Investigation of protein/carbohydrate interactions in the dried state. 2. Diffuse reflectance FTIR studies
Investigation of protein/carbohydrate interactions in the dried state. 2. Diffuse reflectance FTIR studies
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DOI：
10.1016/s0378-5173(01)00987-5
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发表时间：
2002-03-20
影响因子：
5.8
通讯作者：
Rytting, JH
中科院分区：
文献类型：
--
作者：
Souillac, PO;Middaugh, CR;Rytting, JH
在不存在冻干保护剂的情况下冷冻干燥后，傅立叶变换红外（FTIR）光谱检测到蛋白质二级结构的变化。这种FTIR研究通常使用蛋白质/KBr颗粒进行，其中由于压力效应和离液KBr的部分溶解，可能会发生额外的蛋白质降解。漫反射FTIR光谱法，其中没有样品制备是必要的，作为一种替代的光谱方法，以检查蛋白质结构后，冷冻干燥进行了评价。将治疗性蛋白质重组人脱氧核糖核酸酶I（rh-DNase）和重组人胰岛素样生长因子I（rh-IGF-I）分别与甘露醇、蔗糖、海藻糖和两种分子量葡聚糖（69和503 kDa）以0至100%（w/w）的浓度冷冻干燥。冻干后，rh-DNase和rh-IGF-I的二级结构发生了显着变化。对于这两种蛋白质，检测到由于聚集而导致的分子间P-折叠的存在，并且α-螺旋含量显著降低。向制剂中添加碳水化合物以浓度依赖性方式抑制蛋白质二级结构重排。蔗糖和海藻糖似乎是防止二级结构变化的最有效辅料。观察到的两种蛋白质的构象变化似乎是可逆的再水化。(C)2002爱思唯尔科技有限公司。保留所有权利。
Upon freeze-drying in the absence of lyoprotectants, Fourier transform infrared (FTIR) spectroscopy has detected changes in the secondary structures of proteins. Such FTIR studies have been typically conducted using protein/KBr pellets, where additional protein degradation could potentially occur due to pressure effects and partial dissolution of the chaotropic KBr. Diffuse reflectance FTIR spectroscopy, in which no sample preparation is necessary, was evaluated as an alternative spectroscopic method to examine protein structure upon freeze-drying. The therapeutic proteins recombinant human deoxyribonuclease I (rh-DNase) and recombinant human insulin like growth factor I (rh-IGF-I) were freeze-dried with mannitol, sucrose, trehalose, and two molecular weight dextrans (69 and 503 kDa) separately, at concentrations ranging from 0 to 100% (w/w). Upon freeze-drying, rh-DNase and rh-IGF-I underwent significant changes in their secondary structure. For both proteins, the presence of intermolecular P-sheets due to aggregation was detected and the a-helix content decreased significantly. The addition of carbohydrates to the formulations inhibited the protein secondary structure rearrangement in a concentration-dependent manner. Sucrose and trehalose appeared to be the most efficient excipients in preventing secondary structure changes. The conformational changes observed for both proteins appeared to be reversible upon rehydration. (C) 2002 Elsevier Science B.V. All rights reserved.