Characterisation and physical stability of PEGylated glucagon

Glucagon was mono-PEGylated with PEG 5000 at Lys-12 to examine the effect on conformation and physical stability during purification and freeze-drying. The model peptide glucagon is highly unstable and readily forms fibrils in solution. Secondary structure was determined by FTIR and far-UV CD and physical stability was assessed by the Thioflavin T assay.Glucagon samples were included, which underwent the same RP-HPLC purification and/or freeze-drying as glucagon–PEG 5000. After purification and freeze-drying glucagon samples showed formation of intermolecular β-sheet by FTIR, this correlated with shorter lag-times for fibrillation in the Thioflavin T assay. Formation of intermolecular β-sheet was less apparent for glucagon–PEG 5000 and no fibrillation was detected by Thioflavin T assay. Apparently PEGylation significantly improved the physical stability of glucagon after purification and freeze-drying, possibly by steric hindrance of peptide–peptide interactions.Alterations in the secondary structure were observed for freeze-dried and reconstituted peptide samples by liquid FTIR. The peak for α-helix shifted to 1664 cm−1, which could possibly be explained by formation of 310-helix. Neither 310-helix nor intermolecular β-sheet could be detected by far-UV CD, where all peptide samples showed similar spectra.In conclusion, glucagon–PEG 5000 showed a significantly improved physical stability during purification and freeze-drying compared to glucagon.