Freezing of biologic drug substance at large scale is an important unit operation that enables manufacturing flexibility and increased use-period for the material. Stability of the biologic in frozen solutions is associated with a number of issues including potentially destabilizing pH changes. The pH changes arise from temperature-associated change in the pK(a)s, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes for most of the common protein formulation buffers in the frozen state have not been systematically measured. Sodium phosphate buffer, a well-studied system, shows the greatest change in pH when going from +25 to -30 degrees C. Among the other buffers, histidine hydrochloride, sodium acetate, histidine acetate, citrate, and succinate, less than I pH unit change (increase) was observed over the temperature range from +25 to -30 degrees C, whereas Tris-hydrochloride had an similar to 1.2 pH unit increase. In general, a steady increase in pH was observed for all these buffers once cooled below 0 degrees C. A formulated IgG2 monoclonal antibody in histidine buffer with added trehalose showed the same pH behavior as the buffer itself This antibody in various formulations was subject to freeze/thaw cycling representing a wide process (phase transition) time range, reflective of practical situations. Measurement of soluble aggregates after repeated freeze-thaw cycles shows that the change in pH was not a factor for aggregate formation in this case, which instead is governed by the presence or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant, longer phase transition times lead to higher aggregation. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 727-733, 2010