Expression and Purification of the Insulin Icodec Precursor

Abstract Basal insulins are essential for diabetes treatment, yet daily injections often impair patient adherence. To address this limitation, researchers have developed insulin icodec, a once-weekly basal insulin analog; nevertheless, its manufacture requires efficient production of a high-quality recombinant precursor. In this study, we aimed to construct recombinant Pichia pastoris strains expressing the insulin icodec precursor. The key cultivation parameters—including pH, temperature, and induction strategy—were screened in shake-flask experiments. The optimized conditions were subsequently applied in 5-L high-cell-density fermentation to evaluate the effects of C-peptide variants (AMK and AAK) and different media formulations (basal salt medium [BSM], ½ BSM, and ⅓ BSM) on secretion efficiency. A scalable three-step purification process consisting of acid precipitation, cation-exchange chromatography, and preparative reversed-phase chromatography was established. Under optimized conditions (pH 6.5, 28°C, methanol feeding), the AMK variant increased the secreted precursor titer by 38% compared with AAK, and BSM medium supported a maximum titer of 10.97 g/L at 126 hours, representing one of the highest levels reported for insulin precursors in yeast. The final product was obtained with >95% purity and >65% overall recovery. Structural identity was confirmed by high-resolution mass spectrometry and peptide mapping with complete sequence coverage. Overall, this work provides a scalable and high-yield process for industrial production of the insulin icodec precursor.