Allison S D, Manning M C, Randolph T W, Middleton K, Davis A, Carpenter J F
University of Colorado Center for Pharmaceutical Biotechnology, Denver, Colorado 80262, USA.
J Pharm Sci. 2000 Feb;89(2):199-214. doi: 10.1002/(SICI)1520-6017(200002)89:2<199::AID-JPS7>3.0.CO;2-B.
The storage stability of a dry protein depends on the structure of the dried protein, as well as on the storage temperature relative to the glass transition temperature of the dried preparation. Disaccharides are known to preserve the native conformation of a dried protein; however, the resulting T(g) of the sample may be too low ensure adequate storage stability. On the other hand, formulations dried with high molecular weight carbohydrates, such as dextran, have higher glass transition temperatures, but fail to preserve native protein conformation. We tested the hypothesis that optimizing both protein structure and T(g) by freeze-drying actin with mixtures of disaccharides and dextran would result in increased storage stability compared to actin dried with either disaccharide or dextran alone. Protein structure in the dried solid was analyzed immediately after lyophilization and after storage at elevated temperatures with infrared spectroscopy, and after rehydration by infrared and circular dichroism spectroscopy. Structural results were related to the polymerization activity recovered after rehydration. Degradation was noted with storage for formulations containing either sucrose, trehalose, or dextran alone. Slight increases in T(g) observed in trehalose formulations compared to sucrose formulations did not result in appreciable increases in storage stability. Addition of dextran to sucrose or trehalose increased formulation T(g) without affecting the capacity of the sugar to inhibit protein unfolding during lyophilization and resulted in improved storage stability. Also, dextran provides an excellent amorphous bulking agent, which can be lyophilized rapidly with formation of strong, elegant cake structure. These results suggest that the strategy of using a mixture of disaccharide and polymeric carbohydrates can optimize protein storage stability.
干燥蛋白质的储存稳定性取决于干燥蛋白质的结构，以及相对于干燥制剂玻璃化转变温度的储存温度。已知二糖可保持干燥蛋白质的天然构象；然而，所得样品的玻璃化转变温度可能过低，无法确保足够的储存稳定性。另一方面，用高分子量碳水化合物（如葡聚糖）干燥的制剂具有较高的玻璃化转变温度，但无法保持蛋白质的天然构象。我们测试了这样一个假设：与单独用二糖或葡聚糖干燥的肌动蛋白相比，通过用二糖和葡聚糖的混合物冷冻干燥肌动蛋白来优化蛋白质结构和玻璃化转变温度，将提高储存稳定性。冻干后以及在高温下储存后，立即用红外光谱分析干燥固体中的蛋白质结构，复水后用红外光谱和圆二色光谱分析。结构结果与复水后恢复的聚合活性相关。对于单独含有蔗糖、海藻糖或葡聚糖的制剂，储存时会出现降解。与蔗糖制剂相比，海藻糖制剂中观察到的玻璃化转变温度略有升高，但并未导致储存稳定性显著提高。向蔗糖或海藻糖中添加葡聚糖可提高制剂的玻璃化转变温度，同时不影响糖在冻干过程中抑制蛋白质展开的能力，并提高了储存稳定性。此外，葡聚糖提供了一种出色的无定形填充剂，它可以快速冻干，形成坚固、美观的块状结构。这些结果表明，使用二糖和聚合碳水化合物混合物的策略可以优化蛋白质的储存稳定性。
