title: Mechanisms of glucagon degradation at alkaline pH.
abstract: 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.
authors: Oregon Health and Science University, United States.
authors: Nicholas
authors: N
authors: Caputo
authors: Jessica R
authors: JR
authors: Castle
authors: Colin P
authors: CP
authors: Bergstrom
authors: Julie M
authors: JM
authors: Carroll
authors: Parkash A
authors: PA
authors: Bakhtiani
authors: Melanie A
authors: MA
authors: Jackson
authors: Charles T
authors: CT
authors: Roberts
authors: Larry L
authors: LL
authors: David
authors: W Kenneth
authors: WK
authors: Ward
mesh_terms: D011232:Chemical Precipitation; D002853:Chromatography, Liquid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D057075:Enzyme Assays; D005934:Glucagon / Q000031:analogs & derivatives* / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis*; D055550:Protein Stability; D059748:Proteolysis; D012996:Solutions
chemical_list: D010455:Peptides; D012996:Solutions; D005934:Glucagon; D017868:Cyclic AMP-Dependent Protein Kinases
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