Evaluating the Effects of Hinge Flexibility on the Solution Structure of Antibodies at Concentrated Conditions

Abstract

Employing 2 different coarse-grained models, we evaluated the effect of intramolecular domain-domain distances and hinge flexibility on the general solution structure of monoclonal antibodies (mAbs), within the context of protein-protein steric repulsion. These models explicitly account for the hinge region, and represent antibodies at either domain or subdomain levels (i.e., 4-bead and 7-bead representations, respectively). Additionally, different levels of mAb flexibility are also considered. When evaluating mAbs as rigid structures, analysis of small-angle scattering profiles showed that changes in the relative internal distances between Fc and Fab domains significantly alter the local arrangement of neighboring molecules, as well as the molecular packing of the concentrated mAb solutions. Likewise, enabling hinge flexibility in either of the mAb models led to qualitatively similar results, where flexibility increases the spatial molecular arrangement at elevated concentrations. This occurs because fluctuations in mAb quaternary structure are modulated by the close proximity between molecules at elevated concentrations (>50 mg mL^-1), yielding an increased molecular packing and osmotic compressibility. However, our results also showed that the mechanism behind this synergy between flexibility and packing strongly depends on both the level of structural detail and the number of degrees-of-freedom considered in the coarse-grained model.

Keywords: antibodies; coarse-grained model; high concentration; hinge flexibility; molecular simulation; protein stability; small-angle scattering.

Figure 1.

Coarse-grained representation of the different antibody models considered here. (a) 4-bead mAb model. Antibodies are coarse-grained at the domain level, where the Fab, the Fc, and the hinge are represented by a single sphere each. (b) 7-bead mAb model. The different sub-domains on an antibody are modeled by a single sphere, and they correspond to the C_H3, C_H2, C_H1, F_V, and hinge subdomains. (c) Crystal structure of the reference antibody (i.e., IgG2, pdb ID: 1IGT). For visualization purposes, coarse-grained models are overlaid over the structure of the reference antibody.

Figure 2.

Small-angle scattering profiles for the different mAb models as rigid-body representations under different internal configuration. (a) 4-bead mAb; and (b) 7-bead mAb. The black arrow in both panels indicates the direction in which domain-domain distances are decreased. These structures range from a configuration where Fab and Fc domains are at the farthest separation (red line) to that where all domains are in contact with each other (dark-blue line). I(q) profiles are shown at a concentration of 175 mg/mL, and they are normalized by the scattering intensity of a single molecule at q=0 (I₀). For comparison, graphical representations of the different mAb structures are also shown in the figures.

Figure 3.

Small-angle scattering profiles for the different mAb models under different levels of hinge flexibility. (a) 4-bead mAb; and (b) 7-bead mAb. The black arrow in both panels indicates the direction in which the degree of flexibility increases. The rigid configuration for both models corresponds to mAbs in the structure of the reference antibody (pdb: 1IGT). I(q) profiles are shown at a concentration of 175 mg/mL, and they are normalized by the scattering intensity of a single molecule at q=0 (I₀).

Figure 4.

Small-angle scattering profiles for the different mAb models at different mAb concentrations. (a) 4-bead mAb; and (b) 7-bead mAb. In both panels, solid-lines represent fully flexible mAbs, while dashed-lines correspond to rigid mAbs (based on the structure of the reference antibody). Color-coding corresponds to different protein concentrations and is indicated in the legends of each panel. I(q) is normalized by the scattering intensity of a single molecule at q=0 (I₀) and by the average number of molecules 〈N〉 in the scattering volume to facilitate comparison between profiles.

Figure 5.

Hinge–hinge radial distribution function (g_HH(r)) for the different coarse-grained mAb representations as a function of protein concentration. (a) 4-bead mAb; and (b) 7-bead mAb. Color-coding represents different mAb concentrations (20 – 180 mg/mL), and the black arrows indicate the direction in which concentration increases. In both panels, g_HH(r) is shown for the rigid and fully flexible mAb configurations. For clarity, curves from the rigid configuration are shifted up by one unit.

Figure 6.

Osmotic pressure P as a function of mAb concentration for the different mAb models: (a) 4-bead mAb; and (b) 7-bead mAb. In both panels, blue solid-lines correspond to fully flexible mAbs, while red dashed-lines represent rigid mAbs (based on the structure of the reference antibody). For comparison, the pressure from hard-spheres with diameter σ_HS of 10 and 11 nm are also shown in the figures as dot-dashed lines. P is normalized by the pressure of an ideal-gas P^ig=(Nk_BT)/V at equivalent molecular concentration.