Physical factors affecting the storage stability of freeze-dried interleukin-1 receptor antagonist: Glass transition and protein conformation
Physical factors affecting the storage stability of freeze-dried interleukin-1 receptor antagonist: Glass transition and protein conformation
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DOI：
10.1006/abbi.1996.0305
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发表时间：
1996-07-15
影响因子：
3.9
通讯作者：
Carpenter, JF
中科院分区：
文献类型：
--
作者：
Chang, BS;Beauvais, RM;Carpenter, JF
研究了干燥固体的玻璃化转变和蛋白质构象对重组人白细胞介素-1受体拮抗剂（rhIL-1ra）冷冻干燥储存稳定性的影响。玻璃化转变是一种温度相关的现象。非晶材料在低于其特征玻璃化转变温度（Tg）的温度下变得坚硬和脆，使得分子沿着基体的扩散不足以引起大规模的结构变化。为了确定玻璃化转变对蛋白质储存稳定性的重要性，我们比较了10种不同的冻干rhIL-1ra配方，温度范围从20到56摄氏度，在高于和低于样品温度的温度下储存了几周。蛋白质的降解，包括脱酰胺和聚集，在温度高于Tg时大大加速，但对于某些配方，在温度低于Tg时也会加速。因此，将干燥的蛋白质储存在Tg以下是必要的，但不足以确保长期稳定。为了研究干燥固体中蛋白质结构的影响，我们制备了不同蔗糖浓度的配方，所有这些配方的Tg = 66 +/- 2.5℃。通过红外光谱，我们确定了在小于或等于1%蔗糖的情况下冻干的蛋白质在最初的干燥固体中展开。相比之下，在那些大于或等于5%蔗糖的配方中，冻干过程中的构象变化受到抑制。当储存在50℃时，冻干蛋白的降解与蔗糖浓度成反比。这些结果表明，即使储存温度低于Tg，在冻干过程中产生的结构变化也会导致后续储存过程中的损伤。这些研究的结果表明，为了获得干燥的rhIL-1ra的最佳稳定性，有必要在冻干过程中抑制构象变化，并在低于干燥配方Tg的温度下储存。(C) 1996学术出版社，Inc.
The effects of glass transition of, and protein conformation in, the dried solid on the storage stability of freeze-dried recombinant human interleukin-1 receptor antagonist (rhIL-1ra) were examined. Glass transition is a temperature-dependent phenomenon. Amorphous materials become hard and brittle at temperatures below their characteristic glass transition temperatures (Tg) such that diffusion of molecules along the matrix is not sufficient to cause large-scale structural changes. To ascertain the importance of the glass transition in protein storage stability, we compared 10 different lyophilized rhIL-1ra formulations, with Tgs ranging from 20 to 56 degrees C, during several weeks of storage at temperatures above and below the samples' Tgs. Protein degradation, both deamidation and aggregation, was greatly accelerated at temperatures above Tg,but for some formulations also arose below Tg. Thus, storage of dried proteins below the Tg is necessary but not sufficient to ensure long-term stability. To examine the effects of protein structure in the dried solid, we prepared formulations with various sucrose concentrations, all of which had a Tg = 66 +/- 2.5 degrees C. With infrared spectroscopy, we determined that the protein lyophilized with less than or equal to 1% sucrose was unfolded in the initial dried solid. In contrast, in those formulations with greater than or equal to 5% sucrose, conformational change was inhibited during lyophilization. When stored at 50 degrees C, degradation of the freeze-dried protein varied inversely with sucrose concentration. These results indicate that structural changes arising during the lyophilization process led to damage during subsequent storage, even if the storage temperature was less than the Tg. Together the results of these studies document that to obtain optimum stability of dried rhIL-1ra it was necessary to inhibit conformational change during lyophilization and to store at temperatures below the Tg of the dried formulation. (C) 1996 Academic Press, Inc.