Characterization of mRNA Lipid Nanoparticles by Electron Density Mapping Reconstruction: X-ray Scattering with Density from Solution Scattering (DENSS) Algorithm.

PURPOSE: This study aimed to test the feasibility of using Small Angle X-ray Scattering (SAXS) coupled with Density from Solution Scattering (DENSS) algorithm to characterize the internal architecture of messenger RNA-containing lipid nanoparticles (mRNA-LNPs). METHODS: The DENSS algorithm was employed to construct a three-dimensional model of average individual mRNA-LNP. The reconstructed models were cross validated with cryogenic transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS) to assess size, morphology, and internal structure. RESULTS: Cryo-TEM and DLS complemented SAXS, revealed a core-shell mRNA-LNP structure with electron-rich mRNA-rich region at the core, surrounded by lipids. The reconstructed model, utilizing the DENSS algorithm, effectively distinguishes mRNA and lipids via electron density mapping. Notably, DENSS accurately models the morphology of the mRNA-LNPs as an ellipsoidal shape with a "bleb" architecture or a two-compartment structure with contrasting electron densities, corresponding to mRNA-filled and empty lipid compartments, respectively. Finally, subtle changes in the LNP structure after three freeze-thaw cycles were detected by SAXS, demonstrating an increase in radius of gyration (Rg) associated with mRNA leakage. CONCLUSION: Analyzing SAXS profiles based on DENSS algorithm to yield a reconstructed electron density based three-dimensional model can be a useful physicochemical characterization method in the toolbox to study mRNA-LNPs and facilitate their development.

Antibody-Conjugated Polymersomes with Encapsulated Indocyanine Green J-Aggregates and High Near-Infrared Absorption for Molecular Photoacoustic Cancer Imaging.

Imaging plays a critical role in all stages of cancer care from early detection to diagnosis, prognosis, and therapy monitoring. Recently, photoacoustic imaging (PAI) has started to emerge into the clinical realm due to its high sensitivity and ability to penetrate tissues up to several centimeters deep. Herein, we encapsulated indocyanine green J (ICGJ) aggregate, one of the only FDA-approved organic exogenous contrast agents that absorbs in the near-infrared range, at high loadings up to ∼40% w/w within biodegradable polymersomes (ICGJ-Ps) composed of poly(lactide-co-glycolide-b-polyethylene glycol) (PLGA-b-PEG). The small Ps hydrodynamic diameter of 80 nm is advantageous for in vivo applications, while directional conjugation with epidermal growth factor receptor (EGFR) targeting cetuximab antibodies renders molecular specificity. Even when exposed to serum, the ∼11 nm-thick membrane of the Ps prevents dissociation of the encapsulated ICGJ for at least 48 h with a high ratio of ICGJ to monomeric ICG absorbances (i.e., I895/I780 ratio) of approximately 5.0 that enables generation of a strong NIR photoacoustic (PA) signal. The PA signal of polymersome-labeled breast cancer cells is proportional to the level of cellular EGFR expression, indicating the feasibility of molecular PAI with antibody-conjugated ICGJ-Ps. Furthermore, the labeled cells were successfully detected with PAI in highly turbid tissue-mimicking phantoms up to a depth of 5 mm with the PA signal proportional to the amount of cells. These data show the potential of molecular PAI with ICGJ-Ps for clinical applications such as tumor margin detection, evaluation of lymph nodes for the presence of micrometastasis, and laparoscopic imaging procedures.

Characterizing Protein-Protein Interactions and Viscosity of a Monoclonal Antibody from Low to High Concentration Using Small-Angle X-ray Scattering and Molecular Dynamics Simulations.

