The relation between moisture-induced aggregation and structural changes in lyophilized insulin

Abstract

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.