CO₂ and fluorinated solvent-based technologies for protein microparticle precipitation from aqueous solutions

Precipitation with a compressed or supercritical fluid antisolvent (PCA) has been used to produce microparticles of biologically active proteins, pharmaceuticals, and polymers. However, the application of PCA to a wider range of proteins is limited by the low mutual solubility of water (necessary to dissolve most proteins) and CO₂ (traditionally used as the compressed antisolvent). This investigation extends PCA to proteins in aqueous solutions by utilizing ethanol as a cosolvent to enhance the antisolvent properties of CO₂ toward aqueous systems. α-Chymotrypsin, a model protein, was precipitated from both compressed CO₂ and a liquid fluorinated antisolvent, a hydrofluoroether (HFE). The equilibrium phase behavior of the antisolvent/ethanol/water systems was examined to identify a one-phase region suitable for protein precipitation. Spherical protein microparticles with a primary particle size of approximately 0.2-0.6 μm were recovered using both the compressed CO₂ and fluorinated antisolvents. Although the proteins retained significant activity using both antisolvent systems, the HFE-precipitated chymotrypsin retained higher activity than the CO₂-precipitated protein.