A Model Study to Assess Fibrillation and Product Stability to Support Peptide Drug Design.

Area of Science:

• Biochemistry
• Pharmaceutical Sciences
• Materials Science

Background:

• Peptide fibrillation is a significant challenge for therapeutic peptide quality, manufacturing, and storage.
• Understanding fibrillation mechanisms is crucial for designing stable peptide drugs and optimizing formulations.
• Structural modifications can influence peptide stability and fibrillation propensity.

Purpose of the Study:

• To investigate the impact of structural modifications on peptide fibrillation.
• To evaluate the fibrillation kinetics and stability of modified peptides (PepA, PepB, PepC).
• To develop a model for differentiating and characterizing fibrillation risk in therapeutic peptides.

Main Methods:

• Far-UV CD spectroscopy for thermal denaturation studies.
• Fluorescence and turbidity measurements for fibrillation kinetics.
• Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) for fibril structure analysis.
• Real-time and accelerated stability studies under various pH and stress conditions.

Main Results:

• PepA and PepB formed fibrils upon heat-induced conformational changes, while PepC, with an alpha-aminoisobutyric acid (Aib) substitution, did not fibrillate.
• HDX-MS confirmed extensive incorporation of PepA sequence into fibrils.
• Real-time stability studies revealed formulation changes not detected in accelerated studies.
• Stress-induced fibrillation successfully amplified undetected oligomers/pre-fibrillar species.

Conclusions:

• Structural modifications, such as incorporating Aib, can effectively prevent peptide fibrillation.
• Real-time stability studies are essential for detecting subtle changes and assessing fibrillation risk.
• The developed approach allows for rapid, iterative design of stable therapeutic peptides.