Quantifying the impact of an invasive Hornet on Bombus terrestris Colonies.

The invasive hornet Vespa velutina nigrithorax is considered a proliferating threat to pollinators in Europe and Asia. While the impact of this species on managed honey bees is well-documented, effects upon other pollinator populations remain poorly understood. Nonetheless, dietary analyses indicate that the hornets consume a diversity of prey, fuelling concerns for at-risk taxa. Here, we quantify the impact of V. velutina upon standardised commercially-reared colonies of the European bumblebee, Bombus terrestris terrestris. Using a landscape-scale experimental design, we deploy colonies across a gradient of local V. velutina densities, utilising automated tracking to non-invasively observe bee and hornet behaviour, and quantify subsequent effects upon colony outcomes. Our results demonstrate that hornets frequently hunt at B. terrestris colonies, being preferentially attracted to those with high foraging traffic, and engaging in repeated-yet entirely unsuccessful-predation attempts at nest entrances. Notably however, we show that B. terrestris colony weights are negatively associated with local V. velutina densities, indicating potential indirect effects upon colony growth. Taken together, these findings provide the first empirical insight into impacts on bumblebees at the colony level, and inform future mitigation efforts for wild and managed pollinators.

Chemometrics in Protein Formulation: Stability Governed by Repulsion and Protein Unfolding.

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.

The Relationship between Protein-Protein Interactions and Liquid-Liquid Phase Separation for Monoclonal Antibodies.

Being able to predict and control concentrated solution properties for solutions of monoclonal antibodies (mAbs) is critical for developing therapeutic formulations. At higher protein concentrations, undesirable solution properties include high viscosities, opalescence, particle formation, and precipitation. The overall aim of this work is to understand the relationship between commonly measured dilute solution parameters, the reduced osmotic second virial coefficient b22 and the diffusion interaction parameter kD and liquid-liquid phase separation, which occurs at higher protein concentrations. For globular proteins such as lysozyme or γB-crystallin, the location of the liquid-liquid coexistence curve is controlled by the net protein-protein interaction, which is related to b22. Because many mAbs undergo reversible self-association due to forming highly directional interactions, it is not known if b22 can be used as a reliable predictor for LLPS since increasing the anisotropy in the interaction potential causes phase separation to occur at much stonger net protein-protein attractions or lower values of b22. Here, we map the coexistence curves for three mAbs, referred to as COE-01, COE-07, and COE-19, in terms of b22 and kD values. The measurements are carried out at a low salt condition near the pI, where protein-protein interactions are expected to be anisotropic due to the presence of electrostatic attractions, and under salting-out conditions at high ammonium sulfate concentrations, which is expected to reduce the anisotropy by screening electrostatic interactions. We also show that deviations from a linear correlation between b22 and kD can be used as an indicator of reversible self-association. Each of the mAbs under salting-out conditions follows the correlation supporting the hypothesis that protein-protein interactions are nonspecific, while deviations from the correlation occur for COE-01 and COE-19 under low salt conditions indicating the mAbs undergo reversible self-association. For five out of the six conditions, the onset of phase separation, as reflected by the reduced virial coefficient at the critical point b22c occurs in a small window -1.6 > b22c > -2.3, which is similar to what has been observed for lysozyme and for bovine γB-crystallin. Under low salt conditions, b22c ≈ -5.1 for COE-19, which we previously showed to self-associate into small oligomers. Our findings suggest that under conditions where mAb interactions are weakly anisotropic, such as occur at high salt conditions, phase separation will begin to occur in a small window of b22. Deviations from the window can occur when mAbs undergo reversible self-association, although this is not always the case and likely depends upon whether or not highly directional interactions are passivated in the oligomer formation. We expect fitting LLPS measurements to simplified interaction models for mAbs will provide additional insight into the nature of the protein-protein interactions and guide their development for calculating concentrated solution properties.

Enhanced Thermal Stability and Reduced Aggregation in an Antibody Fab Fragment at Elevated Concentrations.

