Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform.

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Adsorption of non-ionic surfactant and monoclonal antibody on siliconized surface studied by neutron reflectometry.

The adsorption of monoclonal antibodies (mAbs) on hydrophobic surfaces is known to cause protein aggregation and degradation. Therefore, surfactants, such as Poloxamer 188, are widely used in therapeutic formulations to stabilize mAbs and protect mAbs from interacting with liquid-solid interfaces. Here, the adsorption of Poloxamer 188, one mAb and their competitive adsorption on a model hydrophobic siliconized surface is investigated with neutron scattering coupled with contrast variation to determine the molecular structure of adsorbed layers for each case. Small angle neutron scattering measurements of the affinity of Poloxamer 188 to this mAb indicate that there is negligible binding at these solution conditions. Neutron reflectometry measurements of the mAb show irreversible adsorption on the siliconized surface, which cannot be washed off with neat buffer. Poloxamer 188 can be adsorbed on the surface already occupied by mAb, which enables partial removal of some adsorbed mAb by washing with buffer. The adsorption of the surfactant introduces significant conformational changes for mAb molecules that remain on the surface. In contrast, if the siliconized surface is first saturated with the surfactant, no adsorption of mAb is observed. Competitive adsorption of mAb and Poloxamer 188 from solution leads to a surface dominantly occupied with surfactant molecules, whereas only a minor amount of mAb absorbs. These findings clearly indicate that Poloxamer 188 can protect against mAb adsorption as well as modify the adsorbed conformation of previously adsorbed mAb.

Characterization of Polysorbate Ester Fractions and Implications in Protein Drug Product Stability.

Polysorbates (PS) are commonly used surfactants in biopharmaceutical protein formulations. However, they are susceptible to a variety of degradation pathways, including chemical hydrolysis, oxidation, and enzymatic hydrolysis. Polysorbates are also heterogeneous mixtures, and it has been observed that the patterns of degradation can be strikingly different between the different pathways. Polysorbates (PS20 and PS80) were fractionated, and the fractions were characterized for their physicochemical properties, such as surface tension, micelle size, critical micelle concentration (CMC), and agitation protection for a monoclonal antibody (mAb). This report seeks to use this information to inform how these properties might change in polysorbates as they degrade in biopharmaceutical formulations. The physicochemical properties examined shed light on some of the differences between PS types and the different chemical components of polysorbates. Differences in physicochemical properties for fractionated polysorbates could help inform biopharmaceutical formulations that use PS surfactants. Importantly, they show that subspecies of PS20 are far more distinct from each other than those of PS80. Fractions of PS20 showed highly different critical micelle concentrations and effects on equilibrium surface tension. These differences, and possibly other untested parameters, led to vastly different protective effects for a model mAb under agitation stress. Additionally, the propensity of various PS fractions to form micelles can impact both polysorbate quantitation measurements, some of which rely on micellization, and the effective solubility of hydrophobic compounds (e.g., fatty acids) in the surfactant solution.

Micellar Morphology of Polysorbate 20 and 80 and Their Ester Fractions in Solution via Small-Angle Neutron Scattering.

Surfactants are commonly used in therapeutic protein formulations in biopharmaceuticals to impart protein stability; however, their solution morphology and the role of the individual components in these structurally heterogeneous commercial grade surfactants at physiologically and pharmaceutically relevant temperatures have not been investigated systematically. The micellar morphologies of Polysorbate 20 and Polysorbate 80 and their primary components monoester fractions, as well as the diester fractions, are evaluated at 4, 22°C, 40°C, and 50°C using small-angle neutron scattering to determine the aggregation number, radius of gyration, core radius, critical micelle concentration, shell thickness, and shell hydration. The sizes and aggregation numbers of the diester fractions of PS20 above 80°C and PS80 above 50°C exhibit significant changes in shape. The analysis of the small-angle neutron scattering data of PS20 confirms that the critical micellar concentration of the monoester fraction is significantly higher at 4°C compared to the diester fraction and their original material, all-laurate PS20. Overall, these experiments identify the dominant components responsible for the temperature-dependent behavior of these surfactants in pharmaceutical protein formulations.

