Understanding the Aggregation Mechanism of and Developing Stabilization Strategies for Recombinant Fibroblast Growth...

Area of Science:

• Biochemistry
• Protein Engineering
• Biotherapeutics

Background:

• Fibroblast Growth Factor 2 (FGF2) shows therapeutic promise for tissue regeneration.
• Clinical success of FGF2 is limited by poor protein stability and aggregation.
• Current strategies to improve FGF2 stability have been largely unsuccessful.

Purpose of the Study:

• Investigate the aggregation propensity of recombinant FGF2.
• Identify conditions and mechanisms leading to FGF2 oligomerization.
• Explore strategies to enhance FGF2 stability for therapeutic applications.

Main Methods:

• Native mass spectrometry (MS) to study FGF2 aggregation.
• Tandem MS of proteolytic fragments to identify disulfide bond formation.
• Molecular modeling to predict heparin binding sites.
• Assessing the effect of fondaparinux on FGF2 stability and aggregation.

Main Results:

• FGF2 aggregation is driven by the formation of external disulfide bonds, particularly involving Cys-31.
• Molecular modeling identified a heparin binding site near Cys-31.
• Fondaparinux, a heparin mimetic, forms a stable complex with FGF2.
• Fondaparinux addition inhibited FGF2 oligomer formation.

Conclusions:

• Cys-31 is a key residue in FGF2-mediated oligomerization.
• Fondaparinux stabilizes FGF2 by forming a complex, preventing aggregation.
• These findings offer insights for optimizing FGF2 formulation and administration.