Coarse-Grained Molecular Dynamics Simulations for Understanding the Impact of Short-Range Anisotropic Attractions on Structure and Viscosity of Concentrated Monoclonal Antibody Solutions.

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.

Self-diffusion of a highly concentrated monoclonal antibody by fluorescence correlation spectroscopy: insight into protein-protein interactions and self-association.

The dynamic behavior of monoclonal antibodies (mAbs) at high concentration provides insight into protein microstructure and protein-protein interactions (PPI) that influence solution viscosity and protein stability. At high concentration, interpretation of the collective-diffusion coefficient Dc, as determined by dynamic light scattering (DLS), is highly challenging given the complex hydrodynamics and PPI at close spacings. In contrast, self-diffusion of a tracer particle by Brownian motion is simpler to understand. Herein, we develop fluorescence correlation spectroscopy (FCS) for the measurement of the long-time self-diffusion of mAb2 over a wide range of concentrations and viscosities in multiple co-solute formulations with varying PPI. The normalized self-diffusion coefficient D0/Ds (equal to the microscopic relative viscosity ηeff/η0) was found to be smaller than η/η0. Smaller ratios of the microscopic to macroscopic viscosity (ηeff/η) are attributed to a combination of weaker PPI and less self-association. The interaction parameters extracted from fits of D0/Ds with a length scale dependent viscosity model agree with previous measurements of PPI by SLS and SAXS. Trends in the degree of self-association, estimated from ηeff/η with a microviscosity model, are consistent with oligomer sizes measured by SLS. Finally, measurements of collective diffusion and osmotic compressibility were combined with FCS data to demonstrate that the changes in self-diffusion between formulations are due primarily to changes in the protein-protein friction in these systems, and not to protein-solvent friction. Thus, FCS is a robust and accessible technique for measuring mAb self-diffusion, and, by extension, microviscosity, PPI and self-association that govern mAb solution dynamics.

Improving Viscosity and Stability of a Highly Concentrated Monoclonal Antibody Solution with Concentrated Proline.

PURPOSE: To explain the effects of the osmolyte proline on the protein-protein interactions (PPI), viscosity and stability of highly concentrated antibody solutions in contrast to other neutral osmolytes. METHODS: The viscosity of ~225 mg/mL mAb solutions was measured with proline, glycine and trehalose as a function of pH and co-solute concentration up to 1.3 M. The stability was assessed via turbidity as well as size exclusion chromatography after 4 weeks storage at 40°C. The PPI strength was assessed qualitatively via the high concentration diffusion rate by dynamic light scattering. RESULTS: Increasing proline significantly reduced the mAb viscosity and increased the colloidal stability at pH 6, but not at pH 5 further from the mAb pI. In contrast, glycine and trehalose did not improve the viscosity nor stability. The normalized diffusion coefficient at high concentration, which is inversely proportional to the attractive PPI strength, increased with proline concentration but decreased with increasing glycine. CONCLUSIONS: Proline demonstrated greater efficacy for improving mAb viscosity and stability in contrast to glycine and trehalose due to its amphipathic structure and partial charge on the pyrrolidine side chain. These properties likely allow proline to screen the attractive electrostatic and hydrophobic interactions that promote self-association and high viscosities. Binary proline-histidine formulations also demonstrated greater viscosity reduction effects than histidine alone at the same total co-solute concentration, while maintaining a lower total solution osmolarity.

Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration.

