILs, environmental conditions such as temperature and pH, and the intrinsic properties of proteins. Hence, this work compiled and analyzed the effect of ILs on non-enzymatic proteins considering the protein properties, ILs classes, type of protein stability, and IL solutions concentrations to find trends that indicate the impact of each variable in protein stability. Considering the top four major IL families in this field, imidazolium and ammonium-based ILs are the predominant classes for protein stabilization studies. However, the most compatible classes with proteins are ammonium and cholinium ILs, followed by imidazolium and pyridinium/pyrrolidinium ILs. As for the concentration range of ILs, IL solutions above 0.1 M appear to be more biocompatible with proteins than very dilute aqueous IL solutions. Moreover, ILs have a great aptitude to prevent protein aggregation (more than half of samples decreased aggregation) and activity (more than 40%), including some IL families that are also adequate for the preservation of structural and thermal stability of proteins (one-third of samples). Finally, we also experimentally evaluated the effect of different concentrations and IL classes on the short and long-term stability of the Green Fluorescent Protein (GFP). For the GFP, imidazolium- and cholinium-based ILs increased GFP short and long-term stabilization at room temperature by decreasing its aggregation. For the imidazolium ILs, the increase in concentration and alkyl chain length of the cation was unfavorable for the maintenance of GFP activity; however, the opposite occurred for cholinium-based ILs. Therefore, ILs can be used as protein stabilizers for a wide range of proteins and purposes, with the potential to help expand the applications of unstable proteins and increase access to biological products. However, it is necessary to apply adequate IL classes, concentrations, and environments to each type of protein for better outcomes.