title: Sorbitol crystallization can lead to protein aggregation in frozen protein formulations.
abstract: OBJECTIVE
  This work examines the cause of aggregation of an Fc-fusion protein formulated in sorbitol upon frozen storage for extended periods of time at -30 degrees C.
  
  
  METHODS
  We designed sub-ambient differential scanning calorimetry (DSC) experiments to capture the effects of long-term frozen storage. The physical stability of formulation samples was monitored by size exclusion high performance liquid chromatography (SE-HPLC).
  
  
  RESULTS
  DSC analysis of non-frozen samples shows the expected glass transitions (Tg') at -45 degrees C for samples in sorbitol and at -32 degrees C in sucrose. In time course studies where sorbitol formulations were stored at -30 degrees C and analyzed by DSC without thawing, two endothermic transitions were observed: a melting endotherm at -20 degrees C dissipated over time, and a second endotherm at -8 degrees C was seen after approximately 2 weeks and persisted in all later time points. Protein aggregation was only seen in the samples formulated in sorbitol and stored at -30 degrees C, correlating aggregation with the aforementioned melts.
  
  
  CONCLUSIONS
  The observed melts are characteristic of crystalline substances and suggest that the sorbitol crystallizes over time. During freezing, the excipient must remain in the same phase as the protein to ensure protein stability. By crystallizing, the sorbitol is phase-separated from the protein, which leads to protein aggregation.
authors.affiliation: Department of Pharmaceutics, Amgen Inc, MS 2-1-A, One Amgen Center Drive, Thousand Oaks, California 91320, USA.
authors.forename: Deirdre Murphy
authors.initials: DM
authors.lastname: Piedmonte
authors.forename: Christie
authors.initials: C
authors.lastname: Summers
authors.forename: Arnold
authors.initials: A
authors.lastname: McAuley
authors.forename: Lejla
authors.initials: L
authors.lastname: Karamujic
authors.forename: Gayathri
authors.initials: G
authors.lastname: Ratnaswamy
mesh_terms: D002152:Calorimetry, Differential Scanning; D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004355:Drug Stability; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments; D010592:Pharmaceutic Aids; D011506:Proteins / Q000737:chemistry*; D013012:Sorbitol / Q000737:chemistry*; D013395:Sucrose / Q000737:chemistry
chemical_list: D007141:Immunoglobulin Fc Fragments; D010592:Pharmaceutic Aids; D011506:Proteins; D013012:Sorbitol; D013395:Sucrose