The aggregation of protein therapeutics such as antibodies remains a major challenge in the biopharmaceutical industry. The present study aimed to characterize the impact of the protein concentration on the mechanisms and potential pathways for aggregation, using the antibody Fab fragment A33 as the model protein. Aggregation kinetics were determined for 0.05 to 100 mg/mL Fab A33, at 65 °C. A surprising trend was observed whereby increasing the concentration decreased the relative aggregation rate, ln(v) (% day-1), from 8.5 at 0.05 mg/mL to 4.4 at 100 mg/mL. The absolute aggregation rate (mol L-1 h-1) increased with the concentration following a rate order of approximately 1 up to a concentration of 25 mg/mL. Above this concentration, there was a transition to an apparently negative rate order of -1.1 up to 100 mg/mL. Several potential mechanisms were examined as possible explanations. A greater apparent conformational stability at 100 mg/mL was observed from an increase in the thermal transition midpoint (Tm) by 7-9 °C, relative to those at 1-4 mg/mL. The associated change in unfolding entropy (△Svh) also increased by 14-18% at 25-100 mg/mL, relative to those at 1-4 mg/mL, indicating reduced conformational flexibility in the native ensemble. Addition of Tween or the crowding agents Ficoll and dextran, showed that neither surface adsorption, diffusion limitations nor simple volume crowding affected the aggregation rate. Fitting of kinetic data to a wide range of mechanistic models implied a reversible two-state conformational switch mechanism from aggregation-prone monomers (N*) into non-aggregating native forms (N) at higher concentrations. kD measurements from DLS data also suggested a weak self-attraction while remaining colloidally stable, consistent with macromolecular self-crowding within weakly associated reversible oligomers. Such a model is also consistent with compaction of the native ensemble observed through changes in Tm and △Svh.

Nonspecific Binding of Adenosine Tripolyphosphate and Tripolyphosphate Modulates the Phase Behavior of Lysozyme.

Adenosine tripolyphosphate (ATP) is a small polyvalent anion that has recently been shown to interact with proteins and have a major impact on assembly processes involved in biomolecular condensate formation and protein aggregation. However, the nature of non-specific protein-ATP interactions and their effects on protein solubility are largely unknown. Here, the binding of ATP to the globular model protein is characterized in detail using X-ray crystallography and nuclear magnetic resonance (NMR). Using NMR, we identified six ATP binding sites on the lysozyme surface, with one known high-affinity nucleic acid binding site and five non-specific previously unknown sites with millimolar affinities that also bind tripolyphosphate (TPP). ATP binding occurs primarily through the polyphosphate moiety, which was confirmed by the X-ray structure of the lysozyme-ATP complex. Importantly, ATP binds preferentially to arginine over lysine in non-specific binding sites. ATP and TPP have similar effects on solution-phase protein-protein interactions. At low salt concentrations, ion binding to lysozyme causes precipitation, while at higher salt concentrations, redissolution occurs. The addition of an equimolar concentration of magnesium to ATP does not alter ATP binding affinities but prevents lysozyme precipitation. These findings have important implications for both protein crystallization and cell biology. Crystallization occurs readily in ATP solutions outside the well-established crystallization window. In the context of cell biology, the findings suggest that ATP binds non-specifically to folded proteins in physiological conditions. Based on the nature of the binding sites identified by NMR, we propose several mechanisms for how ATP binding can prevent the aggregation of natively folded proteins.

Current advances in biopharmaceutical informatics: guidelines, impact and challenges in the computational developability assessment of antibody therapeutics.

Therapeutic monoclonal antibodies and their derivatives are key components of clinical pipelines in the global biopharmaceutical industry. The availability of large datasets of antibody sequences, structures, and biophysical properties is increasingly enabling the development of predictive models and computational tools for the "developability assessment" of antibody drug candidates. Here, we provide an overview of the antibody informatics tools applicable to the prediction of developability issues such as stability, aggregation, immunogenicity, and chemical degradation. We further evaluate the opportunities and challenges of using biopharmaceutical informatics for drug discovery and optimization. Finally, we discuss the potential of developability guidelines based on in silico metrics that can be used for the assessment of antibody stability and manufacturability.