Understanding protein-protein interactions and formation of reversible oligomers (clusters) in concentrated monoclonal antibody (mAb) solutions is necessary for designing stable, low viscosity (η) concentrated formulations for processing and subcutaneous injection. Here we characterize the strength (K) of short-range anisotropic attractions (SRA) for 75-200 mg/mL mAb2 solutions at different pH and cosolute conditions by analyzing structure factors (Seff(q)) from small-angle X-ray scattering (SAXS) using coarse-grained molecular dynamics simulations. Best fit simulations additionally provide cluster size distributions, fractal dimensions, cluster occluded volume, and mAb coordination numbers. These equilibrium properties are utilized in a model to account for increases in viscosity caused by occluded volume in the clusters (packing effects) and dissipation of stress across lubricated fractal clusters. Seff(q) is highly sensitive to K at 75 mg/mL where mAbs can mutually align to form SRA contacts but becomes less sensitive at 200 mg/mL as steric repulsion due to packing becomes dominant. In contrast, η at 200 mg/mL is highly sensitive to SRA and the average cluster size from SAXS/simulation, which is observed to track the cluster relaxation time from shear thinning. By analyzing the distribution of sub-bead hot spots on the 3D mAb surface, we identify a strongly attractive hydrophobic patch in the complementarity determining region (CDR) at pH 4.5 that contributes to the high K and consequently large cluster sizes and high η. Adding NaCl screens electrostatic interactions and increases the impact of hydrophobic attraction on cluster size and raises η, whereas nonspecific binding of Arg attenuates all SRA, reducing η. The hydrophobic patch is absent at higher pH values, leading to smaller K, smaller clusters, and lower η. This work constitutes a first attempt to use SAXS and CG modeling to link both structural and rheological properties of concentrated mAb solutions to the energetics of specific hydrophobic patches on mAb surfaces. As such, our work opens an avenue for future research, including the possibility of designing coarse-grained models with physically meaningful interacting hot spots.

Subclass Effects on Self-Association and Viscosity of Monoclonal Antibodies at High Concentrations.

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 (KCDR-CH3) for vedolizumab IgG1, IgG2, or IgG4 subclasses by fitting small-angle X-ray scattering (SAXS) structure factor Seff(q) data with an extensive library of 12-bead coarse-grained (CG) molecular dynamics simulations. The KCDR-CH3 bead attraction strength was isolated from the strength of long-range 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 (KCDR-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 KCDR-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 short-range 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.

Characterizing Experimental Monoclonal Antibody Interactions and Clustering Using a Coarse-Grained Simulation Library and a Viscosity Model.

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 long-range Coulombic repulsion on cluster properties is investigated by analyzing the cluster-size 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.

Ultra-stable CO2-in-water foam by generating switchable Janus nanoparticles in-situ.

HYPOTHESIS: The surface of silica nanoparticles (NP) may be covalently grafted with two amino ligands to balance colloidal stability and interfacial activity via formation of in situ Janus particles. The modified NP may be combined with a like-charged diamine surfactant to create ultra-stable CO2 foam at low NP concentrations. EXPERIMENTS: The NP colloidal stability was measured up to 80 °C in 230 g/L TDS brine with dynamic light scattering. The NP surface was characterized using zeta potential, TEM, TGA, conductometric and potentiometric titrations, NMR and interfacial measurement. CO2/brine foam was generated at 60-80 °C and 15 MPa and apparent viscosity was measured vs foam quality. The foam stability was measured in-situ with an optical microscope. FINDINGS: Upon adding only 0.1 wt% NP, ultra-stable CO2 foam was observed at 60 °C with a bubble coarsening rate 3 orders of magnitude lower than with surfactant alone. Foam bubbles were spherical with NP present, but became polyhedral for the much less stable surfactant-only foams. For this novel like-charged surfactant-NP system, the limited surfactant adsorption on the NP resulted in NP stabilized CO2 foam, while maintaining NP colloidal stability at high surfactant concentrations and high salinity, providing a new perspective of NP-surfactant design.

Polyzwitterionic Hydrogels for Highly Efficient High Salinity Solar Desalination.

