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.

Interfacial dilatational deformation accelerates particle formation in monoclonal antibody solutions.

Protein molecules are amphiphilic moieties that spontaneously adsorb at the air/solution (A/S) interface to lower the surface energy. Previous studies have shown that hydrodynamic disruptions to these A/S interfaces can result in the formation of protein aggregates that are of concern to the pharmaceutical industry. Interfacial hydrodynamic stresses encountered by protein therapeutic solutions under typical manufacturing, filling, and shipping conditions will impact protein stability, prompting a need to characterize the contribution of basic fluid kinematics to monoclonal antibody (mAb) destabilization. We demonstrate that dilatational surface deformations are more important to antibody stability when compared to constant-area shear of the A/S interface. We have constructed a dilatational interfacial rheometer that utilizes simultaneous pressure and bubble shape measurements to study the mechanical stability of mAbs under interfacial aging. It has a distinct advantage over methods utilizing the Young-Laplace equation, which incorrectly describes viscoelastic interfaces. We provide visual evidence of particle ejection from dilatated A/S interfaces and spectroscopic data of ejected mAb particles. These rheological studies frame a molecular understanding of the protein-protein interactions at the complex-fluid interface.

Critical examination of the colloidal particle model of globular proteins.

Recent studies of globular protein solutions have uniformly adopted a colloidal view of proteins as particles, a perspective that neglects the polymeric primary structure of these biological macromolecules, their intrinsic flexibility, and their ability to sample a large configurational space. While the colloidal perspective often serves as a useful idealization in many cases, the macromolecular identity of proteins must reveal itself under thermodynamic conditions in which the native state is no longer stable, such as denaturing solvents and high protein concentrations where macromolecules tend to have screened excluded volume, charge, and hydrodynamic interactions. Under extreme pH conditions, charge repulsion interactions within the protein chain can overcome the attractive hydrogen-bonding interactions, holding it in its native globular state. Conformational changes can therefore be expected to have great significance on the shear viscosity and other rheological properties of protein solutions. These changes are not envisioned in conventional colloidal protein models and we have initiated an investigation of the scattering and rheological properties of model proteins. We initiate this effort by considering bovine serum albumin because it is a globular protein whose solution properties have also been extensively investigated as a function of pH, temperature, ionic strength, and concentration. As we anticipated, near-ultraviolet circular dichroism measurements and intrinsic viscosity measurements clearly indicate that the bovine serum albumin tertiary structure changes as protein concentration and pH are varied. Our findings point to limited validity of the colloidal protein model and to the need for further consideration and quantification of the effects of conformational changes on protein solution viscosity, protein association, and the phase behavior. Small-angle Neutron Scattering measurements have allowed us to assess how these conformational changes influence protein size, shape, and interprotein interaction strength.

Explaining the non-newtonian character of aggregating monoclonal antibody solutions using small-angle neutron scattering.

A monoclonal antibody solution displays an increase in low shear rate viscosity upon aggregation after prolonged incubation at 40°C. The morphology and interactions leading to the formation of the aggregates responsible for this non-Newtonian character are resolved using small-angle neutron scattering. Our data show a weak repulsive barrier before proteins aggregate reversibly, unless a favorable contact with high binding energy occurs. Two types of aggregates were identified after incubation at 40°C: oligomers with radius of gyration ∼10 nm and fractal submicrometer particles formed by a slow reaction-limited aggregation process, consistent with monomers colliding many times before finding a favorable strong interaction site. Before incubation, these antibody solutions are Newtonian liquids with no increase in low shear rate viscosity and no upturn in scattering at low wavevector, whereas aggregated solutions under the same conditions have both of these features. These results demonstrate that fractal submicrometer particles are responsible for the increase in low shear rate viscosity and low wavevector upturn in scattered intensity of aggregated antibody solutions; both are removed from aggregated samples by filtering.

Both protein adsorption and aggregation contribute to shear yielding and viscosity increase in protein solutions.

