The main goals of this thesis are to understand the supercritical carbon dioxide (scCO2) spray drying mechanisms and parameters that influence the stability of proteins, to evaluate the excipients... Show moreThe main goals of this thesis are to understand the supercritical carbon dioxide (scCO2) spray drying mechanisms and parameters that influence the stability of proteins, to evaluate the excipients to stabilize protein formulations during scCO2 spray drying, and to study the scalability of the scCO2 spray drying process. For this study, lysozyme and myoglobin were used as model proteins. More specifically, the detailed aims are as follows: 1) to study the scCO2 spray drying parameters (i.e., pressure, protein solution and CO2 flow rate, feed volume) without the use of organic solvents, in order to produce dried protein formulations with minimal residual water content in a single drying step, 2) to evaluate the scalability of the scCO2 spray drying process, 3) To gain fundamental insight into the effect of the CO2 spray drying parameters at sub- and supercritical conditions (65-130bar and 25-50°C) on the stability of myoglobin, 4) to understand the effect of the CO2/water interface and pH shift on heme destabilization and aggregation in myoglobin solutions using a gas bubbling method at atmospheric conditions, 5) to evaluate the influence of pharmaceutical excipients on the stability of myoglobin in terms of heme binding and aggregation during scCO2 spray drying. Show less
The aim of this study was to gain fundamental insight into protein destabilization induced by supercritical CO2 spray drying processing parameters. Myoglobin was used as a model protein (5mg/ml... Show moreThe aim of this study was to gain fundamental insight into protein destabilization induced by supercritical CO2 spray drying processing parameters. Myoglobin was used as a model protein (5mg/ml with 50mg/ml trehalose in 10mM phosphate buffer, pH 6.2). The solution was exposed to sub- and supercritical CO2 conditions (65-130bar and 25-50°C), and CO2 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 CO2 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 CO2, these degradation processes were enhanced by increasing temperature. Heme binding destabilization and myoglobin aggregation were also seen after CO2 spray drying, and to a greater extent. Moreover, the CO2 spray drying induced the partial loss of heme. In conclusion, pressurized CO2 destabilizes the myoglobin, leading to heme loss and protein aggregation upon spray drying. Show less
