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Proteins are exposed to hydrostatic pressure (HP) in a variety of ecosystems as well as in processing steps such as freeze–thaw, cell disruption, sterilization, and homogenization, yet pressure effects on protein–protein interactions (PPIs) remain underexplored. With the goal of contributing toward the expanded use of HP as a fundamental control parameter in protein research, processing, and engineering, small-angle X-ray scattering was used to examine the effects of HP and ionic strength on ovalbumin, a model protein. Based on an extensive data set, we develop an empirical method for scaling PPIs to a master curve by combining HP and osmotic effects. We define an effective pressure parameter that has been shown to successfully apply to other model protein data available in the literature, with deviations evident for proteins that do not follow the apparent Hofmeister series. The limitations of the empirical scaling are discussed in the context of the hypothesized underlying mechanisms.

Understanding protein dynamics and conformational stability holds great significance in biopharmaceutical research. Hydrogen–deuterium exchange (HDX) is a quantitative methodology used to examine these fundamental properties of proteins. HDX involves measuring the exchange of solvent-accessible hydrogens with deuterium, which yields valuable insights into conformational fluctuations and conformational stability. While mass spectrometry is commonly used to measure HDX on the peptide level, we explore a different approach using small-angle neutron scattering (SANS). In this work, SANS is demonstrated as a complementary and noninvasive HDX method (HDX-SANS). By assessing subtle changes in the tertiary and quaternary structure during the exchange process in deuterated buffer, along with the influence of added electrolytes on protein stability, SANS is validated as a complementary HDX technique. The HDX of a model therapeutic antibody, NISTmAb, an IgG1κ, is monitored by HDX-SANS over many hours using several different formulations, including salts from the Hofmeister series of anions, such as sodium perchlorate, sodium thiocyanate, and sodium sulfate. The impact of these formulation conditions on the thermal stability of NISTmAb is probed by differential scanning calorimetry. The more destabilizing salts led to heightened conformational dynamics in mAb solutions even at temperatures significantly below the denaturation point. HDX-SANS is demonstrated as a sensitive and noninvasive technique for quantifying HDX kinetics directly in mAb solution, providing novel information about mAb conformational fluctuations. Therefore, HDX-SANS holds promise as a potential tool for assessing protein stability in formulation.

Monoclonal antibodies (mAbs) form viscoelastic gel-like layers at the air–water interface due to their amphiphilic nature, and this same protein characteristic can lead to undesired aggregation of proteins in therapeutic formulations. We hypothesize that the interfacial viscoelasticity and surface pressure of mAbs at the air–water interface will correlate with their long-term stability. To test this hypothesis, the interfacial viscoelastic rheology and surface pressure of five different antibodies with varying visible particle counts from a three-year stability study were measured. We find that both the surface pressures and interfacial elastic moduli correlate well with the long-time mAb solution stability within a class of mAbs with the interfacial elastic moduli being particularly sensitive to discriminate between stable and unstable mAbs across a range of formulations. Furthermore, X-ray reflectivity was used to gain insight into the interfacial structure of mAbs at the air–water interface, providing a possible molecular mechanism to explain the relationship between interfacial elastic moduli and the long-term stability.

Polysorbate 80 (PS80), a nonionic surfactant used in pharmaceutical formulation, is known to be incompatible with m-cresol, an antimicrobial agent for multi-dose injectable formulations. This incompatibility results in increased turbidity caused by micelle aggregation progressing over weeks or longer, where storage temperature, ionic strength, and component concentration influence the aggregation kinetics. Small-angle neutron scattering (SANS) analysis of PS80/m-cresol solutions over a pharmaceutically relevant concentration range of each component reveals the cause of aggregation, the coalescence mechanism, and aggregate structure. PS80 solutions containing m-cresol concentrations below ≈2.0 mg/mL and above ≈4.5 mg/mL are kinetically stable and do not aggregate over a 50 h period. At 5 mg/mL of m-cresol, the mixture forms a kinetically stable microemulsion phase, despite being well below the aqueous solubility limit of m-cresol. Solutions containing intermediate m-cresol concentrations (2.0–4.5 mg/mL) are unstable, resulting in aggregation, coalescence, and eventual phase separation. In unstable solutions, two stages of aggregate growth (nucleation and power-law growth) are observed at m-cresol concentrations at or below ≈3.6 mg/mL. At higher m-cresol concentrations, aggregates experience a third stage of exponential growth. A single kinetic model is developed to explain the stages of aggregate growth observed in both kinetic mechanisms. This work establishes the phase diagram of PS80/m-cresol solution stability and identifies component concentrations necessary for producing stable formulations.

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.

