Aqueous Two-phase Extraction System Research Articles

Two-step process for initial capture of plasmid DNA and partial removal of RNA using aqueous two-phase systems

In this paper, a two-step process for initial capture of plasmid DNA (pDNA) and partial removal of RNA using polyethylene glycol (PEG) and di-potassium hydrogen phosphate aqueous two-phase systems (ATPS) has been investigated. A Kühni-type ATPS extraction column was prepared with 50 ml (12% (w/w) PEG 1450, 12% (w/w) phosphate) of stationary phase and loaded with crude mobile phase (26% (w/w) PEG 1450, 4% (w/w) phosphate and 70% (w/w) lysate) at a flow rate of 6 ml min −1 at an impeller speed of 200 rpm. The experiment was terminated after 100 min, and after complete resettling of the phases, 45 ml of stationary phase was harvested. During a subsequent second extraction step contained 18% (w/w) PEG 300 and 14% (w/w) phosphate, a proportion of RNA, which was also concentrated during the column process, was removed. It was demonstrated that the recovery of pDNA in the second bottom phase was 89.4%, which was similar to the initial recovery after column extraction (92.1%).

Effect of Electrolyte and Temperature on Volatile Organic Compounds Removal from Wastewater Using Aqueous Surfactant Two-Phase System of Cationic and Anionic Surfactant Mixtures

Benzene, toluene, ethylbenzene, and xylene are frequently observed contaminants in industrial wastewaters causing concerns about environmental and health effects. An aqueous surfactant two-phase (ASTP) extraction system using mixtures of cationic and anionic surfactants have been shown to be a promising surfactant-based separation technique to concentrate solutes such as proteins and dyes from aqueous solution. A phase separation of a surfactant solution occurs at certain surfactant compositions and concentrations, forming two isotropic phases. One is rich in surfactant aggregates (surfactant-rich phase) and the other is lean in surfactant aggregates (surfactant-dilute phase). Most of the organic contaminants tend to solubilize and concentrate in the surfactant-rich phase, leaving the surfactant-dilute phase containing only small amounts of contaminants as remediated water. The effect of NaCl addition on the critical micelle concentration (CMC) and the extraction ability of ASTP formed by mixtures of cationic surfactant (dodecyltrimethylammonium bromide; DTAB) and anionic surfactant (alkyl diphenyloxide disulfonate; DPDS) at 50 mM total surfactant concentration with a 2:1 molar ratio of DTAB:DPDS was investigated; the CMC of the mixture slightly decreases with increasing NaCl concentration. The extraction and preconcentration of benzene are greatly enhanced by added NaCl. The higher the degree of hydrophobicity of contaminants, the greater the extraction into the surfactant-rich phases. At 1.0 M NaCl addition, about 95% of xylene, 92% of ethylbenzene, 90% of toluene, and 79% of benzene are extracted into the surfactant-rich phase within a single stage extraction and the contaminant partition ratios can be as high as 395 for xylene, 273 for ethylbenzene, 206 for toluene, and 84 for benzene, which are greater than those obtained from the conventional ASTP extraction system using nonionic surfactants.

Selective separation and enrichment of proteins in aqueous two-phase extraction system

A simple aqueous two-phase extraction system (ATPS) of PEG/phosphate was proposed for selective separation and enrichment of proteins. The combination of ATPE with HPLC was applied to identify the partition of proteins in two phases. Five proteins (bovine serum albumin, Cytochrome C, lysozyme, myoglobin, and trypsin) were used as model proteins to study the effect of phosphate concentration and pH on proteins partition. The PEG/phosphate system was firstly applied to real human saliva and plasma samples, some proteins showed obviously different partition in two phases. The primary results manifest the selective separation and enrichment of proteins in ATPS provided the potential for high abundance proteins depletion in proteomics.

