Elucidation of acid-induced unfolding and aggregation of human immunoglobulin IgG1 and IgG2 Fc.

Understanding the underlying mechanisms of Fc aggregation is an important prerequisite for developing stable and efficacious antibody-based therapeutics. In our study, high resolution two-dimensional nuclear magnetic resonance (NMR) was employed to probe structural changes in the IgG1 Fc. A series of (1)H-(15)N heteronuclear single-quantum correlation NMR spectra were collected between pH 2.5 and 4.7 to assess whether unfolding of C(H)2 domains precedes that of C(H)3 domains. The same pH range was subsequently screened in Fc aggregation experiments that utilized molecules of IgG1 and IgG2 subclasses with varying levels of C(H)2 glycosylation. In addition, differential scanning calorimetry data were collected over a pH range of 3-7 to assess changes in C(H)2 and C(H)3 thermostability. As a result, compelling evidence was gathered that emphasizes the importance of C(H)2 stability in determining the rate and extent of Fc aggregation. In particular, we found that Fc domains of the IgG1 subclass have a lower propensity to aggregate compared with those of the IgG2 subclass. Our data for glycosylated, partially deglycosylated, and fully deglycosylated molecules further revealed the criticality of C(H)2 glycans in modulating Fc aggregation. These findings provide important insights into the stability of Fc-based therapeutics and promote better understanding of their acid-induced aggregation process.

Diffusion and sedimentation interaction parameters for measuring the second virial coefficient and their utility as predictors of protein aggregation.

The concentration-dependence of the diffusion and sedimentation coefficients (k(D) and k(s), respectively) of a protein can be used to determine the second virial coefficient (B2), a parameter valuable in predicting protein-protein interactions. Accurate measurement of B2 under physiologically and pharmaceutically relevant conditions, however, requires independent measurement of k(D) and k(s) via orthogonal techniques. We demonstrate this by utilizing sedimentation velocity (SV) and dynamic light scattering (DLS) to analyze solutions of hen-egg white lysozyme (HEWL) and a monoclonal antibody (mAb1) in different salt solutions. The accuracy of the SV-DLS method was established by comparing measured and literature B2 values for HEWL. In contrast to the assumptions necessary for determining k(D) and k(s) via SV alone, k(D) and ks were of comparable magnitudes, and solution conditions were noted for both HEWL and mAb1 under which 1), k(D) and k(s) assumed opposite signs; and 2), k(D) ≥k(s). Further, we demonstrate the utility of k(D) and k(s) as qualitative predictors of protein aggregation through agitation and accelerated stability studies. Aggregation of mAb1 correlated well with B2, k(D), and k(s), thus establishing the potential for k(D) to serve as a high-throughput predictor of protein aggregation.

High throughput thermostability screening of monoclonal antibody formulations.

Differential scanning fluorimetry (DSF) was employed to increase the throughput of the thermostability screening of monoclonal antibody (mAb) formulations. The method consists of measuring the fluorescence intensity of a polarity sensitive probe at gradually increasing temperatures, and obtaining the transition temperature of exposure of the hydrophobic regions of proteins (T(h)). The change in fluorescence intensity was directly related to protein unfolding levels and temperatures. The results from thermostability measurements were compared with the data acquired using differential scanning calorimetry (DSC), and a good correlation between T(h) and the temperature of protein unfolding or melting (T(m)) was observed. The method was applied to screen four mAb molecules in 84 different buffers. The studies revealed a good correlation of T(h) values with the known effects of pH and excipients on protein stability in solution. Specifically, the elevated aggregation levels induced by salt, low pH, and high protein concentrations could be successfully predicted by this thermal stability screening. This method is efficient, with high throughput capability, and could be widely applied in the biopharmaceutical industry for formulation and process development, and characterization.

Detection of IgG aggregation by a high throughput method based on extrinsic fluorescence.

The utility of extrinsic fluorescence as a tool for high throughput detection of monoclonal antibody aggregates was explored. Several IgG molecules were thermally stressed and the high molecular weight species were fractionated using size-exclusion chromatography (SEC). The isolated aggregates and monomers were studied by following the fluorescence of an extrinsic probe, SYPRO Orange. The dye displayed high sensitivity to structurally altered, aggregated IgG structures compared to the native form, which resulted in very low fluorescence in the presence of the dye. An example of the application is presented here to demonstrate the properties of this detection method. The fluorescence assay was shown to correlate with the SEC method in quantifying IgG aggregates. The fluorescent probe method appears to have potential to detect protein particles that could not be analyzed by SEC. This method may become a powerful high throughput tool to detect IgG aggregates in pharmaceutical solutions and to study other protein properties involving aggregation. It can also be used to study the kinetics of antibody particle formation, and perhaps allow identification of the species, which are the early building blocks of protein particles.

Effect of ions on agitation- and temperature-induced aggregation reactions of antibodies.

PURPOSE: The impact of ions on protein aggregation remains poorly understood. We explored the role of ionic strength and ion identity on the temperature- and agitation-induced aggregation of antibodies. METHODS: Stability studies were used to determine the influence of monovalent Hofmeister anions and cations on aggregation propensity of three IgG(2) mAbs. The C(H)2 domain melting temperature (T (m1)) and reduced valence (z*) of the mAbs were measured. RESULTS: Agitation led to increased solution turbidity, consistent with the formation of insoluble aggregates, while soluble aggregates were formed during high temperature storage. The degree of aggregation increased with anion size (F(-) < Cl(-) < Br(-) < I(-) < SCN(-) ~ ClO(4) (-)) and correlated with a decrease in T (m1) and z*. The aggregation propensity induced by the anions increased with the chaotropic nature of anion. The cation identity (Li(+), Na(+), K(+), Rb(+), or Cs(+)) had no effect on T (m1), z* or aggregation upon agitation. CONCLUSIONS: The results indicate that anion binding mediates aggregation by lowering mAb conformational stability and reduced valence. Our observations support an agitation-induced particulation model in which anions enhance the partitioning and unfolding of mAbs at the air/water interface. Aggregation predominantly occurs at this interface; refreshing of the surface during agitation releases the insoluble aggregates into bulk solution.