Ionic Liquid-Based Strategy for Predicting Protein Aggregation Propensity and Thermodynamic Stability

Abstract

Novel drug candidates are continuously being developed to combat the most life-threatening diseases; however, many promising protein therapeutics are dropped from the pipeline. During biological and industrial processes, protein therapeutics are exposed to various stresses such as fluctuations in temperature, solvent pH, and ionic strength. These can lead to enhanced protein aggregation propensity, one of the greatest challenges in drug development. Recently, ionic liquids (ILs), in particular, biocompatible choline chloride ([Cho]Cl)-based ILs, have been used to hinder stress-induced protein conformational changes. Herein, we develop an IL-based strategy to predict protein aggregation propensity and thermodynamic stability. We examine three key variables influencing protein misfolding: pH, ionic strength, and temperature. Using dynamic light scattering, zeta potential, and variable temperature circular dichroism measurements, we systematically evaluate the structural, thermal, and thermodynamic stability of fresh immunoglobin G4 (IgG4) antibody in water and 10, 30, and 50 wt % [Cho]Cl. Additionally, we conduct molecular dynamics simulations to examine IgG4 aggregation propensity in each system and the relative favorability of different [Cho]Cl-IgG4 packing interactions. We re-evaluate each system following 365 days of storage at 4 °C and demonstrate how to predict the thermodynamic properties and protein aggregation propensity over extended storage, even under stress conditions. We find that increasing [Cho]Cl concentration reduced IgG4 aggregation propensity both fresh and following 365 days of storage and demonstrate the potential of using our predictive IL-based strategy and formulations to radically increase protein stability and storage.

Unveiling the Rational Development of Stimuli-Responsive Silk Fibroin-Based Ionogel Formulations

“…Overall, formulations lacking IL showed less negative zeta potential values compared to the ionogel formulations. This indicated that the inclusion of IL served to reduce the aggregation propensity and induce a more negative surface charge in each system compared to formulations lacking IL, in line with previous work. ,, …”

“…13 In the absence of IL, the D h and polydispersity index (PDI) values were relatively higher for each formulation indicating enhanced aggregation propensity, which can lead to reduced structural stability. 45,46 We found that [Cho][DHP] and [Cho][OAc]-ionogel formulations showed relatively more negative zeta potential values compared to the [Cho][Cl]-ionogel formulations (Figure 3A−D and Table S1). PVP inclusion also resulted in more negative zeta potential values.…”

Impact of ZIF-8, ArgHCl, and Ionic Liquid-Based Formulations on the Conformational and Colloidal Stability of Antibodies

“…The T m of IgG in the buffer is found to be 74 °C which is in corroboration with the literature. 14,15 However, an increment in the T m values can be seen in the presence of formulations F2, F4, F5, and F6 changing from 74 °C (buffer) to 75, 87, 83, and 75 °C respectively. The overall sequence of T m is F4 > F5 > F2 = F6 > control>F7 = F1 > F3.…”

“…ILs have shown great potential for use in medication delivery applications. − However, choline-based ILs, in particular, have stood out as drug carriers owing to their biocompatible nature, accessibility, and cost-effective properties. , Mitragotri and group have illustrated the use of choline-based ILs and deep eutectic solvents (DESs) as an oral delivery platform for local as well as systemic delivery of therapeutic antibodies. Shmool et al have also recently investigated the IgG4’s propensity to aggregation in the presence of choline chloride [Ch]­Cl. Very recently, our research group investigated the stability profile of IgG in the presence of choline-based ILs and found that the protein’s conformational and colloidal stability improved significantly in the presence of choline acetate ([Ch]­[Ac]).…”

Amphiphilic structured PEG derivatives suppressing protein thermal aggregation at extremely low molecular ratio

“…In sharp contrast, stabilization of proteins by artificial means is usually achieved by addition of large stoichiometric excess kosmotropic salts, 5 saccharides and related polyols, 6 polar amino acids, 7-10 polymers, 11-17 nanogels 18 and ionic liquids. [19][20][21] A common characteristic of these additives is that typically their high concentration (>100 mM for small molecules or 10 mg mL -1 for polymers) is required to achieve satisfactory results. In such cases, however, the high concentration of additives changes the general properties of the solution, including viscosity, ionic strength, refractive index and chemical compatibility.…”