Growth of Clusters toward Liquid-Liquid Phase Separation of Monoclonal Antibodies as Characterized by Small-Angle X-ray Scattering and Molecular Dynamics Simulation.

In concentrated protein solutions, short-range attractions (SRAs) contribute to liquid-liquid phase separation (LLPS) as a function of temperature and salinity, particularly when the charge and thus long-range repulsions are low near the isoelectric point pI. Herein, we study how SRA and solution morphology vary with the approach to LLPS from increased SRA for two monoclonal antibodies (mAbs) as salt concentration is reduced near the pI. These properties are quantified using small-angle X-ray scattering (SAXS) interpreted via coarse-grained (CG) molecular dynamics (MD) simulations and compared with less descriptive properties from static and dynamic light scattering.

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 () of short-range anisotropic attractions (SRA) for 75-200 mg/mL mAb2 solutions at different pH and cosolute conditions by analyzing structure factors (()) 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.

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 (K) for vedolizumab IgG1, IgG2, or IgG4 subclasses by fitting small-angle X-ray scattering (SAXS) structure factor () data with an extensive library of 12-bead coarse-grained (CG) molecular dynamics simulations. The K 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.

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.

Patient-tailored algorithm for laparoscopic cruroplasty standardization: comparison with hiatal surface area and medium-term outcomes.

Background: Different methods have been described for laparoscopic hiatoplasty and hiatus hernia (HH) repair. All techniques are not standardized and the choice to reinforce or not the hiatus with a mesh is left to the operating surgeon's preference. Hiatal surface area (HSA) has been described as an attempt at standardization; in case the area is > 4 cm, a mesh is used to reinforce the repair.