Interfacial solar vapor generation (SVG) is regarded as a promising and sustainable strategy for clean water production. While many materials have demonstrated excellent evaporation rates under one sun, it remains challenging to design solar evaporators without compromising SVG performance in high-salinity brines (≥10 wt %). Herein, polyzwitterionic hydrogels (PZHs) are proposed as a novel platform for high-salinity solar desalination. Taking advantage of the unique anti-polyelectrolyte effects, PZHs can trap salt ions from the brine water to form a more hydrated polymer network, leading to enhanced SVG performance. PZHs exhibit an exceptional solar evaporation rate of 4.14 kg m-2 h-1 in 10 wt % brine, which is ≈20 % higher than that in pure water. It is anticipated that salt-responsive PZHs may provide insights for the design of next-generation solar desalination systems.

Ultrastable N2/Water Foams Stabilized by Dilute Nanoparticles and a Surfactant at High Salinity and High Pressure.

The rapid development of unconventional oil and gas resources presents challenges for foam flooding for reservoirs with high salinity and high heterogeneity at elevated temperatures. In this study, hydrophilic anionic sulfonate-modified nanoparticles (NPs) exhibited a synergistic effect with a cationic surfactant in stabilizing N2/water foam in the presence of concentrated divalent ions from ambient temperature up to 70 °C. With low concentrations of both the sulfonated NPs (SNPs) and cationic surfactant, the foams remained stable for 4 days at 50 °C and atmospheric pressure, while the surfactant-stabilized foams collapsed completely in 1 day. This stability mechanism of foams by the SNPs and cationic surfactant is described in terms of phase behavior, bulk shear rheology of the aqueous phase, and the dilational modulus of the gas-brine interface. The high surface elastic dilational modulus E' observed upon addition of the SNP provided stability against coarsening according to the Gibbs criteria. The cryo-SEM images also showed the compact bubble structure of foams provided by the SNPs. Consequently, very minor changes in the foam bubble size were observed at 208 bar (3000 psi) and 50 °C for up to 48 h with only 0.1 wt % or 0.3 wt % SNPs and 0.01 wt % Arquad 12-50, indicating excellent foam stability. The ability of the surfactant and NPs to stabilize foams at low concentrations broadens the application of foams in subsurface reservoirs at high temperatures and salinities.

Highly Elastic Interconnected Porous Hydrogels through Self-Assembled Templating for Solar Water Purification.

Interfacial evaporation using porous hydrogels has demonstrated highly effective solar evaporation performance under natural sunlight to ensure an affordable clean water supply. However, it remains challenging to realize scalable and ready-to-use hydrogel materials with durable mechanical properties. Here, self-assembled templating (SAT) is developed as a simple yet effective method to fabricate large-scale elastic hydrogel evaporators with excellent desalination performance. The highly interconnected porous structure of the hydrogels with low tortuosity and tunable pore size enables high level of tunability on the water transport rate. With superior elasticity, the porous hydrogels are easy to process with a rapid shape recovery after being rolled, folded, and twisted over hundred times, and exhibit highly effective and stable evaporation with an evaporation rate of ≈2.8 kg m-2 h-1 and ≈90 % solar-to-vapor efficiency. It is anticipated that this SAT strategy, without the typical need for freeze-drying, will accelerate the industrialization of hydrogel solar evaporators for practical applications.

Elastic gas/water interface for highly stable foams with modified anionic silica nanoparticles and a like-charged surfactant.

HYPOTHESIS: Surface active anionic nanoparticles (NPs) with strategically designed covalent ligands may be combined with a liked-charged surfactant to form a highly elastic gas-water interface leading to highly stable gas/water foams. EXPERIMENTS: The colloidal stability of the NPs was determined by dynamic light scattering, and the surface elastic dilational modulus E' of the interface by sinusoidal oscillation of a pendant droplet at 0.1 Hz, which was superimposed on large-amplitude compression-expansion cycles. The foam stability was measured with optical microscopy of the bubble size distribution and from the macroscopic foam height. FINDINGS: The NPs played the key role the formation of a highly elastic air-water interface with a high E' despite a surfactant level well above the critical micelle concentration. Unlike the case for most previous studies, the NP amphiphilicity was essentially independent of the surfactant given the very low adsorption of the surfactant on the like-charged NP surfaces. With high E' values, both coalescence and coarsening were reduced leading to highly foam up to 80 °C. However, the surfactant facilitated foam generation at much lower shear rates than with NPs alone. The tuning of NP surfaces with ligands for colloidal stability in brine and simultaneously high amphiphilicity at the gas-water interface, over a wide range in surfactant concentration, is of broad interest for enabling the design of highly stable foams.

