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The Effect of Buffers on Protein Conformational Stability

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The Effect of Buffers on Protein Conformational Stability The Effect of Buffers on Protein Conformational Stability Sydney O. Ugwu and Shireesh P.Apte* uffers used to formulate proteins should not serve as substrates or inhibitors. They should exhibit little or no change in pH with temperature, show insignificant pen- Betration through biological membranes, and have max- imum buffer capacity at a pH where the protein exhibits opti- mal stability. In conformity with the proposition that “Nature designs the optimum molecules,”buffers should mimic the an- tidenaturant properties of nature exhibited by osmolytes (1–5) that are independent of the evolutionary history of the proteins PHOTODISC, INC. (6, 7). Such properties may include preferential exclusion from the protein domain (8–11) and stabilization without changing ⌬ The extent to which a particular protein may the denaturation Gibbs energy ( Gd) (12). be stabilized or destabilized by a buffer Conformational instability refers not only to unfolding, ag- depends on many factors, thereby making gregation, or denaturation but also to subtle changes in local- ized protein domains and the alteration of enzyme catalytic the selection of a buffer for formulating a properties (13) that may result from buffer-component bind- specific protein a formidable challenge.The ing, proton transfer, and metal or substrate binding effects di- authors describe qualitative and rectly or indirectly mediated by buffers or by buffers themselves semiqualitative correlations to help in the acting as pseudosubstrates. selection of a buffer for a particular protein Salts can affect protein conformation to the extent that the and formulation. anions or cations of the salt could be potential buffer compo- nents. 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 com- plex and not well understood. The problem is compounded by the inability to definitively differentiate between various pro- tein stabilization mechanisms, the small free energies of stabi- lization of globular proteins (14–16), and a paucity of review manuscripts on this subject in the literature. The authors ad- dress some of these issues as they relate to buffers used in the Sydney O. Ugwu, PhD, of Baxter IV formulation of proteins. The effect of buffers that may be used Systems in Murray Hill, New Jersey, is in the extraction, purification, dialysis, refolding, or assay of currently at NeoPharm Inc. (Waukegan, IL). proteins on protein conformation is not discussed. Shireesh P. Apte, PhD, is a lead scientist at Baxter IV Systems (Murray Hill, NJ) and may be contacted at Alcon Research Buffer effects on freeze drying Inc., 6201 S. Freeway, Ft. Worth, TX 76134, Change in pH as a result of buffer salt crystallization. When inor- tel. 817.551.4901, fax 817.551.8626, ganic salts are used as buffers, the freezing point of the mono- [email protected]. ionized species (salt) can be different from that of the non- *To whom all correspondence should be addressed. ionized (i.e., free acid or base) species and from its higher ionized species. This difference leads to the freezing of one form before 86 Pharmaceutical Technology MARCH 2004 www.pharmtech.com List of abbreviations ing at Ϫ7 ЊC because of the propensity for KCl, but not NaCl, to form a stable glass at this temperature (26). Also, the specific ACES:N-2-acetamido-2-aminoethane sulfonic acid surface area of freeze-dried bovine IgG from solutions con- 2,3-BPG: 2,3-bis phosphoglycerate taining NaCl was found to be significantly higher than those CHES:1-[N-cyclohecyamino]-ethane sulfonic acid containing KCl (27). Annealing also increases the surface ac- CLARP: caspase-like-apoptosis-regulatory-protein cumulation of proteins at the ice–liquid interface so that the DIPSO:3-[N,N-bis(hydroxyethyl)amino]-2-hydroxypropane sulfonic acid formation of a stable glass at the annealing temperature is es- G-CSF: granulocyte colony stimulating factor pecially important to minimize denaturation caused by such a Good’s buffers: zwitterionic buffers containing aminoalkyl sulfonate (e.g.,DIPSO, mechanism (28). MES,HEPPSO,HEPES) The rate of aggregation of recombinant human interleukin-1 HEPES:4-(2-hydroxyethyl)piperazine-N’-2-ethane sulfonic acid receptor antagonist (rhIl-1ra) was greater in mannitol–phosphate HEPPSO:[N-(2-hydroxyethyl)piperazine-N’-2-hydroxypropane sulfonic acid formulations than in glycine–phosphate formulations, possibly KCl: potassium chloride owing to the inhibition of the selective crystallization of the NaCl: sodium chloride dibasic salt by glycine during freezing, thereby preventing large NADPH: nicotinamide adenine dinucleotide phosphate localized pH changes in the frozen matrix (29). Na HPO :disodium phosphate 2 4 The effect of various buffer solutions on freezing damage to (NH ) SO :ammonium sulfate 4 2 4 rabbit-muscle-derived lactate dehydrogenase, type II (LDH, NTB: nitrothiobenzoate isoionic point [pI] ϭ 4.6) was examined with sodium phos- MES: 2-(N-morpholino)ethane sulfonic acid phate, TRIS-HCl, HEPES, and