title: Perturbation of thermal unfolding and aggregation of human IgG1 Fc fragment by Hofmeister anions.
abstract: The thermal unfolding and subsequent aggregation of the unglycosylated Fc fragment of a human IgG1 antibody (Fc) were studied in the salt solutions of Na(2)SO(4), KF, KCl and KSCN at pH 4.8 and 7.2 below and at its pI of 7.2, respectively, using differential scanning calorimetry (DSC), far ultraviolet circular dichroism (far-UV CD), size exclusion chromatography (SE-HPLC) and light scattering. First, our experimental results demonstrated that the thermal unfolding of the C(H)2 domain of the Fc was sufficient to induce aggregation. Second, at both pH conditions, the anions (except F(-)) destabilized the C(H)2 domain where the effectiveness of SO(4)(2-) > SCN(-) > Cl(-) > F(-) was more apparent at pH 4.8. In addition, the thermal stability of the C(H)2 domain was less sensitive to the change in salt concentration at pH 7.2 than at pH 4.8. Third, at pH 4.8 when the Fc had a net positive charge, the anions accelerated the aggregation reaction with SO(4)(2-) > SCN(-) > Cl(-) > F(-) in effectiveness. But these anions slowed down the aggregation kinetics at pH 7.2 with similar effectiveness when the Fc was net charge neutral. We hypothesize that the effectiveness of the anion on destabilizing the C(H)2 domain could be attributed to its ability to perturb the free energy for both of the native and unfolded states. The effect of the anions on the kinetics of the aggregation reaction could be interpreted based on the modulation of the electrostatic protein-protein interactions by the anions.
authors.affiliation: Department of Analytical and Formulation Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, United States.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang-van Enk
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Mason
authors.forename: Lei
authors.initials: L
authors.lastname: Yu
authors.forename: Le
authors.initials: L
authors.lastname: Zhang
authors.forename: Wael
authors.initials: W
authors.lastname: Hamouda
authors.forename: Gang
authors.initials: G
authors.lastname: Huang
authors.forename: Dingjiang
authors.initials: D
authors.lastname: Liu
authors.forename: Richard L
authors.initials: RL
authors.lastname: Remmele
authors.forename: Jifeng
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000838:Anions / Q000494:pharmacology*; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005459:Fluorides / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D011189:Potassium Chloride / Q000494:pharmacology; D017680:Potassium Compounds / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D013431:Sulfates / Q000494:pharmacology; D013861:Thiocyanates / Q000494:pharmacology
chemical_list: D000838:Anions; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D017680:Potassium Compounds; D013431:Sulfates; D013861:Thiocyanates; C012036:sodium sulfate; D011189:Potassium Chloride; C033320:potassium fluoride; D005459:Fluorides