Towards an improved prediction of concentrated antibody solution viscosity using the Huggins coefficient.

The viscosity of a monoclonal antibody solution must be monitored and controlled as it can adversely affect product processing, packaging and administration. Engineering low viscosity mAb formulations is challenging as prohibitive amounts of material are required for concentrated solution analysis, and it is difficult to predict viscosity from parameters obtained through low-volume, high-throughput measurements such as the interaction parameter, kD, and the second osmotic virial coefficient, B22. As a measure encompassing the effect of intermolecular interactions on dilute solution viscosity, the Huggins coefficient, kh, is a promising candidate as a parameter measureable at low concentrations, but indicative of concentrated solution viscosity. In this study, a differential viscometry technique is developed to measure the intrinsic viscosity, [η], and the Huggins coefficient, kh, of protein solutions. To understand the effect of colloidal protein-protein interactions on the viscosity of concentrated protein formulations, the viscometric parameters are compared to kD and B22 of two mAbs, tuning the contributions of repulsive and attractive forces to the net protein-protein interaction by adjusting solution pH and ionic strength. We find a strong correlation between the concentrated protein solution viscosity and the kh but this was not observed for the kD or the b22, which have been previously used as indicators of high concentration viscosity. Trends observed in [η] and kh values as a function of pH and ionic strength are rationalised in terms of protein-protein interactions.

ATP and Tri-Polyphosphate (TPP) Suppress Protein Aggregate Growth by a Supercharging Mechanism.

A common strategy to increase aggregation resistance is through rational mutagenesis to supercharge proteins, which leads to high colloidal stability, but often has the undesirable effect of lowering conformational stability. We show this trade-off can be overcome by using small multivalent polyphosphate ions, adenosine triphosphate (ATP) and tripolyphosphate (TPP) as excipients. These ions are equally effective at suppressing aggregation of ovalbumin and bovine serum albumin (BSA) upon thermal stress as monitored by dynamic and static light scattering. Monomer loss kinetic studies, combined with measurements of native state protein-protein interactions and ζ-potentials, indicate the ions reduce aggregate growth by increasing the protein colloidal stability through binding and overcharging the protein. Out of three additional proteins studied, ribonuclease A (RNaseA), α-chymotrypsinogen (α-Cgn), and lysozyme, we only observed a reduction in aggregate growth for RNaseA, although overcharging by the poly-phosphate ions still occurs for lysozyme and α-Cgn. Because the salts do not alter protein conformational stability, using them as excipients could be a promising strategy for stabilizing biopharmaceuticals once the protein structural factors that determine whether multivalent ion binding will increase colloidal stability are better elucidated. Our findings also have biological implications. Recently, it has been proposed that ATP also plays an important role in maintaining intracellular biological condensates and preventing protein aggregation in densely packed cellular environments. We expect electrostatic interactions are a significant factor in determining the stabilizing ability of ATP towards maintaining proteins in non-dispersed states in vivo.

Cluster Percolation Causes Shear Thinning Behavior in Concentrated Solutions of Monoclonal Antibodies.

High-concentration (>100 g/L) solutions of monoclonal antibodies (mAbs) are typically characterized by anomalously large solution viscosity and shear thinning behavior for strain rates ≥103 s-1. Here, the link between protein-protein interactions (PPIs) and the rheology of concentrated solutions of COE-03 and COE-19 mAbs is studied by means of static and dynamic light scattering and microfluidic rheometry. By comparing the experimental data with predictions based on the Baxter sticky hard-sphere model, we surprisingly find a connection between the observed shear thinning and the predicted percolation threshold. The longest shear relaxation time of mAbs was much larger than that of model sticky hard spheres within the same region of the phase diagram, which is attributed to the anisotropy of the mAb PPIs. Our results suggest that not only the strength but also the patchiness of short-range attractive PPIs should be explicitly accounted for by theoretical approaches aimed at predicting the shear rate-dependent viscosity of dense mAb solutions.

Thermodynamic Origin of Differential Excipient-Lysozyme Interactions.