Interfacial stresses can destabilize therapeutic formulations containing monoclonal antibodies (mAbs), which is proposed to be a result of adsorption and aggregation at the air–water interface. To increase protein stability, pharmaceutical industries add surfactants, such as Polysorbate 20 (PS20), into protein formulations to minimize mAb adsorption at the interface but rarely quantify this process. We determine that mAb adsorption in surfactant-free solutions creates a monolayer with significant viscoelasticity, which can influence measurements of bulk mAb solution viscosity. In contrast, PS20 absorption leads to an interface with negligible interfacial viscosity that protects the air–water interface from mAb adsorption. These studies were performed through a combined study of surface tensiometry, interfacial rheology, capillary viscometry, and neutron reflectometry to determine the surface activity of a model surfactant, PS20, and mAb system, which can be useful for the successful formulation developments of biotherapeutics.

An ultrastretchable iono-elastomer with resistance sensitive to both elongation strain and temperature has been developed by hierarchical self-assembly of an end functionalized triblock copolymer in a protic ionic liquid (ethylammonium nitrate) followed by cross-linking. Small-angle X-ray scattering experiments in situ with uniaxial elongation reveal a nanoscale microstructural transition of the hierarchically self-assembled cross-linked micelles that is responsible for the material’s remarkable mechanical and ionic conductivity responses. The results show that the intermicelle distance extends along the deformation direction while the micelles organize into a long-range ordered face-centered-cubic structure during the uniaxial elongation. Besides good cyclability and resistance to selected physical damage, the iono-elastomer simultaneously achieves an unprecedented combination of high stretchability (340%), highly linear resistance vs elongation strain (R2= 0.998), and large temperature gauge factor (ΔR/R= 3.24%/°C@30 °C). Human subject testing demonstrates that the iono-elastomer-based wearable thermomechanical sensor is able to effectively and accurately register both body motion and skin temperature simultaneously.

Spatially controlled wrinkle formation on an elastomeric substrate is achieved through flowcoating of a nanometer thick, high modulus thiol–ene film laminated on a millimeter-thick thiol–ene elastomer. The elastomer substrate is formed viaphotopolymerization of thiol and ene monomers with a slight stoichiometric excess of thiol to yield a 2.3 MPa elastomer. Separately, thiol and ene monomers, with a higher degree of functionality, are flow coated onto a glass slide and photopolymerized under ambient conditions to yield a 1.9 GPa film tens of nanometers thick. The photocrosslinked thin film is transferred onto the strained elastomer to spontaneously produce wrinkles upon strain release. Careful selection of thiol and ene monomers facilitate tuning of the film and elastomer mechanical properties, while stoichiometry control (i.e., excess thiols in the elastomer and excess enes in the film) allows covalent attachment of the film to the elastomer to prevent delamination upon elastomer destrain. Photopatterning of a flowcoated thin film is demonstrated, which, when wrinkled, generates a very distinct pattern of aligned wrinkle structures with clearly defined boundaries. Finally, by coating and photopatterning in sequence, spatial wrinkle wavelength control is readily attained by defining the film thickness at any given point.

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.

Slurry based electrodes have shown promise as an energy dense and scalable storage technology for electrochemical flow batteries. Key to their efficient operation is the use of a conductive additive which allows for volumetric charging and discharging of the electrochemically active species contained within the electrodes. Carbon black is commonly used for this purpose due to the relatively low concentrations needed to maintain electrical percolation. While carbon black supplies the desirable electrical properties for the application, it contributes detrimentally to the rheology characteristics of these concentrated suspensions. In this work, we develop a synthesis protocol to produce inorganic oxide particles with electrostatically adsorbed poly(3,4-ethylenedioxithiophene):polystyrenesulfonate (PEDOT:PSS). Using a combination of small angle neutron scattering (SANS), electron microscopy, and thin-film conductivity, we show that the synthesis scheme provides a flexible platform to form conductive PEDOT:PSS-SiO2nanoparticle dispersions. Based on these measurements, we demonstrate that these particles are stable when dispersed in propylene carbonate. Using a combination of rheology and dielectric spectroscopy, we show that these stable dispersions facilitate electrical percolation at concentrations below their mechanical percolation threshold, and this percolation is maintained under flow. These results demonstrate the potential for strategies which seek to decouple mechanical and electrical percolation to allow for the development of higher performance conductive additives for slurry based flow batteries.

Proteins exhibit a variety of dense phases ranging from gels, aggregates, and precipitates to crystalline phases and dense liquids. Although the structure of the crystalline phase is known in atomistic detail, little attention has been paid to noncrystalline protein dense phases, and in many cases the structures of these phases are assumed to be fully amorphous. In this work, we used small-angle neutron scattering, electron microscopy, and electron tomography to measure the structure of ovalbumin precipitate particles salted out with ammonium sulfate. We found that the ovalbumin phase-separates into core-shell particles with a core radius of ∼2 μm and shell thickness of ∼0.5 μm. Within this shell region, nanostructures comprised of crystallites of ovalbumin self-assemble into a well-defined bicontinuous network with branches ∼12 nm thick. These results demonstrate that the protein gel is comprised in part of nanocrystalline protein.

