(W1030-06-32) Probing Microenvironmental Acidity in Pharmaceutical Frozen Solution Using Solid-State NMR Spectroscopy

Purpose: Biopharmaceutical products, for example, monoclonal antibodies, are usually stored in frozen solutions to improve their long-term stability.^1,2 However, the microenvironmental acidity of frozen-state protein formulation can shift significantly during the freezing process, which results in protein instability and diminished efficacy.^3,4 Therefore, a precise measure of pH in the frozen solution can provide insights into the rational development of protein therapeutics. However, techniques that can determine the frozen-state acidity or basicity are very limited.^2,5 The present study aims to develop solid-state nuclear magnetic resonance (ssNMR) as a spectroscopic tool to probe the microenvironmental acidity in frozen solution using histidine as a molecular pH indicator.

Methods: Histidine is a capable molecular pH indicator due to its wide range of protonation states and high sensitivity of chemical shift changes to different microenvironmental pH in aqueous solution, solid crystal, and lyophilized cakes.^2,6,7 A series of sodium phosphate buffer solutions containing 50 mM ¹³C₆, ¹⁵N₃ L-histidine and 5% w/v of trehalose was prepared and the pH was varied from 5.0 to 11.0. ¹³C and ¹⁵N cross polarization (CP) ssNMR experiments were utilized to obtain ¹³C and ¹⁵N chemical shifts of histidine in frozen solution at -35°C and different pHs. Furthermore, a temperature-dependence study was carried out on the pH 7 sample from 25 to -35°C to investigate the phase change of histidine upon freezing. ¹H 1D, ¹³C insensitive nuclei enhancement by polarization transfer (INEPT), ¹³C direct polarization (DP), and ¹³C CP experiments were performed to quantify the content of unfrozen water as well as monitor the concentration change of the mobile and amorphous histidine.

Results: The addition of trehalose in the formulation inhibits the crystallization of disodium hydrogen phosphate buffer (i.e., Na₂HPO₄·12H₂O), and thus the potential pH shift during freezing. The broader peak linewidth indicates that amorphous histidine was formed in the frozen solution. ¹³C and ¹⁵N chemical shifts of histidine demonstrate a strong correlation with the pH of the pre-lyo solutions. Specifically, C_γ, C_δ2, and N_ε2 demonstrate a systematic upfield shift, while C_α and C_β exhibit a downfield shift with increasing pH (Figures 1 & 2). 1D 1H experiments suggest that the content of unfrozen water decreases as the freezing temperature is lowered. Moreover, the CP signal intensity change of amorphous histidine indicates the formation of amorphous histidine is correlated with the mobile water content. Component analyses of amorphous and liquid histidine were derived by combining results from ¹³C INEPT, DP, and CP experiments (Figure 3).

Conclusion: We have developed an ssNMR protocol to probe the microenvironmental pH in frozen solutions using histidine as a molecular pH indicator. ¹³C and ¹⁵N chemical shifts of amorphous histidine are shown to well correlate with the pH of the parent solutions. Considering histidine is a commonly used buffer and stabilizer in formulations, we anticipate this method can be widely applied to gain in-depth information on acidity or basicity for pharmaceutical frozen solutions.

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(5) Pudipeddi, M.; Zannou, E. A.; Vasanthavada, M.; Dontabhaktuni, A.; Royce, A. E.; Joshi, Y. M.; Serajuddin, A. T. M. Measurement of Surface PH of Pharmaceutical Solids: A Critical Evaluation of Indicator Dye-Sorption Method and Its Comparison With Slurry PH Method. J. Pharm. Sci. 2008, 97 (5), 1831–1842.
(6) Li, S.; Hong, M. Protonation, Tautomerization, and Rotameric Structure of Histidine: A Comprehensive Study by Magic-Angle-Spinning Solid-State NMR. J. Am. Chem. Soc. 2011, 133 (5), 1534–1544.
(7) Platzer, G.; Okon, M.; McIntosh, L. P. PH-Dependent Random Coil 1H, 13C, and 15N Chemical Shifts of the Ionizable Amino Acids: A Guide for Protein PK a Measurements. J. Biomol. NMR 2014 602 2014, 60 (2), 109–129.

Acknowledgments: Yong Du is grateful to the Merck Research Laboratory postdoctoral program