GLP-1类似物的体外活性及宿主杂质测定

In vitro activity and host impurity detection of GLP-1 analogues

胰高血糖素样多肽1(glucagon-like peptide-1,GLP-1)通过提高细胞内环磷酸腺苷(cyclic adenosine monophosphate,c AMP)水平刺激胰岛β细胞分泌胰岛素。GLP-1受体(GLP-1 receptor,GLP-1R)是2型糖尿病药物研发的重要靶点,其激动剂也已成为新型抗高血糖药物。本研究基于稳定表达GLP-1受体的GLP-1R-GFP(green fluorescent protein)-HEK293A细胞,通过均相时间分辨荧光法建立了一套适合GLP-1类似物的体外活性检测方法。通过对细胞活性、细胞密度、是否避光等检测条件进行优化后,利用该方法检测了利拉鲁肽等4个GLP-1类似物刺激GLP-1R-GFP-HEK293A细胞后所产生的c AMP含量变化,通过c AMP量效曲线计算其半数有效剂量值(concen‐tration for 50%of maximal effect,EC50),对药物的体外活性进行评价。本研究还利用酶联免疫吸附实验和定量实时聚合酶链式反应实验分别测定了GLP-1类似物宿主细胞蛋白和宿主残留DNA含量,为高通量筛选GLP-1类似物提供了稳定、可靠和灵敏的体外活性分析和宿主杂质检测方法。

Glucagon-like peptide-1(GLP-1)could increase the level of cyclic adenosine monophosphate(c AMP)in cells to stimulate insulin secretion inβcells of pancreas.So GLP-1 analogues,such as liraglutide,have become new anti-hyperglycemia drugs for type 2 diabetes.In this study,a set of in vitro activity detection method suitable for GLP-1 analogues was established using GLP-1R-GFP(green fluorescent protein,GFP)-HEK293A cells which stably expressing GLP-1 receptor(GLP-1R).After optimizing the detection parameters such as assay sensitivity,cell density,and the incubation condition,the c AMP content level of GLP-1R-GFP-HEK293A cells stimulated by four GLP-1 analogues,such as liraglutide,were detected by homogeneous time-resolved fluores‐cence(HTRF).The values of concentration for 50%of maximal effect(EC50)of GLP-1 analogues were calculated by c AMP dose-response curve to evaluate the in vitro activity of those drugs.In addition,enzyme-linked immuno‐sorbent assay and quantitative polymerase chain reaction were applied to determine the content of host cell protein and host residual DNA,respectively.This study provides a stable,reliable,and sensitive in vitro activity analysis and host impurity detection method for high-throughput screening of GLP-1 analogues.