title: Physical instability of a therapeutic Fc fusion protein: domain contributions to conformational and colloidal stability.
abstract: Protein therapeutics made up of artificially combined proteins or protein domains, so-called fusion proteins, are a novel and growing class of biopharmaceuticals. We have studied abatacept (Orencia), a fusion protein that is constructed of a modified IgG Fc domain and the soluble part of the T-cell receptor CTLA-4. In accelerated degradation studies conducted at 40 degrees C, a pH shift from 7.5 to 6.0 yields significantly faster aggregation kinetics, as measured by size-exclusion chromatography. To understand how the fusion domains and their interactions contribute to this result, we considered aggregation in light of the modified Lumry-Eyring reaction pathway. Protein conformational stabilities against chaotropes and temperature were measured. The structural consequences of these perturbations were observed by a variety of experimental techniques, including differential scanning calorimetry, circular dichroism, and intrinsic fluorescence. Abatacept's colloidal stability was studied by measuring zeta potentials and osmotic second virial coefficients, as well as by modeling electrostatic potentials on the protein's surface. The domains of abatacept exhibit different conformational stabilities that are highly pH dependent, whereas abatacept was weakly colloidally unstable at pH 6 or 7.5. These results are ascribed to conformational instability of the CTLA-4 and C(H)2 domains, which unfold to form a molten globule-like structure that is aggregation-prone. We suggest the instability against aggregation is determined by the least stable domains.
authors.affiliation: Department of Chemical and Biological Engineering, Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado 80309, USA. jonas.fast@roche.com
authors.forename: Jonas L
authors.initials: JL
authors.lastname: Fast
authors.forename: Amanda A
authors.initials: AA
authors.lastname: Cordes
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
mesh_terms: D000069594:Abatacept; D000595:Amino Acid Sequence; D003102:Colloids / Q000737:chemistry*; D018360:Crystallography, X-Ray; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000235:genetics / Q000627:therapeutic use; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics / Q000627:therapeutic use; D008027:Light; D008969:Molecular Sequence Data; D011487:Protein Conformation; D055550:Protein Stability; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000627:therapeutic use; D012542:Scattering, Radiation
chemical_list: D003102:Colloids; D018796:Immunoconjugates; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D000069594:Abatacept