Optimizing the Formulation and Lyophilization Process for a Fragment Antigen Binding (Fab) Protein Using Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry (ssHDX-MS).

Solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) was evaluated as an analytical method to rapidly screen and select an optimal lyophilized fragment antigen binding protein (Fab) formulation and the optimal lyophilization cycle. ssHDX-MS in lyophilized Fab formulations, varying in stabilizer type and stabilizer/protein ratio, was conducted under controlled humidity and temperature. The extent of deuterium incorporation was measured using mass spectrometry and correlated with solid-state stress degradation at 50 °C as measured by size exclusion chromatography (SEC) and ion-exchange chromatography (IEC). ssHDX-MS was also used to evaluate the impact of three different types of lyophilization processing on storage stability: controlled ice nucleation (CN), uncontrolled ice nucleation (UCN), and annealing (AN). The extent of deuterium incorporation for different Fab formulations agreed with the order of solid-state stress degradation, with formulations having lower deuterium incorporation showing lower stress-induced degradation (aggregation and charge modifications). For lyophilization processing, no significant effect of ice nucleation was observed in either solid-state stress degradation or in the extent of deuterium incorporation for high concentration Fab formulations (25 mg/mL). In contrast, for low concentration Fab formulations (2.5 mg/mL), solid-state stability from different lyophilization processes correlated with the extent of deuterium incorporation. The order of solid-state degradation (AN < CN < UCN) was the same as the extent of deuterium incorporation on ssHDX-MS (AN < CN < UCN). The extent of deuterium incorporation on ssHDX-MS correlated well with the solid-state stress degradation for different Fab formulations and lyophilization processing methods. Thus, ssHDX-MS can be used to rapidly screen and optimize the formulation and lyophilization process for a lyophilized Fab, reducing the need for time-consuming stress degradation studies.

Data-Driven Development of Predictive Models for Sustained Drug Release.

Mathematical modeling of drug release can aid in the design and development of sustained delivery systems, but the parameter estimation of such models is challenging owing to the nonlinear mathematical structure and complexity and interdependency of the physical processes considered. Highly parameterized models often lead to overfitting, strong parameter correlations, and as a consequence, inaccurate model predictions for systems not explicitly part of the fitting database. Here, we show that an efficient stochastic optimization algorithm can be used not only to find robust estimates of global minima to such complex problems but also to generate metadata that allow quantitative evaluation of parameter sensitivity and correlation, which can be used for further model refinement and development. A practical methodology is described through the analysis of a predictive drug release model on published experimental data sets. The model is then used to design a zeroth-order release profile in an experimental system consisting of an antibody fragment in a poly(lactic-co-glycolic acid) solvent depot, which is validated experimentally. This approach allows rational decision-making when developing new models, selecting models for a specific application, or designing formulations for experimental trials.

In Situ Characterization of the Microstructural Evolution of Biopharmaceutical Solid-State Formulations with Implications for Protein Stability.

Lyophilized and spray-dried biopharmaceutical formulations are used to provide long-term stability for storage and transport, but questions remain about the molecular structure in these solid formulations and how this structure may be responsible for protein stability. Small-angle neutron scattering with a humidity control environment is used to characterize protein-scale microstructural changes in such solid-state formulations as they are humidified and dried in situ. The findings indicate that irreversible protein aggregates of stressed formulations do not form within the solid-state but do emerge upon reconstitution of the formulation. After plasticization of the solid-state matrix by exposure to humidity, the formation of reversibly self-associating aggregates can be detected in situ. The characterization of the protein-scale microstructure in these solid-state formulations facilitates further efforts to understand the underlying mechanisms that promote long-term protein stability.

Adsorption of polysorbate 20 and proteins on hydrophobic polystyrene surfaces studied by neutron reflectometry.

