Surfactant Poloxamer 188 as a New Crystallizing Agent for Urate

Aug 3, 2009 - cedex 9, France, ‡Institut Pasteur, D´epartement de Biologie Structurale et ... Sanofi-Aventis, 371 Rue du Professeur Blayac, 34184 M...
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DOI: 10.1021/cg900473n

Surfactant Poloxamer 188 as a New Crystallizing Agent for Urate Oxidase

2009, Vol. 9 4199–4206

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Marion Giffard,†,^ Vanessa Delfosse,‡,# Giuliano Sciara,§ Claudine Mayer,‡ Christian Cambillau,§ Mohamed El Hajji, Bertrand Castro, and Franc- oise Bonnete*,† †

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CINaM CNRS UPR 3118, Aix Marseille Universit e, 163 Avenue de Luminy, case 913, 13288 Marseille epartement de Biologie Structurale et Chimie, Universit e Paris cedex 9, France, ‡Institut Pasteur, D ecules Diderot, 25 rue du Dr Roux, 75 015 Paris, France, §Architecture et Fonction des Macromol Biologiques, UMR 6098 CNRS Aix Marseille Universit e, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 9, France, and Sanofi-Aventis, 371 Rue du Professeur Blayac, 34184 Montpellier, France. ^ Present address: Novartis Pharma AG, WSJ-340.8.11, Novartis Campus, CH-4056 Basel, e de Montr eal, D epartement de Biochimie, CP6128 Succursale Switzerland. # Present address: Universit Centre-ville, Montr eal QC H3C 3J7, Canada. Received April 29, 2009; Revised Manuscript Received July 21, 2009

ABSTRACT: We characterize a new class of crystallizing agent for soluble protein crystallization compatible with both pharmaceutical processes and high-resolution structure determination in biocrystallography. Poloxamers are amphiphilic nonionic multiblock polymers used in the cosmetic and pharmaceutical industries. Poloxamer P188 (EO75PO31EO75) is generally used as an emulsifier, solubilizer, and dispersing and wetting agent in the preparation of solid dispersions. Here, we divert the surfactant properties of poloxamer P188 at low concentrations into crystallizing properties at high concentrations of poloxamer P188 to crystallize urate oxidase and to control its crystal design.

*To whom correspondence should be addressed. E-mail: bonnete@cinam. univ-mrs.fr.

crystals. Furthermore, biological crystals are generally much smaller, more fragile, and diffract at lower resolutions. Several reasons can be advanced to explain all of these phenomena.8 The first is the large amount of solvent found within a crystal of biological macromolecules (20-80%). The second is the size and complexity of macromolecules, which let them establish different types of possible crystal contacts and populate a variety of conformational states in solution. Finally, the macromolecule solution may contain degradated forms of the protein, which may also adsorb onto the crystal surface. In this case, the nucleation rate and the crystal growth greatly reduce crystal quality. To induce a liquid-solid phase transition, it is necessary to change solvent-macromolecule interactions by solvent-solvent and macromolecule-macromolecule interactions. This can be achieved by introducing chemical agents in the solution or by modifying solution properties via temperature or pressure changes. Inorganic ionic salts have been the most common and effective precipitants used for protein crystallization.9 Because of the strong electric field around these ions, a large number of water molecules are loosely bound around the ions in a sphere of hydration. This, in turn, reduces the amount of water that is free to keep the protein in solution. Thus, the supersaturation level can be effectively increased. Polymers are also used as crystallizing agents.10 To date, polyethylene glycol (PEG) is the most popular polymer used in protein crystallization, because of both its precipitating ability and its cost effectiveness. Like salts, PEG competes with proteins for water and exerts an excluded volume effect. In the case of proteins used in the pharmaceutical industry, the choice of crystallizing additives depends on their compatibility and toxicity. In view of such applications and to avoid any toxicity issue related to the chosen crystallizing agent, preparative purification should ideally be based on a chemical agent already used in or compatible with the production process.

