Mixing properties of lyophilized protein systems: a spectroscopic and calorimetric study

The purpose of this study was to investigate the solid-state properties of lyophilized formulations of protein (ribonuclease A) containing sucrose or trehalose across a wide range of compositions, both in the presence or absence of hydroxyethylstarch (HES). Infrared spectroscopy reveals that the protein forms hydrogen bonds to sugars (sucrose or trehalose) as water is removed from the sample. The strength and/or number of hydrogen bonds in dried samples increase as the weight fraction of sugar increases. Significant deviations of glass transition temperatures (T(g)'s) from those predicted by free volume theory are seen in both protein-sugar systems. The behavior can be explained by formation of protein-sugar hydrogen bonds at the expense of self-interactions between the sugars. Attractive interactions between lyophilized ribonuclease A and HES were detected spectroscopically and from thermodynamic analysis of T(g) values, contrary to the view that HES is sterically hindered from interacting with the protein surface. Sucrose-HES interactions were much less favorable than trehalose-HES interactions, suggesting that phase separation in sugar/HES/protein mixtures would be more likely in the presence of sucrose than trehalose. Finally, the thermodynamics of mixing were investigated using differential scanning calorimetry (DSC) providing some of the first data for such solid protein sugar formulations with and without HES. In nearly all samples, positive excess enthalpy, excess entropy and excess free energy were observed, with the excess free energy being greater for samples containing sucrose rather than trehalose. Analysis of Flory-Huggins chi parameters suggests that phase separation between protein and excipients may be thermodynamically favored in these dried solid preparations.