Understanding the adsorption of protein and surfactant molecules on hydrophobic surfaces is very important for storage stability and delivery of pharmaceutical liquid formulations as many commonly-used devices, such as drug containers and syringes, have hydrophobic surfaces. Neutron reflectometry is used here to investigate the structure information of the adsorption process of non-ionic surfactant (polysorbate 20) and proteins (monoclonal antibody (mAb) and lysozyme) on polystyrene surfaces. Thickness of adsorbed polysorbate 20 thin film is observed to be ≈21 Å, comparable to the radius of gyration of polysorbate 20 micelles in solution. Although no lysozyme adsorption is observed on the polystyrene surface in low solution pH condition, the mAb can be strongly absorbed on the polystyrene surface with a layer thickness of ≈145 Å. The mAb concentration near the surface is about 135 mg/ml significantly larger than the bulk protein concentration. The differences in adsorption behavior are attributed to different protein interactions with a hydrophobic surface. Further, both surfactants and proteins adsorbed on the polystyrene surfaces can not be rinsed off using pure water.

Structure and Relaxation in Solutions of Monoclonal Antibodies.

Reversible self-association of therapeutic antibodies is a key factor in high protein solution viscosities. In the present work, a coarse-grained computational model accounting for electrostatic, dispersion, and long-ranged hydrodynamic interactions of two model monoclonal antibodies is applied to understand the nature of self-association, predicting the solution microstructure and resulting transport properties of the solution. For the proteins investigated, the structure factor across a range of solution conditions shows quantitative agreement with neutron-scattering experiments. We observe a homogeneous, dynamical association of the antibodies with no evidence of phase separation. Calculations of self-diffusivity and viscosity from coarse-grained dynamic simulations show the appropriate trends with concentration but, respectively, over- and under-predict the experimentally measured values. By adding constraints to the self-associated clusters that rigidify them under flow, prediction of the transport properties is significantly improved with respect to experimental measurements. We hypothesize that these rigidity constraints are associated with missing degrees of freedom in the coarse-grained model resulting from patchy and heterogeneous interactions among coarse-grained domains. These results demonstrate how structural anisotropy and anisotropy of interactions generated by features at the 2-5 nm length scale in antibodies are sufficient to recover the dynamics and rheological properties of these important macromolecular solutions.

Impact of polymer geometry on the interactions of protein-PEG conjugates.

The conjugation of high molecular weight polyethylene glycol (PEG) to an active pharmaceutical ingredient (API) is an attractive strategy for the modification of biophysical and biodistribution properties of the API. Indeed, several therapeutic proteins conjugated to PEG have been safely administered in the clinic. While there have been studies on the configuration of these conjugates in solution, investigations on the impact of PEG geometry on protein-PEG conjugate interactions is limited. In this study, we use dynamic light scattering (DLS), rheology, and small-angle neutron scattering (SANS) to investigate the biophysical solution and interaction behavior of a 50kDa Fab protein attached to either a linear or tetrameric (branched) 40kDa PEG molecule. The hydrodynamic radii, diffusivity, viscosity and pair distance distribution function (PDDF) were obtained for the protein-PEG conjugates in solution. An analysis revealed that interactions between unconjugated proteins were quite attractive, however linear PEG-protein conjugates exhibited net repulsive interactions, similar to that of the unconjugated polymer. Tetramer PEG-protein conjugates on the other hand, exhibited a net weak attractive interaction, indicating a more balanced distribution of repulsive and attractive interaction states. Further analysis of the SANS data using geometric models consistent with the PDDF elucidated the conjugates' equilibrium configuration in solution. Insights gained from measurements and analysis used here can also be useful in predicting how conjugate geometries affect viscosity and aggregation behavior, which are important in determining suitable protein-polymer drug formulations.

Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry: Correlation of Deuterium Uptake and Long-Term Stability of Lyophilized Monoclonal Antibody Formulations.

