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Ionic Liquid-Based Strategy for Predicting Protein Aggregation Propensity and Thermodynamic Stability
Talia A Shmool1, Laura K Martin2, Richard P Matthews1
1Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, U.K.
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.
Insights
Ionic liquids, specifically choline chloride, reduce protein aggregation and enhance stability in drug formulations. This strategy predicts and improves the storage of protein therapeutics under stress conditions.
Area of Science:
- Biochemistry and Biophysics
- Materials Science
- Pharmaceutical Sciences
Background:
- Protein therapeutics face aggregation challenges during development and storage due to environmental stresses.
- Ionic liquids (ILs), particularly biocompatible choline chloride ([Cho]Cl)-based ILs, show promise in preventing stress-induced protein conformational changes.
- Predicting and mitigating protein aggregation is crucial for successful drug development.
Purpose of the Study:
- To develop an ionic liquid-based strategy for predicting protein aggregation propensity and thermodynamic stability.
- To evaluate the impact of choline chloride concentration on the stability of immunoglobulin G4 (IgG4) under various stress conditions.
- To assess the long-term storage stability of IgG4 in choline chloride formulations.
Main Methods:
- Systematic evaluation of IgG4 structural, thermal, and thermodynamic stability using dynamic light scattering, zeta potential, and variable temperature circular dichroism.
- Molecular dynamics simulations to analyze IgG4 aggregation propensity and choline chloride-IgG4 interactions.
- Re-evaluation of protein stability after 365 days of storage at 4 °C.
Main Results:
- Increasing choline chloride concentration significantly reduced IgG4 aggregation propensity in fresh and stored samples.
- Choline chloride demonstrated a protective effect against stress-induced protein misfolding and aggregation.
- The study successfully predicted thermodynamic properties and aggregation propensity over extended storage periods.
Conclusions:
- An ionic liquid-based strategy can effectively predict and enhance protein aggregation propensity and thermodynamic stability.
- Choline chloride formulations offer a promising approach to increase the stability and storage life of protein therapeutics.
- This predictive IL-based strategy has the potential to overcome major challenges in protein drug development.
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