Understanding the intricate interplay of interactions between proteins, excipients, ions and water is important to achieve the effective purification and stable formulation of protein therapeutics. The free energy of lysozyme interacting with two kinds of polyanionic excipients, citrate and tripolyphosphate, together with sodium chloride and TRIS-buffer, are analysed in multiple-walker metadynamics simulations to understand why tripolyphosphate causes lysozyme to precipitate but citrate does not. The resulting multiscale decomposition of energy and entropy components for water, sodium chloride, excipients and lysozyme reveals that lysozyme is more stabilised by the interaction of tripolyphosphate with basic residues. This is accompanied by more sodium ions being released into solution from tripolyphosphate than for citrate, whilst the latter instead has more water molecules released into solution. Even though lysozyme aggregation is not directly probed in this study, these different mechanisms are suspected to drive the cross-linking between lysozyme molecules with vacant basic residues, ultimately leading to precipitation.

Comparison of Huggins Coefficients and Osmotic Second Virial Coefficients of Buffered Solutions of Monoclonal Antibodies.

The Huggins coefficient kH is a well-known metric for quantifying the increase in solution viscosity arising from intermolecular interactions in relatively dilute macromolecular solutions, and there has been much interest in this solution property in connection with developing improved antibody therapeutics. While numerous kH measurements have been reported for select monoclonal antibodies (mAbs) solutions, there has been limited study of kH in terms of the fundamental molecular interactions that determine this property. In this paper, we compare measurements of the osmotic second virial coefficient B22, a common metric of intermolecular and interparticle interaction strength, to measurements of kH for model antibody solutions. This comparison is motivated by the seminal work of Russel for hard sphere particles having a short-range "sticky" interparticle interaction, and we also compare our data with known results for uncharged flexible polymers having variable excluded volume interactions because proteins are polypeptide chains. Our observations indicate that neither the adhesive hard sphere model, a common colloidal model of globular proteins, nor the familiar uncharged flexible polymer model, an excellent model of intrinsically disordered proteins, describes the dependence of kH of these antibodies on B22. Clearly, an improved understanding of protein and ion solvation by water as well as dipole-dipole and charge-dipole effects is required to understand the significance of kH from the standpoint of fundamental protein-protein interactions. Despite shortcomings in our theoretical understanding of kH for antibody solutions, this quantity provides a useful practical measure of the strength of interprotein interactions at elevated protein concentrations that is of direct significance for the development of antibody formulations that minimize the solution viscosity.

Determination of Protein-Protein Interactions at High Co-Solvent Concentrations Using Static and Dynamic Light Scattering.

Protein-protein interactions are commonly measured in terms of the second osmotic virial coefficient, B22 from static light scattering (SLS) or the interaction parameter, kD from dynamic light scattering (DLS). Often these measurements are carried out at high co-solvent compositions, where correction factors are required for the light scattering analysis. For lysozyme in aqueous solutions containing the co-solvents NaCl, arginine chloride, urea, sucrose or guanidine chloride, we show that B22 determination requires using in the light scattering equation the refractive index increment of the protein measured at constant solvent chemical potential. Because the increment decreases with increasing co-solvent composition, using a constant value can lead to mis-interpretation of protein-protein interaction trends deduced from the B22 measurements. Furthermore, there is a contribution to the intensity auto-correlation function measured by dynamic light scattering due to co-solvents. This effect is removed by including longer delay times when fitting the cumulant analysis to determine the diffusion coefficients. We show that an experimentally observed correlation between B22 and kD is recovered once these correction factors have been applied. The findings are particularly relevant to biopharmaceutical industry, where B22 and kD measurements are used for screening excipient effects in liquid formulations.

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization.

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.

Determination of Protein-Protein Interactions in a Mixture of Two Monoclonal Antibodies.