r 2009 American Chemical Society

Published on Web 08/03/2009

1. Introduction Protein crystallization is a liquid-solid interfacial process mainly performed nowadays to produce crystals for structure determination by X-ray crystallography. Originally, crystallization was used as an ideal purification method, not only for low molecular weight substances but also for biological macromolecules. Only a few proteins have been purified by crystallization, including hen egg white lysozyme,1 ovalbumin,2 jack bean urease,3,4 and glucose isomerase.5 Chromatography has finally replaced protein crystallization in the protein purification process,6 because crystallization for protein purification requires a good knowledge of the phase diagram and a certain amount of the protein to be crystallized. However, as compared with other protein purification/separation techniques, crystallization has the inherent advantage of higher final purity yields, not denaturing the protein of interest, and often providing some stabilization effects. As such, crystallization can be used as a large-scale, high-yield purification process of industrially important proteins. Properly executed, crystallization is a powerful and more economical protein purification method since high-purity proteins can be obtained in a single-step operation. The mechanisms involved in the crystallization of proteins, that is, nucleation, crystal growth, and cessation of growth, are essentially the same as those for small molecule crystallization such as inorganic salts and organic molecules;7 what can differ are the solvents used. In each case, to precipitate or crystallize a solid phase, the solution has to be supersaturated, that is to say that the concentration of molecules has to be higher than their solubility, which is the concentration in equilibrium with the solid state. However, proteins are known to be more difficult to crystallize. Nucleation usually occurs at supersaturation significantly higher than those for inorganic

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Table 1. Data Processing and Refinement Statistics uox/polox X-ray source wavelength (A˚) temperature (K) space group unit cell parameters (A˚, ) resolution range (A˚)a no. of unique reflections multiplicitya completeness (%)a Rsyma,b ÆI/σ(I)æ a Rwork/Rfreec rms deviation with model 1R51 (A˚)

ESRF FIP-BM30A 0.9797 100 I222 a = 80.06, b = 95.28, c = 104.53, R = β = γ = 90 1.60-29.52 (1.60-1.70) 51746 (8127) 6.9 (4.8) 97.7 (93.1) 0.058 (0.351) 23.6 (4.3) 17.9/19.9 0.178

a Values in parentheses correspond to the highest-resolution shell. Rsym = ΣhΣi|Ih,i - ÆIhæ|/ΣhΣi ÆIhæ, where Ih,i is the ith observation of reflection h and ÆIhæ is the weighted average intensity for all observations i of reflection h. c Rwork = Σ|Fo - Fc|/Σ|Fo|, where Fo and Fc are observed and calculated amplitudes, respectively. Rfree is calculated similarly using a test set of reflections. The test set (5% of reflections) is omitted in the refinement. b

The urate oxidase (uricase, EC 1.7.3.3, uox) gene, inactive in humans and higher primates, encodes for an enzyme that catalyzes the oxidation of uric acid to allantoin, an inactive and soluble metabolite. Uox is used as a protein drug to reduce toxic uric acid accumulation and to treat the hyperuricemic disorders occurring during chemotherapy. It is produced, purified, and made commercially available by Sanofi-Aventis.11,12 Initially, uox was extracted from Aspergillus flavus, but now, the drug is produced by genetic engineering, and the recombinant uox is called rasburicase. Uox crystallization and phase diagrams have been thoroughly studied with PEG.13-16 Among PEGs, PEG 8000 is the most widely used crystallizing agent to produce uox crystals for X-ray crystallography.17-19 Indeed, crystallization of uox by salt addition only is difficult to achieve due to a strong salting-in effect,20 which favors solubility and not crystallization. In some cases, salts and polymers are not efficient for crystallization or cannot be used in pharmaceutical processes. Organic compounds such as ethanol,21 isopropanol, methanol, and 2-methyl 2,4-pentanediol22 are other crystallizing agents, which are less used than other agents. Another polymer, the amphyphatic poloxamer P188, is already used as a surfactant in the resuspension buffer for the solubilization of lyophilized uox prior to administration. To date, its use as a crystallizing agent has never been described. Here, we report the first results on the possibility of using poloxamer P188 as a crystallizing agent. We demonstrate that poloxamer P188 at high concentrations induces the crystallization of uox by a depletion effect, similarly to PEG 8000. Moreover, the crystallographic structure of uox is conserved, without specific interactions of poloxamer with the enzyme in the crystal. 2. Materials and Methods 2.1. Solutions. Purified recombinant uox from A. flavus (also named rasburicase), heterologously expressed in Saccharomyces cerevisiae, was supplied by Sanofi-Aventis in a phosphate buffer. As in previous studies, the protein buffer was replaced with 50 mM Tris buffer, pH 7.5, using gel filtration chromatography on a Superdex S200PG column with an AKTAbasic system, and the protein was concentrated by ultrafiltration on an Amicon cell (Millipore). The resulting uox stock solution was kept at 3 mg mL-1 in a cold room and further concentrated when needed.

