Solid-State NMR Spectroscopy to Probe State and Phase Transitions in Frozen Solutions

Abstract

Freezing is commonly encountered during the processing and storage of biomacromolecule products. Therefore, understanding the phase and state transitions in pharmaceutical frozen solutions is crucial for the rational development of biopharmaceuticals. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) was used to analyze solutions containing sodium phosphate buffer, histidine, and trehalose. Upon freezing, crystallization of disodium phosphate hydrogen dodecahydrate (Na2HPO4·12H2O, DPDH) and histidine was identified using 31P and 13C ssNMR, respectively, and confirmed by synchrotron X-ray diffractometry (SXRD). Using histidine as a molecular probe and based on the chemical shifts of atoms of interest, the pH of the freeze concentrate was measured. The unfrozen water content in freeze concentrates was quantified by 1H single pulse experiments. 13C-insensitive nuclei enhancement by polarization transfer (INEPT) and cross-polarization (CP) experiments were used as orthogonal tools to characterize the solutes in a “mobile” and a more “solid-like” state in the freeze-concentrated solutions, respectively. The above analyses were applied to a commercial monoclonal antibody (mAb) formulation of dupilumab. This work further establishes ssNMR spectroscopy as a highly capable biophysical tool to investigate the attributes of biopharmaceuticals and thereby provide insights into process optimization and formulation development.

Industrial Horizons in Pharmaceutical Science

“…In recent years, these tools have significantly advanced our understanding by pushing the boundaries in various areas. Examples include molecular or atomic level elucidation of key structures and interactions in amorphous solid dispersions within solid dosages, − critical (de)stabilizing interactions in parenteral peptide or protein formulations, − microenvironmental properties in lyophilized , and frozen biologics, and the encapsulation mechanisms of mRNA within LNPs for vaccine delivery. − These new applications exemplify the evolving mission of pharmaceutical sciences: to pose new questions to scientific and technical communities, to enhance collaboration, and to bring back innovative ideas and solutions. This dynamic approach helps drive forward the collective mission of delivering effective drugs to patients.…”

Probing Chemical Equilibrium in Frozen Sodium Phosphate Buffer Solution by 31P Solid-State NMR

“…12 We have recently demonstrated the feasibility of ssNMR, both to detect solute crystallization and to quantify the unfrozen water content, in frozen solutions. 13 Using crosspolarization (CP) and insensitive nuclei enhancement by polarization transfer (INEPT) methods under magic angle spinning (MAS), the state transition of the solutes, from a "mobile" to "solid-like" state, was also revealed.…”

“…Solid-state NMR (ssNMR) spectroscopy has emerged as a useful tool in the characterization of a variety of complex systems. − For example, it has been used to study the conformational stability of proteins in frozen solutions. − ssNMR can distinguish between state and phase transitions in frozen systems . We have recently demonstrated the feasibility of ssNMR, both to detect solute crystallization and to quantify the unfrozen water content, in frozen solutions . Using cross-polarization (CP) and insensitive nuclei enhancement by polarization transfer (INEPT) methods under magic angle spinning (MAS), the state transition of the solutes, from a “mobile” to “solid-like” state, was also revealed.…”