Downstream processing of antibodies: Single-stage versus multi-stage aqueous two-phase extraction

Single-stage and multi-stage strategies have been evaluated and compared for the purification of human antibodies using liquid–liquid extraction in aqueous two-phase systems (ATPSs) composed of polyethylene glycol 3350 (PEG 3350), dextran, and triethylene glycol diglutaric acid (TEG-COOH). The performance of single-stage extraction systems was firstly investigated by studying the effect of pH, TEG-COOH concentration and volume ratio on the partitioning of the different components of a Chinese hamster ovary (CHO) cells supernatant. It was observed that lower pH values and high TEG-COOH concentrations favoured the selective extraction of human immunoglobulin G (IgG) to the PEG-rich phase. Higher recovery yields, purities and percentage of contaminants removal were always achieved in the presence of the ligand, TEG-COOH. The extraction of IgG could be enhanced using higher volume ratios, however with a significant decrease in both purity and percentage of contaminants removal. The best single-stage extraction conditions were achieved for an ATPS containing 1.3% (w/w) TEG-COOH with a volume ratio of 2.2, which allowed the recovery of 96% of IgG in the PEG-rich phase with a final IgG concentration of 0.21 mg/mL, a protein purity of 87% and a total purity of 43%. In order to enhance simultaneously both recovery yield and purity, a four stage cross-current operation was simulated and the corresponding liquid–liquid equilibrium (LLE) data determined. A predicted optimised scheme of a counter-current multi-stage aqueous two-phase extraction was hence described. IgG can be purified in the PEG-rich top phase with a final recovery yield of 95%, a final concentration of 1.04 mg/mL and a protein purity of 93%, if a PEG/dextran ATPS containing 1.3% (w/w) TEG-COOH, 5 stages and volume ratio of 0.4 are used. Moreover, according to the LLE data of all CHO cells supernatant components, it was possible to observe that most of the cells supernatant contaminants can be removed during this extraction step leading to a final total purity of about 85%.

Hydrophobic ionic liquids-assisted polymer recovery during penicillin extraction in aqueous two-phase system

In this study, terminal modified poly(ethylene glycerol) (PEG) was employed as the phase-forming polymer to construct aqueous two-phase (ATP). After the phase equilibrium, 95.8% penicillin could be extracted into imidazole-terminal PEG-rich phase efficiently. Imidazole-terminal PEG (I-PEG) was separated from the aqueous phase containing penicillin to hydrophobic ionic liquid phase at basic pH, and a weak acidic aqueous phase was then employed to recover the I-PEG from the hydrophobic ionic liquid phase into water at pH 5.5–6. The recycle of the polymer was achieved with the aid of hydrophobic ionic liquids.

Ionic liquid-based aqueous two-phase extraction of selected proteins

As an effective extraction method, aqueous two-phase extraction systems based on imidazolium ionic liquids have been used, in this work, to extract proteins of bovine serum albumin, trypsin, cytochrome c and γ-globulins. Effects of the ionic liquids concentration, pH value, and temperature of the systems on the extraction efficiencies have been investigated. Experimental results show that 75–100% of the proteins could be extracted into the ionic liquid-rich phase in a single-step extraction. The extraction efficiency of cytochrome c was changed slightly with the increase of pH values. Extraction efficiencies of the proteins were found to increase with increasing temperature and increasing alkyl chain length of cation of the ionic liquids. Thermodynamic studies indicated that hydrophobic interactions were the main driving force, although electrostatic interactions and salting-out effects were also important for the transfer of the proteins. Importantly, conformation of the proteins was not affected after extraction into the upper ionic liquid-rich phase as determined by UV–visible (UV–vis) and Fourier transform infrared (FT-IR) spectroscopy of the proteins. The enzyme activity of native trypsin and the trypsin in ionic liquids was determined using N-α-benzoyl- l-arginine ethyl ester as a substrate. This novel process is suggested to have important applications for the separation of proteins.

Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction chromatography and size-exclusion chromatography

We have evaluated a process incorporating aqueous two-phase extraction, hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for the purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cell supernatant. These unit operations were chosen not only for allowing the removal of target impurities but also for facilitating the integration of different process units without the need for any conditioning step. Extraction in aqueous two-phase systems (ATPSs), composed of polyethylene glycol (PEG) and sodium citrate, allowed the concentration of the antibodies in the citrate-rich phase and the removal of the most hydrophobic compounds in the PEG-rich phase. An ATPS composed of 10% (w/w) PEG 3350 and 12% (w/w) citrate, at pH 6, allowed the recovery of IgG with a 97% yield, 41% HPLC purity and 72% protein purity. This bottom phase was then directly loaded on a phenyl-Sepharose HIC column. This intermediate purification step allowed the capture of the antibodies using a citrate mobile phase with 99% of the antibody recovered in the elution fractions, with 86% HPLC purity and 91% protein purity. Finally, SEC allowed the final polishing by removing IgG aggregates. HIC-eluted fractions were directly injected in a Superose 6 size-exclusion column affording a 100% pure IgG solution with 90% yield.

Hypoglycaemic activity of saponin fraction extracted from Momordica charantia in PEG/salt aqueous two-phase systems

Momordica charantia (family, Cucurbitaceae), commonly known as karela or bitter melon (Japanese name ‘Tsurureishi’), is used as a folk medicine in China, the Indian subcontinent and South America. In Chinese traditional medicine, the plant is usually used as a hypoglycaemic and anti-diabetic agent. The hypoglycaemic activity of saponin fraction (SF) extracted from M. charantia in PEG/salt aqueous two-phase systems was studied in this article. Alloxan-induced hyperglycaemic mice were used in the study. The blood glucose, insulin secretion, glycogen synthesis and the body weight of the mice were analysed. At the same time, the sugar tolerance of the normal mice was also determined. After the mice were administered (i.g.) with SF (500 mg kg−1), the blood glucose of alloxan-induced hyperglycaemic mice decreased (p < 0.05), the level of insulin secretion and glycogen synthesis of alloxan-induced hyperglycaemic mice elevated (p < 0.05, p < 0.01) and the sugar tolerance of the normal mice was improved. Also, the body weight of the alloxan-induced hyperglycaemic mice was increased gradually. The saponin constituents extracted from M. charantia in an aqueous two-phase extraction system induced significant hypoglycaemic activity in hyperglycaemic and normal mice.

Improving the extraction of intracellular L‐asparaginase by high‐pressure homogenization

AbstractThe effect of high‐pressure homogenization (HPH) on the primary extraction of intracellular L‐asparaginase from Escherichia coli by aqueous two‐phase extraction (ATPE) has been investigated. When HPH was operated under a pressure of 120 MPa for four passages, 17.8% of L‐asparaginase activity was lost. The addition of EOPO (a triblock copolymer of a central hydrophobic poly(propylene oxide) (PPO) block with hydrophilic poly(ethylene oxide) (PEO) blocks on each side) could improve the endurance of L‐asparaginase to high pressure; while the existence of KHP (a mixture of KH2PO4 and K2HPO4) could strongly facilitate such deactivation. When HPH was operated in the ATPE system formed from EOPO and KHP, the recovery of L‐asparaginase was greatly enhanced, as compared to the conventional HPH process. Copyright © 2008 Curtin University of Technology and John Wiley &amp; Sons, Ltd.

Membrane-supported extraction of biomolecules with aqueous two-phase systems

Membrane-supported liquid–liquid extraction is known as an interesting alternative to conventional extraction. With this technology, both liquid phases do not have to be dispersed, formation of emulsions hardly takes place and phase separation is not necessary. In biotechnology, extraction by means of aqueous two-phase systems (ATPS) is known as a very promising tool for the capturing of proteins and peptides. As both involved phases are water based, there is no undesired interaction between bio molecule and liquid phase. There also is only little difference in phase density. Therefore, the phase separation after extraction normally requires centrifugation. By applying an ATPS into a membrane contactor, extraction of biomolecules can be applied without phase separation. It could be shown that BSA and lysozyme could be extracted by means of ATPS with a membrane contactor without mixing and phase separation. However, extraction only took place if traces of a surfactant (Tween-20) were added to the ATPS, since then the used hydrophobic membrane (Liqui-Cel X40) was wetted from at least one of the two aqueous phases. Under these conditions, extraction could be repeated several times without any technical problems. With a dedicated experimental set-up, even kinetic data of the over all extraction could be generated. The combination of ATPS and membrane extraction enhances the advantages of both single technologies and makes this process an interesting alternative to down-stream purification steps such as chromatography.

