Compressed CO2 antisolvent precipitation of lysozyme

The precipitation of proteins using compressed CO2 as antisolvent in the precipitation with a compressed antisolvent (PCA) process has been investigated for lysozyme in its DMSO solution. In the vapor liquid coexistence region of the solvent–antisolvent mixture, particles between 100 and 200 nm were obtained, whereas at supercritical and liquid conditions studied in the 25–40 ° C and 100–150 bar ranges, particle sizes were reproducibly below 100 nm. No effect on particle size was observed for variations of the CO2 and solutions flow rates between 40 and 120 g/min, and between 0.4 and 2 ml/min, respectively. Phase behavior experiments indicate that protein precipitation results from a metastable liquid–liquid phase separation, with the protein rich phase remaining trapped in a gel-like state. Moreover, we suggest that due to the high CO2 concentrations involved in PCA, phase separation takes place by spinodal decomposition. The proposed precipitation mechanism is not limited to the PCA process, but can be extended also to results obtained for the precipitation of lysozyme using the gas antisolvent (GAS) process.