Molecular Engineering of Hydrogels for Rapid Water Disinfection and Sustainable Solar Vapor Generation.

Consumption of unsafe water is a major cause of morbidity and mortality in developing regions. Pasteurizing or boiling water to remove pathogens is energy-intensive and often impractical to off-grid communities. Therefore, low capital cost, rapid and energy-efficient water disinfection methods are urgently required to address global challenges of safe water access. Here, anti-bacterial hydrogels (ABHs) with catechol-enabled molecular-level hydrogen peroxide generators and quinone-anchored activated carbon particles are designed for effective water treatment. The bactericidal effect is attributed to the synergy of hydrogen peroxide and quinone groups to attack essential cell components and disturb bacterial metabolism. ABHs can be directly used as tablets to achieve >99.999% water disinfection efficiency within 60 min without energy input. No harmful byproducts are formed during the treatment process, after which the ABH tablets can be easily removed without residues. Taking advantage of their excellent photothermal and biofouling-resistant properties, ABHs can also be applied as solar evaporators to achieve stable water purification under sunlight (≤1 kW m-2 ) after months of storage and operation in bacteria-containing river water. The ABH platform offers reduced energy and chemical demands for point-of-use water treatment technologies in remote areas and emergency rescue applications.

Tuning Surface Chemistry and Ionic Strength to Control Nanoparticle Adsorption and Elastic Dilational Modulus at Air-Brine Interface.

The relationship between the interfacial rheology of nanoparticle (NP) laden air-brine interfaces and NP adsorption and interparticle interactions is not well understood, particularly as a function of the surface chemistry and salinity. Herein, a nonionic ether diol on the surface of silica NPs provides steric stabilization in bulk brine and at the air-brine interface, whereas a second smaller underlying hydrophobic ligand raises the hydrophobicity to promote NP adsorption. The level of NPs adsorption at steady state is sufficient to produce an interface with a relatively strong elastic dilational modulus E' = dγ/d ln A. However, the interface is ductile with a relatively slow change in E' as the interfacial area is varied over a wide range during compression and expansion. In contrast, for silica NPs stabilized with only a single hydrophobic ligand, the interfaces are often more fragile and may fracture with small changes in area. The presence of concentrated divalent cations improves E' and ductility by screening electrostatic dipolar repulsion and strengthening the attractive forces between nanoparticles. The ability to tune the interfacial rheology with NP surface chemistry is of great interest for designing more stable gas/brine foams.

Tuning Nanoparticle Surface Chemistry and Interfacial Properties for Highly Stable Nitrogen-In-Brine Foams.

The design of surface chemistries on nanoparticles (NPs) to stabilize gas/brine foams with concentrated electrolytes, especially with divalent ions, has been elusive. Herein, we tune the surface of 20 nm silica NPs by grafting a hydrophilic and a hydrophobic ligand to achieve two seemingly contradictory goals of colloidal stability in brine and high NP adsorption to yield a viscoelastic gas-brine interface. Highly stable nitrogen/water (N2/brine) foams are formed with CaCl2 concentrations up to 2% from 25 to 90 °C. The viscoelastic gas-brine interface retards drainage of the lamellae, and the high dilational elasticity arrests coarsening (Ostwald ripening) with no observable change in foam bubble size over 48 h. The ability to design NP-laden viscoelastic interfaces for highly stable foams, even with high divalent ion concentrations, is of fundamental mechanistic interest for a broad range of foam applications and in particular foams for CO2 sequestration and enhanced oil recovery.

