Denaturation and Aggregation of Interferon-τ in Aqueous Solution

Area of Science:

• Biochemistry
• Protein Chemistry
• Physical Chemistry

Background:

• Interferon-τ (IFN-τ) is a therapeutic protein.
• Understanding protein stability and aggregation is crucial for biopharmaceutical development.
• Residual structure in unfolded states influences protein behavior.

Purpose of the Study:

• To evaluate residual structure in unfolded interferon-τ using chemical denaturation.
• To assess the impact of temperature on protein structure.
• To compare urea and guanidinium hydrochloride (GnHCl) as denaturants.

Main Methods:

• Asymmetrical flow field-flow fractionation (AF4) with UV and multi-angle laser light scattering (MALLS).
• Flow Microscopy (FlowCAM) for subvisible particle imaging.
• Chemical denaturation using urea and GnHCl at varying temperatures.

Main Results:

• Urea and GnHCl yielded different estimates of interferon-τ conformational stability.
• Residual structure in the denatured state decreased with increasing temperature above 25°C.
• Histidine buffer stabilized interferon-τ, reducing particle formation and aggregation at 50°C.
• Thermal aggregation followed a nucleation-dependent mechanism.

Conclusions:

• Chemical denaturants (urea, GnHCl) produce distinct residual structures in unfolded interferon-τ.
• AF4 effectively characterized size changes and ligand binding effects.
• Histidine is a suitable buffer for interferon-τ, mitigating aggregation and particle formation.