SEPARATION OF URANIUM(VI) FROM SOILS BY EXTRACTION IN AQUEOUS PEG (1550) - Na2SO4 - Na2CO3 TWO-PHASE SYSTEMS. I. PRELIMINARY RESULTS

In this paper preliminary results of studies concerning the extraction of uranium(VI) from soils in aqueous PEG (1550) 20 % Na2SO4 20 % - Na2CO3 10 -2 -10 -3 M two-phase systems are presented, as well as the spectrophotometric determination of extracted uranium with pirocatechol violet (PV). A special attention was given to the experimental factors (pH, redox potential, CO3 2- anion concentration and initial uranium concentration), which was found to influence both the formation and stability of aqueous two-phase extraction system, and the values of extraction parameters. The experimental values of distribution coefficients, extraction degree and enrichment coefficients for U(VI) extraction indicate an acceptable separation at pH=4-5, and at pH=8-9, respectively. Under certain work conditions, the increasing of U(VI) concentration in initial solution and of CO3 2anion concentration determined an increase of extraction degree and of enrichment coefficient.

In situ extraction of intracellular l-asparaginase using thermoseparating aqueous two-phase systems

The feasibility and generic applicability of directly integrating conventional discrete operations of cell disruption by high pressure homogenizer and the product capture by aqueous two-phase extraction (ATPE) system have been demonstrated for the extraction of intracellular l-asparaginase from E. coli. In a side-by-side comparison with the conventional ATPE process, including cell disruption, centrifugal clarification and following ATPE, purification of l-asparaginase via this novel in situ ATPE process yielded a product of l-asparaginase with a higher specific activity of 94.8 U/(mg protein) and a higher yield of 73.3%, both of which in the conventional ATPE process were 78.6 U/(mg protein) and 52.1%, respectively. In the purification of l-asparaginase (p I = 4.9), product–debris interactions commonly diminish its recovery. It was demonstrated that immediate extraction of l-asparaginase in ATPE systems when it is released at pH 5.0 during cell disruption effectively increased its recovery in the top phase due to the reduced interaction between l-asparaginase and cell debris and the reduced degradation by contaminated protease. In addition, no clarification step and/or disruptate storage are required in this in situ ATPE, which reduced the number of unit operations and thus shortened the overall process time. This novel process has a good potential for the separation of other intracellular biological products.

Improved recovery of B‐phycoerythrin produced by the red microalga Porphyridium cruentum

AbstractA simplified process for the primary recovery and purification of B‐phycoerythrin (BPE) from Porphyridium cruentum exploiting aqueous two‐phase systems (ATPS) and isoelectric precipitation was developed in order to reduce the number of unit operations and benefit from increased purity and yield of the protein product. Evaluation of the partitioning behaviour of BPE in polyethylene glycol (PEG)/sulphate, PEG/dextran and PEG/phosphate ATPS was carried out to determine under what conditions the BPE and contaminants concentrated into opposite phases. An additional stage of isoelectric precipitation at pH 4.0 after cell disruption resulted in an increase in purity of the target protein from the BPE crude extract and enhanced the performance of the subsequent ATPS. PEG1000/phosphate ATPS proved to be suitable after isoelectric precipitation for the recovery of highly purified (defined as absorbance ratio A545 nm/A280 nm &gt; 4.0) BPE with a potential commercial value as high as US$ 50/mg. An ATPS extraction stage comprising 29.5% (w/w) PEG1000, 9.0% (w/w) phosphate, a volume ratio (Vr) equal to 1.0, a system pH of 7.0 and loaded with 40% (w/w) of the BPE extract generated by precipitation allowed BPE recovery with a purity of 4.1±0.2 and an overall product yield of 72% (w/w). The purity of BPE from the crude extract increased 5.9‐fold after isoelectric precipitation and ATPS. The results reported herein demonstrate the benefits of the practical application of isoelectric precipitation together with ATPS for the recovery and purification of BPE produced by P. cruentum as a first step in the development of a commercial purification process. Copyright © 2006 Society of Chemical Industry

