(M1130-06-32) Investigating Pharmaceutical Frozen Solution Using 31P and ¹H Solid-State NMR

Purpose: A substantial number of protein therapeutics and vaccines, for example, Pfizer and Moderna covid-19 vaccines, are stored in a frozen state in order to enhance product stability. However, the freezing process can induce stresses including cold temperature, solute concentration due to the ice formation, eutectic crystallization of buffer solutes, and resultant pH shifts. These microenvironmental conditional changes may result in the instability of proteins. For example, a pH decrease of 3-4 units has been reported in phosphate buffered protein solutions during freezing. Therefore, a mechanistic understanding of freeze-thaw cycling is of vital importance for the rational design of biological drug products. However, the lack of high-resolution techniques often adds challenges to the characterization of frozen solutions. The present study is aimed at using novel and sensitive solid-state nuclear magnetic resonance (ssNMR) spectroscopy to probe the phase behaviours of phosphorous species in the frozen phosphate buffer solution. The inherent quantitative nature of NMR offers the opportunity to quantitate mobile and frozen phosphate ions and unfrozen water content, providing valuable implications for pharmaceutical formulations.

Methods: ³¹P direct-polarization and cross-polarization ssNMR were utilized to identify the phosphorus species in the sodium phosphate buffer solutions, where the ratio of mobile phosphate ions (HPO₄^2- and H₂PO^4-) and crystalline disodium hydrogen phosphate dodecahydrate (Na₂HPO₄·12H₂O) are obtained by spectral analysis. Moreover, ¹H NMR was employed to quantify the unfrozen water and the respective unfrozen content at each freezing temperature was calculated as compared to the liquid water at room temperature. The crystallization of Na₂HPO₄·12H₂O and ice was also determined by differential scanning calorimetry and low-temperature X-ray diffractometry.

Results: ³¹P ssNMR can readily identify the crystallization of disodium hydrogen phosphate as a dodecahydrate (Na₂HPO₄·12H₂O). The crystallization of Na₂HPO₄·12H₂O was inhibited by the addition of trehalose in the sodium phosphate solution. By further increasing trehalose contents, "amorphous-like" ³¹P species were detected at -30 ºC. Moreover, the pH shift as a consequence of Na₂HPO₄·12H₂O crystallization was correlated to the freezing temperatures. As the temperature is lowered, the ratio of mobile HPO₄^2-/H₂PO^4- decreases, indicating the occurrence of a pH shift in sodium phosphate frozen solution. Furthermore, ¹H NMR was shown capable of quantifying the unfrozen water content. The effect of freezing temperature, buffer and trehalose concentration, and freezing speed on the unfrozen content was systematically investigated. For example, higher trehalose and buffer concentrations were shown to promote the unfrozen water content.

Conclusion: We, for the first time, have developed an ssNMR protocol to characterize the phase behaviours of mobile and frozen phosphorous species in sodium phosphate buffered frozen solutions. This study demonstrates that ssNMR is a promising biophysical technique that can be widely applied to obtain critical knowledge of the physicochemical stability of therapeutical proteins in their frozen state.

References: Thorat, A. A.; Munjal, B.; Geders, T. W.; Suryanarayanan, R. Freezing-Induced Protein Aggregation - Role of PH Shift and Potential Mitigation Strategies. J. Control. Release 2020, 323, 591–599.

Acknowledgements: Yong Du is grateful to the Merck Postdoctoral program


31P solid-state NMR cross-polarization spectra of Na2HPO4·xH2O and NaH2PO4 crystalline powders and 100 mM sodium phosphate buffered frozen solution.


Quantification of unfrozen water content and mobile and solid phosphorous 31P species in sodium phosphate frozen solution using 31P and 1H solid-state NMR


31P solid-state NMR cross-polarization spectra of 100 mM sodium phosphate buffered frozen solution with different levels of trehalose.