Solid state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) has been used to assess protein conformation and matrix interactions in lyophilized solids. ssHDX-MS metrics have been previously correlated to the formation of aggregates of lyophilized myoglobin on storage. Here, ssHDX-MS was applied to lyophilized monoclonal antibody (mAb) formulations and correlated to their long-term stability. After exposing lyophilized samples to D2O(g), the amount of deuterium incorporated at various time points was determined by mass spectrometry for four different lyophilized mAb formulations. Hydrogen-deuterium exchange data were then correlated with mAb aggregation and chemical degradation, which was obtained in stability studies of >2.5 years. Deuterium uptake on ssHDX-MS of four lyophilized mAb formulations determined at the initial time point prior to storage in the dry state was directly and strongly correlated with the extent of aggregation and chemical degradation during storage. Other measures of physical and chemical properties of the solids were weakly or poorly correlated with stability. The data demonstrate, for the first time, that ssHDX-MS results are highly correlated with the stability of lyophilized mAb formulations. The findings thus suggest that ssHDX-MS can be used as an early read-out of differences in long-term stability between formulations helping to accelerate formulation screening and selection.

Characterization of Protein-Excipient Microheterogeneity in Biopharmaceutical Solid-State Formulations by Confocal Fluorescence Microscopy.

Protein-stabilizer microheterogeneity is believed to influence long-term protein stability in solid-state biopharmaceutical formulations and its characterization is therefore essential for the rational design of stable formulations. However, the spatial distribution of the protein and the stabilizer in a solid-state formulation is, in general, difficult to characterize because of the lack of a functional, simple, and reliable characterization technique. We demonstrate the use of confocal fluorescence microscopy with fluorescently labeled monoclonal antibodies (mAbs) and antibody fragments (Fabs) to directly visualize three-dimensional particle morphologies and protein distributions in dried biopharmaceutical formulations, without restrictions on processing conditions or the need for extensive data analysis. While industrially relevant lyophilization procedures of a model IgG1 mAb generally lead to uniform protein-excipient distribution, the method shows that specific spray-drying conditions lead to distinct protein-excipient segregation. Therefore, this method can enable more definitive optimization of formulation conditions than has previously been possible.

Impact of mono- and poly-ester fractions on polysorbate quantitation using mixed-mode HPLC-CAD/ELSD and the fluorescence micelle assay.

Determination of excipient content in drug formulation is an important aspect of pharmaceutical formulation development and for analytical testing of the formulation. In this study, the influence of polysorbate subspecies, in particular mono- and poly-esters, for determining polysorbate (PS) content were investigated by comparing three of the most widely used PS quantitation approaches, the Fluorescence Micelle Assay (FMA) and Mixed-Mode High Performance Liquid Chromatography coupled with Charged Aerosol Detection (MM-CAD) or Evaporative Light Scattering Detection (MM-ELSD). FMA and MM-CAD were employed to investigate the quantitation behavior of PS20 and PS80 subspecies and corresponding degradation products in placebo formulations using forced degradation conditions at 40°C for up to 12 weeks. While both methods allowed accurate and comparable quantification of neat PS at the beginning of stress studies, pronounced differences in content determination between the methods were observed at later time points, which were attributable to substantial differences in the contribution of individual mono- and poly-esters to the overall quantitation results. It was particularly surprising to find that the main component of PS20, polyoxyethylene sorbitan monolaurate, did not show a signal at the studied concentration using FMA. Moreover, the degradation of polysorbate poly-esters, was reflected much stronger in FMA than MM-CAD results. Additional experiments employing chemical oxidation and base hydrolysis to degrade PS20, quantified by FMA and MM-ELSD, also show preferential reduction in certain subspecies depending on the degradation pathway involved. For PS20 degraded by chemical oxidation, quantitation results were lower for FMA than MM-ELSD, while the opposite trend was observed with base hydrolysis.

Molecular simulations of micellar aggregation of polysorbate 20 ester fractions and their interaction with N-phenyl-1-naphthylamine dye.

