Precipitation of proteins in supercritical carbon dioxide

Michael A. Winters, Barbara L. Knutson, Pablo G. Debenedetti, H. Gerald Sparks, Todd M. Przybycien, Cynthia L. Stevenson, Steven J. Prestrelski

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192 Citations (SciVal)


Supercritical CO2 was used as an antisolvent to form protein particles that exhibited minimal loss of activity upon reconstitution. Organic protein solutions were sprayed under a variety of operating conditions into the supercritical fluid, causing precipitation of dry, microparticulate (1-5 μm) protein powders. Three proteins were studied: trypsin, lysozyme, and insulin. Amide 1 band Raman spectra were used to estimate the α-helix and β-sheet structural contents of native and precipitate powders of each protein. Analysis of the Raman spectra revealed minimal (lysozyme), intermediate (trypsin), and appreciable (insulin) changes in secondary structure with respect to the commercial starting materials. The perturbations in secondary structure suggest that the most significant event during supercritical fluid-induced precipitation involved the formation of β-sheet structures with concomitant decreases of α- helix. Amide I band Raman and Fourier-transform infrared (FTIR) spectra indicate that higher operating temperatures and pressures lead to more extensive β- sheet-mediated intermolecular interactions in the precipitates. Raman and FTIR spectra of redissolved precipitates are similar to those of aqueous commercial proteins, indicating that conformational changes were reversible upon reconstitution. These results suggest that protein precipitation in supercritical fluids can be used to form particles suitable for controlled release, direct aerosol delivery to the lungs, and long-term storage at ambient conditions.

Original languageEnglish
Pages (from-to)586-594
Number of pages9
JournalJournal of Pharmaceutical Sciences
Issue number6
StatePublished - Jun 1996

Bibliographical note

Funding Information:
We thank Joseph Goodhouse of the Department of Molecular Biology at Princeton for assistance with the electron microscopy and Mohsen A. Khalili of the Dupont Experimental Station for performing the Coulter Counter analysis. This work was supported by the National Science Foundation, Interfacial, Transport, and Separations Processes Program (Grant CTS-9321978 to P.G.D.) and Career Award and Research Infrastructure Programs (Grants BES-9502184 and 9413527 to T.M.P.), and by the Air Force Office of Scientific Research (Grant F49620-93-0040 to P.G.D.).

ASJC Scopus subject areas

  • Pharmaceutical Science


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