Critical processing parameters of carbon dioxide spray drying for the production of dried protein formulations: A study with myoglobin.

The aim of this study was to gain fundamental insight into protein destabilization induced by supercritical CO 2 spray drying processing parameters. Myoglobin was used as a model protein (5 mg/ml with 50 mg/ml trehalose in 10 mM phosphate buffer, pH 6.2). The solution was exposed to sub- and supercritical CO 2 conditions (65–130 bar and 25–50 °C), and CO 2 spray drying under those conditions. The heme binding of myoglobin was determined by UV/Vis, fluorescence, and circular dichroism spectroscopy, while myoglobin aggregation was studied by using size-exclusion chromatography and flow imaging microscopy. It was found that pressure and temperature alone did not influence myoglobin’s integrity. However, when pressurized CO 2 was introduced into myoglobin solutions at any condition, the pH of the myoglobin formulation shifted to about 5 (measured after depressurization), resulting in heme binding destabilization and aggregation of myoglobin. When exposed to CO 2 , these degradation processes were enhanced by increasing temperature. Heme binding destabilization and myoglobin aggregation were also seen after CO 2 spray drying, and to a greater extent. Moreover, the CO 2 spray drying induced the partial loss of heme. In conclusion, pressurized CO 2 destabilizes the myoglobin, leading to heme loss and protein aggregation upon spray drying. [ABSTRACT FROM AUTHOR]