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.

Attractive protein−protein interactions in concentrated monoclonal antibody (mAb) solutions may lead to the formation of clusters that increase viscosity. Here, we propose an analytical model that relates mAb solution viscosity to clustering by accounting for the contributions of suboptimal mAb packing within a cluster and cluster fractal dimension. The influence of short-range, anisotropic attractions and longrange Coulombic repulsion on cluster properties is investigated by analyzing the clustersize distributions, cluster fractal dimensions, radial distribution functions, and static structure factors from a library of coarse-grained molecular dynamics simulations. The library spans a vast range of mAb charges and attractive interactions in solutions of varying ionic strength. We present a framework for combining the viscosity model and simulation library to successfully characterize the attraction, repulsion, and clustering of an experimental mAb in three different pH and cosolute conditions by fitting the measured viscosity or structure factor from small-angle X-ray scattering. At low ionic strength, the cluster-size distribution is impacted by strong charges, and both the viscosity and net charge or structure factor and net charge must be considered to deconvolute the effects of short-range attraction and long-range repulsion.

The ability to design and formulate mAbs to minimize attractive interactions at high concentrations is important for protein processing, stability, and administration, particularly in subcutaneous delivery, where high viscosities are often challenging. The strength of protein−protein interactions (PPIs) of an IgG1 and IgG4 monoclonal antibody (mAb) from low to high concentration was determined by static light scattering (SLS) and used to understand viscosity data. The PPI were tuned using NaCl and five organic ionic co-solutes. The PPI strength was quantified by the normalized structure factor S(0)/S(0) HS and Kirkwood−Buff integral G 22 /G 22,HS (HS = hard sphere) determined from the SLS data and also by fits with (1) a spherical Yukawa potential and (2) an interacting hard sphere (IHS) model, which describes attraction in terms of hypothetical oligomers. The IHS model was better able to capture the scattering behavior of the more strongly interacting systems (mAb and/or co-solute) than the spherical Yukawa potential. For each descriptor of PPI, linear correlations were obtained between the viscosity at high concentration (200 mg/mL) and the interaction strengths evaluated both at low (20 mg/mL) and high concentrations (200 mg/mL) for a given mAb. However, the only parameter that provided a correlation across both mAbs was the oligomer mass ratio (m oligomer /m monomer+dimer ) from the IHS model, indicating the importance of self-association (in addition to the direct influence of the attractive PPI) on the viscosity.

Understanding protein–protein interactions in concentrated therapeutic monoclonal antibody (mAb) solutions is desirable for improved drug discovery, processing, and administration. Here, we deduce both the net protein charge and the magnitude and geometry of short-ranged, anisotropic attractions of a mAb across multiple concentrations and cosolute conditions by comparing structure factors S(q) obtained from small-angle X-ray scattering experiments with those from molecular dynamics (MD) simulations. The simulations, which utilize coarse-grained 12-bead models exhibiting a uniform van der Waals attraction, uniform electrostatic repulsion, and short-range attractions between specific beads, are versatile enough to fit S(q) of a wide range of protein concentrations and ionic strength with the same charge on each bead and a single anisotropic short-range attraction strength. Cluster size distributions (CSDs) obtained from best fit simulations reveal that the experimental structure is consistent with small reversible oligomers in even low viscosity systems and help quantify the impact of these clusters on viscosity. The ability to systematically use experimental S(q) data together with MD simulations to discriminate between different possible protein–protein interactions, as well as to predict viscosities from protein CSDs, is beneficial for designing mAbs and developing formulation strategies that avoid high viscosities and aggregation at high concentration.

The effects of a subclass of monoclonal antibodies (mAbs) on protein−protein interactions, formation of reversible oligomers (clusters), and viscosity (η) are not well understood at high concentrations. Herein, we quantify a short-range anisotropic attraction between the complementarity-determining region (CDR) and CH3 domains (K CDR-CH3 ) for vedolizumab IgG1, IgG2, or IgG4 subclasses by fitting small-angle X-ray scattering (SAXS) structure factor S eff (q) data with an extensive library of 12-bead coarse-grained (CG) molecular dynamics simulations. The K CDR-CH3 bead attraction strength was isolated from the strength of longrange electrostatic repulsion for the full mAb, which was determined from the theoretical net charge and a scaling parameter ψ to account for solvent accessibility and ion pairing. At low ionic strength (IS), the strongest short-range attraction (K CDR-CH3 ) and consequently the largest clusters and highest η were observed with IgG1, the subclass with the most positively charged CH3 domain. Furthermore, the trend in K CDR-CH3 with the subclass followed the electrostatic interaction energy between the CDR and CH3 regions calculated with the BioLuminate software using the 3D mAb structure and molecular interaction potentials. Whereas the equilibrium cluster size distributions and fractal dimensions were determined from fits of SAXS with the MD simulations, the degree of cluster rigidity under flow was estimated from the experimental η with a phenomenological model. For the systems with the largest clusters, especially IgG1, the inefficient packing of mAbs in the clusters played the largest role in increasing η, whereas for other systems, the relative contribution from stress produced by the clusters was more significant. The ability to relate η to shortrange attraction from SAXS measurements at high concentrations and to theoretical characterization of electrostatic patches on the 3D surface is not only of fundamental interest but also of practical value for mAb discovery, processing, formulation, and subcutaneous delivery.