The critical role of solvation forces in dispersing and stabilizing nanoparticles and colloids in 1-butyl-3-methylimidazolium tetrafluoroborate [C4mim][BF4] is demonstrated. Stable silica nanoparticle suspensions over 60 wt % solids are achieved by particle surface chemical functionalization with a fluorinated alcohol. A combination of techniques including rheology, dynamic light scattering (DLS), transmission electron microscopy (TEM), and small angle neutron scattering (SANS) are employed to determine the mechanism of colloidal stability. Solvation layers of ∼5 nm at room temperature are measured by multiple techniques and are thought to be initiated by hydrogen bonds between the anion [BF4]−and the fluorinated group on the surface coating. Inducing structured solvation layering at particle surfaces through hydrogen bonding is demonstrated as a method to stabilize particles in ionic liquids.

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.

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.

Although reconstitution of membrane proteins within protein detergent complexes is often used to enable their structural or biophysical characterization, it is unclear how one should rationally choose the appropriate micellar environment to preserve native protein folding. Here, we investigated model mixed micelles consisting of a nonionic glucosylated alkane surfactant from the maltoside and thiomaltoside families, bile salt surfactant, and the steryl derivative cholesteryl hemisuccinate. We correlated several key attributes of these micelles with the in vitro ligand-binding activity of hA2aR in these systems. Through small-angle neutron scattering and radioligand-binding analysis, we found several key aspects of mixed micellar systems that preserve the activity of hA2aR, including a critical amount of cholesteryl hemisuccinate per micelle, and an optimal hydrophobic thickness of the micelle that is analogous to the thickness of native mammalian bilayers. These features are closely linked to the headgroup chemistry of the surfactant and the hydrocarbon chain length, which influence both the morphology and composition of resulting micelles. This study should serve as a general guide for selecting the appropriate mixed surfactant systems to stabilize membrane proteins for biophysical analysis.

The penetration behavior of Kevlar fabric intercalated with dry particles and shear thickening fluids (STF), highly concentrated fluid−particle suspensions, is presented. In particular, the role of particle hardness is explored by comparing fabric treatments containing SiO2particles, which are significantly harder than Kevlar, to treatments containing softer poly(methyl methacrylate) (PMMA) particles. The fabric testing includes yarn pull-out, quasi-static spike puncture, and ballistic penetration resistance, performed on single fabric layers. It was found that both dry particle and STF treatments resulted in improvements in fabric properties relative to neat or poly(ethylene glycol) (PEG) treated fabrics. On comparison of treatments with different particle hardness, the SiO2materials performed better in all tests than comparable PMMA materials, although the SiO2treatments caused yarn failure in pull-out testing, reducing the total pull-out energy. In addition, resistance to yarn pull-out was found to be substantially higher for STF-treated fabrics than for dry particle treated fabrics. However, both dry particle addition and STF treatments exhibited comparable enhancements in puncture and ballistic resistance. These observations suggest that viscous stress transfer, friction, and physical entrainment of hard particles into filaments contribute to the demonstrated improvements in the properties of protective fabrics treated with shear thickening fluids.

The molecular structure of poly(amido amine) dendrimers is investigated with molecular dynamics simulations using the Amber 7 modeling package. A method for defining residues for complex molecules is developed, and it enables the study of the effects of protonation of the primary and tertiary amines. The effects of implicit solvents versus explicit solvents as well as the pH of the solution on the molecular structure are calculated. Good agreement with experimental results for the radius of gyration measured in methanol by X‐ray scattering is observed for simulations with an explicit solvent and protonation of the primary amines. Calculations of the intramolecular atomistic pair correlation function show a dense core, as well as the presence of voids filled with the solvent inside the molecule. The primary amines (end groups) are shown to access the molecular interior by backfolding. Comparisons with experiments and other reported simulation results highlight the advantages of the approach developed here. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 3062–3077, 2006

Electrospinning is reported for viscoelastic aqueous solutions of poly(ethylene oxide) (PEO) and with added nanoclay (laponite). A weak correlation between fiber diameter and the spinning solution's zero‐shear viscosity is observed and compared with previous work reported by McKee et al. (McKee et al., Macromolecules 2004, 37, 1760). A new analysis of electrospinning results for PEO indicates a universal correlation between fiber diameter and solution properties that does not include shear viscosity as a primary variable. The addition of laponite nanoclay to the PEO solutions, which has been previously shown to result in rheologically simple solutions, leads to different fiber morphologies for the same shear viscosity in contradiction with reports for titania particles (Drew et al., J Macromol Sci Part A: Pure Appl Chem 2003, 40, 1415). The research identifies additional physicochemical properties that are important in determining electrospun fiber morphology. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 1608–1617, 2006

