Department of Pharmaceutics and Biopharmaceutics, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CH-1211 Geneva 4, Switzerland.
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.
本研究考察了两种不同 pH 值对重组人骨形态发生蛋白-2（rhBMP-2）在水溶液中物理稳定性的影响。通过内源（Nile Red 和 1,8-ANS）和外源荧光光谱、90 度光散射和透射电子显微镜（TEM）对 pH 值为 4.5 或 6.5 的 rhBMP-2 溶液进行了表征。与 pH 4.5 溶液相比，pH 6.5 溶液中的 rhBMP-2 具有以下特征：（i）内源荧光强度更强；（ii）荧光寿命更长；（iii）90 度光散射强度更强；（iv）Nile Red 荧光强度更强；（v）Nile Red 荧光各向异性更高；（vi）1,8-ANS 荧光强度更低；（vii）1,8-ANS 荧光各向异性更高；（viii）1,8-ANS 荧光寿命更长。电子显微镜显示，pH 4.5 下的 rhBMP-2 包含约 100nm 直径的聚集体。在 pH 6.5 下的溶液中观察到更多和更大的蛋白质聚集体（0.1-2μm）。综上所述，与 pH 4.5 相比，pH 6.5 下 rhBMP-2 的构象发生变化并增加了聚集，表明 pH 对 rhBMP-2 物理稳定性有很大影响。在开发 rhBMP-2 递送系统时必须考虑这些观察结果。
