Costantino H R, Langer R, Klibanov A M
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139.
Pharm Res. 1994 Jan;11(1):21-9. doi: 10.1023/a:1018981208076.
A critical problem in the storage and delivery of pharmaceutical proteins is aggregation in the solid state induced by elevated temperature and moisture. These conditions are particularly relevant for studies of protein stability during accelerated storage or for proteins loaded in polymeric delivery devices in vivo. In the present investigation, we have found that, when exposed to an environment simulating these conditions, lyophilized insulin undergoes both covalent and noncovalent aggregation. The covalent process has been elucidated to be intermolecular thiol-catalyzed disulfide interchange following beta-elimination of an intact disulfide bridge in the insulin molecule. This process is accelerated by increasing the temperature and water content of the insulin powder or by performing lyophilization and/or dissolution of insulin in alkaline media. The aggregation can be ameliorated by the presence of Cu2+, which presumably catalyzes the oxidization of free thiols. The water sorption isotherm for insulin reveals that the extent of aggregation directly correlates with the water uptake by the lyophilized insulin powder, thus pointing to the critical role of protein conformational mobility in the aggregation process.
药物蛋白质储存与递送中的一个关键问题是高温和湿度引起的固态聚集。这些条件对于加速储存期间的蛋白质稳定性研究或体内加载到聚合物递送装置中的蛋白质而言尤为重要。在本研究中，我们发现，当冻干胰岛素暴露于模拟这些条件的环境时，会发生共价和非共价聚集。共价过程已被阐明为胰岛素分子中完整二硫键的β-消除后分子间硫醇催化的二硫键交换。通过提高胰岛素粉末的温度和含水量，或在碱性介质中进行胰岛素冻干和/或溶解，该过程会加速。Cu2+ 的存在可改善聚集，Cu2+ 可能催化游离硫醇的氧化。胰岛素的水吸附等温线表明，聚集程度与冻干胰岛素粉末的吸水量直接相关，从而表明蛋白质构象流动性在聚集过程中的关键作用。
