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A Novel GLP-1R/fHCS Method to Determine the Bioactivity of Glucagon-- Like Peptide 1(GLP-1) Analogous Drugs In Vitro

Author(s):

Jie Wang, Ping Lu, Shengting Zhang, Wanwan Li, Xiuling Ji, Jing Li, Hui Zhang, Yunlin Wei* and Chenggang Liang*   Pages 1 - 10 ( 10 )

Abstract:


Background: Current methods for assaying bioactivity of GLP-1 analogues are costly and time consuming, mainly depend on AlphaScreen luminous beads, based on fluorescent resonance energy transfer technology, and cAMPGloTM Max assay kit, based on ATP to generate a negative-association curve.

Objective: To establish a sensitive, stable, and low-cost method for the determination of biological activity of therapeutic glucagon-like peptide-1(GLP-1) and their analogous drugs in vitro in quality control.

Methods: An applicable fluorescent high-content system (fHCS) method was developed in U2OS cell which stably transfected with green fluorescent protein (GFP) labeled at the C-terminal of glucagon-like peptide-1 receptor (GLP-1R). HCS was firstly used to measure per unit area of fluorescent intensity changes resulting from drug-stimulated receptor endocytosis.

Results: The detection sensitivity improved at least 30 folds than other reported methods. This method showed a wide detection range with a good linear regression of R2 >0.98. In five scgs from 50~150% SCG, the accuracy achieved the standard of the 41th United States Pharmacopoeia with all biases being less than 7.5%. The precision attained to the 9.0th European Pharmacopoeia standard with all relative standard deviations (RSD)s below 10%. This assay result can keep stable over 100 h at room temperature. Furthermore, the U2OS/GLP-1R cells showed a sharp specificity towards GLP-1 analogues during detecting activities in a mixture of different ligands.

Conclusion: These results suggested that the GLP-1R/fHCS method can be employed broadly in bioactivity determination of GLP-1 analogous drugs and as a platform for screening GLP-1 similar active ingredients in natural products.

Keywords:

GFP labeled cell, high content system, determination, GLP-1 analogues, bioactivity, quality control.

Affiliation:

Kunming University of Science and Technology, Kunming, Yunnan, 650500, National Institutes for Food and Drug Control, Beijing, 102269, Kunming University of Science and Technology, Kunming, Yunnan, 650500, Kunming University of Science and Technology, Kunming, Yunnan, 650500, Kunming University of Science and Technology, Kunming, Yunnan, 650500, National Institutes for Food and Drug Control, Beijing, 102269, National Institutes for Food and Drug Control, Beijing, 102269, Kunming University of Science and Technology, Kunming, Yunnan, 650500, National Institutes for Food and Drug Control, Beijing, 102269



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