Suppression of Electrostatic Mediated Antibody Liquid–Liquid Phase Separation by Charged and Noncharged Preferentially Excluded Excipients

Abstract

Isotonic concentrations of inert cosolutes or excipients are routinely used in protein therapeutic formulations to minimize physical instabilities including aggregation, particulation, and precipitation that are often manifested during drug substance/product manufacture and long-term storage. Despite their prevalent use within the biopharmaceutical industry, a more detailed understanding for how excipients modulate the specific protein–protein interactions responsible for these instabilities is still needed so that informed formulation decisions can be made at the earliest stages of development when protein supply and time are limited. In the present report, subisotonic concentrations of the five common formulation excipients, sucrose, proline, sorbitol, glycerol, arginine hydrochloride, and the denaturant urea, were studied for their effect on the room temperature liquid–liquid phase separation of a model monoclonal antibody (mAb-B). Although each excipient lowered the onset temperatures of mAb-B liquid–liquid phase separation to different extents, all six were found to be preferentially excluded from the native state monomer by vapor pressure osmometry, and no apparent correlations to the excipient dependence of mAb-B melting temperatures were observed. These results and those of the effects of solution pH, addition of salt, and impact of a small number of charge mutations were most consistent with a mechanism of local excipient accumulation, to an extent dependent on their type, with the specific residues that mediate mAb-B electrostatic protein–protein interactions. These findings suggest that selection of excipients on the basis of their interaction with the solvent exposed residues of the native state may at times be a more effective strategy for limiting protein–protein interactions at pharmaceutically relevant storage conditions than choosing those that are excluded from the residues of the native state interior.

Suppression of Liquid‐Liquid Phase Separation and Aggregation of Antibodies by Modest Pressure Application

“…Conversely, addition of TMAO lead to a slight destabilization of the droplet phase of the system. A similar observation was made by Banks and Cordia, who also found a destabilizing effect of multiple cosolutes on the temperature‐dependent LLPS of monoclonal antibodies which did not correlate with the structural temperature stability as determined by DSC [10] …”

“…A similar observation was made by Banks and Cordia, who also found a destabilizing effect of multiple cosolutes on the temperaturedependent LLPS of monoclonal antibodies which did not correlate with the structural temperature stability as determined by DSC. [10] To yield a better understanding of the mechanism by which pressure and the cosolvent affect the dissolution of the droplet phase of γ-globulin, steady-state fluorescence anisotropy measurements were employed to determine the binding constant, K b , of γ-globulin molecules upon complex formation. The pressure-dependent binding assay was performed by measuring the fluorescence anisotropy of dansyl-labeled γ-globulin in pure buffer and in the presence of 0.5 M TMAO.…”

“…The mechanisms by which excipients affect the stability of protein solutions can be very complex, changing attractive interactions between protein molecules directly through excipient-protein interactions or indirectly by altering the hydration properties of the protein and the activity (coefficients) of all components, but their effects are generally difficult to predict. [6,10] Measured excipient preferential interaction coefficients revealed interaction parameters that are generally indicative of thermodynamically unfavorable proteinexcipient interactions (negative preferential binding coefficients, Γ PC , between protein and cosolvent), although specific interactions with the protein interface might still occur. [6,27,28] Here, we have seen that the compatible cosolvent TMAO had no drastic effect on the temperature and pressure stability of the droplet phase even at the high concentration of 0.5 M, leading only to a slight destabilization of the droplet phase.…”

“…The LLPS formation of antibodies implies strong attractive enthalpic protein-protein interactions through multiple chargepatch interactions of complementary surface charge, which require greater than room temperature (here, T > 30 °C) for the entropic contribution to the Gibbs free energy of mixing to dominate and favor a homogeneous solution. [4,5,10] Abs have a more branched and flexible structure than many other proteins, such as the globular lysozyme molecule. As a result, the binodal of LLPS of antibodies has a much lower critical volume fraction than globular proteins because of their nonspherical and anisotropic nature.…”

“…[4][5][6] At high concentrations, immunoglobulins undergo liquidliquid phase separation (LLPS) at low temperatures, that is, they phase separate into protein-poor and protein-rich liquid phases, in particular when formulated at low ionic strength and buffered at neutral pH near their isoelectric point. [2,[4][5][6][7][8][9][10] It is often observed that such fluid-like droplet phases undergo liquid-to-solid gel-like phase transitions over time, which upon maturation (or expedited by disease-associated mutations) lead to fibril formation and the development of pathological diseases, such as Parkinson's, Alzheimer's, cataract, and antibody light-chain (AL) amyloidosis. [11,12] In AL amyloidosis, fibrils are deposited in various organs, most often in the heart and kidney, and impair their function.…”