title: Non-native intermediate conformational states of human growth hormone in the presence of organic solvents.
abstract: OBJECTIVE
  Manufacturing processes expose protein pharmaceuticals to organic solvents that may perturb the native folded state, increasing the potential for irreversible aggregation or surface adsorption. The aim of this study was to characterize the conformational states of human growth hormone (hGH) in aqueous ethanolic solutions.
  
  
  METHODS
  The higher order structure of hGH was investigated using far- and near-UV circular dichroism (CD) and fluorescence spectroscopy as orthogonal techniques, and the hydrodynamic size was monitored using dynamic light scattering.
  
  
  RESULTS
  CD data suggested that the secondary structure of hGH remained unchanged up to 50\% (v/v) ethanol, but the tertiary structure was perturbed at a20% ethanol. Fluorescence anisotropy, however, showed that the mobility of the buried Trp residue was restricted even at 30% ethanol, suggesting a differently packed structural core in 30% ethanol relative to the native structure. Consistent with this result, thermal unfolding of hGH in 30% ethanol was more facile compared to that in 0% and 20% ethanol. At >40% ethanol, fluorescence data were consistent with increased solvent exposure of the tryptophan.
  
  
  CONCLUSIONS
  The results point to progressive unfolding of hGH that increases solvent exposure of the hydrophobic core as a function of ethanol concentration and suggest that non-native intermediate states are populated in 30-60% ethanol.
authors.affiliation: Biopharmaceutical Research and Development, Eli Lilly and Company, Indianapolis, Indiana, USA. sukumar@lilly.com
authors.forename: Muppalla
authors.initials: M
authors.lastname: Sukumar
authors.forename: Sacha M
authors.initials: SM
authors.lastname: Storms
authors.forename: Michael R
authors.initials: MR
authors.lastname: De Felippis
mesh_terms: D002626:Chemistry, Pharmaceutical / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D000431:Ethanol / Q000032:analysis; D005454:Fluorescence Polarization / Q000379:methods; D019382:Human Growth Hormone / Q000032:analysis* / Q000737:chemistry / Q000502:physiology; D006801:Humans; D008027:Light; D008968:Molecular Conformation; D011487:Protein Conformation / Q000187:drug effects*; D011489:Protein Denaturation; D012542:Scattering, Radiation; D012997:Solvents / Q000032:analysis* / Q000145:classification; D013678:Technology, Pharmaceutical / Q000379:methods; D013696:Temperature
chemical_list: D012997:Solvents; D019382:Human Growth Hormone; D000431:Ethanol