Next-Generation GLP-1 Agonist Bioassays: Integrating Precision with Biological Relevance

Glucagon-Like Peptide-1 (GLP-1) is an incretin family peptide hormone with significant therapeutic applications. This study evaluated multiple functional methodologies for assessing GLP-1 agonist biological activity, encompassing in vitro cell-based bioassays and in vivo systems. We developed and comparatively analyzed cell-based bioassays measuring distinct cellular signaling events, such as β-arrestin recruitment, cyclic adenosine monophosphate (cAMP) elevation, and calcium (Ca²⁺) mobilization. Each assay was systematically evaluated for robustness and quality control applicability. All assay systems demonstrated functionality independent of the cellular mechanism employed. Real-time Ca²⁺ flux measurements confirmed the cell-based assay endpoint data, while cross-verification analyses using the oral glucose tolerance test in mice confirmed the representativeness of the cAMP formats. Initial validation studies revealed inter-assay variations, leading to the selection of the cAMP HTRF assay as the preferred quality control methodology. Further assay refinement and subsequent validation studies confirmed the cAMP HTRF assay's suitability for quality control applications. This methodology has been successfully implemented in routine release testing of a commercially available GLP-1 agonist.