Mechanisms of glucagon degradation at alkaline pH

• Published in: Peptides (New York, N.Y. : 1980) Vol. 45; pp. 40 - 47
• Main Authors: Caputo, Nicholas, Castle, Jessica R., Bergstrom, Colin P., Carroll, Julie M., Bakhtiani, Parkash A., Jackson, Melanie A., Roberts, Charles T., David, Larry L., Ward, W. Kenneth
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2013
• Subjects: Agglomeration; Alkaline; aqueous solutions; bioactive properties; bioassays; Biochemistry; Chemical Precipitation; Chromatography, Liquid; Cyclic AMP-Dependent Protein Kinases - chemistry; Deamidation; Degradation; diabetes; Diabetes mellitus; Enzyme Assays; Fibrillation; Glucagon; Glucagon - analogs & derivatives; Glucagon - chemistry; Humans; Hydrogen-Ion Concentration; Isomerization; Kinases; liquid chromatography; Mass Spectrometry; Oxidation; Peptides; Peptides - analysis; Protein Stability; Proteolysis; pumps; Solutions; RT; Glucagon; MS; CHO-K1; Diabetes mellitus; PKA; MW; Deamidation; EGFP; Degradation; Alkaline; EC50; Fmoc; Oxidation; Da; HPLC; rp
• ISSN: 0196-9781; 1873-5169
• DOI: 10.1016/j.peptides.2013.04.005

•Because of its ability to prevent and treat hypoglycemia, glucagon shows great promise in closed loop treatment of diabetes.•However, glucagon degrades in liquid solution, and the mechanisms of degradation are not well understood.•We found that deamidation and isomerization were common, and in many cases, impair the bioactivity of glucagon.•Glucagon also undergoes spontaneous oxidation, but oxidized glucagon retains its bioactivity.•An understanding of glucagon's inactivating degradation mechanisms might help in the search for stabilizing excipients. Glucagon is unstable and undergoes degradation and aggregation in aqueous solution. For this reason, its use in portable pumps for closed loop management of diabetes is limited to very short periods. In this study, we sought to identify the degradation mechanisms and the bioactivity of specific degradation products. We studied degradation in the alkaline range, a range at which aggregation is minimized. Native glucagon and analogs identical to glucagon degradation products were synthesized. To quantify biological activity in glucagon and in the degradation peptides, a protein kinase A-based bioassay was used. Aged, fresh, and modified peptides were analyzed by liquid chromatography with mass spectrometry (LCMS). Oxidation of glucagon at the Met residue was common but did not reduce bioactivity. Deamidation and isomerization were also common and were more prevalent at pH 10 than 9. The biological effects of deamidation and isomerization were unpredictable; deamidation at some sites did not reduce bioactivity. Deamidation of Gln 3, isomerization of Asp 9, and deamidation with isomerization at Asn 28 all caused marked potency loss. Studies with molecular-weight-cutoff membranes and LCMS revealed much greater fibrillation at pH 9 than 10. Further work is necessary to determine formulations of glucagon that minimize degradation and fibrillation.