Selective domain stabilization as a strategy to reduce fusion protein aggregation

Area of Science:

• Biochemistry
• Protein Science
• Colloid Science

Background:

• Understanding protein aggregation is crucial for biopharmaceutical development and stability.
• Fusion proteins, like human serum albumin-human growth hormone (HSA-hGH), present complex aggregation behaviors.
• The interplay between conformational stability and colloidal interactions in protein aggregation requires further elucidation.

Purpose of the Study:

• To investigate the aggregation mechanisms of a model HSA-hGH fusion protein.
• To determine the relative contributions of conformational stability and protein-protein interactions to aggregation.
• To assess the impact of pH and ligand binding on the aggregation behavior of HSA-hGH.

Main Methods:

• Studied HSA-hGH aggregation at pH 5 and pH 7.
• Modulated human serum albumin (HSA) domain stability using octanoic acid.
• Measured conformational stability via free energies of unfolding (ΔG(unf)) and apparent unfolding temperatures (T(m)).
• Assessed protein-protein interactions using osmotic second virial coefficients.

Main Results:

• Octanoic acid binding increased the apparent T(m) of both HSA and HSA-hGH, indicating enhanced conformational stability.
• Reduced aggregation rates correlated with increased protein-protein repulsive interactions.
• These reductions occurred even without detectable changes in conformational stability.

Conclusions:

• Colloidal instabilities, rather than conformational stability of the HSA domain, are the primary drivers of HSA-hGH aggregation.
• Protein-protein repulsive forces play a significant role in mitigating aggregation.
• The findings provide insights into controlling protein aggregation in biotechnological applications.