Division of Pharmaceutics, Freeze-Drying Focus Group, University of Erlangen, 91058 Erlangen, Germany.
J Pharm Sci. 2012 Jul;101(7):2534-44. doi: 10.1002/jps.23173. Epub 2012 Apr 25.
The objective of this study was to investigate the influence of mannitol crystallization and the effect of annealing on the stability of lactate dehydrogenase (LDH) during freeze-drying. For this purpose, protein formulations with different weight ratios of mannitol to sucrose were freeze-dried with and without annealing. Product crystallinity was calculated based on differential scanning calorimetry data. Protein stability was evaluated both functionally by measuring the activity recovery of the model protein LDH after freeze-drying, and structurally by analyzing the protein secondary structure. LDH showed lower stability in annealed samples, and a correlation could be established between the extent of product crystallinity and the stability of the model protein. The destabilizing effect of mannitol crystallization on LDH during freeze-drying can likely be attributed to removal of mannitol from the amorphous phase containing the protein. In addition, the formation of mannitol crystals seemed to enhance the interfacial denaturation of protein during freeze-drying. The use of 0.01% of Tween 80 in the formulation greatly improved the structural and functional stability of LDH against stresses induced by mannitol crystallization.
本研究旨在探讨甘露醇结晶和退火对冻干过程中乳酸脱氢酶（LDH）稳定性的影响。为此，采用不同甘露醇与蔗糖重量比的蛋白质配方进行了有退火和无退火的冷冻干燥实验。根据差示扫描量热法数据计算了产物结晶度。通过测量模型蛋白 LDH 冻干后的活性回收率，从功能上评估蛋白质稳定性，通过分析蛋白质二级结构，从结构上评估蛋白质稳定性。结果表明，退火样品中 LDH 的稳定性较低，并且产物结晶度的程度与模型蛋白的稳定性之间存在相关性。甘露醇结晶在冻干过程中对 LDH 的失稳作用可能归因于从含有蛋白质的无定形相中去除甘露醇。此外，甘露醇晶体的形成似乎在冻干过程中增强了蛋白质的界面变性。配方中使用 0.01%的吐温 80 可大大提高 LDH 的结构和功能稳定性，使其免受甘露醇结晶引起的应激。
