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.

Relation of Colloidal and Conformational Stabilities to Aggregate Formation in a Monoclonal Antibody

“…46,47 The difference in the stability due to the presence of anions could be interpreted in terms of change in the hydration level and ion interaction. 48 Based on the previous and this research, the free energy diagram between native state, N, and denatured state, D, of MAb-T in AB5 and CB5 was depicted in Figure 5b. When MAb-T exists in the electrolyte solution, the Gibbs free energy is required to hydrate while a removal of hydration water by ion interaction decreased the free energy state.…”

Acetate- and Citrate-Specific Ion Effects on Unfolding and Temperature-Dependent Aggregation Rates of Anti-Streptavidin IgG1

“…However, such effects are usually observed at much higher salt concentrations. 44 An alternative hypothesis is that preferential accumulation of citrate ions (compared to acetate) results in reduced repulsions between proteins, and this increases aggregation rates at a given temperature. Increased aggregation rates will also shift T m app values to lower temperatures.…”

“…Those questions notwithstanding, the results in Figure 3, Figure 6 and Supporting Information highlight that statistically precise E a values are needed to be able to better assess different contributions to aggregation rates occur at ion concentrations much lower than those typically reported for so-called Hofmeister effects. 44,52 Future work will focus on determining whether this behavior holds for a wider range of proteins and salts in the Hofmeister series at low salt concentrations.…”

Conformational and Colloidal Stabilities of Isolated Constant Domains of Human Immunoglobulin G and Their Impact on Antibody Aggregation under Acidic Conditions

“…13,14 Enk et al reported that thermally unfolded aglycosylated CH2 region led to aggregation of Fc, and that the presence of anions destabilized the CH2 region and accelerated the aggregation reaction. 13 Kim et al reported that aggregation rates for intact antibody were strongly influenced by the conformational stability of the Fab region. 14 Furthermore, Buchner and coworkers showed that murine IgG1 domains (i.e., whole-IgG1, Fab, CH3, VH, VL, CH1, and CL) form molten-globule-like intermediate structures under specific acidic conditions (pH 2 and 100 mM NaCl; CL: pH 2, 175 mM NaCl).…”