Forbes Robert T, Barry Brian W, Elkordy Amal A
Drug Delivery Group, School of Pharmacy, University of Bradford, Bradford BD7 1DP, UK.
Eur J Pharm Sci. 2007 Mar;30(3-4):315-23. doi: 10.1016/j.ejps.2006.11.019. Epub 2006 Dec 10.
The production of stable protein formulations is difficult due to unique properties of proteins. Accordingly, spray drying and crystallisation techniques were assessed for their effects on trypsin, a model protein. Samples were investigated using polarising microscopy, thermogravimetry, differential scanning calorimetry (DSC), FT-Raman spectroscopy and enzymatic assay. Unprocessed, spray-dried and crystallised trypsin were evaluated in solution for secondary structure in low and high protein concentrations using aqueous state FT-Raman spectroscopy and for folding reversibility employing high sensitivity differential scanning calorimetry. Spray-dried trypsin showed FT-Raman spectral changes and less biological activity, after rehydration, compared with unprocessed and crystallised trypsin. Crystals maintained activity better than did the spray-dried form and retained a higher folding reversibility compared to unprocessed and spray-dried protein. Proteins may denature with structural changes under thermal stress and lose their activities. Thus, this research studied the effect of heating solid unprocessed, spray-dried and crystallised trypsin samples on their secondary structures, using FT-Raman spectroscopy, to identify the influence of the initial solid form on its propensity for thermal denaturation and whether this can be correlated with catalytic activity. DSC heated protein samples to two temperatures, one before the apparent denaturation temperature (T(m)) and the other after the T(m). Samples heated below their T(m) showed some perturbations of the secondary structure and some activity, whilst materials rose to the higher temperature were insoluble with complete loss of activity.
由于蛋白质具有独特性质，因此难以生产出稳定的蛋白质制剂。相应地，对喷雾干燥和结晶技术对模型蛋白胰蛋白酶的影响进行了评估。使用偏光显微镜、热重分析法、差示扫描量热法（DSC）、傅里叶变换拉曼光谱法和酶活性测定对样品进行了研究。使用水相傅里叶变换拉曼光谱法在低蛋白浓度和高蛋白浓度下评估未处理、喷雾干燥和结晶的胰蛋白酶在溶液中的二级结构，并使用高灵敏度差示扫描量热法评估其折叠可逆性。与未处理和结晶的胰蛋白酶相比，喷雾干燥的胰蛋白酶在复水后显示出傅里叶变换拉曼光谱变化且生物活性较低。晶体比喷雾干燥形式更好地保持了活性，并且与未处理和喷雾干燥的蛋白质相比保留了更高的折叠可逆性。蛋白质可能在热应力下因结构变化而变性并失去活性。因此，本研究使用傅里叶变换拉曼光谱法研究了加热固体未处理、喷雾干燥和结晶的胰蛋白酶样品对其二级结构的影响，以确定初始固体形式对其热变性倾向的影响以及这是否与催化活性相关。差示扫描量热法将蛋白质样品加热到两个温度，一个在表观变性温度（T(m)）之前，另一个在T(m)之后。在其T(m)以下加热的样品显示出二级结构的一些扰动和一些活性，而升温至较高温度的材料不溶且活性完全丧失。
