title: Utilization of Zwitterion-based solutions to dissect the relative effects of solution pH and ionic strength on the aggregation behavior and conformational stability of a fusion protein.
abstract: Solution pH and ionic strength (I) have complex effects on protein stability. We developed an experimental approach based on exploitation of the zwitterionic characteristic of amino acid molecules to probe the relative contribution from each. A variety of types of amino acid solutions were adopted to investigate the effects of pH and I in a manner that allows independent evaluation of each factor. The same effect could not be achieved using conventional buffer solutions. Size-exclusion chromatography, capillary differential scanning calorimetry, and fluorescence spectroscopy were utilized to probe the protein aggregation and conformation. The results suggested that, in addition to pH, solution ionic strength as a function of ionization state of the amino acid molecules and the ions introduced by pH adjustment played an important role in the aggregation and conformation of the protein studied. This experimental approach offers a useful tool to aid fundamental understanding of the relative effects of solution pH and ionic strength on protein stability.
authors.affiliation: Drug Product Science & Technology, Bristol-Myers Squibb, New Brunswick, New Jersey, 08903.
authors.forename: Rong
authors.initials: R
authors.lastname: Zhou
authors.forename: Vishal
authors.initials: V
authors.lastname: Nashine
authors.forename: Thomas
authors.initials: T
authors.lastname: Palm
authors.forename: Rajesh
authors.initials: R
authors.lastname: Gandhi
authors.forename: Monica
authors.initials: M
authors.lastname: Adams
mesh_terms: D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012996:Solutions
chemical_list: D011993:Recombinant Fusion Proteins; D012996:Solutions
keywords: calorimetry (DSC); chromatography; fluorescence spectroscopy; ionic strength; pH; protein aggregation; protein structure; zwitterion