Pilot-scale production and characterization of PEGylated human FGF-21 analog

FGF-21 has become a potential drug candidate for the treatment of type 2 diabetes. Previous studies have demonstrated that PEGylation of FGF-21 could significantly increase its in vivo half-life and provide its long-lasting blood glucose-lowering effect. To accelerate the development of PEGylated FGF-21 for clinical application as a long-acting antidiabetes drug, we prepared ahmFGF-21 (FGF-21 mutant) and PEGylated ahmFGF-21 in Escherichia coli Rosetta (DE3) by high cell density fermentation at a 50-L scale and pilot-scale purification. The physical and chemical properties of the purified proteins were analyzed in this study, including purity, molecular weight, isoelectric point, bacterial endotoxin, PEGylated site and second structure. As well as the in vitro glucose uptake activity and in vivo anti-diabetic effect were evaluated. Under the optimal fermentation and purification conditions, the average bacterial yield and expression level of target protein of three batches attained 52.2±4.6g/L and 223.92±5.41mg/L, respectively. The purity of pilot product was above 98% by SDS-PAGE (non-reducing or reducing) and HPLC (SEC or RPC) analysis and the final yield of PEGylated ahmFGF-21 was 87.91±1.49mg/L, which indicated that the pilot-scale production process was relatively stable. N-terminal sequencing and circular dichroism (CD) spectroscopy results showed that modification site of PEGylated ahmFGF-21 was alanine at N-terminal and the second structure of ahmFGF-21 had no obvious changes after PEGylation. Compared with ahmFGF-21, the long-acting hypoglycemic effect of PEGylated ahmFGF-21 prepared in the pilot-scale production was significantly improved in type 2 diabetic db/db mice. Our results demonstrated that the pilot-scale production process of PEGylated ahmFGF-21 was successfully established, which was very important for the clinical application.