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.

Role of Cosolute-Protein Interactions in the Dissociation of Monoclonal Antibody Clusters.

The solution thermodynamics and interactions of a reversibly self-associating monoclonal IgG1 antibody (mAb1) have been investigated as a function of cosolute type (NaCl, NaSCN, Arginine-HCl) and cosolute concentration over a wide range of protein concentrations (1-275 mg/mL) using static light scattering. Within the framework of multicomponent solution thermodynamic theory, the preferential interactions of cosolutes with mAb1 were evaluated in the concentration limit of the system. The overall interactions of cosolutes with mAb1 relative to the bulk solutions appear to be very weak, but preferential interactions alone are insufficient to account for the cosolutes' dissociation of mAb1 clusters. As a complementary approach to understanding cosolute interactions at high concentrations, mAb1 concentration dependent light scattering was also analyzed with models of interacting hard spheres (IHS). Evaluating the cosolute concentration dependence of association constants and states as specific binding interactions to dissociate mAb1 oligomers, numerical estimates of cosolute molecules required to dissociate oligomers were obtained. A descending order of cosolute binding number per mAb oligomer was found: arginine-Cl (∼17) > NaSCN (∼12) > NaCl (∼8). Differences in the cosolute effectiveness in reducing mAb1 equilibrium oligomer formation (self-association) are in part attributable to ion binding and salt identity (Hofmeister series) effects that reduce the attractive electrostatic interactions of mAb1. However, only Arg-Cl effectively disrupts mAb1 dimer formation, likely through interactions with the hydrophobic surface features on mAb1. Results indicate that localized cosolute-protein binding interactions have an important role in modulating nonspecific protein self- or heteroassociations at high concentrations.

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.

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.

Cosolute effects on the chemical potential and interactions of an IgG1 monoclonal antibody at high concentrations.

The solution thermodynamics and interactions of a reversibly self-associating IgG1 monoclonal antibody have been investigated as a function of cosolute type (NaCl, NaSCN, arginine-HCl) and cosolute concentration over a wide range of protein concentrations (1-235 mg/mL) using static light scattering. The nonideality of mAb solutions is analyzed within the simplifying framework of a two-component system to obtain the dependencies of the excess chemical potential of the mAb on protein and cosolute concentrations. Using hard spheres as a model of mAbs in the absence of intermolecular interactions, the mean interparticle distances can be estimated as a function of antibody concentration. Analysis of MAb1 excess chemical potential and mean intermolecular distance results in a potential function representing the sum of protein-protein interactions and their contributions to solution nonideality. This approach facilitates evaluation of the relative contributions of attractive/repulsive intermolecular interactions and excluded volume effects, as well as the effects of cosolutes on protein multiparticle interactions in crowded conditions. Underlying the dominant effect of volume exclusion at high protein concentrations, attractive interactions were found to be amplified with decreasing intermolecular distances by the MAb1 many-body correlations. Comparison of the cosolute concentration dependence of the protein chemical potential, dµ2(ex)/dC3, across the mAb concentrations demonstrates that MAb1 self-association is reduced with increasing ionic strength and in a series based on cosolute identity; Arg-Cl > NaSCN > NaCl. The effectiveness of arginine-HCl and NaSCN in modulating MAb1 excess chemical potential in concentrated solutions is ascribed to the cosolute's ability to mitigate both electrostatic as well as weaker hydrophobic attractive interactions between MAb1 molecules. This investigation presents the first direct analysis of cosolute specific effects on protein-protein interactions at high concentrations, and provides a novel approach for characterizing the many-body effects that contribute to solution nonideality.

Assessment and significance of protein-protein interactions during development of protein biopharmaceuticals.

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein-protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein-protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody-antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.

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.

Issues and challenges of subvisible and submicron particulate analysis in protein solutions.

The analysis of particulates has been a longstanding challenge in biopharmaceutical drug product development and quality control because the active constituents themselves may form particulate matter as a degradation product that may be difficult to quantify. These analytical challenges were met with success as long as the definition of particulate matter remained well within the capabilities of the instruments and methods used to measure it. The current testing as per USP <788> for parenterals at ≤100 mL stipulates that the sample "passes" the test if the average number of particles present does not exceed 6,000 per container at ≥10 µm and does not exceed 600 per container at ≥25 µm. The new challenge, posed by regulatory direction and academic research, is to count and to characterize subvisible particulates that are ≤10 µm with the goal of providing higher resolution information about the particulate levels and potential consequences of this product quality attribute in vivo. The present discussion focuses on two parallel efforts: (a) to develop a model system for protein subvisible particulates in samples with high protein concentrations and (b) to evaluate the capabilities and limitations of different technologies available (at the time these studies were conducted) for subvisible and submicron particle (<1 µm in diameter) sizing and counting. Our findings illustrate the importance of using appropriate instrumentation that is adapted to the characteristics of the samples to be analyzed. Any sample manipulation to meet the capabilities and to accommodate the limitations of the analytical technique should be carefully evaluated.

Use of dynamic light scattering to determine second virial coefficient in a semidilute concentration regime.

The present work discusses an alternative procedure to obtain static light scattering (SLS) parameters in a dilute and semidilute concentration regime from a dynamic light scattering (DLS) instrument that uses an avalanche photodiode (APD) for recording the scattered intensity signal. An APD enables one to perform both SLS and DLS measurements by photon counting and photon correlation, respectively. However, due to the associated recovery time, the APDs are susceptible to saturation (above 1000 kcps), which may limit the measurements in systems that scatter too much light. We propose an alternative way of obtaining the SLS parameters with instruments that use APD for recording signal intensities.

Intermolecular interactions of IgG1 monoclonal antibodies at high concentrations characterized by light scattering.

Light scattering intensity measurements of solutions of two purified monoclonal antibodies were performed over a wide range of concentrations (0.5-275 mg/mL) and ionic strengths (0.02 to 0.6 M). Despite extensive sequence homology between these mAbs, alteration of ∼20 amino acids in the complementarity determining regions resulted in different net intermolecular interactions and responses to solution ionic strength. The concentration dependence of scattering was analyzed by comparison with the predictions of three models, allowing for intermolecular interaction of various types. In order of increasing complexity, the three models account for: (1) steric repulsions (simple hard-sphere model), (2) steric repulsion with short-ranged attractive interactions of varying magnitude (adhesive hard-sphere model), and (3) steric and nonsteric repulsive interactions between several species whose relative concentrations may change as a function of total protein concentration as dictated by equilibrium self-association (effective hard-sphere mixture model). Simple scattering models of noninteracting and adhesive hard-sphere species permitted qualitative interpretation of contributions from excluded volume, electrostatic, and van der Waals interactions on net mAb interactions at high concentration as a function of ionic strength. mAb2 electrostatic interactions were repulsive, whereas mAb1 interactions were net attractive at low ionic strengths, attributed to an anisotropic distribution of molecular charge. The effective hard-sphere mixture model can account quantitatively for the dependence of scattering for both antibodies over the entire concentration range and at salt concentrations exceeding 40 mM. This analysis showed that at high ionic strength both mAbs self-associate weakly to form dimer with an affinity that varies little with salt concentration at concentrations exceeding 75 mM. In addition, mAb1 appears to self-associate further to form oligomers with stoichiometry of 4-6 and an affinity that declines substantially with increasing ionic strength. All three models lead to the conclusion that at high concentrations repulsive interactions are predominantly due to excluded volume, whereas additional features are salt-dependent and reflect a substantial electrostatic contribution to intermolecular interactions of both mAbs.