Micellar aggregation behavior of polysorbate 20 (PS20) has generated significant interest because of the wide use of PS20 as a surfactant to minimize protein surface adsorption and mitigate protein aggregation. Thus, there is a need for better molecular understanding of what drives the biophysical behavior of PS20 in solution. We observe that a complex amphipathic PS20 molecule, which contains both hydrophobic tail and relatively large hydrophilic head, self-associates strongly within the course of a molecular dynamics simulation performed with a fully atomistic representation of the molecule and an explicit water solvent model. The in silico behavior is consistent with micellar models of PS20 in solution. The dynamics of this self-association is rather complex involving both internal reorganization of the molecule and diffusion to form stable micelle-like aggregates. The micellar aggregates of PS20 are long-lived and are formed by the balance between the large hydrophobic interactions associated with the aliphatic tail of PS20, and the steric repulsion of the hydrophilic sorbitan head structure. In the present work, molecular models of PS20 that represent naturally occurring PS20 fractions were produced and characterized in silico. The study investigated the monoester and diester fractions: PS20M, and PS20D. These fractions present differences in the strength of their hydrophobic effect, which influences the aggregation behavior. Adaptive biasing force (ABF) simulations were carried out with the PS20M and PS20D molecular constructs to calculate the free energy of their pairwise interaction. The free energy barrier for the dissociation is higher for PS20D compared with PS20M. The results show that hydrogen bonds can form when head groups are in close proximity, such as in the PS20 aggregate assembly, and the free energy of interaction can be used to predict the morphology of the micellar aggregate for the different PS20 fractions. We were also able to simulate PS20 in the presence of N-phenyl-1-naphthylamine (NPN) to study the solution behavior of the hydrophobic molecule and of the mechanism in which it is sequestered in the hydrophobic core of the PS20 micellar aggregate.

Effect of Hierarchical Cluster Formation on the Viscosity of Concentrated Monoclonal Antibody Formulations Studied by Neutron Scattering.

Recently, reversible cluster formation was identified as an underlying cause of anomalously large solution viscosities observed in some concentrated monoclonal antibody (mAb) formulations, which poses a major challenge to the use of subcutaneous injection for some mAbs. A fundamental understanding of the structural and dynamic origins of high viscosities in concentrated mAb solutions is thus of significant relevance to mAb applications in human health care, as well as being of scientific interest. Herein, we present a detailed investigation of an IgG1-based mAb to relate the short-time dynamics and microstructure to significant viscosity changes over a range of pharmaceutically relevant physiochemical conditions. The combination of light scattering, small-angle neutron scattering, and neutron spin echo measurement techniques conclusively demonstrates that, upon addition of Na2SO4, these antibodies form strongly bound reversible dimers at dilute concentrations that interact with each other to form large, loosely bound, transient clusters when concentrated. This hierarchical structure formation in solution causes a significant increase in the solution viscosity.

Modeling of protein-anion exchange resin interaction for the human growth hormone charge variants.

Modeling ion exchange chromatography (IEC) behavior has generated significant interest because of the wide use of IEC as an analytical technique as well as a preparative protein purification process; indeed there is a need for better understanding of what drives the unique behavior of protein charge variants. We hypothesize that a complex protein molecule, which contains both hydrophobic and charged moieties, would interact strongly with an in silico designed resin through charged electrostatic patches on the surface of the protein. In the present work, variants of recombinant human growth hormone that mimic naturally-occurring deamidation products were produced and characterized in silico. The study included these four variants: rhGH, N149D, N152D, and N149D/N152D. Poisson-Boltzmann calculations were used to determine surface electrostatic potential. Metropolis Monte Carlo simulations were carried out with the resulting variants to simulate IEC systems, examining the free energy of the interaction of the protein with an in silico anion exchange column represented by polylysine polypeptide. The results show that the charge variants have different average binding energies and the free energy of interaction can be used to predict the retention time for the different variants.

Molecular simulations of the pairwise interaction of monoclonal antibodies.

Molecular simulations are employed to compute the free energy of pairwise monoclonal antibodies (mAbs) association using a conformational sampling algorithm with a scoring function. The work reported here is aimed at investigating the mAb-mAb association driven by weak interactions with a computational method capable of predicting experimental observations of low binding affinity. The simulations are able to explore the free energy landscape. A steric interaction component, electrostatic interactions, and a nonpolar component of the free energy form the energy scoring function. Electrostatic interactions are calculated by solving the Poisson-Boltzmann equation. The nonpolar component is derived from the van der Waals interactions upon close contact of the protein surfaces. Two mAbs with similar IgG1 framework but with small sequence differences, mAb1 and mAb2, are considered for their different viscosity and propensity to form a weak interacting dimer. mAb1 presents favorable free energy of association at pH 6 with 15 mM of ion concentration reproducing experimental trends of high viscosity and dimer formation at high concentration. Free energy landscape and minimum free energy configurations of the dimer, as well as the second virial coefficient (B22) values are calculated. The energy distributions for mAb1 are obtained, and the most probable configurations are seen to be consistent with experimental measurements. In contrast, mAb2 shows an unfavorable average free energy at the same buffer conditions due to poor electrostatic complementarity, and reversible dimer configurations with favorable free energy are found to be unlikely. Finally, the simulations of the mAb association dynamics provide insights on the self-association responsible for bulk solution behavior and aggregation, which are important to the processing and the quality of biopharmaceuticals.

