Division of Pharmaceutics, Freeze Drying Focus Group, University of Erlangen, Erlangen 91058, Germany.
J Pharm Sci. 2013 Mar;102(3):813-26. doi: 10.1002/jps.23427. Epub 2012 Dec 20.
The objective of this study was to investigate the effect of histidine on the stability of the model protein lactate dehydrogenase (LDH) during freeze-drying. Several parameters were varied including pH of the bulk solution, histidine concentration, and performance of an annealing step during freezing. First, histidine was used as a buffer in the protein formulations and compared with "conventional" potassium phosphate and citrate buffer systems. For this purpose, sucrose or mannitol was used as stabilizers. Second, the possibility of using histidine as both buffer and stabilizer (cryoprotectant and lyoprotectant) in the protein formulations was evaluated with focus on protein stability and the physical state of histidine in the final product, in addition to cake elegance. 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 improved stability in histidine buffer in comparison with other buffers. Protein stability and the tendency of histidine to crystallize during freeze-drying were pH dependent. Annealing destabilized LDH and resulted in a decrease of the activity recovery. However, the extent of protein destabilization caused by annealing appears to be also pH dependent.
本研究旨在探讨组氨酸对模型蛋白乳酸脱氢酶（LDH）在冷冻干燥过程中稳定性的影响。考察了多个参数，包括本体溶液的 pH 值、组氨酸浓度和冷冻过程中退火步骤的性能。首先，组氨酸在蛋白配方中被用作缓冲液，并与“传统”的磷酸钾和柠檬酸盐缓冲体系进行了比较。为此，使用蔗糖或甘露醇作为稳定剂。其次，评估了组氨酸在蛋白配方中同时作为缓冲液和稳定剂（保护剂和冻干保护剂）的可能性，重点关注蛋白稳定性以及最终产品中组氨酸的物理状态和外观。蛋白稳定性通过测量模型蛋白 LDH 冷冻干燥后的活性回收率进行功能评估，通过分析蛋白二级结构进行结构评估。与其他缓冲液相比，LDH 在组氨酸缓冲液中表现出更好的稳定性。蛋白稳定性和组氨酸在冷冻干燥过程中结晶的趋势与 pH 值有关。退火会使 LDH 失稳，并导致活性回收率降低。然而，退火引起的蛋白失稳程度似乎也与 pH 值有关。