The coformulation of monoclonal antibody (mAb) mixtures provides an attractive route to achieving therapeutic efficacy where the targeting of multiple epitopes is necessary. Controlling and predicting the behavior of such mixtures requires elucidating the molecular basis for the self- and cross-protein-protein interactions and how they depend on solution variables. While self-interactions are now beginning to be well understood, systematic studies of cross-interactions between mAbs in solution do not exist. Here, we have used static light scattering to measure the set of self- and cross-osmotic second virial coefficients in a solution containing a mixture of two mAbs, mAbA and mAbB, as a function of ionic strength and pH. mAbB exhibits strong association at a low ionic strength, which is attributed to an electrostatic attraction that is enhanced by the presence of a strong short-ranged attraction of nonelectrostatic origin. Under all solution conditions, the measured cross-interactions are intermediate self-interactions and follow similar patterns of behavior. There is a strong electrostatic attraction at higher pH values, reflecting the behavior of mAbB. Protein-protein interactions become more attractive with an increasing pH due to reducing the overall protein net charges, an effect that is attenuated with an increasing ionic strength due to the screening of electrostatic interactions. Under moderate ionic strength conditions, the reduced cross-virial coefficient, which reflects only the energetic contribution to protein-protein interactions, is given by a geometric average of the corresponding self-coefficients. We show the relationship can be rationalized using a patchy sphere model, where the interaction energy between sites i and j is given by the arithmetic mean of the i-i and j-j interactions. The geometric mean does not necessarily apply to all mAb mixtures and is expected to break down at a lower ionic strength due to the nonadditivity of electrostatic interactions.

Arginine to Lysine Mutations Increase the Aggregation Stability of a Single-Chain Variable Fragment through Unfolded-State Interactions.

Increased protein solubility is known to correlate with an increase in the proportion of lysine over arginine residues. Previous work has shown that the aggregation propensity of a single-chain variable fragment (scFv) does not correlate with its conformational stability or native-state protein-protein interactions. Here, we test the hypothesis that aggregation is driven by the colloidal stability of partially unfolded states, studying the behavior of scFv mutants harboring single or multiple site-specific arginine to lysine mutations in denaturing buffers. In 6 M guanidine hydrochloride (GdmCl) or 8 M urea, repulsive protein-protein interactions were measured for the wild-type and lysine-enriched (4RK) scFvs reflecting weakened short-range attractions and increased excluded volume. In contrast to the arginine-enriched mutant (7KR) scFv exhibited strong reversible association. In 3 M GdmCl, the minimum concentration at which the scFvs were unfolded, the hydrodynamic radius of 4RK remained constant but increased for the wild type and especially for 7KR. Studies of single-point arginine to lysine scFv mutants indicated that the observed aggregation propensity of arginine under denaturing conditions was nonspecific. Interestingly, one such swap generated a scFv with especially low aggregation rates under low/high ionic strengths and denaturing buffers; molecular modeling identified hydrogen bonding between the arginine side chain and main chain peptide groups, stabilizing the structure. The arginine/lysine ratio is not routinely considered in biopharmaceutical scaffold design or current amyloid prediction methods. This work therefore suggests a simple method for increasing the stability of a biopharmaceutical protein against aggregation.

Measuring Nonspecific Protein-Protein Interactions by Dynamic Light Scattering.

Dynamic light scattering has become a method of choice for measuring and quantifying weak, nonspecific protein-protein interactions due to its ease of use, minimal sample consumption, and amenability to high-throughput screening via plate readers. A procedure is given on how to prepare protein samples, carry out measurements by commonly used experimental setups including flow through systems, plate readers, and cuvettes, and analyze the correlation functions to obtain diffusion coefficient data. The chapter concludes by a theoretical section that derives and rationalizes the correlation between diffusion coefficient measurements and protein-protein interactions.

The Macromolecular Femtosecond Crystallography Instrument at the Linac Coherent Light Source.

The Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS) is the seventh and newest instrument at the world's first hard X-ray free-electron laser. It was designed with a primary focus on structural biology, employing the ultrafast pulses of X-rays from LCLS at atmospheric conditions to overcome radiation damage limitations in biological measurements. It is also capable of performing various time-resolved measurements. The MFX design consists of a versatile base system capable of supporting multiple methods, techniques and experimental endstations. The primary techniques supported are forward scattering and crystallography, with capabilities for various spectroscopic methods and time-resolved measurements. The location of the MFX instrument allows for utilization of multiplexing methods, increasing user access to LCLS by running multiple experiments simultaneously.

