Protein purification with vapor-phase carbon dioxide

Abstract

Gaseous CO2 was used as an antisolvent to induce the fractional precipitation of alkaline phosphatase, insulin, lysozyme, ribonuclease, trypsin, and their mixtures from dimethylsulfoxide (DMSO). Compressed CO2 was added continuously and isothermally to stationary DMSO solutions (gaseous antisolvent, GAS). Dissolution of CO2 was accompanied by a pronounced, pressure‐dependent volumetric expansion of DMSO and a consequent reduction in solvent strength of DMSO towards dissolved proteins. View cell experiments were conducted to determine the pressures at which various proteins precipitate from DMSO. The solubility of each protein in CO2‐expanded DMSO was different, illustrating the potential to separate and purify proteins using gaseous antisolvents. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS‐PAGE) was used to quantify the separation of lysozyme from ribonuclease, alkaline phosphatase from insulin, and trypsin from catalase. Lysozyme biological activity assays were also performed to determine the composition of precipitates from DMSO initially containing lysozyme and ribonuclease. SDS‐PAGE characterizations suggest that the composition and purity of solid‐phase precipitated from a solution containing multiple proteins may be accurately controlled through the antisolvent's pressure. Insulin, lysozyme, ribonuclease, and trypsin precipitates recovered substantial amounts of biological activity upon redissolution in aqueous media. Alkaline phosphatase, however, was irreversibly denaturated. Vapor‐phase antisolvents, which are easily separated and recovered from proteins and liquid solvents upon depressurization, appear to be a reliable and effective means of selectively precipitating proteins. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 247–258, 1999.

Preparation of silica and TiO2–SiO2 core–shell nanoparticles in water-in-oil microemulsion using compressed CO2 as reactant and antisolvent

“…One of the useful techniques in material science is the gas antisolvent process [24], which has been used in extraction and fractionation [25], recrystallization of chemicals [26], micronization [27] and production of polymeric particles [28]. These methods have some potential advantages.…”

The formation of plasmid DNA loaded pharmaceutical powders using supercritical fluid technology

“…While a variety of proteins has been successfully processed with supercritical fluid technology (Sloan et al, 1998;Winters et al, 1999), the formulation of nucleic acids has so far not been addressed. Nucleic acids, mainly in the form of plasmid DNA, are a class of biological macromolecules that play an important role in the treatment of genetically caused diseases like Cystic Fibrosis (Crystal, 1995;Marquet et al, 1995;Prazeres et al, 1999).…”

Supercritical Fluid Application in Food and Bioprocess Technology

“…SC antisolvent method has great potential for processing of pharmaceuticals (Mosqueira et al 1981;Steckel et al 1997) and labile compounds such as proteins Winters et al 1999;Yeo et al 1994;Yeo et al 1993) and to obtain various morphologies of biopolymers (Bleich et al 1996;Debenedetti et al 1993;Dixon and Johnstone 1993;Reverchon 1999;Subramanian et al 1997), such as microspheres (Falk et al 1997) threads, fibers, networks (Dixon and Johnstone 1993), sponges, foams, and films. One of the advantages of using SCF in polymer processing is the possibility of producing different solid shapes and structures at low temperature with a minimum amount of residual organic solvents.…”