Abstract
Liquid-liquid phase separation (LLPS) of monoclonal antibody (mAb) formulations involves spontaneous separation into dense (protein-rich) and diluted (protein-lean) phases and should be avoided in the final drug product. Understanding the factors leading to LLPS and ways to predict and prevent it would therefore be highly beneficial. Here we describe the link between LLPS behavior of an IgG1 mAb (mAb5), its solubility, and parameters extracted using 1H NMR spectroscopy, for various formulations. We show that the formulations demonstrating least LLPS lead to the largest mAb5 NMR signal intensities. In the formulations exhibiting the highest propensity to phase-separate the mAb NMR signal intensities are the lowest, even at higher temperatures without visible phase separation, suggesting a high degree of self-association prior to distinct phase separation. Addition of arginine glutamate prevented LLPS and led to a significant increase in the observed mAb signal intensity, whereas the effect of arginine hydrochloride was only marginal. Solution NMR spectroscopy was further used to characterize the protein-lean and protein-rich phases separately and demonstrated that protein self-association in the protein-rich phase can be significantly reduced by arginine glutamate. Solution NMR spectroscopy may be useful as a tool to assess the propensity of mAb solutions to phase-separate.
Highlights
We have previously demonstrated that 1H NMR spectroscopy is highly sensitive to the association state of monoclonal antibody (mAb) in solution and can contribute to selection of the best formulation for monomeric content and protein stability.[23]
Distinct phase separation of mAb5 was achieved by increasing ionic strength upon addition of 30, 50, or 100 mM NaCl to acetate buffer in the positive control group of samples (Figure 1)
Four formulations showing mixed opalescence and liquid−liquid phase separation (LLPS) behavior were chosen for further analysis and compared to the behavior of mAb5 in succinate buffer, each being subjected to eight freeze−thaw cycles (Figure 2)
Summary
During LLPS the initially uniform solution separates spontaneously into a lower, dense, protein-rich phase with high protein concentration and an upper, less dense, proteinlean ( often referred to as protein-poor) phase with a lower protein concentration. The lower protein-rich phase may become clear or may retain some opalescence due to density inhomogeneity leading to light scatter; while high protein concentration in this phase may eventually lead to further physical instabilities such as irreversible protein aggregation or precipitation.[16] LLPS is unacceptable in the final drug product since it presents an inhomogeneous formulation with different layer concentrations of protein and buffer components.[17] LLPS may occur spontaneously, especially if triggered by lowering the solution temperature, its appearance may be somewhat variable due to the metastable nature of the process.
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