title: Characterizing Reversible Protein Association at Moderately High Concentration Via Composition-Gradient Static Light Scattering.
abstract: Analysis of weakly self-associating macromolecules at concentrations beyond a few g/L is challenging on account of the confounding effect of thermodynamic nonideality on the association signal. When the reversible association comprises only 1 or 2 oligomeric species in equilibrium with the monomer, the nonideality may be accounted for in a relatively rigorous manner, but if more association states are involved, the analysis becomes quite complex. We show that under reasonable assumptions, the nonideality in a composition-gradient static light scattering measurement may be accounted for in a simple fashion. The correction is applied to determining the stoichiometry and binding affinity of a protein previously characterized via sedimentation equilibrium and dynamic light scattering. The results of the new analysis are remarkably self-consistent and in line with the expectations for the form of self-association predicted previously from analysis of the surface residuals, establishing composition-gradient multi-angle static light scattering with nonideality corrections as a critical technology for characterizing associative interactions in concentrated solutions.
authors.affiliation: Wyatt Technology Corporation, Santa Barbara, California 93117.
authors.forename: Daniel
authors.initials: D
authors.lastname: Some
authors.affiliation: Process Development, Amgen, Thousand Oaks, California 91320.
authors.forename: Joseph
authors.initials: J
authors.lastname: Pollastrini
authors.affiliation: Process Development, Amgen, Thousand Oaks, California 91320. Electronic address: scao@amgen.com.
authors.forename: Shawn
authors.initials: S
authors.lastname: Cao
mesh_terms: D001702:Biopharmaceutics / Q000379:methods*; D008027:Light; D008956:Models, Chemical; D046912:Multiprotein Complexes / Q000737:chemistry*; D011485:Protein Binding; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D012965:Sodium Chloride / Q000737:chemistry; D012996:Solutions; D013816:Thermodynamics
chemical_list: D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D012996:Solutions; C539318:peptibody A; D012965:Sodium Chloride
keywords: biopharmaceuticals characterization; biotechnology; light-scattering; protein aggregation; protein formulation; stability