Remmele R L, Nightlinger N S, Srinivasan S, Gombotz W R
Immunex Corporation, Seattle, WA 98101, USA.
Pharm Res. 1998 Feb;15(2):200-8. doi: 10.1023/a:1011902215383.
To elucidate the solution conditions that confer stability of aqueous IL-1R using differential scanning calorimetry (DSC).
Optimal pH conditions were determined by monitoring degradation products encountered during accelerated studies (at elevated temperatures) using SDS-PAGE. At the pH optimum, DSC screened for excipients that enhanced thermal stability by shifting the Tm to higher values. Using SEC the relationship between thermal unfolding and stability was investigated by considering if lower Tm's in the presence of preservatives correlated with degradation products at 37 degrees C over time. The degree of aggregation relative to that of a control determined the level of stability achieved.
Circular dichroism (CD) measurements confirmed molecular modeling studies showing IL-1R to be about 39% beta-sheet. Two major transitions characterized the DSC data with Tm's observed near 47 degrees C and 66 degrees C. Among 21 excipients screened, NaCl exhibited the greatest stabilizing influences based on shifting the low temperature transition to 53 degrees C. The low temperature transition was later found to comprise two transitions, yielding a total of three melting transitions for IL-1R. High Tm's arising from the presence of preservatives correlated with the order of stability (i.e., 0.065% phenol > 0.1% m-Cresol > 0.9% benzyl alcohol).
The three melting transitions are consistent in origin with the cooperative unfolding of three unique immunoglobulin-like domains of IL-1R. Optimal stability was achieved in 20 mM sodium citrate at pH 6 with sufficient NaCl to attain the tonicity of human serum. A correlation between the predicted ranking of stability and the extent of aggregation was demonstrated using DSC.
使用差示扫描量热法（DSC）阐明赋予水性白细胞介素-1受体（IL-1R）稳定性的溶液条件。
通过使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）监测加速研究（在高温下）过程中遇到的降解产物来确定最佳pH条件。在最佳pH值下，DSC筛选能够通过将熔点（Tm）移至更高值来提高热稳定性的辅料。使用尺寸排阻色谱法（SEC），通过考虑在防腐剂存在下较低的Tm是否与37℃下随时间的降解产物相关，研究热解折叠与稳定性之间的关系。相对于对照的聚集程度决定了所达到的稳定性水平。
圆二色性（CD）测量证实了分子模拟研究，表明IL-1R约39%为β-折叠。DSC数据有两个主要转变，观察到的Tm值接近47℃和66℃。在筛选的21种辅料中，基于将低温转变移至53℃，氯化钠表现出最大的稳定作用。后来发现低温转变包括两个转变过程，使得IL-1R共有三个熔融转变过程。由防腐剂导致的高Tm值与稳定性顺序相关（即0.065%苯酚>0.1%间甲酚>0.9%苯甲醇）。
这三个熔融转变在起源上与IL-1R的三个独特免疫球蛋白样结构域的协同解折叠一致。在pH 6的20 mM柠檬酸钠中加入足够的氯化钠以达到人血清的张力时可实现最佳稳定性。使用DSC证明了稳定性的预测排名与聚集程度之间的相关性。
