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

Abstract

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° 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° 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 μm) 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.