Buffer salt is typically used to maintain the pH of the formulation to control the stability of proteins. A commonly encountered issue is the pH shift during the lyophilization process that can occur due to the undesired crystallization of a buffer component, resulting in loss of protein activities. The buffer ionization constant, pKa and crystallization of buffer components determine the pH changes in the freezing cycle. Phosphate, histidine, acetate, citrate, succinate, glycine, and tris are the common buffers. Phosphate and succinate are well-known examples of a buffer that can crystallize. On the other hand, buffers like acetate and tris buffer are generally considered non-crystallizing under a representative lyophilization cycle. Aside from the buffer type, researchers have demonstrated that the formulation and freezing condition including sample size and cooling rate can impact the buffer crystallization behavior, such as selective crystallization of disodium hydrogen phosphate. The acidity of lyophilized materials can be evaluated using the Hammett acidity function, which measures the activity of H + ions in the solid. This function utilizes an acid-sensitive dye to determine the state of acidity. Hammett acidity has been proven to accurately predict the rates of acid-catalyzed chemical reactions in the solid materials and a correlation between stability and measured acidity in the solid state was established from several studies. As a result, it has been proposed as a valuable tool for characterizing and ranking the stability of formulations containing acid-sensitive molecules in dry formats. 236 DSC, SEM, and low-temperature X-ray powder diffractometry provided evidence of the crystallization of disodium hydrogen phosphate. The pH shift as a consequence of the crystallization of phosphate buffer was detected during the freezing step. The pH values of a frozen solution can be determined using low-temperature pH electrodes operating at sub-zero temperatures, by adding a universal pH indicator solution, or by synchrotron radiation. Studies have shown that the extent of pH shift from phosphate buffer is concentration dependent, where an initial buffer at a pH 7.4 drops to pH 5.2 with a lower concentration of phosphate buffer at 8mM in contrast to pH 4 with a higher concentration of 50 and 100mM buffers. 238 However, even when protein was formulated with a very low concentration of phosphate buffer at 1mM, the crystallization of disodium hydrogen phosphate as a dodecahydrate was still detected by high sensitivity synchrotron radiation technique. Furthermore, when proteins are formulated in the lyophilized state, they exhibit something known as "pH memory", which refers to lyophilized proteins retaining the characteristics and behavior related to pH that they had prior to being freeze-dried. It is crucial to understand that the impact of pH shift will vary among different proteins and may not elicit the same effect universally. Though IgG2 and bovine IgG were not sensitive to the pH shift, the impact on βgalactosidase and lactate dehydrogenase protein can be detrimental. Furthermore, the protein itself or another excipient can prohibit the buffer-induced pH shift. Many methods have been attempted to measure the pH of protein formulation in the solid state including Hammett acidity function. An indicator dye-sorption method is widely used to measure the solid surface acidity based on the ionization state of the indicator dye molecule. 240 However, discrepancies have been observed comparing the dye-sorption method and slurry pH method depending on the compound and indicator. As such, it is critical to develop a reliable and non-invasive technique to examine the microenvironmental acidity which serves as an indicator of stability and establishes the correlation with the stability of solid biological products after water removal. Recently, an approach based on solid-state NMR was developed to noninvasively investigate the microenvironmental acidity in the lyophilized matrix and the first reported use of histidine as an indicator for measuring pH changes in the solid protein formulation by ssNMR. Histidine is commonly used in buffer and has unique acid-base properties making it a suitable choice of a pH indicator. Histidine contains four potential sites for protonation: the carboxylate end, two nitrogen atoms of the imidazole ring, and the amine terminal. The sequential deprotonation of these sites, including the carboxylic acid group, imidazolium cation, and amine nitrogen, occurs at specific pKa values of 1.9, 6.1, and 9.1, respectively and it can regulate shifts of pH as protonated τ tautomer and the Nδ1-protonated π tautomer in . This ssNMR pH measurement approach was further applied to understand the crystallization of disodium phosphate hydrogen dodecahydrate-induced phase transition and the pH of the freeze concentration using histidine as a molecular probe and based on the chemical shifts of atoms of interest. It is worth noting that the methodology using histidine as a molecular probe to identify microenvironmental pH has also been utilized in quantifying acidity in pharmaceutical frozen solutions.