Understanding protein stability is central to combatting protein aggregation diseases and developing new protein therapeutics. At the high concentrations often present in biological systems, purified proteins can exhibit undesirable high solution viscosities and poor solubilities mediated by short-range electrostatic and hydrophobic protein-protein interactions. The interplay between protein amino acid sequence, protein structure, and solvent conditions to minimize protein-protein interactions is key to designing well-behaved pharmaceutical proteins. However, theoretical approaches have yet to yield a general framework to address these problems. Here, we analyzed the high concentration behavior of superfolder GFP (sfGFP) and two supercharged sfGFP variants engineered to have formal charges of -18 or +15. Under low cosolute conditions, sfGFP and the -18 variant formed a gel or phase separated at ∼10 mg/mL. Under conditions that screen surface charges, including formulations with high histidine or high NaCl concentrations, all three variants attained concentrations up to 250 mg/mL with moderate viscosities. Moreover, all three variants exhibited very similar viscosity-concentration profiles over this range. This effect was not mimicked by high sugar concentrations that exert excluded-volume effects without shielding charge. Collectively, these data demonstrate that charge shielding neutralizes not only long-range electrostatic interactions but also, surprisingly, short-range electrostatic effects due to surface charge anisotropy. This work shows that supercharged sfGFP behavior under high ionic strength is largely determined by particle geometry, a conclusion that is supported by colloid models and may be applicable to pharmaceutically relevant proteins.

Contrasting the Influence of Cationic Amino Acids on the Viscosity and Stability of a Highly Concentrated Monoclonal Antibody.

PURPOSE: To explain the effects of cationic amino acids and other co-solutes on the viscosity, stability and protein-protein interactions (PPI) of highly concentrated (≥200 mg/ml) monoclonal antibody (mAb) solutions to advance subcutaneous injection. METHODS: The viscosities of ≥200 mg/ml mAb1 solutions with various co-solutes and pH were measured by capillary rheometry in some cases up to 70,000 s-1. The viscosities are analyzed in terms of dilute PPI characterized by diffusion interaction parameters (k D ) from dynamic light scattering (DLS). MAb stability was measured by turbidity and size exclusion chromatography (SEC) after 4 weeks of 40°C storage. RESULTS: Viscosity reductions were achieved by reducing the pH, or adding histidine, arginine, imidazole or camphorsulfonic acid, each of which contains a hydrophobic moiety. The addition of inorganic electrolytes or neutral osmolytes only weakly affected viscosity. Systems with reduced viscosities also tended to be Newtonian, while more viscous systems were shear thinning. CONCLUSIONS: 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 D , only after normalization to remove the effect of charge screening. Shear rate profiles demonstrate that select co-solutes reduce protein network formation.

Understanding the Impact of Microstructures on Reconstitution and Drying Kinetics of Lyophilized Cake Using X-ray Microscopy and Image-Based Simulation.

The drying time of lyophilization and resultant cake microstructure are dependent on each other as water and solvent leave a lyophilized cake. The drying rate affects the size, distribution, and tortuosity of the pores as these macropores evolve during the primary drying phase, which in return impact the further removal of water and solvent from the cake throughout the drying period. This interplay results in a microstructure that determines the reconstitution time for a given formulation. The current study employs advanced X-ray Microscopy (XRM) coupled with mathematical models to correlate the microstructure with the drying kinetics and the reconstitution time. The normalized diffusion coefficients, derived from the reconstructed 3D microstructure of the cake, correlate with the solid content of the pre-lyophilization solution and agree with the mass transfer coefficients from a semi-empirical drying model built with lyophilization process data. Specifically, a solution with less solid content leads to a lyophilized cake with larger pores, thinner walls, and a greater pore volume compared to a solution with more solid content. Consequently, models from the microstructure and drying experiments reveals faster mass transfer independently. While the mass transfer models from the cake structure and the lyophilization process data accurately represents the drying kinetics, both models are inadequate to describe the reconstitution process due to the significant impact from formulation ingredients that alter the mass transfer mechanism via solubility and wettability. In summary, X-ray microscopy imaging and mathematical models are powerful tools that provide insights into the lyophilization process from a new angle.

Ranking mAb-excipient interactions in biologics formulations by NMR spectroscopy and computational approaches.

Excipients are added to biopharmaceutical formulations to enhance protein stability and enable the development of robust formulations with acceptable physicochemical properties, but the mechanism by which they confer stability is not fully understood. Here, we aimed to elucidate the mechanism through direct experimental evidence of the binding affinity of an excipient to a monoclonal antibody (mAb), using saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopic method. We ranked a series of excipients with respect to their dissociation constant (KD) and nonspecific binding constants (Ns). In parallel, molecular dynamic and site identification by ligand competitive saturation (SILCS)-Monte Carlo simulations were done to rank the excipient proximity to the proteins, thereby corroborating the ranking by STD NMR. Finally, the excipient ranking by NMR was correlated with mAb conformational and colloidal stability. Our approach can aid excipient selection in biologic formulations by providing insights into mAb-excipient affinities before conventional and time-consuming excipient screening studies are conducted.

