Maintaining pH control with an appropriate buffer system is crucial in lyophilized formulations to minimize protein degradation and aggregation. One factor to consider is freezing can concentrate the buffer salt and drive the buffer salt precipitation and pH shift which subsequently induces protein aggregation. Therefore, the pH in the final dry protein may not retain the pH value as it is prepared in the solution. It has been reported that a large pH shift was seen for several buffers when the temperature was lowered. The activity and stability of a protein can be affected by the ionization state of the protein itself. The stability of lyophilized proteins can be effectively regulated through the thorough selection of buffers and controlling the initial pH. Evidence has indicated that buffers play a significant role in influencing the characteristics of freeze-dried protein formulations, which can be molecule-specific. It was found that histidine outperformed phosphate and citrate as a buffer for stabilizing lyophilized lactate dehydrogenase. In another study, it was discovered that sodium citrate buffer offered superior stability compared to sodium phosphate buffer for freeze-dried IL-1 receptor antagonists (IL-1ra) at a pH of 6.5. The stability of IL-1ra was found to be higher when sodium citrate buffer was used as a stabilizer as opposed to sodium phosphate buffer. As we discussed in Section 4.4, the microenvironmental pH of lyophilized formulation plays a critical role in protein stability. Buffer pH shift is typically caused by the certain buffer species precipitated from the solution. As such, there are formulation strategies to inhibit the buffer crystallization during the freezing process. For example, the buffer salt crystallization-induced pH shift can be adversely affected by adding non-crystallizing solutes e.g., sugar, polymer and proteins. In a recent study, the role of pH shift from phosphate buffer on the aggregation behavior of two model proteins BSA and β-galactosidase (β-gal) was evaluated. Phosphate buffer (100 mM) demonstrated a pH swing of 3.1 and 2.7 for BSA and β-gal respectively when cooling from 20 to -25°C. Moreover, it was also found that the addition of cellobiose not only mitigates the pH shifts but also inhibits buffer salt crystallization. Similar inhibition of buffer salt using trehalose and mannitol was observed. Trehalose can completely inhibit the buffer salt crystallization and render the lyophilized solid completely amorphous, whereas mannitol can partially suppress the buffer salt crystallization and only can attenuate the magnitude of pH shift. The co-solutes of buffer salt with sugar can potentially mitigate the aggregation of proteins caused by pH shifts in phosphate buffered saline Strategies and knowledge enabling pH stability during the process are critical to the drug development of proteins and vaccines. Most recently, 31 P solid-state NMR was used to probe the physicochemical properties of phosphate salt species in equilibrium at subzero temperatures. Phosphate species in frozen solution were directly quantified which provides insights into ionic strength and pH in the freeze concentration. Briefly, the crystallization of disodium phosphate hydrogen dodecahydrate (Na2HPO4•12H2O, DPDH) can be probed by SXRD and readily identified by P ssNMR (Figure ). All phosphate species in solution and the frozen state in freeze concentrates can be quantified, enabling the calculation of pH and ionic strength (Figure ). In addition, the type of buffer can alter the thermodynamic properties of the glass matrix of the protein formulation. Phosphate ion was found to increase the Tg values of both trehalose and sucrose, but the extent of this increase varied depending on the pH conditions. 271 A similar observation was seen with the increase in the Tg of trehalose in citrate buffer. In contrast, a lowering effect of Tg of trehalose was observed with the presence of tris and other buffers. Moreover, there is spectroscopic evidence that phosphate salt crystallization can be mitigated by trehalose in the formulation. The physical forms of excipients in the frozen matrix, by influencing their functionality, can significantly impact drug stability. For example, stabilizers (also called cryoprotectants) need to be in the amorphous state to exert their function. Therefore, cryoprotectant crystallization can be a major destabilizing factor in protein formulations. Thus, understanding the crystallization tendency of excipients is necessary to ensure stability and maintain the biological activity of proteins. As shown in Figure , the amorphization of trehalose has been spectroscopically identified, as evidenced by the peak linewidth change from a narrow peak in solution at room temperature to a broad peak upon freezing. Very importantly, the in-situ ssNMR analysis enables the characterization of molecular dynamics and phase transitions of individual components in protein formulations upon freezing (Figure ). Specifically, the protein pembrolizumab has exhibited global and local motions at room temperature and, at temperatures below ~173 K, is nearing a globally rigid-lattice state.