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Characterisation and physical stability of PEGylated glucagon |
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| Authors: | Stigsnaes Pernille Frokjaer Sven Bjerregaard Simon van de Weert Marco Kingshott Peter Moeller E Horn |
| Affiliation: | aDepartment of Pharmaceutics, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, 2100 Copenhagen, Denmark bPreformulation and Delivery, Novo Nordisk A/S, Novo Nordisk Allé, 2880 Bagsvaerd, Denmark cThe Danish Polymer Centre, Risø National Laboratory, Frederiksborgvej 399, 4000 Roskilde, Denmark |
| 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 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 -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. |
| Keywords: | PEGylation Glucagon Freeze-drying Secondary structure Fibrillation Physical stability |
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