CO 2 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 CO 2 (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 CO 2 toward aqueous systems. α‐Chymotrypsin, a model protein, was precipitated from both compressed CO 2 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 CO 2 and fluorinated antisolvents. Although the proteins retained significant activity using both antisolvent systems, the HFE‐precipitated chymotrypsin retained higher activity than the CO 2 ‐precipitated protein.