Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, Puerto Rico.
J Pharm Pharmacol. 2009 Nov;61(11):1555-61. doi: 10.1211/jpp/61.11.0016.
Long-term stability is a critical factor in the successful development of protein pharmaceuticals. Due to the relative instability of proteins in aqueous solutions, they are formulated frequently and stored as lyophilized powders. Exposure of such powders to moisture constitutes a substantial storage problem leading to aggregation and inactivation. We have investigated the structural consequences of moisture sorption by lyophilized insulin under controlled humidity conditions by employing Fourier transform-infrared (FT-IR) microscopy.
Lyophilized insulin samples were stored in humidity chambers under controlled conditions at 50(o)C. Protein aggregation studies were carried out by redissolving the insulin samples and measuring the amount of both soluble protein and insoluble aggregates. Near-UV circular dichroism spectra were collected to assess the tertiary structure. FT-IR microscopy studies were carried out to investigate secondary structural changes in solid-state insulin after incubation at different relative humidities.
It was found that sorption of moisture was accompanied by small structural changes in lyophilized insulin at low levels of relative humidity (i.e. 11%). At higher relative humidity levels, structural changes were becoming more pronounced and were characterized by a loss in the alpha-helix and increase in beta-sheet content. The magnitude of the structural changes in tendency paralleled the solid-state instability data (i.e. formation of buffer-insoluble aggregates and loss in tertiary structure upon reconstitution).
The results support the hypothesis that water sorption by lyophilized proteins enables structural transitions which can lead to protein aggregation and other deleterious phenomena.
蛋白质类药物的成功开发，其长期稳定性是一个关键因素。由于蛋白质在水溶液中相对不稳定，因此常将其配制成冻干粉末并储存。这些粉末暴露于水分中会构成严重的储存问题，导致聚集和失活。我们通过傅里叶变换红外（FT-IR）显微镜研究了在控制湿度条件下，冻干胰岛素吸收水分后的结构变化。
将冻干胰岛素样品储存在湿度受控的 50（o）C 恒温箱中。通过重新溶解胰岛素样品并测量可溶性蛋白和不溶性聚集体的量来进行蛋白质聚集研究。收集近紫外圆二色性光谱以评估三级结构。FT-IR 显微镜研究用于研究在不同相对湿度下孵育后固态胰岛素的二级结构变化。
结果发现，在相对湿度较低（即 11%）时，水分的吸收伴随着冻干胰岛素的微小结构变化。在较高的相对湿度水平下，结构变化变得更加明显，特征是α-螺旋的损失和β-折叠含量的增加。结构变化的程度与固态不稳定性数据（即缓冲液不溶性聚集体的形成和重构后三级结构的丧失）大致平行。
结果支持了这样的假设，即冻干蛋白质吸收水分能够引发结构转变，从而导致蛋白质聚集和其他有害现象。
