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

Attractive protein?protein interactions (PPI) in concentrated monoclonal antibody (mAb) solutions may lead to reversible oligomers (clusters) that impact colloidal stability and viscosity. Herein, the PPI are tuned for two mAbs via the addition of arginine (Arg), NaCl, or ZnSO4 as characterized by the structure factor ( Seff( q)) with small-angle X-ray scattering (SAXS). The SAXS data are fit with molecular dynamics simulations by placing a physically relevant short-range attractive interaction on selected beads in coarse-grained 12-bead models of the mAb shape. The optimized 12-bead models are then used to differentiate key microstructural properties, including center of mass radial distribution functions ( gCOM( r)), coordination numbers, and cluster size distributions (CSD). The addition of cosolutes results in more attractive Seff( q) relative to the no cosolute control for all systems tested, with the most attractive systems showing an upturn at low q. Only the All1 model with an attractive site in each Fab and Fc region (possessing Fab?Fab, Fab?Fc, and Fc?Fc interactions) can reproduce this upturn, and the corresponding CSDs show the presence of larger clusters compared to the control. In general, for models with similar net attractions, i.e., second osmotic virial coefficients, the size of the clusters increases as the attraction is concentrated on a smaller number of evenly distributed beads. The cluster size distributions from simulations are used to improve the understanding and prediction of experimental viscosities. The ability to discriminate between models with bead interactions at particular Fab and Fc bead sites from SAXS simulations, and to provide real-space properties (CSD and gCOM( r)), will be of interest in engineering protein sequence and formulating protein solutions for weak PPI to minimize aggregation and viscosities.

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

The ability to design and formulate mAbs to minimize attractive interactions at high concentrations is important for protein processing, stability, and administration, particularly in subcutaneous delivery, where high viscosities are often challenging. The strength of protein-protein interactions (PPIs) of an IgG1 and IgG4 monoclonal antibody (mAb) from low to high concentration was determined by static light scattering (SLS) and used to understand viscosity data. The PPI were tuned using NaCl and five organic ionic co-solutes. The PPI strength was quantified by the normalized structure factor S(0)/ S(0)HS and Kirkwood-Buff integral G22/ G22,HS (HS = hard sphere) determined from the SLS data and also by fits with (1) a spherical Yukawa potential and (2) an interacting hard sphere (IHS) model, which describes attraction in terms of hypothetical oligomers. The IHS model was better able to capture the scattering behavior of the more strongly interacting systems (mAb and/or co-solute) than the spherical Yukawa potential. For each descriptor of PPI, linear correlations were obtained between the viscosity at high concentration (200 mg/mL) and the interaction strengths evaluated both at low (20 mg/mL) and high concentrations (200 mg/mL) for a given mAb. However, the only parameter that provided a correlation across both mAbs was the oligomer mass ratio ( moligomer/ mmonomer+dimer) from the IHS model, indicating the importance of self-association (in addition to the direct influence of the attractive PPI) on the viscosity.

Thermal processing of thermogelling nanoemulsions as a route to tune material properties.

Many soft matter systems have properties which depend on their processing history. It is generally accepted that material properties can be finely tuned by carefully directing self-assembly. However, for gelling colloidal systems, it is difficult to characterize such path-dependent effects since the colloidal attraction is often provided by adding another component to the system such as salts or depletants. Therefore, studies of and an understanding of the role of processing on the material properties of attractive colloidal systems are largely lacking. In this work, we systematically studied how processing greatly influences the properties and the microstructures of model attractive colloidal systems. We perform experiments using a thermogelling nanoemulsion as a model system where the isotropic attraction can be precisely tuned via the temperature. The effects of processing conditions on gel formation and properties is tested by performing well-designed sequential temperature jumps. By properly controlling the thermal history, we demonstrate that properties of colloidal gels can be beyond the limit set by direct quenching, which has been a major focus in literature, and that otherwise slow aging of the system associated with a decrease in elasticity can be prevented. Our results provide new experimental evidence of path-dependent rheology and associated microstructures in attractive colloidal systems and provide guidance to future applications in manufacturing complex colloid-based materials.

