Preferential exclusion mechanism by carbohydrates on protein stabilization using thermodynamic evaluation

Area of Science:

• Biochemistry
• Biophysics
• Pharmaceutical Sciences

Background:

• Carbohydrates are common additives in biopharmaceutical formulations.
• Their precise mechanisms for enhancing protein stability are not fully understood.

Purpose of the Study:

• To elucidate the thermodynamic and biophysical mechanisms by which carbohydrates stabilize proteins.
• To investigate the effects of specific carbohydrates on the stability of etanercept.

Main Methods:

• Isothermal titration calorimetry (ITC) and micro-differential scanning calorimetry (mDSC) for thermodynamic analysis.
• Circular dichroism (CD), dynamic light scattering (DLS), and size-exclusion chromatography (SEC) for biophysical characterization.
• Evaluation of sucrose, fructose, mannitol, and trehalose at various concentrations using etanercept.

Main Results:

• Sucrose and trehalose increased exothermic enthalpy, suggesting preferential hydration.
• No specific non-covalent interactions were detected between proteins and carbohydrates.
• SEC results post-accelerated storage confirmed the stabilizing effects.
• Maximal stabilizing energy observed at 250 mM sucrose and trehalose.

Conclusions:

• Carbohydrates, particularly sucrose and trehalose, significantly improve the aggregation and conformational stability of etanercept.
• The primary stabilizing mechanism involves the preferential exclusion of water molecules.
• This study proposes a detailed mechanism for carbohydrate-mediated protein stabilization.