Maschke Angelika, Calí Nadia, Appel Bernhard, Kiermaier Josef, Blunk Torsten, Göpferich Achim
Department of Pharmaceutical Technology, University of Regensburg, 93040 Regensburg, Germany.
Pharm Res. 2006 Sep;23(9):2220-9. doi: 10.1007/s11095-006-9019-0. Epub 2006 Aug 12.
The aim of this study was to establish the high pressure homogenization of proteins in non-aqueous suspension as an alternative method for classical micronization strategies and to investigate the effect of high pressure on protein stability and bioactivity.
The influence of drug loading, homogenization pressure and cycles on particle size reduction was investigated by experimental design using a Box Behnken matrix with insulin as a model compound. Particle size measurements were performed by laser light scattering. Protein stability was investigated by HPLC and HPLC-MS analysis and the bioactivity of insulin was tested in a chondrocyte proliferation assay. For investigations into the effect of temperature on protein stability, insulin was micronized in molten lipid at 75 degrees C in one cycle at 1,000 bar.
Within one homogenization cycle at 1,500 bar, the particle size of insulin could be reduced from 15.8 to 7.3 microm, six cycles resulted in a particle size of 3.7 microm d(0.5) (50% of the particles are smaller than the indicated value). Evaluation of the response surface diagram revealed that the homogenization pressure had the highest impact on micronization efficiency, followed by the number of homogenization cycles. Protein stability was maintained during the micronization process as well as bioactivity. Micronization at elevated temperature (75 degrees C) had no effect on protein stability.
High pressure homogenization of protein suspensions can be used as an alternative method for the micronization of proteins without affecting protein stability or bioactivity.
本研究旨在确立非水悬浮液中蛋白质的高压均质化作为经典微粉化策略的替代方法，并研究高压对蛋白质稳定性和生物活性的影响。
以胰岛素为模型化合物，采用Box-Behnken矩阵实验设计，研究载药量、均质化压力和循环次数对粒径减小的影响。通过激光散射进行粒径测量。通过高效液相色谱（HPLC）和HPLC-质谱（HPLC-MS）分析研究蛋白质稳定性，并在软骨细胞增殖试验中检测胰岛素的生物活性。为研究温度对蛋白质稳定性的影响，将胰岛素在75℃的熔融脂质中于1000巴压力下进行一个循环的微粉化处理。
在1500巴压力下进行一次均质化循环，胰岛素的粒径可从15.8微米减小至7.3微米，六次循环后粒径为3.7微米d(0.5)（50%的颗粒小于所示值）。响应面图评估显示，均质化压力对微粉化效率影响最大，其次是均质化循环次数。在微粉化过程中蛋白质稳定性和生物活性均得以保持。高温（75℃）下的微粉化对蛋白质稳定性无影响。
蛋白质悬浮液的高压均质化可作为蛋白质微粉化的替代方法，且不影响蛋白质稳定性或生物活性。
