Investigating Liquid-Liquid Phase Separation of a Monoclonal Antibody Using Solution-State NMR Spectroscopy: Effect of Arg·Glu and Arg·HCl.

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.

Characterizing monoclonal antibody formulations in arginine glutamate solutions using 1H NMR spectroscopy.

Assessing how excipients affect the self-association of monoclonal antibodies (mAbs) requires informative and direct in situ measurements for highly concentrated solutions, without sample dilution or perturbation. This study explores the application of solution nuclear magnetic resonance (NMR) spectroscopy for characterization of typical mAb behavior in formulations containing arginine glutamate. The data show that the analysis of signal intensities in 1D 1H NMR spectra, when compensated for changes in buffer viscosity, is invaluable for identifying conditions where protein-protein interactions are minimized. NMR-derived molecular translational diffusion rates for concentrated solutions are less useful than transverse relaxation rates as parameters defining optimal formulation. Furthermore, NMR reports on the solution viscosity and mAb aggregation during accelerated stability study assessment, generating data consistent with that acquired by size-exclusion chromatography. The methodology developed here offers NMR spectroscopy as a new tool providing complementary information useful to formulation development of mAbs and other large therapeutic proteins.

The effects of arginine glutamate, a promising excipient for protein formulation, on cell viability: Comparisons with NaCl.

The effects of an equimolar mixture of l-arginine and l-glutamate (Arg·Glu) on cell viability and cellular stress using in vitro cell culture systems are examined with reference to NaCl, in the context of monoclonal antibody formulation. Cells relevant to subcutaneous administration were selected: the human monocyte cell line THP-1, grown as a single cell suspension, and adherent human primary fibroblasts. For THP-1 cells, the mechanism of cell death caused by relatively high salt concentrations was investigated and effects on cell activation/stress assessed as a function of changes in membrane marker and cytokine (interleukin-8) expression. These studies demonstrated that Arg·Glu does not have any further detrimental effects on THP-1 viability in comparison to NaCl at equivalent osmolalities, and that both salts at higher concentrations cause cell death by apoptosis; there was no significant effect on measures of THP-1 cellular stress/activation. For adherent fibroblasts, both salts caused significant toxicity at ~400 mOsm/kg, although Arg·Glu caused a more precipitous subsequent decline in viability than did NaCl. These data indicate that Arg·Glu is of equivalent toxicity to NaCl and that the mechanism of toxicity is such that cell death is unlikely to trigger inflammation upon subcutaneous injection in vivo.

The effect of arginine glutamate on the stability of monoclonal antibodies in solution.

Finding excipients which mitigate protein self-association and aggregation is an important task during formulation. Here, the effect of an equimolar mixture of l-Arg and l-Glu (Arg·Glu) on colloidal and conformational stability of four monoclonal antibodies (mAb1-mAb4) at different pH is explored, with the temperatures of the on-set of aggregation (Tagg) and unfolding (Tm1) measured by static light scattering and intrinsic fluorescence, respectively. Arg·Glu increased the Tagg of all four mAbs in concentration-dependent manner, especially as pH increased to neutral. Arg·Glu also increased Tm1 of the least thermally stable mAb3, but without similar direct effect on the Tm1 of other mAbs. Raising pH itself from 5 to 7 increased Tm1 for all four mAbs. Selected mAb formulations were assessed under accelerated stability conditions for the monomer fraction remaining in solution after storage. The aggregation of mAb3 was suppressed to a greater extent by Arg·Glu than by Arg·HCl. Furthermore, Arg·Glu suppressed the aggregation of mAb1 at neutral pH such that the fraction monomer was near to that at the more typical formulation pH of 5.5. We conclude that Arg·Glu can suppress mAb aggregation with increasing temperature/pH and, importantly, under accelerated stability conditions at weakly acidic to neutral pH.