Characterisation and physical stability of PEGylated glucagon
Characterisation and physical stability of PEGylated glucagon
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DOI：
10.1016/j.ijpharm.2006.09.002
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发表时间：
2007-02-07
影响因子：
5.8
通讯作者：
Moeller, E. Horn
中科院分区：
文献类型：
--
作者：
Stigsnaes, Pernille;Frokjaer, Sven;Moeller, E. Horn
使用 PEG 5000 在 Lys-12 处对胰高血糖素进行单聚乙二醇化，以检查纯化和冷冻干燥过程中对构象和物理稳定性的影响。模型肽胰高血糖素高度不稳定，很容易在溶液中形成原纤维。通过 FTIR 和远紫外 CD 测定二级结构，并通过硫黄素 T 测定评估物理稳定性。包括胰高血糖素样品，其经过与胰高血糖素-PEG 5000 相同的 RP-HPLC 纯化和/或冷冻干燥。纯化和冷冻干燥后胰高血糖素样品通过 FTIR 显示分子间 P-片层的形成，这与硫黄素 T 测定中纤维颤动的较短滞后时间相关。对于胰高血糖素-PEG 5000，分子间 P-片层的形成不太明显，并且通过硫磺素 T 测定未检测到原纤维化。显然，聚乙二醇化在纯化和冷冻干燥后显着提高了胰高血糖素的物理稳定性，可能是通过肽-肽相互作用的空间位阻。通过液体 FTIR 观察到冷冻干燥和重构的肽样品的二级结构的变化。 α-螺旋的峰移至1664 cm(-1)，这可能是通过3(10)-螺旋的形成来解释的。远紫外 CD 无法检测到 310 螺旋和分子间 β-折叠，所有肽样品都显示相似的光谱。 总之，与胰高血糖素相比，胰高血糖素-PEG 5000 在纯化和冷冻干燥过程中显示出显着改善的物理稳定性。 (c) 2006 Elsevier B.V. 保留所有权利。
Glucagon was mono-PEGylated with PEG 5000 at Lys-12 to examine the effect on conformation and physical stability during purification and freeze-drying. The model peptide glucagon is highly unstable and readily forms fibrils in solution. Secondary structure was determined by FTIR and far-UV CD and physical stability was assessed by the Thioflavin T assay.Glucagon samples were included, which underwent the same RP-HPLC purification and/or freeze-drying as glucagon-PEG 5000. After purification and freeze-drying glucagon samples showed formation of intermolecular P-sheet by FTIR, this correlated with shorter lag-times for fibrillation in the Thioflavin T assay. Formation of intermolecular P-sheet was less apparent for glucagon-PEG 5000 and no fibrillation was detected by Thioflavin T assay. Apparently PEGylation significantly improved the physical stability of glucagon after purification and freeze-drying, possibly by steric hindrance of peptide-peptide interactions.Alterations in the secondary structure were observed for freeze-dried and reconstituted peptide samples by liquid FTIR. The peak for alpha-helix shifted to 1664 cm(-1), which could possibly be explained by formation of 3(10)-helix. Neither 310-helix nor intermolecular beta-sheet could be detected by far-UV CD, where all peptide samples showed similar spectra.In conclusion, glucagon-PEG 5000 showed a significantly improved physical stability during purification and freeze-drying compared to glucagon. (c) 2006 Elsevier B.V. All rights reserved.