Short-time dynamics of lysozyme solutions with competing short-range attraction and long-range repulsion: Experiment and theory.

Recently, atypical static features of microstructural ordering in low-salinity lysozyme protein solutions have been extensively explored experimentally and explained theoretically based on a short-range attractive plus long-range repulsive (SALR) interaction potential. However, the protein dynamics and the relationship to the atypical SALR structure remain to be demonstrated. Here, the applicability of semi-analytic theoretical methods predicting diffusion properties and viscosity in isotropic particle suspensions to low-salinity lysozyme protein solutions is tested. Using the interaction potential parameters previously obtained from static structure factor measurements, our results of Monte Carlo simulations representing seven experimental lysoyzme samples indicate that they exist either in dispersed fluid or random percolated states. The self-consistent Zerah-Hansen scheme is used to describe the static structure factor, S(q), which is the input to our calculation schemes for the short-time hydrodynamic function, H(q), and the zero-frequency viscosity η. The schemes account for hydrodynamic interactions included on an approximate level. Theoretical predictions for H(q) as a function of the wavenumber q quantitatively agree with experimental results at small protein concentrations obtained using neutron spin echo measurements. At higher concentrations, qualitative agreement is preserved although the calculated hydrodynamic functions are overestimated. We attribute the differences for higher concentrations and lower temperatures to translational-rotational diffusion coupling induced by the shape and interaction anisotropy of particles and clusters, patchiness of the lysozyme particle surfaces, and the intra-cluster dynamics, features not included in our simple globular particle model. The theoretical results for the solution viscosity, η, are in qualitative agreement with our experimental data even at higher concentrations. We demonstrate that semi-quantitative predictions of diffusion properties and viscosity of solutions of globular proteins are possible given only the equilibrium structure factor of proteins. Furthermore, we explore the effects of changing the attraction strength on H(q) and η.

Rheology of clustering protein solutions.

High viscosity is a major challenge with protein therapeutics at extremely high concentrations. To overcome this obstacle, it is essential to understand the relationship between the concentration of a protein solution and its viscosity as a function of shear rate and temperature. Here, lysozyme is a model charged globular protein having both short-ranged attraction (SA) and long-ranged repulsion (LR) that promote the formation of dynamic clusters at high concentrations. We report viscosity measurements from a micro-capillary rheometer (using only several microliters of solution) over a wide range of lysozyme solution concentrations, shear rates, and temperatures. Solution structural relaxation dynamics are also probed by dynamic light scattering (DLS). As a result of lysozyme's SALR interactions, the viscosity increased dramatically across all shear rates with increasing concentration and decreasing temperature. While most of the solutions exhibited Newtonian behavior, shear thinning was exhibited at the highest concentration (480 g/l) and lowest temperatures at shear rates above approximately 10(4 )s(-1). The onset shear rate for thinning and a structural relaxation rate estimated from a slow-mode measured by DLS are compared. These measurements provide insights into the properties of protein solutions and their microscopic structural origins.

Core-Shell Composite Hydrogels for Controlled Nanocrystal Formation and Release of Hydrophobic Active Pharmaceutical Ingredients.

Although roughly 40% of pharmaceuticals being developed are poorly water soluble, this class of drugs lacks a formulation strategy capable of producing high loads, fast dissolution kinetics, and low energy input. In this work, a novel bottom-up approach is developed for producing and formulating nanocrystals of poorly water-soluble active pharmaceutical ingredients (APIs) using core-shell composite hydrogel beads. Organic phase nanoemulsion droplets stabilized by polyvinyl alcohol (PVA) and containing a model hydrophobic API (fenofibrate) are embedded in the alginate hydrogel matrix and subsequently act as crystallization reactors. Controlled evaporation of this composite material produces core-shell structured alginate-PVA hydrogels with drug nanocrystals (500-650 nm) embedded within the core. Adjustable loading of API nanocrystals up to 83% by weight is achieved with dissolution (of 80% of the drug) occurring in as little as 30 min. A quantitative model is also developed and experimentally validated that the drug release patterns of the fenofibrate nanocrystals can be modulated by controlling the thickness of the PVA shell and drug loading. Thus, these composite materials offer a "designer" drug delivery system. Overall, our approach enables a novel means of simultaneous controlled crystallization and formulation of hydrophobic drugs that circumvents energy intensive top-down processes in traditional manufacturing.

