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.

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.

Viscosity reduction down to 20 cP at 220 mg/ml mAb1 was achieved with co-solutes that are both charged and contain a hydrophobic interaction domain for sufficient binding to the protein surface. These reductions are related to the DLS diffusion interaction parameter, k , only after normalization to remove the effect of charge screening. Shear rate profiles demonstrate that select co-solutes reduce protein network formation.

During production of concentrated monoclonal antibody formulations by tangential flow ultrafiltration (TFF), high viscosities and aggregation often cause extensive membrane fouling, flux decay and low product yields. To address these challenges, the co-solutes histidine or imidazole were added at high concentrations from 250 to 320 mM to reduce the viscosity by up to tenfold relative to conventional low co-solute formulations, to as low as 40 cP at 250 mg/mL. At high mAb concentrations of up to 280 mg/mL, the transmembrane flux was increased threefold by adding high concentrations of co-solutes that also lowered the viscosity. Furthermore, the co-solutes also increased the mAb gel point concentration c g by up to 100 mg/mL mAb and thus enhanced concentration polarization-driven back-diffusion of the mAb at the membrane wall, which led to increased fluxes. The low viscosity and hollow fiber filter modules with straight flow paths enabled more uniform TMP and wall shear stress τ w profiles, which mitigated the reversible flux decay that results from an axial decline in the local TMP. The concentrated mAb was stable by SEC before and after extended storage at 4°C and 37°C.