MOISTURE-INDUCED AGGREGATION OF LYOPHILIZED INSULIN

A critical problem in the storage and delivery of pharmaceutical prote ins is aggregation in the solid state induced by elevated temperature and moisture. These conditions are particularly relevant for studies o f protein stability during accelerated storage or for proteins loaded in polymeric delivery devices in vivo. In the present investigation, w e have found that, when exposed to an environment simulating these con ditions, lyophilized insulin undergoes both covalent and noncovalent a ggregation. The covalent process has been elucidated to be intermolecu lar thiol-catalyzed disulfide interchange following beta-elimination o f an intact disulfide bridge in the insulin molecule. This process is accelerated by increasing the temperature and water content of the ins ulin powder or by performing lyophilization and/or dissolution of insu lin in alkaline media. The aggregation can be ameliorated by the prese nce of Cu2+, which presumably catalyzes the oxidization of free thiols . The water sorption isotherm for insulin reveals that the extent of a ggregation directly correlates with the water uptake by the lyophilize d insulin powder, thus pointing to the critical role of protein confor mational mobility in the aggregation process.