X-ray Scattering and Coarse-Grained Simulations for Clustering and Interactions of Monoclonal Antibodies at High Concentrations

Abstract

Attractive protein–protein interactions (PPI) in concentrated monoclonal antibody (mAb) solutions may lead to reversible oligomers (clusters) that impact colloidal stability and viscosity. Herein, the PPI are tuned for two mAbs via the addition of arginine (Arg), NaCl, or ZnSO4 as characterized by the structure factor (S eff(q)) with small-angle X-ray scattering (SAXS). The SAXS data are fit with molecular dynamics simulations by placing a physically relevant short-range attractive interaction on selected beads in coarse-grained 12-bead models of the mAb shape. The optimized 12-bead models are then used to differentiate key microstructural properties, including center of mass radial distribution functions (g COM(r)), coordination numbers, and cluster size distributions (CSD). The addition of cosolutes results in more attractive S eff(q) relative to the no cosolute control for all systems tested, with the most attractive systems showing an upturn at low q. Only the All1 model with an attractive site in each Fab and Fc region (possessing Fab–Fab, Fab–Fc, and Fc–Fc interactions) can reproduce this upturn, and the corresponding CSDs show the presence of larger clusters compared to the control. In general, for models with similar net attractions, i.e., second osmotic virial coefficients, the size of the clusters increases as the attraction is concentrated on a smaller number of evenly distributed beads. The cluster size distributions from simulations are used to improve the understanding and prediction of experimental viscosities. The ability to discriminate between models with bead interactions at particular Fab and Fc bead sites from SAXS simulations, and to provide real-space properties (CSD and g COM(r)), will be of interest in engineering protein sequence and formulating protein solutions for weak PPI to minimize aggregation and viscosities.

A single molecular descriptor to predict solution behavior of therapeutic antibodies

“…While the measurement of k D has great utilitarian value in identifying poorly and well-behaved mAbs based on relevant criteria, it may be limited in providing quantitative regressions for viscosity-and opalescence-mAb concentration relationships. In this regard, studies of empirical measurements of solution microstructure at high concentrations coupled with coarse-grain modeling of protein-protein interactions hold promise for providing rich additional insight (17,18). Deeper study and validation of such approaches would present a powerful, practical approach for predicting antibody solution behavior.…”

Protein-Protein Interactions, Clustering, and Rheology for Bovine IgG up to High Concentrations Characterized by Small Angle X-Ray Scattering and Molecular Dynamics Simulations

“…25,26 The same upturn and corresponding increase in viscosity were also observed for GmAbG with the addition of Na 2 SO 4 26 and mAb4 with the addition of NaCl. 27 Bovine gamma globulin (BGG), a polyvalent mixture of IgG, IgM, and IgA obtained from pooled bovine sera, 28 is a commonly used model system for studying PPI in complex protein solutions. Of particular interest is the IgG fraction (bIgG) which is the primary subtype of antibodies used in therapeutics.…”

“…36 In a recent complementary approach, the strength of attraction and type of attraction anisotropy (Fab-Fab interactions vs. Fab-Fab, Fab-Fc, and Fc-Fc interactions) were regressed by comparing molecular dynamics (MD) simulations of 12-bead models to experimental SAXS S(q) without knowing the sequence. 27 This approach was further refined by adding a van der Waals (vdW) background on each bead to achieve better fits of S(q) and more physically meaningful attractive interaction parameters and cluster sizes (Chowdhury A, et al Unpublished results). With these methods, the S(q) profile of a weakly interacting system such as mAb2 with 250-mM Arg is described well by weak attraction spread uniformly between all 12 beads of the mAbs, resulting in weak selfassociation and low viscosities.…”

“…In contrast, the upturn in S(q) at low q for a strongly interacting system such as mAb4 with 250-mM NaCl could only be described by a model with strong specific attraction between the Fab-Fab, Fab-Fc, and Fc-Fc domains yielding large transient cluster formations and high viscosities. 27 In this study, we characterize the PPI, cluster size, and viscosity response of bIgG to changes in pH and cosolute using SLS and SAXS for mAb concentrations from 10 to 200 mg/mL. Following our recent work (Chowdhury A, et al Unpublished results), 27 the strength and locations of anisotropic attractions were quantified by comparing I(q) from SAXS to MD simulations of CG 12-bead models exhibiting a short-range attraction on 1 bead in each Fab domain and in the Fc domain in addition to a uniform vdW background.…”

“…27 In this study, we characterize the PPI, cluster size, and viscosity response of bIgG to changes in pH and cosolute using SLS and SAXS for mAb concentrations from 10 to 200 mg/mL. Following our recent work (Chowdhury A, et al Unpublished results), 27 the strength and locations of anisotropic attractions were quantified by comparing I(q) from SAXS to MD simulations of CG 12-bead models exhibiting a short-range attraction on 1 bead in each Fab domain and in the Fc domain in addition to a uniform vdW background. To achieve a broader systematic understanding of PPI behavior, pH is varied to change the charge distribution and protein structural stability, whereas nonbinding and binding cosolutes, NaCl and Arg, are used to screen the short-range attraction.…”