Sorbitol crystallization can lead to protein aggregation in frozen protein formulations.
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.
Piedmonte
Deirdre Murphy
DM
Department of Pharmaceutics, Amgen Inc, MS 2-1-A, One Amgen Center Drive, Thousand Oaks, California 91320, USA.
Summers
Christie
C
McAuley
Arnold
A
Karamujic
Lejla
L
Ratnaswamy
Gayathri
G
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
D007141:Immunoglobulin Fc Fragments; D010592:Pharmaceutic Aids; D011506:Proteins; D013012:Sorbitol; D013395:Sucrose