Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregation

Abstract

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 1 pH unit change (increase) was observed over the temperature range from +25 to -30 degrees C, whereas Tris-hydrochloride had an approximately 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.

Advance Understanding of Buffer Behavior during Lyophilization

“…Alternative buffers to carboxylic acids which are known to have minor to moderate pH changes (around one pH unit change) during freezing include histidine and Tris [25]. The temperature-pH profile of 20 mM histidine has been studied and shown to have less than one pH unit increase (5.4 to 6.1) upon freezing to − 30 °C [25].…”

“…The temperature-pH profile of 20 mM histidine has been studied and shown to have less than one pH unit increase (5.4 to 6.1) upon freezing to − 30 °C [25]. The crystallization behavior of histidine during freeze/thaw has been shown to be pH dependent [35].…”

Stability of monoclonal antibodies (mAbs)

“…Several buffer systems such as Tris-HCl and sodium phosphate have large dpKa/ dT, which can result in change in pH during cooling and freezing. Recent studies have suggested that pH changes due to buffer during cooling and freezing can be ruled out as a factor in aggregation behavior (Kolhe, Amend, & Singh, 2010). However, buffer systems where the pH changes due to crystallization of a buffer salt upon freezing can impact protein conformation as shown by studies on the freeze-drying of γ interferon (Lam, Costantino, Overcashier, Nguyen, & Hsu, 1996).…”

Non-Arrhenius Protein Aggregation

“…In a relatively narrow pH range, IgG1 aggregates were shown to grow primarily by monomer addition and remained small and soluble at pH 3.5 but grew by both chain polymerization and condensation and become large and insoluble at pH 4.5 and higher pH (36). Changing temperature also changes the pH of certain formulation buffers due to temperature-dependent changes in the degree of ionization (74). Chemical degradation could lead to formation of basic or acidic species, altering the solution pH, such as deamidation of asparagines (75).…”