title: Long-term and high-temperature storage of supercritically-processed microparticulate protein powders.
abstract: OBJECTIVE
  The long-term and high-temperature storage of dry, micron-sized particles of lysozyme, trypsin, and insulin was investigated. Subsequent to using supercritical carbon dioxide as an antisolvent to induce their precipitation from a dimethylsulfoxide solution, protein microparticles were stored in sealed containers at -25, -15, 0, 3, 20, 22, and 60 degrees C. The purpose of this study was to investigate the suitability of supercritical antisolvent precipitation as a finishing step in protein processing.
  
  
  METHODS
  Karl Fisher titrations were used to determine the residual moisture content of commercial and supercritically-processed protein powders. The secondary structure of the dry protein particles was determined periodically during storage using Raman spectroscopy. The proteins were also redissolved periodically in aqueous buffers and assayed spectrophotometrically for biological activity and by circular dichroism for structural conformation in solution.
  
  
  RESULTS
  Amide I band Raman spectra indicate that the secondary structure of the protein particles, while perturbed from that of the solution state, remained constant in time, regardless of the storage temperature. The recoverable biological activity upon reconstitution for the supercritically-processed lysozyme and trypsin microparticles was also preserved and found to be independent of storage temperature. Far UV circular dichroism spectra support the bioactivity assays and further suggest that adverse structural changes, with potential to hinder renaturation upon redissolution, do not take place during storage.
  
  
  CONCLUSIONS
  The present study suggests that protein precipitation using supercritical fluids may yield particles suitable for long-term storage at ambient conditions.
authors.affiliation: Department of Chemical Engineering, Princeton University, New Jersey 08544-5263, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Winters
authors.forename: P G
authors.initials: PG
authors.lastname: Debenedetti
authors.forename: J
authors.initials: J
authors.lastname: Carey
authors.forename: H G
authors.initials: HG
authors.lastname: Sparks
authors.forename: S U
authors.initials: SU
authors.lastname: Sane
authors.forename: T M
authors.initials: TM
authors.lastname: Przybycien
mesh_terms: D000818:Animals; D002245:Carbon Dioxide; D002417:Cattle; D011232:Chemical Precipitation; D002942:Circular Dichroism; D004356:Drug Storage; D007328:Insulin / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D010316:Particle Size; D011208:Powders; D017433:Protein Structure, Secondary; D012996:Solutions; D013059:Spectrum Analysis, Raman; D013696:Temperature; D014357:Trypsin / Q000737:chemistry*
chemical_list: D007328:Insulin; D011208:Powders; D012996:Solutions; D002245:Carbon Dioxide; D009113:Muramidase; D014357:Trypsin