Liquid-liquid phase separation was studied for a monoclonal antibody in the monovalent salt solutions of KF, KCl, and KSCN under different pH conditions. A modified Carnahan-Starling hard-sphere model was utilized to fit the experimental data, establish the liquid-liquid coexistence curve, and determine antibody-antibody interactions in the form of T(c) (critical temperature) under the different solution conditions. The liquid-liquid phase separation revealed the complex relationships between antibody-antibody interactions and different solution conditions, such as pH, ionic strength, and the type of anion. At pH 7.1, close to the pI of the antibody, a decrease of T(c) versus ionic strength was observed at low salt conditions, suggesting that the protein-protein interactions became less attractive. At a pH value below the pI of the antibody, a nonmonotonic relationship of T(c) versus ionic strength was apparent: initially as the ionic strength increased, protein-protein interactions became more attractive with the effectiveness of the anions following the inverse Hofmeister series; then the interactions became less attractive following the direct Hofmeister series. This nonmonotonic relationship may be explained by combining the charge neutralization by the anions, perhaps with the ion-correlation force for polarizable anions, and their preferential interactions with the antibody.

Proteins destined to circulate in the blood are first folded and assembled in the endoplasmic reticulum of secretory cells. For antibodies, like many other serum proteins, the folding and assembly steps involve the formation of disulfide bonds. Such bonds have been thought to be static features of proteins, stabilizing domains, and linking polypeptide chains, although some cases of extracellular disulfide bond cleavage have been noted. Recently, the human IgG2 antibody subclass was found to possess multiple structures differing in specific disulfide linkages. These structures are naturally occurring and can, in some cases, affect the activity of the antibody. Here we show that these IgG2 disulfide linkages interconvert while circulating in humans. Secretory cells initially produce primarily one form (IgG2-A), which is rapidly converted to a second form (IgG2-A/B) while circulating in the blood, followed by a slower conversion to a third form (IgG2-B). This work demonstrates that the disulfide structure of the IgG2 antibody is dynamic in vivo, on a time scale similar to that of the protein's lifetime. Thus, changes to the IgG2 disulfide structure provide a marker of the protein's age and may alter its activity over its lifetime.

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.

The role of thermal unfolding as it pertains to thermodynamic properties of proteins and their stability has been the subject of study for more than 50 years. Moreover, exactly how the unfolding properties of a given protein system may influence the kinetics of aggregation has not been fully characterized. In the study of recombinant human Interleukin-1 receptor type II (rhuIL-1R(II)) aggregation, data obtained from size exclusion chromatography and differential scanning calorimetry (DSC) were used to model the thermodynamic and kinetic properties of irreversible denaturation. A break from linearity in the initial aggregation rates as a function of 1/T was observed in the vicinity of the melting transition temperature (T(m) approximately 53.5 degrees C), suggesting significant involvement of protein unfolding in the reaction pathway. A scan-rate dependence in the DSC experiment testifies to the nonequilibrium influences of the aggregation process. A mechanistic model was developed to extract meaningful thermodynamic and kinetic parameters from an irreversibly denatured process. The model was used to simulate how unfolding properties could be used to predict aggregation rates at different temperatures above and below the T(m) and to account for concentration dependence of reaction rates. The model was shown to uniquely identify the thermodynamic parameters DeltaC(P) (1.3 +/- 0.7 kcal/mol-K), DeltaH(m) (74.3 +/- 6.8 kcal/mol), and T(m) with reasonable variances.

Thermal stability of the C2 domain for an IgG1 monoclonal antibody and its aggregation kinetics were systematically studied at pH 4.8, below its pI of 8.8 in individual solutions of arginine salts with acetate, glutamate (Glu), chloride, and sulfate as the anion, in comparison to sodium chloride and sodium sulfate. Thermal unfolding temperature, T, an indicator of thermal stability, was measured by both differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF). The aggregation kinetics was determined by assessing reversibility for the C2 domain in the DSC repetitive scans and then cross-examined by the isothermal aggregation study measured by size exclusion chromatography. The effect of Arg on the thermal stability and aggregation kinetics of the antibody is shown to be strongly anion-dependent: both ArgAceate and ArgGlu improve the stability, while both ArgSO and ArgCl decrease it. Furthermore, the addition of ArgCl and ArgSO accelerates the aggregation kinetic, but to a lesser extent than the respective Na salt, suggesting that Arg binds to the antibody more strongly than Na. However, the binding of Arg did not lead to more destabilization of the C2 domain by the Arg salts at low concentrations, comparing to the respective Na salt. This finding indicates that Arg prefers the protein surface, rather than the exposed backbone upon unfolding. Furthermore, the change in the ranking for affecting the thermal stability and aggregation kinetics as the salt concentration increases implies the presence of other multiple mechanisms, e.g., cluster formation through the homoion pairing between Arg molecules and their preferential exclusion from the protein surface, and heteroion pairing between Arg and SO.