Figure 1. Chemical structures of poloxamer P188 (A) and PEG (B). Scheme of micellization of the poloxamer at a high concentration (C). A 1 M concentration of potassium chloride and 0.015 mg mL-1 uric acid stock solutions were prepared by dilution of the appropriate amount of the two salts (purchased from Sigma-Aldrich) in 50 mM Tris buffer, pH 7.5, and 50 mM Tris buffer, pH 8.5, respectively. Forty percent w/v of PEG 8000 and 25% w/v of poloxamer P188 solutions in 50 mM Tris buffer, pH 7.5, were, respectively, prepared from a 50% w/v solution (Hampton Research) and from powder (supplied by BASF). All salt and uox solutions for crystallization trials were filtered on 0.22 μm Millipore filters. 2.2. Phase Diagram Determination. Crystallization trials prior to crystal growth studies were performed in an air-conditioned room (20 C) using the microbatch technique.23 Droplets were prepared by mixing the concentrated purified protein solution with the precipitant agent (40% PEG 8000 or 25% poloxamer P188) and salt (1 M KCl). The droplets were pipetted under a layer of paraffin oil in a 72 well microbatch plate (paraffin oil and plates from Hampton Research). The final volumes were 10 μL. To favor the crystal growth process, droplets were seeded with a crystallized solution at higher supersaturation under identical precipitant conditions. Crystals were observed with an inverted optical microscope (TE 200 Nikon), recorded to a digital video camera. Photographs were taken with an 200 enlargement using the Replay software (Microvision). Pictures were taken 1 day after droplet preparation and seeding. Crystal growth studies were performed in a temperature-controlled Peltier device using small glass cells ( CMCpolox, increase in concentration of poloxamer induces a depletion attraction increasing the accessible volume to micelles. scattering vector q, q = 4πλ-1 sin θ, by: Iðc, qÞ ¼ Ið0, qÞ 3 Sðc, qÞ I(0, q), the intensity scattered by one particle and usually called the particle form factor, is the Fourier transform of the spherically averaged autocorrelation function of the electron density contrast associated with the particle. The form factor is generally obtained from curves recorded at low concentrations to avoid interaction effects. Assuming interacting spherical particles, departure from ideality may be simply accounted for by a multiplying factor or interference term, S(c, q), usually called the solution structure factor. The nature of the net interactions, either attractive or repulsive, can be simply determined by the intercept at the origin, S(c, 0), of the plot of the structure factor as a function of the particle concentration, since it is related to the osmotic pressure Π by:   RT DΠ -1 Sðc, 0Þ ¼ M Dc

Poloxamer P188 is a nonionic copolymer surfactant with a triblock structure, comprised of two hydrophilic segments, the poly(oxyethylene) (PEO), and a central hydrophobic segment, the poly(oxypropylene) (PPO), taken together by ether bonds. The resulting construct can be represented as HO(C2H4O)a(C3H6O)b(C2H4O)aH, where a is about 75 and b is about 3133 (Figure 1). Its average molecular weight is around 8400 g mol-1, and its cmc (critical micellar concentration) is about 0.1% w/v.34 Poloxamer is similar to the usual crystallizing agent for uox, PEG 8000 [H(OCH2CH2)nOH], which is a linear hydrophilic polymer consisting of approximately n = 180 PEO units and has a molecular weight of about 8000 g mol-1. Because of their amphiphilic structure, poloxamers have surfactant properties that make them useful in pharmaceutical applications. They can be used to increase the water solubility of hydrophobic, oily substances as well as to increase the miscibility of two substances with different hydrophobicities. For this reason, these polymers are used in cosmetics and pharmaceuticals. They have also been used as model systems for drug delivery35 applications. Their tendency to aggregate into micelles makes them appealing candidates for the encapsulation and delivery of hydrophobic drugs. Recently, they have also been shown to function as artificial chaperones to facilitate refolding of denatured proteins in solution or to suppress aggregation.36-39 In general, all of these applications

