Department of Chemical Engineering, University of Virginia, 102 Engineer's Way, Charlottesville, Virginia 22904, USA.
Pharm Res. 2011 May;28(5):1179-93. doi: 10.1007/s11095-011-0383-z. Epub 2011 Feb 8.
The impact of freeze-thaw (F/T) on structure integrity of protein therapeutics is poorly understood, partially due to lack of methods to detect protein structural perturbations during F/T processing in the frozen state.
A new approach of hydrogen/deuterium exchange was developed to separate and distinguish the specific impact of single freezing and F/T cycling on protein structure, using lactate dehydrogenase (LDH) as model system.
In the freezing process, a fraction of LDH molecules that was inversely dependent on protein concentration was observed to partially denature its structure. Local structural perturbations were localized by peptide level HX analysis to the surface residues in segments 91-132, 170-237 and 288-331. In contrast, F/T cycling led to irreversible LDH aggregation with global structural unfolding. Residual solvent-protected structure was only detected in the aggregates for three segments, 13-31, 109-117 and 133-143, that were coincident with the consensus aggregation hotspots predicted by four different algorithms.
Results indicate freezing preferentially disturbs local structure at the surface residues, consistent with ice-solution interface-mediated denaturation mechanism. F/T-induced aggregation begins as partial denaturation during freezing, but is accompanied by more comprehensive structural rearrangement during F/T cycling.
冻融（F/T）对蛋白质治疗药物结构完整性的影响尚未被充分理解，部分原因是缺乏在冷冻状态下检测 F/T 处理过程中蛋白质结构变化的方法。
开发了一种新的氢/氘交换方法，用于分离和区分单次冷冻和 F/T 循环对蛋白质结构的特定影响，以乳酸脱氢酶（LDH）为模型系统。
在冷冻过程中，观察到与蛋白质浓度成反比的 LDH 分子部分变性，其结构发生局部结构变化。通过肽水平 HX 分析将局部结构变化定位到残基 91-132、170-237 和 288-331 表面残基。相比之下，F/T 循环导致 LDH 不可逆聚集，结构全面展开。在三个片段（13-31、109-117 和 133-143）的聚集物中仅检测到残留的溶剂保护结构，这些片段与四种不同算法预测的共识聚集热点一致。
结果表明，冷冻优先在表面残基处扰乱局部结构，这与冰-溶液界面介导的变性机制一致。F/T 诱导的聚集首先在冷冻过程中发生部分变性，但在 F/T 循环过程中伴随着更全面的结构重排。