Effect of surface chemistry of silica nanoparticles on contact angle of oil on calcite surfaces in concentrated brine with divalent ions.

HYPOTHESIS: For an oil droplet on calcite with an intervening brine film, the water contact angle θw may be reduced markedly (greater water wetness) with surface modified silica nanoparticles (NP). Modification with cationic, anionic, and nonionic ligands may be used to control the nanoparticle adsorption and interactions at the oil-brine and brine-calcite interfaces to influence the rate and degree of reduction in θw. EXPERIMENTS: The colloidal stability at 25 °C was determined in concentrated divalent brine (8 wt% NaCl and 2 wt% CaCl2) with dynamic light scattering, and the NP adsorption was determined on calcite. The NP adsorption at the oil-brine interface was characterized with the elastic dilational modulus. θw was measured for model decane-stearic acid droplets and crude oil droplets on calcite from 25 to 80 °C. FINDINGS: The fastest rate and greatest extent of reduction in θw for grafted ligands followed the order: cationic quaternary trimethylamine > sulfonate > methyl phosphonate > gluconamide. New mechanisms for reduction in θw were demonstrated on the basis of changes in interactions from NP adsorption at each interface. The greatest efficacy for the cationic NPs results from the weakest adsorption on calcite, steric repulsion at the three-phase contact line and the greatest desorption of carboxylate surfactants from the calcite.

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.

Protein-Protein Interactions, Clustering, and Rheology for Bovine IgG up to High Concentrations Characterized by Small Angle X-Ray Scattering and Molecular Dynamics Simulations.

A systematic understanding of intermolecular interactions is necessary for designing concentrated monoclonal and polyclonal antibody solutions with reduced viscosity and enhanced stability. Here, we determine the effects of pH and cosolute on the strength and geometry of short-range anisotropic protein-protein attractions for a polyclonal bovine IgG by comparing intensities [I(q)] obtained from small-angle X-ray scattering to those computed in molecular dynamics simulations with 12-bead models. As our model embodies key features of the protein shape, it can describe the experimental I(q) for solutions of 10-200 mg/mL protein with only a small (<1 kBT) variation in the model's well depth. At high concentration, small changes in the interaction potential produce large increases in clustering given the close interprotein spacing. Reducing the pH below the pI or adding NaCl weakens short-range anisotropic attractions but not enough to remove large reversible oligomers that raise viscosity. In contrast, for arginine added at pH 5.5, a uniform attraction model is sufficient to describe the I(q) that plateaus at low q. With primarily monomers and dimers, the viscosity is reduced relative to the other systems that have larger clusters as described with a model that includes the cluster size distribution.

Indocyanine Green J Aggregates in Polymersomes for Near-Infrared Photoacoustic Imaging.

Clinical translation of photoacoustic imaging (PAI) has been limited by the lack of near-infrared (NIR) contrast agents with low toxicity required for regulatory approval. Herein, J aggregates of indocyanine green (ICG) with strong NIR absorbance were encapsulated at high loadings within small 77 nm polymersomes (nanocapsules) composed of poly(lactide-co-glycolide-b-poly(ethylene glycol)) (PLGA-b-PEG) bilayers, thus enabling PAI of of breast and ovarian cancer cells with high specificity and a sensitivity at the level of ∼100 total cells. All of the major components of the polymersomes are FDA approved and used in the clinic. During formation of polymersomes with a water-in-oil-in-water double emulsion process, loss of ICG from the ICG J aggregates was minimized by coating them with a layer of branched polyethylenimine and by providing excess "sacrificial" ICG to adsorb at the oil-water interfaces. The encapsulated J aggregates were protected against dissociation by the polymersome shell for 24 h in 100% fetal bovine serum, after which the polymersomes biodegraded and the J aggregates dissociated to ICG monomers.

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.

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.

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.