Enhancing Stability and Reducing Viscosity of a Monoclonal Antibody With Cosolutes by Weakening Protein-Protein Interactions.

An understanding of how cosolutes affect the viscosity and storage stability of highly concentrated mAbs as a function of protein-protein interactions (PPIs) would be desirable for improving processing and administration of protein therapeutics. The effects of inorganic and organic cosolutes on the viscosity and stability of mAb5 were determined for concentrations up to 250 mg/mL. Organic electrolytes Arg(HCl) and His(HCl) produced the largest viscosity reductions, indicating screening of local anisotropic short-ranged attractive and hydrophobic interactions. These cosolutes significantly reduced mAb5 aggregate concentration as measured by size-exclusion chromatography after 4 weeks of 40°C storage at 200 mg/mL, with the largest reduction for Arg(Glu). The effects of the cosolutes on storage stability and viscosity are related to their ability to reduce attractive PPIs at high concentration (200 mg/mL), as shown by comparing measurements of structure factor (by small-angle X-ray scattering) and collective diffusion (by dynamic light scattering) with models of hard and attractive spheres. The improved stability of Arg(Glu) over Arg(HCl) despite similar PPI by small-angle X-ray scattering at high concentration is consistent with higher protein conformational stability as determined by differential scanning fluorimetry and differential scanning light scattering.

Protein-Protein Interactions of Highly Concentrated Monoclonal Antibody Solutions via Static Light Scattering and Influence on the Viscosity.

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 G22/ G22,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 ( moligomer/ mmonomer+dimer) from the IHS model, indicating the importance of self-association (in addition to the direct influence of the attractive PPI) on the viscosity.

X-ray Scattering and Coarse-Grained Simulations for Clustering and Interactions of Monoclonal Antibodies at High Concentrations.

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 ( Seff( 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 ( gCOM( r)), coordination numbers, and cluster size distributions (CSD). The addition of cosolutes results in more attractive Seff( 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 gCOM( r)), will be of interest in engineering protein sequence and formulating protein solutions for weak PPI to minimize aggregation and viscosities.

Quenched Assembly of NIR-Active Gold Nanoclusters Capped with Strongly Bound Ligands by Tuning Particle Charge via pH and Salinity.

Gold nanospheres coated with a binary monolayer of bound citrate and cysteine ligands were assembled into nanoclusters, in which the size and near-infrared (NIR) extinction were tuned by varying the pH and concentration of added NaCl. During full evaporation of an aqueous dispersion of 4.5 ± 1.8 nm Au primary particles, the nanoclusters were formed and quenched by the triblock copolymer polylactic acid (PLA)(1K)-b-poly(ethylene glycol) (PEG)(10K)-b-PLA(1K), which also provided steric stabilization. The short-ranged depletion and van der Waals attractive forces were balanced against longer ranged electrostatic repulsion to tune the nanocluster diameter and NIR extinction. Upon lowering the pH from 7 to 5 at a given salinity, the magnitude of the charge on the primary particles decreased, such that the weaker electrostatic repulsion increased the hydrodynamic diameter and, consequently, NIR extinction of the clusters. At a given pH, as the concentration of NaCl was increased, the NIR extinction decreased monotonically. Furthermore, the greater screening of the charges on the nanoclusters weakened the interactions with PLA(1K)-b-PEG(10K)-b-PLA(1K) and thus lowered the amount of adsorbed polymer on the nanocluster surface. The generalization of the concept of self-assembly of small NIR-active nanoclusters to include a strongly bound thiol and the manipulation of the morphologies and NIR extinction by variation of pH and salinity not only is of fundamental interest but also is important for optical biomedical imaging and therapy.