The effect of buffers on protein conformational stability

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The effect of buffers on protein conformational stability
March 2004
Sydney O. Ugwu, Shireesh P. Apte
Pharmaceutical Technology
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Buffers used to formulate proteins should not serve as substrates or inhibitors. They should exhibit little or no change in pH with temperature, show insignificant penetration through biological membranes, and have maximum buffer capacity at a pH where the protein exhibits optimal stability. In conformity with the proposition that "Nature designs the optimum molecules," buffers should mimic the antidenaturant properties of nature exhibited by osmolytes (1-5) that are independent of the evolutionary history of the proteins (6, 7). Such properties may include preferential exclusion from the protein domain (8-11) and stabilization without changing the denaturation Gibbs energy ([DELTA][G.sub.d]) (12).
Conformational instability refers not only to unfolding, aggregation, or denaturation but also to subtle changes in localized protein domains and the alteration of enzyme catalytic properties (13) that may result from buffer-component binding, proton transfer, and metal or substrate binding effects directly or indirectly mediated by buffers or by buffers themselves acting as pseudosubstrates.
Salts can affect protein conformation to the extent that the anions or cations of the salt could be potential buffer components. When the salt concentration is much larger than that of the buffer, the salt becomes the effective buffer in the reaction.
The mechanisms or combinations thereof by which buffers may cause protein stabilization (or destabilization) are complex and not well understood. The problem is compounded by the inability to definitively differentiate between various protein stabilization mechanisms, the small free energies of stabilization of globular proteins (14-16), and a paucity of review manuscripts on this subject in the literature. The authors address some of these issues as they relate to buffers used in the formulation of proteins. The effect of buffers that may be used in the extraction, purification, dialysis, refolding, or assay of proteins on protein conformation is not discussed.
Buffer effects on freeze drying
Change in pH as a result of buffer salt crystallization. When inorganic salts are used as buffers, the freezing point of the mono-ionized species (salt) can be different from that of the non-ionized (i.e., free acid or base) species and from its higher ionized species. This difference leads to the freezing of one form before the other during the freezing phase of lyophilization (17). Such a phenomenon has been linked to drastic changes in pH of the liquid medium during freezing, which can lead to the denaturation of the protein being lyophilized (18, 19). If an amphoteric molecule were to function as a buffer containing both acidic and basic groups on one molecule, one would expect negligible pH shifts to occur during the crystallization of this zwitterionic molecule (20). Such is indeed the case for various organic buffers broadly categorized as aminoalkylsulfonate zwitterions (21). Good et. al. prepared and disclosed such buffers in their classic publication (22).
Researchers have shown that replacing the [Na.sup.+] cation with the [K.sup.+] cation in a phosphate buffer could significantly decrease the pH shift during the freezing stage of lyophilization (23). A potassium phosphate buffer at pH 7.2 exhibited a eutectic point at a temperature greater than -10[degrees]C. However, the sodium cation counterpart showed a eutectic point at a temperature below -20[degrees]C. Monoclonal antibodies against HBV and L-selectin, humanized IgG, as well as monomeric and tetrameric [beta]-galactosidase exhibited less aggregation when subjected to freeze-thaw cycles with a potassium phosphate buffer than with a sodium phosphate buffer (24). Similarly, the propensity of recombinant hemoglobin to denature as a result of phase separation from a polyethylene glycol-dextran matrix was reduced when NaCl was replaced with KCl in the formulation buffer. In this case, the sodium phosphate buffer did not exhibit a pH shift during freezing owing to inhibition of crystallization of disodium phosphate by the polymer (25). However, replacing NaCl with KCl did decrease the phase separation caused by annealing at -7[degrees]C because of the propensity for KCl, but not NaCl, to form a stable glass at this temperature (26). Also, the specific surface area of freeze-dried bovine IgG from solutions containing NaCl was found to be significantly higher than those containing KCl (27). Annealing also increases the surface accumulation of proteins at the ice-liquid interface so that the formation of a stable glass at the annealing temperature is especially important to minimize denaturation caused by such a mechanism (28).
The rate of aggregation of recombinant human interleukin-1 receptor antagonist (rhIl-1ra) was greater in mannitol-phosphate formulations than in glycine-phosphate formulations, possibly owing to the inhibition of the selective crystallization of the dibasic salt by glycine during freezing, thereby preventing large localized pH changes in the frozen matrix (29).
The effect of various buffer solutions on freezing damage to rabbit-muscle-derived lactate dehydrogenase, type II (LDH, isoionic point [pI] = 4.6) was examined with sodium phosphate, TRIS-HCl, HEPES, and citrate buffers (50 mM, pH 7.0) and pH 7.4 (30). The activity recovery was directly proportional to enzyme concentration and was the lowest in the sodium phosphate buffer (31). The activity increased in the following order: citrate < Tris [approximately equal to] potassium phosphate < HEPES. The low activity recovery in the sodium phosphate buffer was attributed to its significant pH shift on freezing (32). The study revealed no clear pattern relating recovery of activity after freezing to the freezing rate because an intermediate freezing rate gave the highest recovery of activity (31). The researchers hypothesized that the slowest freezing method actually resulted in a greater degree of supercooling and better thermal equilibration throughout the volume of liquid such that, when ice crystals nucleated, the freezing rate actually was faster than designed. Therefore, the study illustrates the need to control the extent of supercooling by seeding the cooling liquid when comparing the effects of buffers or lyoprotectants on the stability of freeze-dried proteins.