Unveiling the potential of deep eutectic solvents to improve the conformational and colloidal stability of immunoglobulin G antibodies

Abstract

Antibodies, such as immunoglobulin G (IgG), have a pivotal role as biopharmaceuticals capable of treating a wide variety of diseases. However, IgG is a protein, hence, it can easily lose stability and therapeutic efficiency, which may happen during its handling, transportation and preservation. The current work shows, for the first time, the positive effect of deep eutectic solvents (DESs) on the conformational and colloidal stability of IgG antibodies, thus opening the door for their use as novel solvents in IgG formulations. Here, aqueous solutions of cholinium-based DESs were applied to enhance the conformational and colloidal stability of IgG, with no need to add excipients. A series of DESs were prepared through the combination of cholinium chloride ([Ch]Cl), as a hydrogen-bond acceptor (HBA), and various hydrogen-bond donors (HBD), such as urea, glycerol (Gly) and ethylene glycol (EG), and investigated in detail. The effect of [Ch]Cl–urea at different molar ratios (1 : 1, 1 : 2, 1 : 3 and 2 : 1) was also analysed. Conformational stability was checked by thermal fluorescence spectrometry, and it was found that selected DESs allowed increasing the transition temperature (T_m) of IgG by ca. 4 °C. The observed increase in the conformational stability of IgG in the presence of DESs was in agreement with the results of other spectroscopic studies, including FTIR and Raman spectroscopies. In the presence of DESs, there was a minimum exposed surface of IgG with water molecules, thereby improving its stability. Dynamic light scattering (DLS), size-exclusion high-pressure liquid chromatography (SE-HPLC) and sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) experiments were additionally performed to analyse the aggregation rate of IgG, which was found to decrease in the presence of appropriate DESs. Finally, the long-term stability of IgG in the presence of DESs was investigated at room temperature. All the results obtained from the conformational and colloidal studies of IgG demonstrated the outstanding potential of cholinium-based DESs as novel solvents for IgG formulations, with the DESs comprising [Ch]Cl–urea or [Ch]Cl–Gly noted as the most promising candidates. All the described studies were also performed with the DESs’ individual components, demonstrating that the full DESs (HBD + HDA) are needed to improve the stability of IgG.