Characterizing Protein-Protein Interactions and Viscosity of a Monoclonal Antibody from Low to High Concentration Using Small-Angle X-ray Scattering and Molecular Dynamics Simulations.

Understanding protein-protein interactions and formation of reversible oligomers (clusters) in concentrated monoclonal antibody (mAb) solutions is necessary for designing stable, low viscosity (η) concentrated formulations for processing and subcutaneous injection. Here we characterize the strength (K) of short-range anisotropic attractions (SRA) for 75-200 mg/mL mAb2 solutions at different pH and cosolute conditions by analyzing structure factors (Seff(q)) from small-angle X-ray scattering (SAXS) using coarse-grained molecular dynamics simulations. Best fit simulations additionally provide cluster size distributions, fractal dimensions, cluster occluded volume, and mAb coordination numbers. These equilibrium properties are utilized in a model to account for increases in viscosity caused by occluded volume in the clusters (packing effects) and dissipation of stress across lubricated fractal clusters. Seff(q) is highly sensitive to K at 75 mg/mL where mAbs can mutually align to form SRA contacts but becomes less sensitive at 200 mg/mL as steric repulsion due to packing becomes dominant. In contrast, η at 200 mg/mL is highly sensitive to SRA and the average cluster size from SAXS/simulation, which is observed to track the cluster relaxation time from shear thinning. By analyzing the distribution of sub-bead hot spots on the 3D mAb surface, we identify a strongly attractive hydrophobic patch in the complementarity determining region (CDR) at pH 4.5 that contributes to the high K and consequently large cluster sizes and high η. Adding NaCl screens electrostatic interactions and increases the impact of hydrophobic attraction on cluster size and raises η, whereas nonspecific binding of Arg attenuates all SRA, reducing η. The hydrophobic patch is absent at higher pH values, leading to smaller K, smaller clusters, and lower η. This work constitutes a first attempt to use SAXS and CG modeling to link both structural and rheological properties of concentrated mAb solutions to the energetics of specific hydrophobic patches on mAb surfaces. As such, our work opens an avenue for future research, including the possibility of designing coarse-grained models with physically meaningful interacting hot spots.

Subclass Effects on Self-Association and Viscosity of Monoclonal Antibodies at High Concentrations.

The effects of a subclass of monoclonal antibodies (mAbs) on protein-protein interactions, formation of reversible oligomers (clusters), and viscosity (η) are not well understood at high concentrations. Herein, we quantify a short-range anisotropic attraction between the complementarity-determining region (CDR) and CH3 domains (KCDR-CH3) for vedolizumab IgG1, IgG2, or IgG4 subclasses by fitting small-angle X-ray scattering (SAXS) structure factor Seff(q) data with an extensive library of 12-bead coarse-grained (CG) molecular dynamics simulations. The KCDR-CH3 bead attraction strength was isolated from the strength of long-range electrostatic repulsion for the full mAb, which was determined from the theoretical net charge and a scaling parameter ψ to account for solvent accessibility and ion pairing. At low ionic strength (IS), the strongest short-range attraction (KCDR-CH3) and consequently the largest clusters and highest η were observed with IgG1, the subclass with the most positively charged CH3 domain. Furthermore, the trend in KCDR-CH3 with the subclass followed the electrostatic interaction energy between the CDR and CH3 regions calculated with the BioLuminate software using the 3D mAb structure and molecular interaction potentials. Whereas the equilibrium cluster size distributions and fractal dimensions were determined from fits of SAXS with the MD simulations, the degree of cluster rigidity under flow was estimated from the experimental η with a phenomenological model. For the systems with the largest clusters, especially IgG1, the inefficient packing of mAbs in the clusters played the largest role in increasing η, whereas for other systems, the relative contribution from stress produced by the clusters was more significant. The ability to relate η to short-range attraction from SAXS measurements at high concentrations and to theoretical characterization of electrostatic patches on the 3D surface is not only of fundamental interest but also of practical value for mAb discovery, processing, formulation, and subcutaneous delivery.

Characterizing Experimental Monoclonal Antibody Interactions and Clustering Using a Coarse-Grained Simulation Library and a Viscosity Model.

Attractive protein-protein interactions in concentrated monoclonal antibody (mAb) solutions may lead to the formation of clusters that increase viscosity. Here, we propose an analytical model that relates mAb solution viscosity to clustering by accounting for the contributions of suboptimal mAb packing within a cluster and cluster fractal dimension. The influence of short-range, anisotropic attractions and long-range Coulombic repulsion on cluster properties is investigated by analyzing the cluster-size distributions, cluster fractal dimensions, radial distribution functions, and static structure factors from a library of coarse-grained molecular dynamics simulations. The library spans a vast range of mAb charges and attractive interactions in solutions of varying ionic strength. We present a framework for combining the viscosity model and simulation library to successfully characterize the attraction, repulsion, and clustering of an experimental mAb in three different pH and cosolute conditions by fitting the measured viscosity or structure factor from small-angle X-ray scattering. At low ionic strength, the cluster-size distribution is impacted by strong charges, and both the viscosity and net charge or structure factor and net charge must be considered to deconvolute the effects of short-range attraction and long-range repulsion.

A Microfluidic Prototype for High-Frequency, Large Strain Oscillatory Flow Rheometry.

We introduce a "Rheo-chip" prototypical rheometer which is able to characterise model fluids under oscillatory flow at frequencies f up to 80 Hz and nominal strain up to 350, with sample consumption of less than 1 mL, and with minimum inertial effects. Experiments carried out with deionized (DI) water demonstrate that the amplitude of the measured pressure drop ΔPM falls below the Newtonian prediction at f≥ 3 Hz. By introducing a simple model which assumes a linear dependence between the back force and the dead volume within the fluid chambers, the frequency response of both ΔPM and of the phase delay could be modeled more efficiently. Such effects need to be taken into account when using this type of technology for characterising the frequency response of non-Newtonian fluids.

A microfluidic approach to studying the injection flow of concentrated albumin solutions.

ABSTRACT: Subcutaneous injection by means of prefilled syringes allows patients to self-administrate high-concentration (100 g/L or more) protein-based drugs. Although the shear flow of concentrated globulins or monoclonal antibodies has been intensively studied and related to the injection force proper of SC processes, very small attention has been paid to the extensional behavior of this category of complex fluids. This work focuses on the flow of concentrated bovine serum albumin (BSA) solutions through a microfluidic "syringe-on-chip" contraction device which shares some similarities with the geometry of syringes used in SC self-injection. By comparing the velocity and pressure measurements in complex flow with rheometric shear measurements obtained by means of the "Rheo-chip" device, it is shown that the extensional viscosity plays an important role in the injection process of protinaceous drugs. ARTICLE HIGHLIGHTS: A microfluidic "syringe on chip" device mimicking the injection flow of protinaceous drugs has been developed.The velocity field of concentrated BSA solutions through the "syringe on chip" is Newtonian-like.The extensional viscosity of concentrated protein solutions should also be considered when computing injection forces through needles.

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.