Abstract The influence of medium viscosity on the onset of shear thickening of silica dispersions is investigated with two different methods. In the first method, the sample temperature is varied over a narrow range for two different suspensions. For the first suspension, the stress at the onset of shear thickening, or the critical stress, was found to be independent of sample viscosity, and the shear viscosity scaled with Peclet number, as expected. The critical stress for the second suspension was not independent of sample viscosity, and the Peclet number scaling was only moderately successful. The differences were attributed to changes in particle interactions with temperature. In the second method, the molecular weight of an oligomeric silicone oil medium is varied. In principle, this method should maintain constant chemical interactions as medium viscosity varies; however the polymer is found to adsorb onto the silica surface and delay shear thickening to higher stresses with increasing molecular weight. The critical stress for the highest molecular weight systems, which is highly dependent on particle loading, overlays with an effective volume fraction based on the hydrodynamic diameter of the polymer-stabilized colloids. The results of both methods suggest that if all other properties of the dispersion are held constant, critical stress is independent of medium viscosity.

The effect of carbon topology on nitrogen adsorption at zero coverage on a variety of carbonaceous materials: graphite, a nanotube bundle, C60fullerite, C168schwarzite, and a nanoporous carbon is studied systematically using full atomistic Monte Carlo simulations. The pairwise additive site–site Lennard-Jones potential of Bojan and Steele is used to model the carbon−nitrogen interaction of adsorbent−adsorbate. The Henry constant is found to increase in the order of graphite < nanotube bundle < C60fullerite < C168schwarzite < nanoporous carbon, and the isosteric heat of adsorption increases in the order of graphite < nanotube bundle < C168schwarzite < C60fullerite < nanoporous carbon. The latter order is consistent with the degree of curvature, as characterized by the carbon coordination number around nitrogen in favorable adsorption sites. With increasing temperature, the Henry constant significantly decreases on all the carbonaceous materials studied, while the isosteric heat remains nearly constant on graphite, nanotube bundle, and C60fullerite, decreases slightly on C168schwarzite, and to a greater extent on nanoporous carbon.

A novel electrorheological (ER) effect is presented where the application of an electric field, orthogonal to the vorticity-flow plane, increases the critical hydrodynamic stress required to shear thicken concentrated, colloidal dispersions (the E-FiRST effect). The shear thickening behavior of a Brownian charge stabilized dispersion (226 nm silica in 4-methylcyclohexanol at 53, 50. and 41 vol.%) is studied in the presence of an electric field as a function of the field strength and coupling parameter ( ß) where the latter is a function of a.c. field frequency due to diffusion limitations on the polarization of the particles' double layer. A mechanism is proposed whereby the applied electric field suppresses the formation of the self-organized hydrocluster microstructure responsible for shear thickening, thus delaying the onset of shear thickening to higher applied shear stresses. A Mason-number type scaling law is found to scale the effect, which supports the proposed mechanism.

Abstract The transient shear rheology (i.e., frequency and strain dependence) is compared to the steady rheology for a model colloidal dispersion through the shear thickening transition. Reversible shear thickening is observed and the transition stress compares well to theoretical predictions. Steady and transient shear thickening are observed to occur at the same value of the average stress. The critical strain for shear thickening is found to depend inversely on the frequency at fixed applied stress for low frequencies (high strains), but is limited to an apparent minimum critical strain at higher frequencies. This minimum critical strain is shown to be an artifact of slip. Lissajous plots illustrate the transition in material properties through the shear thickening transition, and the energy dissipated by a shear thickening suspension is analyzed as a function of strain amplitude.

Atomic force microscopy (AFM) was used to study the effect of gravitational forces on the deposition of submicrometer colloidal particles onto solid surfaces to test the usual assumption that the contribution of gravity to the behavior of particles with diameters <1 μm is negligible. The effects of both particle size and density were examined using polystyrene sulfate latex, silica, and colloidal gold particles of several sizes ranging from 10 nm to 1 μm. The results show that significant differences are observed in the deposition of colloidal particles onto horizontal and vertical surfaces, under identical suspension conditions and exposure times, showing that gravitational forces can have a considerable effect. This effect was quantified by analysis of the AFM images. The experimental results are compared to calculations of the expected coverage and particle surface concentration assuming diffusion-limited adsorption and deposition by sedimentation. Gravity can be negligible for low-density particles with diameters considerably smaller than 1 μm. However, if the density of the colloidal particles is high, as in the case of colloidal gold, gravity can become a significant driving force for particle transport to the surface, even for particles with diameters much smaller than 100 nm.