Stabilization of Bone Morphogenetic Protein‐2 at Physiological pH : Contrasting Roles of CHAPS and Arginine in Aggregation Inhibition

Bone morphogenetic protein-2 (BMP-2) is a key osteoinductive growth factor employed clinically in spinal fusion and fracture repair where bone regeneration is insufficient. However, its therapeutic efficacy is limited by low solubility and aggregation at physiological pH. This study investigates BMP-2 aggregation and identifies additives that stabilize its native, biologically active dimer form under physiological conditions. We demonstrate that 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) efficiently refolded monomeric BMP-2 from inclusion bodies into dimeric form but failed to prevent aggregation of the folded dimer. In contrast, arginine did not promote refolding but significantly enhanced solubility and stability of the native dimer against aggregation as evidenced by turbidity, Rayleigh scattering, nanoparticle tracking analysis (NTA), dynamic light scattering (DLS) and microscopic analyzes. Functional assays, including alkaline phosphatase (ALP) activity, calcium deposition, and native PAGE, verified that BMP-2 retained its biological activity in presence of arginine. Tryptophan fluorescence and in silico analysis revealed distinct interaction mechanisms to BMP-2: CHAPS interacts with aromatic residues, enhancing flexibility and stabilizes open conformation, whereas arginine binds preferentially to acidic residues, promoting a compact, closed conformation. Collectively, arginine confers robust stabilization of BMP-2 at physiological pH, offering a rational framework for developing stable and therapeutically effective BMP-2 formulations.