A combination of sensitive rotational rheometry and surface rheometry with a double-wall ring were used to identify the origins of the viscosity increase at low shear rates in protein solutions. The rheology of two high molecular weight proteins is discussed: Bovine Serum Albumin (BSA) in a Phosphate Buffered Saline solution and an IgG1 monoclonal antibody (mAb) in a formulation buffer containing small quantities of a non-ionic surfactant. For surfactant-free BSA solutions, the interfacial viscosity dominates the low shear viscosity measured in rotational rheometers, while the surfactant-laden mAb solution has an interfacial viscosity that is small compared to that from aggregation in the bulk. A viscoelastic film forms at the air/water interface in the absence of surfactant, contributing to an apparent yield stress (thus a low shear viscosity increase) in conventional bulk rheology measurements. Addition of surfactant eliminates the interfacial yield stress. Evidence of a bulk yield stress arising from protein aggregation is presented, and correlated with results from standard characterization techniques used in the bio-pharmaceutical industry. The protein film at the air/water interface and bulk aggregates both lead to an apparent viscosity increase and their contributions are quantified using a dimensionless ratio of the interfacial and total yield stress. While steady shear viscosities at shear rates below ∼1 s(-1) contain rich information about the stability of protein solutions, embodied in the measured yield stress, such low shear rate data are regrettably often not measured and reported in the literature.

Do clustering monoclonal antibody solutions really have a concentration dependence of viscosity?

Protein solution rheology data in the biophysics literature have incompletely identified factors that govern hydrodynamics. Whereas spontaneous protein adsorption at the air/water (A/W) interface increases the apparent viscosity of surfactant-free globular protein solutions, it is demonstrated here that irreversible clusters also increase system viscosity in the zero shear limit. Solution rheology measured with double gap geometry in a stress-controlled rheometer on a surfactant-free Immunoglobulin solution demonstrated that both irreversible clusters and the A/W interface increased the apparent low shear rate viscosity. Interfacial shear rheology data showed that the A/W interface yields, i.e., shows solid-like behavior. The A/W interface contribution was smaller, yet nonnegligible, in double gap compared to cone-plate geometry. Apparent nonmonotonic composition dependence of viscosity at low shear rates due to irreversible (nonequilibrium) clusters was resolved by filtration to recover a monotonically increasing viscosity-concentration curve, as expected. Although smaller equilibrium clusters also existed, their size and effective volume fraction were unaffected by filtration, rendering their contribution to viscosity invariant. Surfactant-free antibody systems containing clusters have complex hydrodynamic response, reflecting distinct bulk and interface-adsorbed protein as well as irreversible cluster contributions. Literature models for solution viscosity lack the appropriate physics to describe the bulk shear viscosity of unstable surfactant-free antibody solutions.

The limitations of an exclusively colloidal view of protein solution hydrodynamics and rheology.

Proteins are complex macromolecules with dynamic conformations. They are charged like colloids, but unlike colloids, charge is heterogeneously distributed on their surfaces. Here we overturn entrenched doctrine that uncritically treats bovine serum albumin (BSA) as a colloidal hard sphere by elucidating the complex pH and surface hydration-dependence of solution viscosity. We measure the infinite shear viscosity of buffered BSA solutions in a parameter space chosen to tune competing long-range repulsions and short-range attractions (2 mg/mL ≤ [BSA] ≤ 500 mg/mL and 3.0 ≤ pH ≤ 7.4). We account for surface hydration through partial specific volume to define volume fraction and determine that the pH-dependent BSA intrinsic viscosity never equals the classical hard sphere result (2.5). We attempt to fit our data to the colloidal rheology models of Russel, Saville, and Schowalter (RSS) and Krieger-Dougherty (KD), which are each routinely and successfully applied to uniformly charged suspensions and to hard-sphere suspensions, respectively. We discover that the RSS model accurately describes our data at pH 3.0, 4.0, and 5.0, but fails at pH 6.0 and 7.4, due to steeply rising solution viscosity at high concentration. When we implement the KD model with the maximum packing volume fraction as the sole floating parameter while holding the intrinsic viscosity constant, we conclude that the model only succeeds at pH 6.0 and 7.4. These findings lead us to define a minimal framework for models of crowded protein solution viscosity wherein critical protein-specific attributes (namely, conformation, surface hydration, and surface charge distribution) are addressed.

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.

Investigation on phase separation kinetics of polyolefin blends through combination of viscoelasticity and morphology.

