title: Biophysical signatures of noncovalent aggregates formed by a glucagonlike peptide-1 analog: a prototypical example of biopharmaceutical aggregation.
abstract: LY307161 is a 31 amino acid analog of glucagonlike peptide-1(7-37)OH susceptible to physical instability associated with pharmaceutical processing. Orthogonal biophysical studies were conducted to explore the origins of this physical instability and to distinguish pharmaceutically desirable states of this aggregating peptide from undesirable ones. Equilibrium sedimentation analysis established that LY307161 exists as a monomer at pH 3, and reversibly self-associates in the pH range 7.5-10.5. Causative factors for physical instability related to lyophilization conditions were investigated. Solution pH, acetonitrile content, and concentration of the peptide prior to lyophilization each impacted physicochemical properties of the resultant powders. A comparative study of two powder samples exhibiting physicochemically disparate properties established that LY307161 forms soluble noncovalent aggregates. FT-IR analyses in the solid and solution states identified a prominent band at 1657-1659 cm(-1) attributed to alpha-helix structure. Noncovalent soluble aggregate exhibited characteristic bands at 1615 and 1698 cm(-1) indicative of intermolecular beta-sheet structure. An agitation-induced, precipitated solid form of LY307161 exhibited a different FT-IR signature indicative of a conformationally distinct species. Circular dichroism and fluorescence spectroscopy, together with dynamic light scattering measurements and dye-aggregate complexation, provided additional insights into the distinctions between aggregated and native LY307161.
authors.affiliation: Biopharmaceutical Research and Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA.
authors.forename: Brandon L
authors.initials: BL
authors.lastname: Doyle
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Pollo
authors.forename: Allen H
authors.initials: AH
authors.lastname: Pekar
authors.forename: Michael L
authors.initials: ML
authors.lastname: Roy
authors.forename: Beth Ann
authors.initials: BA
authors.lastname: Thomas
authors.forename: Mark L
authors.initials: ML
authors.lastname: Brader
mesh_terms: D055592:Biophysical Phenomena; D001703:Biophysics; D002942:Circular Dichroism; D005612:Freeze Drying; D052216:Glucagon-Like Peptide 1 / Q000031:analogs & derivatives* / Q000737:chemistry; D008956:Models, Chemical; D010446:Peptide Fragments / Q000737:chemistry*; D011208:Powders; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: C508112:LY307161; D010446:Peptide Fragments; D011208:Powders; D011994:Recombinant Proteins; D052216:Glucagon-Like Peptide 1