Lysozyme particle formation during supercritical fluid drying: Particle morphology and molecular integrity

Studies have shown that diverse types of particles can be obtained when processing aqueous protein solutions into powders by using supercritical fluids, however, without identifying the mechanism behind these variations. Therefore, the particle formation of lysozyme by supercritical fluid drying was more systemically studied by varying the flow rates of protein solution, supercritical carbon dioxide and ethanol, co-currently sprayed through a coaxial nozzle. Three different morphologies were identified: agglomerated nanoparticles, microspheres and irregular microparticles. These morphologies could be related to the process conditions, in particular to the fraction of ethanol in the extraction medium: agglomerated nanoparticles were produced under anti-solvent precipitation conditions; microspheres under water extraction conditions; and microparticles under competitive rates of both mechanisms. A slight increase in intermolecular β-sheets was observed in powders (<5% residual water content) produced under anti-solvent conditions. Nevertheless, the protein integrity was recovered after rehydration. In conclusion, the alcohol fraction in the extractant has shown to influence both the particle morphology and molecular integrity. The selection of ethanol fraction could be especially important when more labile proteins are to be processed using this technique.