Cholinium-Based Ionic Liquids as Efficient Media for Improving the Structural and Thermal Stability of Immunoglobulin G Antibodies
Diksha Dhiman, Meena Bisht, Ana P. M. Tavares, Mara G. Freire, Pannuru Venkatesu
Abstract
Among antibody-based biopharmaceuticals, immunoglobulin G (IgG) plays a central role. However, IgG is a protein and as such easily loses stability, thus compromising its use as an effective therapeutic. To overcome this drawback, in this work, we evaluated cholinium-based ionic liquids (ILs) as effective stabilizers of IgG. We have investigated four ILs in aqueous solution, namely, cholinium acetate ([Ch][Ac]), cholinium chloride ([Ch]Cl), cholinium dihydrogen citrate ([Ch][Dhc]), and cholinium dihydrogen phosphate ([Ch][Dhp]). We used a quantitative approach using different spectroscopic and chromatographic techniques to evaluate the thermal and structural stability of IgG. Thermal fluorescence spectroscopy studies were performed to obtain the transition temperature (Tm) and the change in Gibbs free energy (ΔG) of IgG in all cholinium-based IL aqueous solutions. The results indicate an appreciable increase in Tm in the presence of [Ch][Ac] and [Ch]Cl. The thermodynamic parameters obtained from thermal fluorescence are compared with the structural stability of IgG by UV, fluorescence, CD, and FT-IR spectroscopies, as well as with size exclusion high-performance liquid chromatography and sodium dodecyl-sulfate polyacrylamide gel electrophoresis, showing that all results are in good agreement. Furthermore, the hydrodynamic diameter (dH) of the IgG as a function of the concentration of IL was analyzed using dynamic light scattering, showing favorable interactions between protein residues. Finally, molecular docking studies for IgG in ILs using Molegro virtual docker have been performed, reinforcing the main interactions ruling the IgG stability. Time dependent studies were also performed for one month to study the long-term stability of IgG at room temperature in presence of ILs. This work highlights the potential validity of using cholinium-based ILs in IgG formulations for enhancing its thermal and structural stability and thus the preservation of IgG antibodies.