Observation of small cluster formation in concentrated monoclonal antibody solutions and its implications to solution viscosity.

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals. It is hypothesized that some concentrated mAb solutions exhibit formation of a solution phase consisting of reversibly self-associated aggregates (or reversible clusters), which is speculated to be responsible for their distinct solution properties. Here, we report direct observation of reversible clusters in concentrated solutions of mAbs using neutron spin echo. Specifically, a stable mAb solution is studied across a transition from dispersed monomers in dilute solution to clustered states at more concentrated conditions, where clusters of a preferred size are observed. Once mAb clusters have formed, their size, in contrast to that observed in typical globular protein solutions, is observed to remain nearly constant over a wide range of concentrations. Our results not only conclusively establish a clear relationship between the undesirable high viscosity of some mAb solutions and the formation of reversible clusters with extended open structures, but also directly observe self-assembled mAb protein clusters of preferred small finite size similar to that in micelle formation that dominate the properties of concentrated mAb solutions.

Rheological characterization and injection forces of concentrated protein formulations: an alternative predictive model for non-Newtonian solutions.

Development of injection devices for subcutaneous drug administration requires a detailed understanding of user capability and forces occurring during the drug administration process. Injection forces of concentrated protein therapeutics are influenced by syringe properties (e.g., needle diameter) and injection speed, and are driven by solution properties such as rheology. In the present study, it is demonstrated that concentrated protein therapeutics may show significantly reduced injection forces because of shear-thinning (non-Newtonian) behavior. A mathematical model was thus established to predict/correlate injection forces of Newtonian and non-Newtonian solutions with viscosity data from plate/cone rheometry. The model was verified experimentally by glide-force measurements of reference and surrogate solutions. Application of the suggested model was demonstrated for injection force measurements of concentrated protein solutions to determine viscosity data at high shear rates (3 × 10(4)-1.6 × 10(5)s(-1)). By combining these data with viscosity data obtained by different viscosity methods (plate/cone and capillary rheometry), a viscosity-shear rate profile of the protein solution between 10(2) and 1.6 × 10(5)s(-1) was obtained, which was mathematically described by the Carreau model. Characterization of rheological properties allows to accurately predict injection forces for different syringe-needle combinations as well as injection rates, thus supporting the development of injection devices for combination products.

Small-angle neutron scattering characterization of monoclonal antibody conformations and interactions at high concentrations.

Small-angle neutron scattering (SANS) is used to probe the solution structure of two protein therapeutics (monoclonal antibodies 1 and 2 (MAb1 and MAb2)) and their protein-protein interaction (PPI) at high concentrations. These MAbs differ by small sequence alterations in the complementarity-determining region but show very large differences in solution viscosity. The analyses of SANS patterns as a function of different solution conditions suggest that the average intramolecular structure of both MAbs in solution is not significantly altered over the studied protein concentrations and experimental conditions. Even though a strong repulsive interaction is expected for both MAbs due to their net charges and low solvent ionic strength, analysis of the SANS data shows that the effective PPI for MAb1 is dominated by a very strong attraction at small volume fraction that becomes negligible at large concentrations. The MAb1 PPI cannot be modeled simply by a spherically symmetric central forces model. It is proposed that an anisotropic attraction strongly affects the local interprotein structure and leads to an anomalously large viscosity of concentrated MAb1 solutions. Conversely, MAb2 displays a repulsive interaction potential throughout the concentration series probed and a comparatively small solution viscosity.