So-called "weak" protein-protein interactions are important for the control of solution properties and stability at elevated protein concentrations (c2) but are not practical to capture in atomistic simulations. This report focuses on a series of coarse-grained models for predicting second osmotic virial coefficients (B22) and high-concentration Rayleigh scattering (osmotic compressibility) as a function of c2 for monoclonal antibodies (MAbs) that are of interest in biotechnology. B22 and molecular volume along with c2-dependent osmotic compressibility were calculated for a series of models with increasing structural detail. Models were refined to include contributions from sterics, short-ranged van der Waals and hydrophobic attractions, screened electrostatics, and the flexibility of the mAb hinge region. The results highlight shortcomings for spherical models of MAbs and a useful balance between numerical accuracy and computational burden offered by models based on 6 or 12 spherical, partly overlapping domains. The results provide bounds for realistic values of effective charges on variable domains in order for MAbs to be stable in solution and more generally illustrate semiquantitative bounds for the space of model parameters that can reproduce experimental behavior and provide a basis for future development of computationally efficient and accurate CG mAb models to predict both low- and high-c2 behavior.

Protein-protein interactions for solutions of an IgG1 molecule were quantified using static light scattering (SLS) measurements from low to high protein concentrations (c). SLS was used to determine second osmotic virial coefficients (B) at low c, and excess Rayleigh profiles (R/K vs. c) and zero-q structure factors (S) as a function of c at higher c for a series of conditions (pH, sucrose concentration, and total ionic strength [TIS]). Repulsive (attractive) interactions were observed at low TIS (high TIS) for pH 5 and 6.5, with increasing repulsions when 5% w/w sucrose was also present. Previously developed and refined coarse-grained antibody models were used to fit model parameters from B versus TIS data. The resulting parameters from low-c conditions were used as the sole input to multiprotein Monte Carlo simulations to predict high-cR/K and S behavior up to 150 g/L. Experimental results at high-c conditions were quantitatively predicted by the simulations for the coarse-grained models that treated antibody molecules as either 6 or 12 (sub) domains, which preserved the basic shape of a monoclonal antibody. Finally, preferential accumulation of sucrose around the protein surface was identified via high-precision density measurements, which self-consistently explained the simulation and experimental SLS results.

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The concentration-dependence of the diffusion and sedimentation coefficients (k(D) and k(s), respectively) of a protein can be used to determine the second virial coefficient (B₂), a parameter valuable in predicting protein-protein interactions. Accurate measurement of B₂ under physiologically and pharmaceutically relevant conditions, however, requires independent measurement of k(D) and k(s) via orthogonal techniques. We demonstrate this by utilizing sedimentation velocity (SV) and dynamic light scattering (DLS) to analyze solutions of hen-egg white lysozyme (HEWL) and a monoclonal antibody (mAb1) in different salt solutions. The accuracy of the SV-DLS method was established by comparing measured and literature B₂ values for HEWL. In contrast to the assumptions necessary for determining k(D) and k(s) via SV alone, k(D) and ks were of comparable magnitudes, and solution conditions were noted for both HEWL and mAb1 under which 1), k(D) and k(s) assumed opposite signs; and 2), k(D) ≥k(s). Further, we demonstrate the utility of k(D) and k(s) as qualitative predictors of protein aggregation through agitation and accelerated stability studies. Aggregation of mAb1 correlated well with B₂, k(D), and k(s), thus establishing the potential for k(D) to serve as a high-throughput predictor of protein aggregation.

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3M) salt concentrations, in dilute (<0.1M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li 1 , Na 1 , K 1 , Rb 1 , Cs 1 , GdnH 1 , and Ca 21 ), identity. The Q* decreased in the order, demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO 2À 4 anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.