An integrated bioprocess for the production of insulin icodec in Pichia pastoris

The global prevalence of diabetes highlights the urgent need for efficient and scalable insulin manufacturing. Insulin icodec, the first once-weekly basal insulin analog approved for clinical use, offers significant therapeutic benefits but presents manufacturing challenges due to its structural complexity. In this study, an integrated process was developed to produce insulin icodec in Pichia pastoris. A high-expression GS115 strain was engineered and cultivated under high-cell-density fermentation, yielding 7.53 g/L proinsulin in the supernatant (5.04 g/L in whole broth). The downstream process combined cation-exchange chromatography capture, enzymatic conversion to the desB30 insulin analog using lysyl endopeptidase, and optimization of fatty acid acylation through response surface methodology (RSM). Final purification by a sequential two-step reversed-phase chromatography polishing achieved a purity of 99.90%. The structural and functional equivalence to commercial insulin icodec was verified by RP-HPLC, HR-LC-MS, peptide mapping, and cell-based proliferation assays. Collectively, this study establishes a comprehensive and high-yield platform for insulin icodec biosimilar production and provides a generalizable framework for the manufacturing of other long-acting or structurally complex insulin analogs.