## 1 Introduction  
Lyophilization, also known as freeze-drying or cryodesiccation, is a dehydration process performed under low-temperature and -pressure conditions. Freeze-drying yields high-quality products because there is minimal thermal damage [1]. Lyophilization has become a standard practice in the life science industry because lyophilized products retain their original properties; by adding water or a suitable solvent, they can be easily and quickly reconstituted. However, some biomolecules like serum, vaccines, and proteins may get damaged by the stress associated with freezing, freeze-drying, or both. Thus, the activity of biomolecules after lyophilization often decreases, in comparison to non-lyophilized materials [2]. The development of methods aiming for the renaturation and recovery of the activity of lyophilized biomolecules is necessary to extend the shelf-life of these macromolecules.  
Ionic liquids (ILs) are salts with melting points below 100 \({}^{\circ}\)C. The properties of ILs can be changed by ensuring the appropriate structural design of their component ions. ILs have been gathering attention as green [3], multi-use reaction media [4, 5] and solvents for a variety of applications. For example, ILs have been studied as potential candidates for substituents of reaction media in electrochemistry [6], enzymatic catalysis [7, 8], and green chemistry [9] because of their excellent stability [10] and unusual solvent properties [11, 12, 13, 14]. They have also been investigated for their potential to assist renaturation of proteins [15]. Some ILs, e.g., triethyl ammonium phosphate and imidazolium type cation with halide anion, have shown to be as effective as other small molecular additives when used as refolding agents in aqueous phase [16, 17]. On the other hand, hydrated ILs, which are a mixture of ILs and a small amount of water, have showed great potential as solvents for the dissolution and refolding of aggregated and inactive proteins [18, 19]. By selecting the suitable component ion and water content, inclusion bodies and thermally aggregated proteins can be dissolved and subsequently refolded in hydrated ILs.  
In this study, we investigated the effect of hydrated ILs as a treatment solvent for the renaturation of lyophilized inactive biomolecules. ConA has been selected as the target biomolecule to assess the capability of hydrated ILs on the recovery of protein functionality. ConA is a type of lectin with a specific affinity for mannose and glucose. In the past, ConA has been used in the medical field as a lymphocyte transformation test, and as a tumor marker [20]. We have previously reported the improvement in the shelf life and thermal stability of proteins (including ConA) after dissolution in hydrated ILs, especially in cholinium dihydrogen phosphate ([ch][dhp]) [21; 22]. Furthermore, thermally aggregated ConA has shown a greater solubility and refolding behavior in some selected hydrated ILs [18]. Hydrated ILs composed of kosmotropic anions, such as dihydrogen phosphate anions, have been reported to be effective at dissolving proteins and maintaining their original higher-order structure [23]. In this study, we prepared several ILs composed of different cations and dhp anion or bromide anion (Figure 1). The mannose binding ability of ConA was investigated after treatment with these hydrated ILs with different water content.