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Default Investigating liquid-liquid phase separation of a monoclonal antibody using solution-state NMR spectroscopy: effect of Arg·Glu and Arg·HCl.

Investigating liquid-liquid phase separation of a monoclonal antibody using solution-state NMR spectroscopy: effect of Arg·Glu and Arg·HCl.

Investigating liquid-liquid phase separation of a monoclonal antibody using solution-state NMR spectroscopy: effect of Arg·Glu and Arg·HCl.

Mol Pharm. 2017 Jun 14;:

Authors: Kheddo P, Bramham JE, Dearman RJ, Uddin S, van der Walle CF, Golovanov AP

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 (1)H 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 lead 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.


PMID: 28614662 [PubMed - as supplied by publisher]



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