Protein purification with vapor-phase carbon dioxide

Michael A. Winters, David Z. Frankel, Pablo G. Debenedetti, Jannette Carey, Margaret Devaney, Todd M. Przybycien

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47 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)247-258
Number of pages12
JournalBiotechnology and Bioengineering
Issue number3
StatePublished - Feb 5 1999

All Science Journal Classification (ASJC) codes

  • Applied Microbiology and Biotechnology
  • Bioengineering
  • Biotechnology


  • CO
  • Fractional precipitation
  • GAS
  • Gaseous antisolvent
  • Proteins


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