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<121> bioidentity test, rabbit blood sugar method to test insulin and its analogs.