TY - JOUR
T1 - Distinct effects of sucrose and trehalose on protein stability during supercritical fluid drying and freeze-drying
AU - Jovanović, Nataša
AU - Bouchard, Andréanne
AU - Hofland, Gerard W.
AU - Witkamp, Geert Jan
AU - Crommelin, Daan J.A.
AU - Jiskoot, Wim
PY - 2006/3
Y1 - 2006/3
N2 - Supercritical fluid (SCF) drying has been proposed as an alternative for freeze-drying to stabilize proteins. Here we studied the influence of sucrose and trehalose during SCF drying on the protein stability and the physical powder characteristics of lysozyme and myoglobin formulations. The results obtained with SCF drying were compared with the results after freeze-drying of the same solutions. Aqueous protein solutions, with or without sugar, were sprayed into a SCF mixture of carbon dioxide and ethanol. The dried products were analyzed by residual water measurements, scanning electron microscopy, X-ray powder diffraction and differential scanning calorimetry. After reconstitution the protein structure was studied by UV/VIS, circular dichroism and fluorescence spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioactivity assay (lysozyme). The SCF dried and freeze-dried formulations showed comparable water contents, but their physical properties were substantially different. All freeze-dried cakes were amorphous with fully preserved protein structure. SCF dried sucrose-containing formulations showed agglomerated crystalline particles, whereas SCF dried trehalose-containing formulations appeared to consist of amorphous spherical particles. Particle morphology of excipients-free proteins was protein specific. Nearly all SCF dried lysozyme could be readily reconstituted, but for myoglobin significant fractions of SCF protein did not dissolve, especially in the absence of sugars. Covalent aggregation was not observed for the two proteins. For the recovered soluble fractions, the secondary protein structure was preserved. The tertiary structure was preserved for lysozyme, but not entirely for myoglobin. Surprisingly, during SCF drying trehalose was less protective than sucrose for myoglobin.
AB - Supercritical fluid (SCF) drying has been proposed as an alternative for freeze-drying to stabilize proteins. Here we studied the influence of sucrose and trehalose during SCF drying on the protein stability and the physical powder characteristics of lysozyme and myoglobin formulations. The results obtained with SCF drying were compared with the results after freeze-drying of the same solutions. Aqueous protein solutions, with or without sugar, were sprayed into a SCF mixture of carbon dioxide and ethanol. The dried products were analyzed by residual water measurements, scanning electron microscopy, X-ray powder diffraction and differential scanning calorimetry. After reconstitution the protein structure was studied by UV/VIS, circular dichroism and fluorescence spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioactivity assay (lysozyme). The SCF dried and freeze-dried formulations showed comparable water contents, but their physical properties were substantially different. All freeze-dried cakes were amorphous with fully preserved protein structure. SCF dried sucrose-containing formulations showed agglomerated crystalline particles, whereas SCF dried trehalose-containing formulations appeared to consist of amorphous spherical particles. Particle morphology of excipients-free proteins was protein specific. Nearly all SCF dried lysozyme could be readily reconstituted, but for myoglobin significant fractions of SCF protein did not dissolve, especially in the absence of sugars. Covalent aggregation was not observed for the two proteins. For the recovered soluble fractions, the secondary protein structure was preserved. The tertiary structure was preserved for lysozyme, but not entirely for myoglobin. Surprisingly, during SCF drying trehalose was less protective than sucrose for myoglobin.
KW - Freeze-drying
KW - Protein formulation
KW - Protein stability
KW - Sucrose
KW - Supercritical fluid drying
KW - Trehalose
UR - http://www.scopus.com/inward/record.url?scp=31344451903&partnerID=8YFLogxK
U2 - 10.1016/j.ejps.2005.11.003
DO - 10.1016/j.ejps.2005.11.003
M3 - Article
C2 - 16338123
AN - SCOPUS:31344451903
SN - 0928-0987
VL - 27
SP - 336
EP - 345
JO - European Journal of Pharmaceutical Sciences
JF - European Journal of Pharmaceutical Sciences
IS - 4
ER -