Coarse-grained computational models of two therapeutic monoclonal antibodies are constructed to understand the effect of domain-level charge-charge electrostatics on the self-association phenomena at high protein concentrations. The coarse-grained representations of the individual antibodies are constructed using an elastic network normal-mode analysis. Two different models are constructed for each antibody for a compact Y-shaped and an extended Y-shaped configuration. The resulting simulations of these coarse-grained antibodies that interact through screened electrostatics are done at six different concentrations. It is observed that a particular monoclonal antibody (hereafter referred to as MAb1) forms three-dimensional heterogeneous structures with dense regions or clusters compared to a different monoclonal antibody (hereafter referred to as MAb2) that forms more homogeneous structures (no clusters). These structures, together with the potential mean force (PMF) and radial distribution functions (RDF) between pairs of coarse-grained regions on the MAbs, are qualitatively consistent with the experimental observation that MAb1 has a significantly higher viscosity compared to MAb2, especially at concentrations >50 mg/mL, even though the only difference between the MAbs lies with a few amino acids at the antigen-binding loops (CDRs). It is also observed that the structures in MAb1 are formed due to stronger Fab-Fab interactions in corroboration with experimental observations. Evidence is also shown that Fab-Fc interactions can be equally important in addition to Fab-Fab interactions. The coarse-grained representations are effective in picking up differences based on local charge distributions of domains and make predictions on the self-association characteristics of these protein solutions. This is the first computational study of its kind to show that there are differences in structures formed by two different monoclonal antibodies at high concentrations.
Therapeutic monoclonal antibody (mAb) candidates that form highly viscous solutions at concentrations above 100 mg/mL can lead to challenges in bioprocessing, formulation development, and subcutaneous drug delivery. Earlier studies of mAbs with concentration-dependent high viscosity have indicated that mAbs with negatively charged Fv regions have a dipole-like quality that increases the likelihood of reversible self-association. This suggests that weak electrostatic intermolecular interactions can form transient antibody networks that participate in resistance to solution deformation under shear stress. Here this hypothesis is explored by parametrizing a coarse-grained (CG) model of an antibody using the domain charges from four different mAbs that have had their concentration-dependent viscosity behaviors previously determined. Multicopy molecular dynamics simulations were performed for these four CG mAbs at several concentrations to understand the effect of surface charge on mass diffusivity, pairwise interactions, and electrostatic network formation. Diffusion coefficients computed from simulations were in qualitative agreement with experimentally determined viscosities for all four mAbs. Contact analysis revealed an overall greater number of pairwise interactions for the two mAbs in this study with high concentration viscosity issues. Further, using equilibrated solution trajectories, the two mAbs with high concentration viscosity issues quantitatively formed more features of an electrostatic network than the other mAbs. The change in the number of these network features as a function of concentration is related to the number of pairwise interactions formed by electrostatic complementarities between antibody domains. Thus, transient antibody network formation caused by domain-domain electrostatic complementarities is the most probable origin of high concentration viscosity for mAbs in this study.
Coarse-grained computational models of therapeutic monoclonal antibodies and their mutants can be used to understand the effect of domain-level charge-charge electrostatics on the self-association phenomena at high protein concentrations. The coarse-grained models are constructed for two antibodies at different coarse-grained resolutions by using six different concentrations. It is observed that a particular monoclonal antibody (hereafter referred to as MAb1) forms three-dimensional heterogeneous structures with dense regions or clusters compared to a different monoclonal antibody (hereafter referred to as MAb2) that forms homogeneous structures without clusters. The potential of mean force (PMF) and radial distribution functions (RDF) plots for the mutants (hereafter referred to as M1, M5, M7, and M10) show trends consistent with previously reported experimental observation of viscosities. The mutant referred to as M6 shows strongly attractive interactions that are consistent with previously reported negative second virial coefficients (B(22)) obtained from light-scattering experiments (Yadav et al. Pharm. Res. 2011, 28, 1750-1764; Yadav et al. Mol. Pharmaceutics. 2012, 9, 791-802). Clustering data on MAb1 reveal a small number of large clusters that are hypothesized to be the reason for the high experimental viscosity. This is in contrast with M6 (that differs from MAb1 in only a few amino acids), where cluster analysis reveals the formation of a large number of smaller clusters that is hypothesized to be the reason for the observed lower viscosity. The coarse-grained representations are effective in picking up differences based on local charge distributions of domains to make predictions on the self-association characteristics of these protein solutions.
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