Huus Kasper, Havelund Svend, Olsen Helle B, van de Weert Marco, Frokjaer Sven
Department of Pharmaceutics and Analytical Chemistry, The Danish University of Pharmaceutical Sciences, 2100 Copenhagen, Denmark.
Pharm Res. 2006 Nov;23(11):2611-20. doi: 10.1007/s11095-006-9098-y. Epub 2006 Sep 13.
To study the correlation between the thermal and chemical stability of insulin formulations with various insulin hexamer ligands.
The thermal stability was investigated using differential scanning calorimetry (DSC) and near-UV circular dichroism (NUV-CD). The formation of chemical degradation products was studied with reversed-phase and size-exclusion chromatography and mass spectrometry.
An excellent correlation between the thermal stabilization by ligand binding and the deamidation of Asn(B3) was observed. The correlation between thermal stability and the formation of covalent dimer and other insulin related products was less clear. Zinc was found to specifically increase the deamidation and covalent dimer formation rate when the insulin hexamer was not further stabilized by phenolic ligand. Thiocyanate alone had no effect on the thermal stability of the insulin zinc-hexamer but significantly improved the chemical stability at 37 degrees C. At low temperatures thiocyanate induced a conformational change in the insulin hexamer. NUV-CD thermal scans revealed that this effect decreased with temperature; when the thermal denaturation temperature was reached, the effect was eliminated.
Thermal stability can be used to predict the rate of Asn(B3) deamidation in human insulin. Chemical degradation processes that do not rely on the structural stability of the protein do not necessarily correlate to the thermal stability.
研究不同胰岛素六聚体配体的胰岛素制剂的热稳定性和化学稳定性之间的相关性。
采用差示扫描量热法（DSC）和近紫外圆二色光谱法（NUV-CD）研究热稳定性。用反相色谱法、尺寸排阻色谱法和质谱法研究化学降解产物的形成。
观察到配体结合引起的热稳定性与天冬酰胺（B3）脱酰胺作用之间存在良好的相关性。热稳定性与共价二聚体及其他胰岛素相关产物形成之间的相关性不太明显。当胰岛素六聚体未被酚类配体进一步稳定时，发现锌会特异性地提高脱酰胺作用和共价二聚体的形成速率。单独的硫氰酸盐对胰岛素锌六聚体的热稳定性没有影响，但在37℃时显著提高了化学稳定性。在低温下，硫氰酸盐会引起胰岛素六聚体的构象变化。NUV-CD热扫描显示，这种效应随温度降低；当达到热变性温度时，这种效应消失。
热稳定性可用于预测人胰岛素中天冬酰胺（B3）的脱酰胺速率。不依赖蛋白质结构稳定性的化学降解过程不一定与热稳定性相关。
