Physical instability, aggregation and conformational changes of recombinant human bone morphogenetic protein-2 (rhBMP-2).
The influence of two different pH values on the physical stability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in aqueous solution was evaluated in the present work. RhBMP-2 in solution at pH 4.5 or 6.5 was characterized by intrinsic and extrinsic (Nile Red and 1,8-ANS) fluorescence spectroscopy, 90 degrees light-scattering and transmission electron microscopy (TEM). Compared to the pH 4.5 solution, rhBMP-2 at pH 6.5 had (i) a stronger intrinsic fluorescence intensity, (ii) a longer fluorescence lifetime, (iii) a stronger 90 degrees light-scattering intensity, (iv) a stronger Nile Red fluorescence intensity, (v) a higher Nile Red fluorescence anisotropy, (vi) a lower 1,8-ANS fluorescence intensity, (vii) a higher 1,8-ANS fluorescence anisotropy and (viii) a longer 1,8-ANS fluorescence lifetime. Electron microscopy showed that rhBMP-2 at pH 4.5 contained aggregates of about 100 nm in diameter. More and larger protein aggregates (0.1-2 microm) were observed in solution at pH 6.5. Taken together, these results indicate conformational changes and increased aggregation of rhBMP-2 at pH 6.5 compared to pH 4.5, demonstrating a strong influence of pH on rhBMP-2 physical stability. These observations must be considered when developing a delivery system for rhBMP-2.
Luca
Ludmila
L
Department of Pharmaceutics and Biopharmaceutics, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CH-1211 Geneva 4, Switzerland.
Capelle
Martinus A H
MA
Machaidze
Gia
G
Arvinte
Tudor
T
Jordan
Olivier
O
Gurny
Robert
R
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000032:analysis / Q000737:chemistry*; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D004355:Drug Stability; D005454:Fluorescence Polarization / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry*; D012995:Solubility; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000737:chemistry*
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2