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

Abstract

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.