High-yield recombinant production of the semaglutide main chain P29 intermediate using SNAC-tagged enterokinase-cleavable fusion peptides

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by insufficient insulin secretion or impaired cellular response to insulin, resulting in increased blood sugar levels and persistent hyperglycemia. The diabetes-related health expenditure worldwide is estimated to have surpassed 1,000 billion USD according to the new data from the International Diabetes Federation. Semaglutide, a long-acting glucagon-like peptide-1 (GLP-1) receptor agonist, has demonstrated high efficacy in glycemic control and body weight loss, is approved for T2DM and obesity treatment. However, up to now, industrial production of semaglutide remains constrained by limited yield and high cost associated with conventional approaches. Therefore, improving production efficiency while reducing manufacturing cost remains a challenge. In this study, we report a new strategy for obtaining semaglutide main chain P29, also known as Arg34GLP-1 (9-37), which is a key intermediate precursor in semaglutide synthesis. The method employs a series of semaglutide-derived helical fusion pro-peptides containing the sequence GSHHWHHHSSGDDDDK, which could be cleaved by a 2-step processing via sequence-specific nickel-assisted chemical protein cleavage followed by enterokinase cleavage. Using this strategy, semaglutide main chain P29 was highly expressed and purified to 98% purity, with yields exceeding 5 grams per liter of broth. This process provides improved productivity compared with previously reported strategies. The work establishes an efficient and scalable platform for semaglutide intermediate production and shows potential for large-scale industrial production.