Effects of tween 80 and sucrose on acute short-term stability and long-term storage at -20 °C of a recombinant hemoglobin
Effects of tween 80 and sucrose on acute short-term stability and long-term storage at -20 °C of a recombinant hemoglobin
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DOI：
10.1021/js980140v
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发表时间：
1998-09-01
影响因子：
3.8
通讯作者：
Randolph, TW
中科院分区：
文献类型：
--
作者：
Kerwin, BA;Heller, MC;Randolph, TW
在磷酸盐缓冲盐水中向重组血红蛋白中加入低水平的表面活性剂聚氧乙烯20脱水山梨糖醇单油酸酯（吐温80），可最大限度地降低急性冻融研究期间的蛋白质聚集水平。仅向磷酸盐缓冲盐水制剂中添加蔗糖（高达0.5 M），可提供最低限度的抗冻融诱导聚集的保护。与急性稳定性研究相反，在磷酸盐缓冲盐水中仅含有吐温80的那些制剂中，在-20 ℃下长期储存诱导聚集和高铁血红蛋白形成。在制剂中加入0.1至0.5 M的蔗糖可防止聚集体的形成，并在长期-20 ℃储存期间严重阻止高铁血红蛋白的形成。使用16-doxyl硬脂酸分配技术和电子顺磁共振，未观察到吐温80与血红蛋白的特异性结合。用傅里叶变换红外光谱法观察到在不存在和存在吐温80的情况下冷冻过程中蛋白质二级结构的微小结构变化。通过添加蔗糖，可部分防止这些变化。很可能吐温80在急性稳定性研究期间通过防止血红蛋白到达空气-液体界面或液体-表面界面而严重减少了蛋白质聚集。在长期储存过程中观察到的高铁血红蛋白形成和聚集的减少可以基于蔗糖以类似于优先排除理论的方式减少蛋白质的局部解折叠的前提来解释（Arakawa，T.;和Timasheff，S. N. 1982，Biochemistry 1982，21，6536-6544）。这些研究表明，急性制剂筛选研究虽然有用，但不一定能预测长期储存期间的蛋白质稳定性。
The addition of low levels of surfactant polyoxyethylene 20 sorbitan monooleate, Tween 80, to recombinant hemoglobin in phosphate-buffered saline minimized the level of protein aggregation during acute freeze-thaw studies. Addition of sucrose alone to the phosphate-buffered saline formulation, up to 0.5 M, provided minimal protection against freeze-thaw induced aggregation. In contrast to the acute stability studies, long-term storage at -20 degrees C induced aggregation and methemoglobin formation in those formulations containing only Tween 80 in phosphate-buffered saline. Addition of sucrose between 0.1 and 0.5 M to the formulation prevented formation of aggregates and severely arrested methemoglobin formation during the long-term -20 degrees C storage. Specific binding of Tween 80 to the hemoglobin was not observed using 16-doxyl stearic acid partitioning techniques with electron paramagnetic resonance. Minor structural changes to the protein secondary structure during freezing in the absence and presence of Tween 80 were observed with Fourier transform infrared spectroscopy. The alterations were partially prevented by addition of the sucrose. it is likely that the Tween 80 severely reduced protein aggregation during the acute stability studies by preventing the hemoglobin from reaching the air-liquid interface or the liquid-surface interfaces. The reduction in methemoglobin formation and aggregation observed during long-term storage can be accounted for on the premise that the sucrose reduced localized unfolding of the protein in a manner similar to the preferential exclusion theory (Arakawa, T.; and Timasheff, S. N. 1982, Biochemistry 1982, 21, 6536-6544). These studies demonstrate that acute formulation screening studies, albeit useful, may not necessarily predict protein stability during long-term storage.