Quantification of Residual Water in Pharmaceutical Frozen Solutions Via ¹H Solid-State NMR

Freezing is essential for the stability of biological drug substances and products, particularly in frozen solution formulations and during the primary drying of lyophilized preparations. However, the unfrozen segment within the frozen matrix can alter solute concentration, ionic strength, and stabilizer crystallization, posing risks of increased biophysical instability and faster chemical degradation. While quantifying the unfrozen water content is important for designing stable biopharmaceuticals, there is a lack of analytical techniques for in situ quantitative measurements. In this study, we introduce a ¹H magic angle spinning NMR technique to identify the freezing point (T_ice) and quantify mobile water content in frozen biologics, applying this method to analyze the freezing of a commercial high-concentration drug product, Dupixent®. Our results demonstrate that water freezing is influenced by buffer salt properties and formulation composition, including the presence of sugar cryoprotectants and protein concentration. Additionally, the ¹H chemical shift can probe pH in the unfrozen phase, potentially predicting the microenvironmental acidity in the frozen state. Our proposed methodology provides fresh insights into the analysis of freeze-concentrated solutions, enhancing our understanding of the stability of frozen and lyophilized biopharmaceuticals.