Liposomes as formulation excipients for protein pharmaceuticals: a model protein study

Area of Science:

• Biochemistry
• Pharmaceutical Sciences
• Protein Chemistry

Background:

• Recombinant DNA technology enables pharmaceutical use of proteins and peptides.
• Proteins are prone to physical instabilities like denaturation, aggregation, and precipitation due to their complex structures.
• Understanding and mitigating protein instability is crucial for developing stable pharmaceutical formulations.

Purpose of the Study:

• To test the hypothesis that partial unfolding and exposed hydrophobic domains cause protein physical instability.
• To investigate formulation approaches for stabilizing proteins against denaturation and aggregation.
• To explore the role of liposomes in stabilizing partially unfolded protein structures.

Main Methods:

• Utilized KP6 beta, a killer toxin from Ustilago maydis, as a model protein.
• Employed circular dichroism (CD) and fluorescence spectroscopy to analyze temperature-dependent protein folding/unfolding.
• Used 1,8 anilinonaphthalene sulfonate (ANS) as a fluorescent probe to detect hydrophobic domain exposure.

Main Results:

• Elevated temperatures caused loss of tertiary structure but preserved secondary structure in KP6 beta, indicating partial unfolding.
• Increased exposure of hydrophobic domains was observed, confirmed by ANS fluorescence.
• Liposomes effectively bound to partially unfolded protein structures, preventing aggregation and stabilizing the protein.

Conclusions:

• Partial unfolding exposes hydrophobic domains, leading to aggregation while maintaining secondary structure.
• Liposomes stabilize partially unfolded proteins by interacting with intermediate states, acting as molecular chaperones.
• This suggests a general formulation strategy using excipients to stabilize proteins and prevent physical instability.