title: Raffinose crystallization during freeze-drying and its impact on recovery of protein activity.
abstract: OBJECTIVE
  To study i) phase transitions in raffinose solution in the frozen state and during freeze-drying and ii) evaluate the impact of raffinose crystallization on the recovery of protein activity in reconstituted lyophiles.
  
  
  METHODS
  X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to study the frozen aqueous solutions of raffinose pentahydrate. Phase transitions during primary and secondary drying were monitored by simulating the entire freeze-drying process, in situ, in the sample chamber of the diffractometer. The activity of lactate dehydrogenase (LDH) in reconstituted lyophiles was determined spectrophotometrically.
  
  
  RESULTS
  Raffinose formed a kinetically stable amorphous freeze-concentrated phase when aqueous solutions were frozen at different cooling rates. When these solutions were subjected to primary drying without annealing, raffinose remained amorphous. Raffinose crystallized as the pentahydrate when the solutions were annealed at a shelf temperature of -10 degrees C. Primary drying of these annealed systems resulted in the dehydration of raffinose pentahydrate to an amorphous phase. The phase separation of the protein from the amorphous raffinose in these two systems during freeze-drying resulted in a significant reduction in the recovery of LDH activity, even though the lyophile was amorphous.
  
  
  CONCLUSIONS
  Annealing of frozen aqueous raffinose solutions can result in solute crystallization, possibly as the pentahydrate. The crystalline pentahydrate dehydrates during primary drying to yield an amorphous lyophile. Raffinose crystallization during freeze-drying is accompanied by a significant loss of protein activity.
authors.affiliation: College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Koustuv
authors.initials: K
authors.lastname: Chatterjee
authors.forename: Evgenyi Y
authors.initials: EY
authors.lastname: Shalaev
authors.forename: Raj
authors.initials: R
authors.lastname: Suryanarayanan
mesh_terms: D002152:Calorimetry, Differential Scanning / Q000379:methods; D003460:Crystallization; D005612:Freeze Drying / Q000379:methods; D011208:Powders; D011506:Proteins / Q000737:chemistry*; D011887:Raffinose / Q000737:chemistry*; D014961:X-Ray Diffraction
chemical_list: D011208:Powders; D011506:Proteins; D011887:Raffinose