Drug formulation and development, drug discovery, X-ray and light scattering, molecular dynamics, statistical mechanics, protein stability, phase equilibria, rheology.
Abstract
Short-ranged attractions (SRA) in high concentration monoclonal antibody (mAb) formulations lead to unfavorable properties such as liquid-liquid phase separation (LLPS) [1] with changes in added salt or temperature. Further complications from SRA include high viscosity, and clustering [2], [3] which affect injectability. In this work, the SRA and solution structure are studied for set of mAbs as LLPS is approached by decreasing added salt concentration at the isoelectric point (pI). A second set of mAbs focused on formulations highly relevant to industry and clinical use were also studied. These mAbs were formulated far from the pI and at low ionic strength, where LRR are strong, to understand the role of buffer type, pH, and added surfactant on SRA, LRR, and viscosity. Both mAb sets were characterized via small angle X-ray scattering (SAXS) and coarse-grained molecular dynamics simulation (CG-MD). The results were compared to low concentration methods including light scattering. A library of CG-MD simulations based on a 12-bead CG mAb model were used to fit experimentally measured structure factors and evaluate the magnitude of SRA (K) and cluster size distributions. Closeness to LLPS was indicated by an increase in clustering. The clustering was suppressed when the mAb net charge increased by changing the buffer pH, and when anisotropic electrostatic SRA (for example, hydrogen bonding, charge-charge, charge-dipole, and dipole-dipole interactions) were screened from added salt. The reduced diffusion interaction parameter kD/B22,ex from low concentration light scattering measurements agreed with the trends from SAXS. For the higher LRR formulations, it was found that the solution viscosity and clustering increased as LRR were weakened through screening from buffer moieties and by increasing the pH closer to the pI.
References
[1] W. D. Kimball et al., “Growth of Clusters toward Liquid–Liquid Phase Separation of Monoclonal Antibodies as Characterized by Small-Angle X-ray Scattering and Molecular Dynamics Simulation,” J. Phys. Chem. B, vol. 129, no. 11, pp. 2856–2871, Mar. 2025.
[2] A. A. Chowdhury et al., “Subclass Effects on Self-Association and Viscosity of Monoclonal Antibodies at High Concentrations,” Mol. Pharmaceutics, vol. 20, no. 6, Art. no. 6, Jun. 2023.
[3] A. A. Chowdhury et al., “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,” Mol Pharm, vol. 20, no. 11, Art. no. 11, Nov. 2023.