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Figure 7. Size exclusion chromatography of a solution of pure uox, a solution of poloxamer P188, uox crystallized with poloxamer P188 and then dissolved, and uox crystal supernatant. Uox and poloxamer P188 are detected by both refractive index (left scale) and static light scattering (right scale). Table 2. Uox Solubility with Various Polymers (in 30 mM KCl and 50 mM Tris, pH 7.5, at 20 C) polymer nature and concentration (%) PEG 8000 (4%) poloxamer P188 (4%) poloxamer P407 (4%) polysorbate 20 (4%) polysorbate 80 (16.5%)

uox solubility (mg mL-1) 3 6 10 20 8

require low concentrations of poloxamer, typically below its cmc and implying monomeric poloxamer in solution. Below the cmc, the hydrophobic segment of polymer can nonspecifically interact with exposed hydrophobic domains, preventing aggregation and aiding in the refolding of proteins.37 However, high concentrations of poloxamer have also been reported to induce protein aggregation.40 Indeed, the addition of 12-16% of the block copolymer to plasma or serum induces the precipitation of high molecular weight proteins including antibodies. This suggests that it would be possible to use high concentrations of poloxamer, thus probably in its micelle form, to induce protein crystallization by depletion, as it was observed with PEG. In such a context, we have studied crystallization and interactions of uox by the addition of poloxamer P188. Uox interactions in solution have already been studied as a function of salt and PEG.13,20,41 Salt has been shown to increase repulsive interactions and solubility by a specific salting-in interaction, while PEG induces attractive interactions by a depletion effect and decrease in solubility, favoring crystallization. The control of these two opposite effects allows us to design uox crystals.20 The solubility of uox has thus been measured in 30 mM KCl and 50 mM Tris, pH 7.5, at 20 C, where the solubility is high, as a function of concentration of poloxamer P188. The solubility decreases as the concentration of poloxamer increases and is higher with poloxamer P188 than with PEG 8000 for a concentration lower than 6% (Figure 2).

Figure 8. Chemical structures of polysorbate 20 (A), 40 (B), and 80 (C) (from top to level); x þ y þ z þ w = 20.

Crystals of uncomplexed recombinant uox, obtained at 8 mg mL-1 with 10% poloxamer P188 in Tris buffer, pH 7, and 20 mM KCl, remain stable for several weeks (Figure 3A) without the appearance of other polymorphs. They have been tested for single crystal diffraction but were found to badly diffract, and the data collection was not complete. However, the space group P21 (a = 82.73 A˚, b = 141.76 A˚, and c = 135.03 A˚) has been obtained by powder diffraction (Margiolaki, personal communication) and is the same as for crystals obtained with PEG 8000.17

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Figure 9. Crystals of uox in 30 mM KCl and 50 mM Tris, pH 7.5, at 20 C: (A) 12.5 mg mL-1 uox with 7.5% poloxamer P407, (B) 10.6 mg mL-1 uox with 10% polysorbate 20, and (C) 8.2 mg mL-1 uox with 12.5% polysorbate 80.

While low concentrations of polymer (0), the solution is stable, and when interactions are attractive (A2 cmc), uox from dissolved crystals, supernatant, and the masses calculated for the eluted species. Because of sample dilution on the SEC column, no poloxamer P188 micelles are detected. Calculated masses of species in solution are in agreement with a tetrameric form for uox (135000 g mol-1) and a monomeric form of poloxamer P188 (8400 g mol-1), respectively, without specific interaction between uox and poloxamer. Moreover, this experiment shows that significant amounts of poloxamer P188 are detected in the supernatant but not in the dissolved crystal sample. The shift of poloxamer P188 retention time in the supernatant sample also points out attractive interactions between this polymer and uox.

