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 (K CDR-CH3 ) for vedolizumab IgG1, IgG2, or IgG4 subclasses by fitting small-angle X-ray scattering (SAXS) structure factor S eff (q) data with an extensive library of 12-bead coarse-grained (CG) molecular dynamics simulations. The K CDR-CH3 bead attraction strength was isolated from the strength of longrange 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 (K CDR-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 K CDR-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 shortrange 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 Protein–Protein Interactions and Viscosity of a Monoclonal Antibody from Low to High Concentration Using Small-Angle X-ray Scattering and Molecular Dynamics Simulations

“…An alternative approach is to approximate the LRR of a mAb molecule by placing an average bead charge Q = (net charge/number of beads) on each bead and then assigning additional bead-specific SRA on strategic beads by fitting experimental SAXS data. This approach has yielded similar S eff ( q ) to bead-specific charge models for IgG1 and IgG4 subclasses of vedolizumab, as well as four IgG1 mAbs ,, with published bead-specific charges. Additionally, it effectively decouples SRA and LRR , and enables the construction of a reasonably sized simulation library to treat new mAbs, which would become impractical with individual bead charges. , …”

“…Several CG strategies, especially 12-bead models, have been developed for quantifying SRA and LRR from experimental scattering data. ,,,, A typical approach is to determine the charge on each bead by adding amino acid contributions from sequence information ,− to calculate the Coulombic interactions. Typically, the same SRA is associated with each bead to mimic van der Waals forces.…”

“…Typically, the same SRA is associated with each bead to mimic van der Waals forces. While this approach correctly approximated the S (0) vs C p behavior of a weakly attractive mAb, it is often unsuccessful for mAbs with strong SRA as it does not capture the effect of short-range anisotropic charge–charge, charge–dipole, dipole–dipole, and hydrophobic attractions , at the bead and sub-bead levels. An alternative approach is to approximate the LRR of a mAb molecule by placing an average bead charge Q = (net charge/number of beads) on each bead and then assigning additional bead-specific SRA on strategic beads by fitting experimental SAXS data.…”

“…In a series of studies, we have quantified SRA by fitting the bead-specific attraction strength, K , in our CG model directly from experimental S eff ( q ). ,,,, While this approach yielded the Fab–Fab attraction strength at 200 mg/mL, the sensitivity of the fits was often quite low as S eff ( q ) was dominated by packing effects. Recently, the CDR–CDR and CDR–CH3 attractions were independently quantified using S eff ( q ) at 130 mg/mL for a set of IgG1, IgG2, or IgG4 proteins to understand variations in the Fc region with a fixed Fab .…”

Role of Domain–Domain Interactions on the Self-Association and Physical Stability of Monoclonal Antibodies: Effect of pH and Salt

“…Small-angle X-ray/neutron scattering (SAXS/SANS) have been widely used in recent years to characterize PPI directly from concentrated mAb formulations. ,,, Therefore, in this study, the PPI of NISTmAb molecules was also characterized using SAXS (Figure ). SAXS profiles of NISTmAb prepared in pH 6 buffer conditions are shown in Figure a and c. No upturn is observed in the low- q region from any of the SAXS profiles, indicating that there was no high-molecular-weight species formed in either 5 or 300 mM ionic strength at such a pH condition.…”