NMR detects clustering and ultra-weak excipient interactions governing monoclonal antibody viscosity in formulation-relevant conditions

Overview & Synopsis

High-concentration formulations of monoclonal antibodies (mAbs) are required for subcutaneous administration but are frequently challenging to develop due to elevated viscosity and colloidal instability. These properties are governed by mAb-mAb interactions that are regulated by additional mAb-excipient interactions. These ultra-weak interactions remain difficult to characterize using conventional techniques. Here, we combined nuclear magnetic resonance (NMR) spectroscopy with rheometry and dynamic light scattering to investigate mAb interactions under formulation-relevant conditions. Rheological measurements showed that, in the case of the chosen mAb, the excipients arginine and lysine strongly reduce the macroscopic viscosity, whereas other excipients display a moderate effect by themselves. Characterization of the mAb oligomeric state by 1 H NMR confirmed that arginine and lysine are the most efficient at reducing mAb self-assembly, while proline and glycine promote clustering. Interactions between mAb and excipients were first analyzed by measuring excipient diffusion coefficients, but these are only weakly affected by the addition of concentrated mAb. The mAb-excipient interactions were further detected by measuring excipient 1 H and 13 C chemical shifts, mAb-excipient saturation transfers, and excipient 1 H transverse relaxation rates. These experiments provided complementary information on excipient interactions with the different mAb oligomers. Lysine was identified as the best mAb binder. However, several excipients such as sucrose that do not reduce the macroscopic viscosity also bind to concentrated mAb, highlighting that excipient binding can have various consequences on the transient interactions between mAb species. Altogether, this work proposes a powerful NMR pipeline to dissect ultra-weak molecular interactions that govern viscosity and developability in therapeutic antibody formulations.