Fourier transform infared spectroscopy investigation of protein conformation in spray-dried protein/trehalose powders

Spray drying is a way to generate protein solids (powders), which is also true for lyophilization. Sugars are used to protect proteins from conformational changes and chemical degradations arising from drying processes and storage conditions such as the humidity. The influence of trehalose and humidity on the conformation and hydration of spraydried recombinant human granolucyte colony stimulating factor (rhG-CSF) and recombinant consensus interferon(alpha) (rConIFN) was investigated using Fourier transform IR spectroscopy. The spectral analysis of spray-dried powders in the amide I region demonstrated that trehalose stabilized the (alpha)-helical conformation of both rhGCSF and rConIFN proteins. Exposure of the pure protein powders to 33% relative humidity (RH) resulted in the formation of (beta) sheets and loss of turns but no change in (alpha)-helical structure. Trehalose reduced the magnitude of the changes in (beta) sheets and turns. Exposure of the pure protein powders to 75% RH resulted in the loss of (alpha)-helical conformation with a corresponding increase in (beta) structures ((beta) sheets and turns). Trehalose did not protect proteins from the loss of (alpha)-helical structures, but it reduced the formation of antiparallel (beta) sheets. Hydrogen-deuterium exchange (H-D exchange) was used to further characterize these hydration-induced conformational changes. At 33% RH the percent exchange of the protein decreased with increasing trehalose content, indicating a greater protection of the protein from H-D exchange by a higher concentration of trehalose. Such protection correlates with decreased conformational changes of the protein by trehalose at this humidity. At 75% RH the degree of H-D exchange of the protein was insensitive to the powder composition in all powders. Surprisingly, the H-D exchange of trehalose was low at about 20-25%, which was nearly independent of the protein/trehalose ratio and humidity, indicating that the exchangeable protons on trehalose molecules are highly protected in protein-containing powders. The observed protein hydration is related to the effect of trehalose on the conformational changes of the protein under humidity.