Physical instability, aggregation and conformational changes of recombinant human bone morphogenetic protein-2 (rhBMP-2)

Int J Pharm. 2010 May 31;391(1-2):48-54. doi: 10.1016/j.ijpharm.2010.02.015. Epub 2010 Feb 13.


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.

MeSH terms

  • Bone Morphogenetic Protein 2
  • Bone Morphogenetic Proteins / analysis
  • Bone Morphogenetic Proteins / chemistry*
  • Chemistry, Pharmaceutical / methods*
  • Drug Stability*
  • Fluorescence Polarization / methods
  • Humans
  • Hydrogen-Ion Concentration
  • Protein Conformation
  • Recombinant Proteins / analysis
  • Recombinant Proteins / chemistry*
  • Solubility
  • Transforming Growth Factor beta / analysis
  • Transforming Growth Factor beta / chemistry*


  • Bone Morphogenetic Protein 2
  • Bone Morphogenetic Proteins
  • Recombinant Proteins
  • Transforming Growth Factor beta
  • recombinant human bone morphogenetic protein-2