title: Solution behavior of IFN-beta-1a: an empirical phase diagram based approach.
abstract: An empirical phase diagram approach has been developed as a practical tool to aid macromolecular preformulation/formulation studies. This method employs an eigenvector based procedure to visualize and interpret complex data sets. Human Inteferon-beta-1a, an important therapeutic protein, was used to further develop the method and test its utility. The protein was characterized in solution as a function of pH (2-8), temperature (10 degrees C-85 degrees C) and ionic strength (I = 0.1 and 1.0) using intrinsic and ANS fluorescence, Far-UV circular dichroism (Far-UV CD), Fourier Transform Infrared spectroscopy (FTIR) and derivative UV absorbance spectroscopies, as well as differential scanning calorimetry (DSC) to supplement spectroscopic thermal stability studies. Derivative UV absorbance data were initially used to construct a pH-temperature phase diagram at each ionic strength. Three distinctive phases at I = 0.1 and two major phases at I = 1.0 were identified corresponding to different conformation/aggregation states of the protein. For the first time, heterogeneous data sets (i.e., data from different techniques) including Far-UV CD, fluorescence and UV absorbance results were used to generate empirical phase diagrams. Results from different data sets are compared; precautions in applying the method and its overall utility are discussed.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, USA.
authors.forename: Haihong
authors.initials: H
authors.lastname: Fan
authors.forename: John
authors.initials: J
authors.lastname: Ralston
authors.forename: Mary
authors.initials: M
authors.lastname: Dibiase
authors.forename: Eric
authors.initials: E
authors.lastname: Faulkner
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
mesh_terms: D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000068556:Interferon beta-1a; D016899:Interferon-beta / Q000737:chemistry*; D015233:Models, Statistical; D009994:Osmolar Concentration; D019999:Pharmaceutical Solutions / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D019999:Pharmaceutical Solutions; D011994:Recombinant Proteins; D012996:Solutions; D016899:Interferon-beta; D000068556:Interferon beta-1a