Purification of papain from Carica papaya latex: Aqueous two-phase extraction versus two-step salt precipitation

Purification of papain from wet Carica papaya latex by extraction in aqueous two-phase system was studied and compared with the traditional procedure involving a two-step salt precipitation. The papain obtained by the latter method was usually contaminated with other proteins, and its purity was dependent on the initial protein concentration in the material used for processing. Highly pure papain was obtained in a much shorter processing time directly from unclarified latex with the use of an aqueous two-phase system consisting of 8% (w/w) polyethylene glycol and 15% (w/w) ammonium sulfate.

Aqueous two-phase systems extraction of α-toxin from Clostridium perfringens type A

Two sequential half-fraction designs were applied to studying the alpha-toxin partition produced by Clostridium perfringens type A in aqueous two phase systems (ATPS), as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows, in a single step, an alpha-toxin purification of 4.6-fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase.

Partition of alkaline protease in aqueous two-phase systems of polyethylene glycol 1000 and potassium phosphate

This article presents a study of polyethylene glycol 1000 (PEG1000)/potassium phosphate aqueous two-phase systems (ATPSs) forBacillus subtilis NS99 alkaline protease extraction. The objectives were to evaluate effects of system pH (7.5, 8.5,9.5, and 10.5), and NaCl concentration (0,4,7, and 10% (w/w)) on ATPS binodal curves, effects of system pH, NaCl concentration, and tie-line length (TLL) on alkaline protease partition coefficient (K) and yield (Y%) at room temperature (30±2 ‡C). Casein hydrolysis was used for determination of alkaline protease activity. It was revealed that system pH had the slightest effect on locations of binodal curves (except at pH 10.5). In contrast, addition of NaCl appeared to have a significant effect on phase characteristics since binodal curves of systems with NaCl (4-10% (w/w)) shifted significantly towards the origin in comparison to the ones without NaCl. Increased NaCl concentration from 4 to 10% (w/w), however, showed trivial influence on locations of the binodal curves. Changes of system compositions due to variation in system pH, TLL, and NaCl concentrations obviously resulted in varied obtainable K and Y% of alkaline proteases. Longer TLL and higher pH generally resulted in higher K. In contrast, the lower NaCl concentration, the higher K. Since the same phase volume ration (1:1) was used throughout the experiments, Y% depended solely on K. The most suitable PEG1000/potassium phosphate ATPS was determined at pH 9.5, and comprised PEG1000, potassium phosphate, and NaCl 18.0,13.0, and 0% (w/w), respectively. This system resulted in considerably high K, and Y% of 20.0, and 95.1%, respectively. Information from this study will be important for further development of an ATPS extraction unit for alkaline protease recovery.

Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography

The current study explores the possibility of using a polyethyleneglycol(PEG)–ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400 and 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20% (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16%, w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35%, w/w PEG 600, 6%, w/w NH 4) 2SO 4), it was possible to recover 100% of pDNA in the bottom phase with a three-fold increase in concentration. Further increase in the lysate load up to 40% (w/w) with this system resulted in a eight-fold increase in pDNA concentration, but with a yield loss of 15%. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with iso-propanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.

