Cluster Size and Quinary Structure Determine the Rheological Effects of Antibody Self-Association at High Concentrations.

The question of how nonspecific reversible intermolecular protein interactions affect solution rheology at high concentrations is fundamentally rooted in the translation of nanometer-scale interactions into macroscopic properties. Well-defined solutions of purified monoclonal antibodies (mAbs) provide a useful system with which to investigate the manifold intricacies of weak protein interactions at high concentrations. Recently, characterization of self-associating IgG1 antibody (mAb2) solutions has established the direct role of protein clusters on concentrated mAb rheology. Expanding on our earlier work with three additional mAbs (mAb1, mAb3, and mAb4), the observed concentration-dependent static light scattering and rheological data present a substantially more complex relationship between protein interactions and solution viscosity at high concentrations. The four mAb systems exhibited divergent correlations between cluster formation (size) and concentrated solution viscosities dependent on mAb primary sequence and solution conditions. To address this challenge, well-established features of colloidal cluster phenomena could be applied as a framework for interpreting our observations. The initial stages of mAb cluster formation were investigated with small-angle X-ray scattering (SAXS) and ensemble-optimized fit methods, to uncover shifts in the dimer structure populations which are produced by changes in mAb interaction modes and association valence under the different solution conditions. Analysis of mAb average cluster number and effective hydrodynamic radii at high concentrations revealed cluster architectures can have a wide range of fractal dimensions. Collectively, the static light scattering, SAXS, and rheological characterization demonstrate that nonspecific and anisotropic attractive intermolecular interactions produce antibody clusters with different quinary structures to regulate the rheological properties of concentrated mAb solutions.

Monoclonal antibody self-association, cluster formation, and rheology at high concentrations.

The rheological properties of macromolecular and colloidal suspensions are dependent on the thermodynamic and kinetic parameters that define viscous flow, and remain an active field of study with broad implications in cellular biophysics, soft-matter theory, and biopharmaceutical technology. Here we use static light scattering, small-angle X-ray scattering, and viscosity measurements as a function of protein concentration to semiquantitatively correlate the oligomeric state of an IgG1 antibody (mAb1) with its rheological behavior at solution pH 6.0 and varying ionic strength (modified by 0.01-0.1 M Na2SO4). Solution SAXS characterization of 100 mM Na2SO4 solutions confirmed that mAb1 forms reversible dimers with extended structures in dilute solutions. Light-scattering measurements over a wide range of concentrations (1-175 mg/mL) provide detailed information on the equilibrium thermodynamic mAb1 interactions and their modulation by modest increases of Na2SO4. Through the use of interacting hard sphere models to fit light-scattering data, we establish that protein cluster formations consisting of 2-9 mAb1 molecules also increase the viscosity of 175 mg/mL IgG solutions from 52 up to 450 cP. The analysis of dilute and semidilute mAb1 solution rheology correlates linearly with the thermodynamic equilibrium cluster size, consistent with the viscosity behavior of elongated oligomeric structures that are not significantly dendrimeric or in a state of globular collapse. Furthermore, SAXS- and rheology-based structural modeling illustrate that only a small set of anisotropic interactions between complementary surfaces are required to nucleate and propagate protein clusters.

Influence of the cosolute environment on IgG solution structure analyzed by small-angle X-ray scattering.

Small-angle X-ray scattering experiments of two monoclonal antibodies (mAbs) were performed as a function of Hofmeister salt type and concentration including 100 mM Na(2)SO(4), 100-600 mM of NaSCN, or 100-600 mM arginine chloride at pH 6.0 to yield information on the effects of cosolutes on mAb solution conformation and flexibility. Minimal selected ensemble (MSE) procedures used to reconstruct the SAXS form factors revealed that both IgG1 mAbs exist in a conformational equilibrium with two subpopulations that vary in overall shape and size. The "closed" mAb conformation is characterized by a maximum dimension of ∼155 Šand shorter distances between Fab-Fab and Fab-FC domains. The "open" mAb conformation has a maximum dimension of ∼175 Šand an increase in the interdomain distances with concomitant increases in overall mAb flexibility. Analysis of the distribution of shapes and sizes of mAb structures within the conformational equilibrium indicates that they remain essentially unchanged under conditions with a broad range of chaotropic and kosmotropic salts including 100-600 mM NaSCN and 100 mM Na(2)SO(4). Analysis of the conformations within each MSE population under various conditions reveals a striking similarity between many of the MSE structures, IgG crystal structures, and single-molecule imaging studies; MSE analysis of mAb form factors also identified an overall relaxation of the mAb structure unique to solution conditions containing arginine chloride, characterized by an increased maximum dimension and a shift toward the population of the "open" mAb conformation. Our results provide the first comprehensive characterization of mAb conformational diversity in solution and are of direct relevance to understanding the effects of solution conditions on protein structural dynamics and stability.