Published on Web Date: November 30, 2009 r2009 American Chemical Society 265 DOI: 10.1021/jz900164q | J. Phys. Chem. Lett. 2010, 1, 265–268 pubs.acs.org/JPCL “pH Swing” in Frozen Solutions;Consequence of Sequential Crystallization of Buffer Components Prakash Sundaramurthi, † Evgenyi Shalaev, ‡ and Raj Suryanarayanan* ,† † Department of Pharmaceutics, University of Minnesota, Minneapolis, Minnesota 55455, and ‡ Pfizer Inc., Groton, Connecticut 06349 ABSTRACT Succinate buffer solutions of different initial pH values and concen- trations were cooled. The solution pH and the phases crystallizing from solution were monitored as a function of temperature. In a solution buffered to pH 4.0 (200 mM), the freeze-concentrate pH initially increased to 8.0 and then decreased to 2.2. On the basis of X-ray diffractometry (synchrotron source) , the “pH swing” was attributed to the sequential crystallization of succinic acid, monosodium succinate, and disodium succinate. A similar swing, but in the opposite direction, was seen when a solution with an initial pH of 6.0 was cooled. In this case, crystallization of the basic buffer component occurred first. The direction and magnitude of the pH shift depended on both the initial pH and the buffer concentration. In light of the pH-sensitive nature of a significant fraction of pharmaceuticals (especially proteins), extreme care is needed, both in the buffer selection and in its concentration. SECTION Biophysical Chemistry T he freezing behavior of aqueous solutions is a subject of both fundamental and technological importance and attracts attention from numerous disciplines including physical chemistry, biotechnology, cryobiology, pharmaceutical, and food science. 1-3 Freezing is a critical step, both in the cryostabilization of biologicals and in the lyophilization (freeze-drying) of pharmaceuticals and food products. 4,5 However, it is widely recognized that freezing can induce solute destabilization. 1,6-9 Characterization of the freeze-concentrated liquid provides an avenue for under- standing the destabilization mechanisms. In protein solutions, changes in pH, ionic strength, and solute concentration brought about by ice crystallization are known to cause destabilization. When a buffered solution is cooled, ice crystallization can be followed by selective crystallization of a buffer component, resulting in a significant pH shift (up to ∼3 pH units) in the freeze-concentrate. 10 Such pH shifts are known to accelerate drug degradation in frozen solutions. 11 van den Berg had investigated the pH shift under equilibrium conditions by seeding the frozen systems. 12 However, during freeze-drying of pharmaceuticals, supersaturated solutions are often formed, and the systems are far from equilibrium. 13 As a result, there is potential for crystallization of more than one buffering species with the potential to cause a “pH swing” . Here, we report such a phenomenon of an increase in pH followed by a decrease, or vice versa, in frozen succinate buffer systems due to sequential crystallization of buffer components. This observation was based on direct pH mea- surement coupled with X-ray diffractometry of the frozen systems. A low-temperature pH electrode, with a working tempera- ture range of 80 to -30 °C, was placed in the center of a beaker. The buffer solutions, containing succinic acid and sodium succinate, were equilibrated at 0 °C and then cooled to -25 at 0.5 °C/min. Both the temperature and solution pH were monitored continuously. The phases crystallizing from the frozen solution were monitored by X-ray diffractometry (XRD), using both laboratory (Cu KR radiation) and synchro- tron (Advanced Photon Source, Argonne National Laboratory) sources. Succinic acid [(CH 2 COOH) 2 ] is a dicarboxylic acid with pK a values of 4.21 and 5.64 at room temperature (RT) . Figure 1a shows the solution temperature and pH when a succinate buffer solution (200 mM; buffered to pH 4 at RT) was cooled. At ∼-8 °C, ice crystallization was evident from the abrupt increase in sample temperature. After a substantial fraction of the water had crystallized as ice, the sample temperature decreased again. The pH of the freeze-concentrate increased, slightly at first, and in a more pronounced manner as the sample temperature reached ∼-23 °C. By this time, the cooling was complete, and annealing had been initiated. While the sample and bath temperature remained approxi- mately constant, the pH first exhibited an abrupt increase and then a sharp decrease. The small initial increase in pH, from 4.0 to ∼4.3, may be attributed to the temperature effect on pK a . When the temperature was decreased from 50 to 0 °C, the pK a1 of succinic acid increased from 4.19 to 4.29, while Received Date: October 20, 2009 Accepted Date: November 16, 2009