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.

Short-Time Glassy Dynamics in Viscous Protein Solutions with Competing Interactions.

The glass transition of colloidal dispersions interacting with both a short-ranged attraction and long-ranged repulsion is studied using highly purified lysozyme solutions. Newtonian liquid behavior is observed at all conditions while measurements of the dynamics in the short-time limit show features typical of glassy colloidal systems at high protein concentrations. This interesting behavior is due to the competition of the attraction and repulsion that produces a heterogeneous microstructure only at intermediate range length scales. The results demonstrate that theories for the macroscopic properties of systems with competing interactions need to include intermediate range order.

Generalized phase behavior of cluster formation in colloidal dispersions with competing interactions.

Colloidal liquids interacting with short range attraction and long range repulsion, such as proposed for some protein solutions, have been found to exhibit novel states consisting of equilibrium particle clusters. Monte Carlo simulations are performed for two physically meaningful inter-particle potentials across a broad range of interaction parameters, temperatures and volume fractions to locate the conditions where clustered states are found. A corresponding states phase behavior is identified when normalized by the critical point of an appropriately selected reference attractive fluid. Clustered fluid states and cluster percolated states are found exclusively within the two phase region of the state diagram for a reference attractive fluid, confirming the underlying intrinsic relation between clustered states and bulk phase separation. Clustered and cluster percolated states consistently exhibit an intermediate range order peak in their structure factors with a magnitude above 2.7, leading to a semi-empirical rule for identifying clustered fluids in scattering experiments.

Measuring material microstructure under flow using 1-2 plane flow-small angle neutron scattering.

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is presented. The SANS shear cell consists of a concentric cylinder Couette geometry that is sealed and rotating about a horizontal axis so that the vorticity direction of the flow field is aligned with the neutron beam enabling scattering from the 1-2 plane of shear (velocity-velocity gradient, respectively). This approach is an advance over previous shear cell sample environments as there is a strong coupling between the bulk rheology and microstructural features in the 1-2 plane of shear. Flow-instabilities, such as shear banding, can also be studied by spatially resolved measurements. This is accomplished in this sample environment by using a narrow aperture for the neutron beam and scanning along the velocity gradient direction. Time resolved experiments, such as flow start-ups and large amplitude oscillatory shear flow are also possible by synchronization of the shear motion and time-resolved detection of scattered neutrons. Representative results using the methods outlined here demonstrate the useful nature of spatial resolution for measuring the microstructure of a wormlike micelle solution that exhibits shear banding, a phenomenon that can only be investigated by resolving the structure along the velocity gradient direction. Finally, potential improvements to the current design are discussed along with suggestions for supplementary experiments as motivation for future experiments on a broad range of complex fluids in a variety of shear motions.

Intermediate range order and structure in colloidal dispersions with competing interactions.

Colloidal dispersions with a short-range attraction and long-range repulsion can exhibit an intriguing intermediate range order, manifested in scattering experiments as a low-q peak in the structure factor. Monte Carlo simulations are performed on fluids that exhibit intermediate range order to explicitly determine its connection to a possible state of microphase separation, equilibrium clustering. This is accomplished by decomposing the structure factor into cluster-cluster, monomer-monomer, and cross-correlations that cannot be extracted from experimental scattering patterns. Our simulation results indicate that the intermediate range order arises from either monomeric or cluster species, depending on solution conditions, and reflects the presence of a preferred length scale that is not trivially related to the interparticle potential. Further, criteria are established to define monomer, cluster, and percolated states in these systems that facilitate further studies. Combining scattering techniques with simulations provides an effective method for identifying clustered states in complex fluids.