citrate buffers (50 mM, pH 7.0) MOPS: 3-(N-morpholino-2-hydroxypropane sulfonic acid and pH 7.4 (30). The activity recovery was directly proportional PIPES: [piperazine-N,N’-bis(ethane sulfonic acid)] to enzyme concentration and was the lowest in the sodium POPSO: piperazine-N,N’-bis(2-hydeoxypropane sulfonic acid) phosphate buffer (31). The activity increased in the following PBS: phosphate buffered salineTAPS: N-tris[hydroxymethyl]methyl-3- order: citrate Tris Ϸ potassium phosphate Ͻ HEPES. The aminopropane sulfonic acid low activity recovery in the sodium phosphate buffer was at- TEA: triethylamine tributed to its significant pH shift on freezing (32). The study ϩ ϩ TES: 1% sodium dodecyl sulfate 5mM EDTA 10 mM TRIS-HCL revealed no clear pattern relating recovery of activity after freez- TRIS-HCL: Tris-(hydroxymethyl)aminomethane hydrochloride ing to the freezing rate because an intermediate freezing rate gave the highest recovery of activity (31). The researchers hy- the other during the freezing phase of lyophilization (17). Such pothesized that the slowest freezing method actually resulted a phenomenon has been linked to drastic changes in pH of the in a greater degree of supercooling and better thermal equili- liquid medium during freezing, which can lead to the denatu- bration throughout the volume of liquid such that, when ice ration of the protein being lyophilized (18, 19). If an ampho- crystals nucleated, the freezing rate actually was faster than de- teric molecule were to function as a buffer containing both signed. Therefore, the study illustrates the need to control the acidic and basic groups on one molecule, one would expect neg- extent of supercooling by seeding the cooling liquid when com- ligible pH shifts to occur during the crystallization of this zwit- paring the effects of buffers or lyoprotectants on the stability terionic molecule (20). Such is indeed the case for various or- of freeze-dried proteins. ganic buffers broadly categorized as aminoalkylsulfonate In another study, the decreased solid-state stability of zwitterions (21). Good et. al. prepared and disclosed such buffers lyophillized recombinant human interferon ␥ (pI ϭ 10.3) in in their classic publication (22). sodium succinate buffer as compared with sodium glycolate Researchers have shown that replacing the Naϩ cation with buffer was attributed to a pH shift occuring in the former (19). the Kϩ cation in a phosphate buffer could significantly decrease In this case, however, the decrease in pH on freezing was at- the pH shift during the freezing stage of lyophilization (23). A tributed to crystallization of the monosodium form of succinic potassium phosphate buffer at pH 7.2 exhibited a eutectic point acid. In addition, it was not immediately clear why a similar at a temperature greater than Ϫ10 ЊC. However, the sodium crystallization effect would not be observed with the use of cation counterpart showed a eutectic point at a temperature sodium salt of the glycolic acid. In any event, the Naϩ cation below Ϫ20 ЊC. Monoclonal antibodies against HBV and L-se- can be replaced with the Kϩ cation in inorganic buffers as a first lectin, humanized IgG, as well as monomeric and tetrameric ␤- approximation to potentially increase the stability of proteins galactosidase exhibited less aggregation when subjected to during the freezing stage of lyophillization. freeze–thaw cycles with a potassium phosphate buffer than with Influence on specific surface areas of lyophillized cakes. An ag- a sodium phosphate buffer (24). Similarly, the propensity of re- gregation mechanism involving partial denaturation at the combinant hemoglobin to denature as a result of phase sepa- ice–freeze concentrate interface has also been linked to an in- ration from a polyethylene glycol–dextran matrix was reduced crease in protein degradation (27). Denaturation induced by when NaCl was replaced with KCl in the formulation buffer. In this mechanism can be reduced by incorporating surface active this case, the sodium phosphate buffer did not exhibit a pH shift agents in the formulation (33). Studies have shown the copper during freezing owing to inhibition of crystallization of dis- complexing ability of several zwitterionic N-substituted odium phosphate by the polymer (25). However, replacing NaCl aminosulfonic acid buffers to correlate with surface activity at with KCl did decrease the phase separation caused by anneal- pH 8.0 as measured by alternating current polarography (34). 88 Pharmaceutical Technology MARCH 2004 www.pharmtech.com Increasing surface activity was correlated to the number of hy- concluded that the sulfonate was more chaotropic than the droxyl groups; thus, the following order: DIPSO (three hydroxyl phosphate anion (44). groups) Ͼ HEPPSO, POPSO (two) Ͼ PIPES (no hydroxy The solution and thermal stability of the tetrameric enzyme, groups), showing no surface activity. phosphoenolpyruvate carboxylase (PEPC, pI ϭ 6.0) was de- In another study, the increase of Cuϩ2 toxicity observed in termined at pH 6.2 in MES buffer in the presence of various the marine dinoflagellate A.
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