TY - JOUR
T1 - Precipitation of proteins in supercritical carbon dioxide
AU - Winters, Michael A.
AU - Knutson, Barbara L.
AU - Debenedetti, Pablo G.
AU - Sparks, H. Gerald
AU - Przybycien, Todd M.
AU - Stevenson, Cynthia L.
AU - Prestrelski, Steven J.
PY - 1996/6
Y1 - 1996/6
N2 - 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.
AB - 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.
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U2 - 10.1021/js950482q
DO - 10.1021/js950482q
M3 - Article
C2 - 8773954
AN - SCOPUS:0029891659
VL - 85
SP - 586
EP - 594
JO - Journal of Pharmaceutical Sciences
JF - Journal of Pharmaceutical Sciences
SN - 0022-3549
IS - 6
ER -