Phase separation kinetics of polyethylene copolymer blends polyethylene-co-hexene (PEH)/polyethylene-co-butene (PEB) at a phase separation temperature of 130 degrees C have been investigated through the combination of rheological measurements and optical microscope observation. When the blends are located in the unstable region, i.e., PEH/PEB 40/60 blend (H40), 50/50 blend (H50), and 60/40 blend (H60), due to the coeffect of the fast decay of concentration fluctuations and the reduced interfacial area, the stroage modulus, G', behaves dramatically, decreasing at the early or intermediate stages; while when the blends are located in the metastable region, i.e., PEH/PEB 70/30 blend (H70), G' decreases slightly and slowly during the whole time sweep process. During the cyclic frequency sweeps, G' evolutions of H50 and H70 show similar trends. Obviously different from the strong phase segregation systems, the increase of G' with time in the metastable region has not been observed, possibly due to the entanglement effects and weak interaction between the components of polyethylene blends. The interfacial tension-driven or diffusion-limited morphological evolutions of H50 and H70 during phase separation give direct interpretations to the viscoelastic difference between the two blends, which is dominated by different phase separation kinetics. The relatively low interfacial tensions at the late stage of phase separation for H50 (0.5-0.38 mN/m varying with time) and H70 (1.2 mN/m) can be estimated by using the Gramespacher-Meissner model.

Both Reversible Self-Association and Structural Changes Underpin Molecular Viscoelasticity of mAb Solutions.

The role of antibody structure (conformation) in solution rheology is probed. It is demonstrated here that pH-dependent changes in the tertiary structure of 2 mAb solutions lead to viscoelasticity and not merely a shear viscosity (η) increase. Steady shear flow curves on mAb solutions are reported over broad pH (3.0 ≤ pH ≤ 8.7) and concentration (2 mg/mL ≤ c ≤ 120 mg/mL) ranges to comprehensively characterize their rheology. Results are interpreted using size exclusion chromatography, differential scanning calorimetry, analytical ultracentrifugation, near-UV circular dichroism, and dynamic light scattering. Changes in tertiary structure with concentration lead to elastic yield stress and increased solution viscosity in solution of "mAb1." These findings are supported by dynamic light scattering and differential scanning calorimetry, which show increased hydrodynamic radius of mAb1 at low pH and a reduced melting temperature Tm, respectively. Conversely, another molecule at 120 mg/mL solution concentration is a strong viscoelastic gel due to perturbed tertiary structure (seen in circular dichroism) at pH 3.0, but the same molecule responds as a viscous liquid due to reversible self-association at pH 7.4 (verified by analytical ultracentrifugation). Both protein-protein interactions and structural perturbations govern pH-dependent viscoelasticity of mAb solutions.

A microliter capillary rheometer for characterization of protein solutions.

Rheometry is an important characterization tool for therapeutic protein solutions because it determines syringeability and relates indirectly to solution stability and thermodynamic interactions. Despite the maturity of rheometry, there remains a need for a rheometer that meets the following three needs of the biopharamaceutical industry: small volume; large dynamic range of shear rates; and no air-sample interface. Here, we report the development of a miniaturized capillary rheometer that meets these needs and is potentially scalable to a multiwell format. These measurements consume only a few microliters of sample and have an uncertainty of a few percent. We demonstrate its performance on monoclonal antibody solutions at different concentrations and temperatures. The instrument has a dynamic range of approximately three decades (in shear rate) and can measure Newtonian, shear thinning, and yielding behaviors, which are representative of the different solution behaviors typically encountered. We compare our microliter capillary rheometer with existing instruments to describe the range of parameter space covered by our device.

Layered droplet microstructures in sheared emulsions: finite-size effects.

We investigate the influence of confinement on the steady state microstructure of emulsions sheared between parallel plates, in a regime where the average droplet dimension is comparable to the gap width between the confining walls. Utilizing droplet velocimetry, we find that the droplets can organize into discrete layers under the influence of shear. The number of layers decreases from two (at relatively higher shear rates) to one (at lower shear rates), as the drops grow slightly larger due to coalescence. We argue that the layering and overall composition profile may be controlled by the interplay of droplet collisions (which can cause separation of droplet centers in the velocity gradient direction), droplet migration toward the centerline (due to wall effects), and droplet packing constraints. We also study the effects of mixture composition on droplet microstructure, and summarize these results in the form of a morphology diagram in the parameter space of mass fraction and shear rate. We find that formation of strings of the suspended phase (reported earlier by our group in flow-visualization studies on confined emulsions) is observed over a broad composition window. We also find a stable (nontransient) morphology wherein the droplets are arranged in highly ordered pearl-necklace chain structures.