Sorbitol crystallization can lead to protein aggregation in frozen protein formulations
Sorbitol crystallization can lead to protein aggregation in frozen protein formulations
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DOI：
10.1007/s11095-006-9131-1
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发表时间：
2007-01-01
影响因子：
3.7
通讯作者：
Ratnaswamy, Gayathri
中科院分区：
文献类型：
--
作者：
Piedmonte, Deirdre Murphy;Summers, Christie;Ratnaswamy, Gayathri
目的。这项工作检查了山梨醇中配制的 Fc 融合蛋白在 -30 摄氏度下长期冷冻储存后发生聚集的原因。材料和方法。我们设计了低于环境的差示扫描量热法 (DSC) 实验来捕捉长期冷冻储存的影响。通过尺寸排阻高效液相色谱 (SE-HPLC) 监测制剂样品的物理稳定性。 结果。非冷冻样品的 DSC 分析显示，山梨糖醇中的样品在 -45℃ 下和蔗糖中的样品在 -32℃ 下具有预期的玻璃化转变温度 (T-g')。在时间过程研究中，山梨醇制剂储存在-30℃下，并在不解冻的情况下通过DSC进行分析，观察到两个吸热转变：-20℃下的熔化吸热随着时间的推移而消散，大约两周后在-8℃下出现第二次吸热，并在所有后续时间点持续存在。仅在用山梨醇配制并储存于-30℃的样品中观察到蛋白质聚集，将聚集与上述熔解相关联。结论。观察到的熔化物是结晶物质的特征，表明山梨醇随着时间的推移而结晶。在冷冻过程中，赋形剂必须与蛋白质保持在同一相，以确保蛋白质的稳定性。通过结晶，山梨醇与蛋白质发生相分离，从而导致蛋白质聚集。
Purpose. 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.Materials and 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 (T-g') 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.