Development of a robust functional cell-based assay for replacing the rabbit blood sugar bioidentity test of insulin glargine drug substance

•A cell-based assay, H4IIE G6P-Luc reporter assay, was developed by utilizing insulin’s role in regulating hepatic gluconeogenesis pathway.•This assay was optimized to measure insulin glargine potency, and demonstrated good linearity, accuracy and precision in a qualification study.•A DoE study demonstrated the robustness of this assay with multiple critical factors evaluated.•This assay is comparable to an established phosphor-IR assay with advantages including lower variation, lower cost and ease of handling.•This reporter assay is suitable to replace current USP bioidentity test, rabbit blood sugar method to test insulin and its analogs. A rabbit blood sugar bioidentity assay is required by the FDA to evaluate biological activity for all insulin and its analogs per USP guideline. Not only are a large number of live animals used, but the rabbit blood sugar method is also highly variable and expensive. Our goal is to develop a functional cell-based assay to replace rabbit blood sugar method. An H4IIE G6P-Luc reporter assay was developed by utilizing insulin’s role in regulating hepatic gluconeogenesis pathway. It is known that Glucose 6-phosphatase is a rate-limiting enzyme in the gluconeogenesis pathway, and the mRNA expression of its catalytic subunit, G6PC, is highly regulated by insulin. A G6P-Luc stable cell line in H4IIE hepatocytes was first generated by stably expressing luciferase reporter gene driven by human G6PC promoter via lentivirus technology. The cell-based assay was developed and optimized to demonstrate good dose-dependent responsiveness to insulin. We further qualified the assay with two analysts through multiple runs, and demonstrated excellent performance characteristics of linearity, accuracy, and precision. A robustness study was then conducted to define critical factors for assay performance. We compared this newly developed assay with a previously established cell-based pIR MSD assay, which measures insulin receptor phosphorylation (pIR) in HepG2 cell line using Meso-Scale Discovery (MSD) technology. The comparability study was conducted to compare the two assays using samples generated from forced degradation. This study showed high correlation between assays, and both are stability indicating. Compared with the pIR MSD assay, the G6P-Luc assay not only has a significantly lower variability in qualification studies, but also offers many other advantages, including ease of use in a quality control laboratory with fewer steps, low