Solid-State NMR Characterization of Lyophilized Formulations of Monoclonal Antibody Therapeutics.

Monoclonal antibodies (mAbs) are an important and growing class of biotherapeutic drugs. Method development for the characterization of critical quality attributes, including higher-order structure (HOS), of mAbs remains an area of active inquiry. Recently, solution-state nuclear magnetic resonance (NMR) spectroscopy has received increased attention and is a means for reliable, high-resolution HOS characterization of aqueous-based preparations of mAbs. While mAbs are predominantly formulated in solution, up to 20% are prepared as solid amorphous powders and techniques for the robust characterization of HOS in the solid state remain limited. We propose here the use of solid-state NMR (ssNMR) fingerprinting to inform directly on the HOS of solid preparations of mAbs. Using lyophilized samples of the NISTmAb reference material prepared with different formulation conditions, we demonstrate that 1 H- 13 C cross-polarization (hC-CP) buildup spectral series at natural isotopic abundance mAb samples are sensitive to differences in formulation. We also demonstrate that principal component analysis (PCA) can be used to differentiate the samples from one another in a user-independent manner while also highlighting areas where expert analysis can provide structural details about important molecular interactions in solid-phase protein formulations. Results from this study contribute to establishing the foundation for the use of ssNMR for HOS characterization of solid-phase biotherapeutics.