title: Characterisation and physical stability of PEGylated glucagon.
abstract: 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 beta-sheet by FTIR, this correlated with shorter lag-times for fibrillation in the Thioflavin T assay. Formation of intermolecular beta-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 alpha-helix shifted to 1664 cm(-1), which could possibly be explained by formation of 3(10)-helix. Neither 3(10)-helix nor intermolecular beta-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.
authors.affiliation: Department of Pharmaceutics, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, 2100 Copenhagen, Denmark. pgu@dfuni.dk
authors.forename: Pernille
authors.initials: P
authors.lastname: Stigsnaes
authors.forename: Sven
authors.initials: S
authors.lastname: Frokjaer
authors.forename: Simon
authors.initials: S
authors.lastname: Bjerregaard
authors.forename: Marco
authors.initials: M
authors.lastname: van de Weert
authors.forename: Peter
authors.initials: P
authors.lastname: Kingshott
authors.forename: E Horn
authors.initials: EH
authors.lastname: Moeller
mesh_terms: D052160:Benzothiazoles; D002942:Circular Dichroism; D004355:Drug Stability; D005934:Glucagon / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013844:Thiazoles / Q000737:chemistry
chemical_list: D052160:Benzothiazoles; D013844:Thiazoles; C009462:thioflavin T; D011092:Polyethylene Glycols; D005934:Glucagon