An integrated bioprocess for the production of 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.