Potential Aqueous Two-Phase Processes for the Primary Recovery of Colored Protein from Microbial Origin

The primary recovery of c-phycocyanin and b-phycoerythrin from Spirulina maxima and Porphyridium cruentum, respectively, using an established extraction strategy was selected as a practical model system to study the generic application of polyethylene glycol (PEG)-phosphate aqueous two-phase systems (ATPS). The generic practical implementation of ATPS extraction was evaluated for the recovery of colored proteins from microbial origin. A comparison of the influence of system parameters, such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio, on the partition behavior of c-phycocyanin and b-phycoerythrin was carried out to determine under which conditions target colored protein and contaminants concentrate to opposite phases. One-stage processes are proposed for the primary recovery of the colored proteins. PEG1450-phosphate ATPS extraction (volume ratio (VR) equal to 0.3, tie-line length (TLL) of 34 % w/w and system pH 7.0) for the recovery of c-phycocyanin from Spirulina maxima resulted in a primary recovery process that produced a protein purity of 2.1 ± 0.2 (defined as the relationship of 620 nm to 280 nm absorbance) and a product yield of 98 % [w/w]. PEG1000-phosphate ATPS extraction (i.e., VR = 1.0, PEG 1000, TLL 50 % w/w and system pH 7.0) was preferred for the recovery of b-phycoerythrin from Porphyridium cruentum, which resulted in a protein purity of 2.8 ± 0.2 (defined as the relationship of 545 nm to 280 nm absorbance) and a product yield of 82 % [w/w]. The purity of c-phycocyanin and b-phycoerythrin from the crude extract increased 3- and 4-fold, respectively, after ATPS. The results reported herein demonstrated the benefits of the practical generic application of ATPS for the primary recovery of colored proteins from microbial origin as a first step for the development of purification processes.

Evaluation of different primary recovery methods forE. coli-derived recombinant human growth hormone and compatibility with further down-stream purification

Due to advances in fermentation technology, it is now possible to obtain fermentation broth with over 30% solids. The high solid content makes the clarification step difficult, especially at large scale. The primary protein recovery step is challenging due to the heterogeneous solution of soluble and insoluble material. In this study, we compare different primary recovery routes and the compatibility with the initial capture chromatography step. The primary recovery routes studied are standard clarification by centrifugation and extraction in aqueous two-phase systems. The compatibility of the feed streams from the different primary recovery steps with the first chromatography step is addressed. An anion-exchange column was used as the first capture column in the purification process. The aqueous two-phase system was composed of a random copolymer of ethylene oxide and propylene oxide (EOPO) in combination with a waxy starch. The target protein in this study was human growth hormone (hGH) produced in recombinant Escherichia coli. The purity of hGH in the top phase after aqueous two-phase extraction was found to be significantly higher than in clarified homogenate supernatant and increased as the EOPO polymer concentration in the aqueous two-phase system increased. Stability of the supernatant and EOPO top phases and hGH were determined by turbidity measurements and LC-MS assay. All of the feed-streams from the primary recovery steps were compatible with the anion-exchange chromatography step; however, the capacity of the resin was strongly dependent on the purity of the load. Different process aspects, e.g., resin capacity, viscosity, purification, and yield of hGH and scalability are compared.

Recovery in aqueous two-phase systems of lutein produced by the green microalga Chlorella protothecoides

In this study the potential use of aqueous two-phase system (ATPS) to establish a viable process for the recovery of lutein from the green microalga Chlorella protothecoides is evaluated. The partitioning behaviour of lutein, a representative model of natural compounds of commercial interest, was investigated in a polyethylene glycol (PEG)-phosphate system. An evaluation of system parameters including PEG molecular mass, the concentrations of PEG, phosphate and product concentration was conducted, to estimate conditions under which lutein partitions preferentially to the top phase whilst cell debris partition to the opposite phase. The necessary addition of ethanol to the ATPS for the dissolution of lutein affected the phase formation and such effect was evaluated using the change in the volume ratio produced. ATPS extraction comprising Vr=1.0, PEG 8000 22.9% (w/w) and phosphate 10.3% (w/w), pH 7.0 provided the conditions for the concentration of lutein into the upper phase and the cell debris preferentially to the bottom phase. The use of ATPS resulted in a primary recovery process to obtain lutein with an overall product yield of 81.0±2.8%. The findings reported here demonstrate the potential of ATPS for the further development of a prototype process to recover lutein from C. protothecoides as a first step for the generic application of this technique.