Models for Antibody Behavior in Hydrophobic Interaction Chromatography and in Self-Association.

Monoclonal antibodies (mAbs) form an increasingly important sector of the pharmaceutical market, and their behavior in production, processing, and formulation is a key factor in development. With data sets of solution properties for mAbs becoming available, and with amino acid sequences, and structures for many Fabs, it is timely to examine what features correlate with measured data. Here, previously published data for hydrophobic interaction chromatography and the formation of high molecular weight species are studied. Unsurprisingly, aromatic sidechain content of complementarity-determining regions (CDRs), underpins much of the variability in hydrophobic interaction chromatography data. However, this is not reflected in nonpolar solvent accessible surface enrichment at the antigen-combining site, consistent with a view in which hydrophobic interaction strength is dependent on curvature as well as on the extent of an interface. Sequence properties are also superior to surface-based structural properties in correlations with the high molecular weight species data. Combined length of CDRs is the most important factor, which could be an indication of flexibility that facilitates CDR-CDR interactions in mAb self-association. These observations couple to our understanding of protein physicochemical properties, laying the groundwork for improved developability models.

Controlling Phase Separation of Lysozyme with Polyvalent Anions.

The ability of polyvalent anions to influence protein-protein interactions and protein net charge was investigated through solubility and turbidity experiments, determination of osmotic second virial coefficients ( B22), and ζ-potential values for lysozyme solutions.  B22 values showed that all anions reduce protein-protein repulsion between positively charged lysozyme molecules, and those anions with higher net valencies are more effective. The polyvalent anions pyrophosphate and tripolyphosphate were observed to induce protein reentrant condensation, which has been previously observed with negatively charged proteins in the presence of trivalent cations. Reentrant condensation is a phenomenon in which low concentrations of polyvalent ions induce protein precipitation, but further increasing polyvalent ion concentration causes the protein precipitate to resolubilize. Interestingly, citrate does not induce lysozyme reentrant condensation despite having a similar charge, size, and shape to pyrophosphate. We observe qualitative differences in protein behavior when compared against negatively charged proteins in solutions of trivalent cations. The polyphosphate ions induce a much stronger protein-protein attraction, which correlates with the occurrence of a liquid-gel transition that replaces the liquid-liquid transition observed with trivalent cations. The results indicate that solutions of polyphosphate ions provide a model system for exploring the link between the protein-phase diagram and model interaction potentials and also highlight the importance that ion-specific effects can have on protein solubility.

Self-Interaction of Human Serum Albumin: A Formulation Perspective.

In the present study, small-angle X-ray scattering (SAXS) and static light scattering (SLS) have been used to study the solution properties and self-interaction of recombinant human serum albumin (rHSA) molecules in three pharmaceutically relevant buffer systems. Measurements are carried out up to high protein concentrations and as a function of ionic strength by adding sodium chloride to probe the role of electrostatic interactions. The effective structure factors (S eff) as a function of the scattering vector magnitude q have been extracted from the scattering profiles and fit to the solution of the Ornstein-Zernike equation using a screened Yukawa potential to describe the double-layer force. Although only a limited q range is used, accurate fits required including an electrostatic repulsion element in the model at low ionic strength, while only a hard sphere model with a tunable diameter is necessary for fitting to high-ionic-strength data. The fit values of net charge agree with available data from potentiometric titrations. Osmotic compressibility data obtained by extrapolating the SAXS profiles or directly from SLS measurements has been fit to a 10-term virial expansion for hard spheres and an equation of state for hard biaxial ellipsoids. We show that modeling rHSA as an ellipsoid, rather than a sphere, provides a much more accurate fit for the thermodynamic data over the entire concentration range. Osmotic virial coefficient data, derived at low protein concentration, can be used to parameterize the model for predicting the behavior up to concentrations as high as 450 g/L. The findings are especially important for the biopharmaceutical sector, which require approaches for predicting concentrated protein solution behavior using minimal sample consumption.