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As a final validation of the use of such a new polymer in view of a pharmaceutical process, we verified the uox 3D structure by X-ray crystallography. Because crystals of uncomplexed uox (i.e., without substrate or inhibitor) diffracted poorly, uox has been cocrystallized in poloxamer P188 with a purine type inhibitor, 8-azaxanthine (Figure 3B and Table 1). No structural changes have been observed as compared to the uox structure from crystals obtained in PEG 8000,17 with a rmsd of 0.178 A˚ between the 296 CR coordinates of the two models, proving that poloxamer P188 does not affect uox structure (Table 1). In this study, we show that poloxamer P188, routinely used as a surfactant in the pharmaceutical industry, can also be used as a crystallizing agent for uox. This study was preliminary extended to other amphiphilic surfactants such as poloxamer P407, polysorbate 20, and polysorbate 80. Poloxamer P407 has a similar structure and molecular formula as compared to poloxamer P188, HO(C2H4O)a(C3H6O)b(C2H4O)aH, where a is about 100 and b is about 60. The molecular weight of the hydrophobic core is 4000 g mol-1, which represents 30% of the total mass of the polymer. The P407 average molecular weight is about 13300 g mol-1, and its cmc varies with temperature from 0.008 to 1.75%.44,45 As shown in Table 2, uox solubility in poloxamer P407 is higher than in either poloxamer P188 or PEG 8000, while keeping fixed the remaining solution components (i.e., 50 mM Tris, pH 7.5, and 30 mM KCl). Unlike with PEG, uox solubility increases with the mass of the poloxamer used. This result points toward a solubilizing effect of poloxamer, proportional to the percentage of its hydrophobic core content and compensating its precipitating action by depletion. The effect of polysorbates, another class of emulsifiers used in pharmaceutics and food preparation, has also been tested. These compounds are used in cosmetics to solubilize essential oils into water-based products. Polysorbates are oily liquids derived from PEGylated sorbitan (a sorbitol derivative) esterified with fatty acids. They all share a hydrophilic moiety characterized by 20 oxyethylene--(CH2CH2O)- groups, whereas the hydrophobic segments change depending on the polysorbate compound (Figure 8). Uox crystallization has not been tested with polysorbate 40 because of its extremely low solubility in buffer, which did not allow us to prepare stock solutions. However, polysorbates 20 and 80 efficiently induced crystallization of uox (Figure 9), which has been confirmed by the decrease in uox concentration in the supernatant. Uox solubility is higher with polysorbates 20 and 80 than with PEG 8000 and poloxamers P188 and P407 (Table 2). As for poloxamers, it seems that the longer the polysorbate hydrophobic chain, the higher the solubility of uox. Nevertheless, the modification in the habit of the uox crystals with 16% polysorbate 80 suggests that this concentration of surfactant could interact with the crystal growth and possibly with the protein structure. 4. Conclusion We show that poloxamer P188, which is commonly used as a surfactant to stabilize protein solutions, can be used as a crystallizing agent for uox. The crystals produced using poloxamer P188 are of comparable quality as those grown in PEG 8000. The presence of the central hydrophobic polypropylene glycol moiety in poloxamer P188 does not affect the crystal structure of protein nor uox structure in solution and activity. On the other side, it seems responsible for its solubilizing and

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stabilizing effect on uox solutions at low concentrations of poloxamer. The poloxamer concentration has to be greater than ca. 0.25% to switch from its surfacting to its crystallizing agent role. This same versatility is shared with other polymeric surfactants routinely used in the pharmaceutical industry, such as poloxamer P407, polysorbate 20, and polysorbate 80. 5. Data Deposition Atomic coordinates and structure factors of the uox crystallized with poloxamer P188 have been deposited in the Protein Data Bank with accession code 3GKO. Acknowledgment. We gratefully acknowledge SanofiAventis and CNRS for financial support of this work and F. Ragot for providing us with rasburicase. We thank Dr. M. Pirrochi from the FIP-BM30A beamline at the ESRF for assistance with data collection and V. Heresanu, F. Quintric, and N. Ferte from CINaM-CNRS for technical assistance. We thank Paul Wynblatt, Professor of Materials Science at Carnegie Mellon University, Pittsburgh, for English revision.

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