Perturbation of Thermal Unfolding and Aggregation of Human IgG1 Fc Fragment by Hofmeister Anions

The thermal unfolding and subsequent aggregation of the unglycosylated Fc fragment of a human IgG1 antibody (Fc) were studied in the salt solutions of Na2SO4, 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 CH2 domain of the Fc was sufficient to induce aggregation. Second, at both pH conditions, the anions (except F–) destabilized the CH2 domain where the effectiveness of SO4 2– > SCN– > Cl– > F– was more apparent at pH 4.8. In addition, the thermal stability of the CH2 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 SO4 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 CH2 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.