CN105153284B - Method for purifying linaclotide - Google Patents
Method for purifying linaclotide Download PDFInfo
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- CN105153284B CN105153284B CN201510504867.5A CN201510504867A CN105153284B CN 105153284 B CN105153284 B CN 105153284B CN 201510504867 A CN201510504867 A CN 201510504867A CN 105153284 B CN105153284 B CN 105153284B
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- 108010024409 linaclotide Proteins 0.000 title claims abstract description 56
- 229960000812 linaclotide Drugs 0.000 title claims abstract description 54
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- AHDSRXYHVZECER-UHFFFAOYSA-N 2,4,6-tris[(dimethylamino)methyl]phenol Chemical compound CN(C)CC1=CC(CN(C)C)=C(O)C(CN(C)C)=C1 AHDSRXYHVZECER-UHFFFAOYSA-N 0.000 description 2
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Abstract
The invention relates to a method for purifying linaclotide, which is characterized by comprising the following steps of: (1) adjusting the pH value of the crude linaclotide solution to 3.5 +/-0.2 by using trifluoroacetic acid; (2) setting gradient according to volume fraction, washing the reversed phase packed column with 50% mobile phase B for 10min, and isocratically eluting with 5% mobile phase B for 10 min; (3) loading the solution of (1) into a reverse phase packing; (4) setting a gradient according to volume fraction, keeping the mobile phase B at 5% in an initial state of an elution gradient for 5 minutes, increasing the proportion of the mobile phase B to 16% in 1 minute, increasing the proportion of the mobile phase B to 26% in 80 minutes, and collecting elution fraction; (5) and (4) transferring salt, concentrating and freeze-drying the eluted fractions to obtain the linaclotide refined peptide.
Description
Technical Field
The invention relates to a purification method of a polypeptide drug, in particular to a purification method of linaclotide.
Background
Linaclotide (linaclotide) is a guanylate cyclase C (GC-C) agonist developed by Ironwood in the united states and is approved by FDA in the united states for marketing at 8/30/2012 under the trade name Linzess. The medicine is a capsule, is used for treating constipation irritable bowel syndrome (IBS-C) and Chronic Idiopathic Constipation (CIC), and is the first medicine with the action mechanism for treating constipation.
Linaclotide is a 14-amino acid peptide with the sequence Cys-Glu-Tyr-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH (1-6), (2-10), (5-13) -tris (disulfide) as a white to off-white amorphous powder, slightly soluble in water and aqueous sodium chloride (0.9%).
Linaclotide is a guanylate cyclase C agonist that, upon binding to intestinal GC-C, results in increased intracellular and extracellular cyclic guanylic acid (cGMP) concentrations. The increase of intracellular cGMP can stimulate intestinal juice secretion and accelerate gastrointestinal transit, so that the defecation frequency is increased, and the increase of extracellular cGMP concentration can reduce the sensitivity of pain nerve and reduce the intestinal pain.
There are also few literature reports on the purification process of linaclotide. Only patent application CN103848893A mentions that the separation and purification of linaclotide crude peptide is performed by using high performance liquid chromatography with porous graphitized carbon as stationary phase and gamma-cyclodextrin as stereoselective agent with a certain concentration added in combination with mobile phase. The gamma-cyclodextrin is brought into a purification system, so that the residual risk exists, and the process is high in cost and is not suitable for industrial production.
The existence of multiple pairs of disulfide bonds brings great difficulty to the purification of linaclotide, which is shown in the following steps: 1. during the synthesis of linaclotide, a plurality of impurities with mismatched disulfide bonds are generated, and the impurities are difficult to separate from the product, so that the purity of the final product is low; 2. due to the existence of a plurality of pairs of disulfide bonds, the difference of the linaclotide molecule spatial structure and the common polypeptide is large, and a product with better purity is difficult to obtain by using common fillers; 3. many pairs of disulfide bonds are easy to generate more polymers in the production process, the polymers are one of the most main impurities, and the polymers are extremely unstable and easy to color, seriously affect the color, content and safety of linaclotide. The inventor researches and purifies by using the prior art to find that the content of polymer impurities is difficult to control below 0.1 percent, and the yield of linaclotide is easy to reduce.
The invention provides a linaclotide purification process, which can well solve the problems, can obtain high-yield and high-purity linaclotide, can well control polymer impurities to be below 0.1 percent, and is suitable for industrial production.
Disclosure of Invention
The invention aims to solve the technical problems that the existing method for purifying the polypeptide containing a plurality of pairs of disulfide bonds, in particular linaclotide, is difficult to remove impurities, and has low total product yield and low purity.
When the polypeptide is purified by using the reverse phase high performance liquid chromatography, the specific surface area of the filler with small pore diameter is larger, and the purification effect is better. However, in practical application, the fact that the polypeptide containing a plurality of pairs of disulfide bonds has the particularity of spatial structure leads to that the effective specific surface area obtained on the small-aperture filler is smaller than that of the large-aperture filler, polymer impurities are difficult to remove, and the purification effect is not ideal.
The technical scheme of the invention is to provide a method for purifying linaclotide. The purification method uses a filler with a larger pore diameter in a certain range, and purifies linaclotide under proper preparation conditions. It is shown in particular that the filler with a 200 a-300 a pore diameter is effective, and it is further shown that the filler with a 300 a pore diameter is more effective, especially for polymeric impurities.
Some common abbreviations used in the present invention have the following meanings;
fmoc: fmoc group
Fmoc-AA: fmoc-protected amino acids
DIC: n, N' -diisopropylcarbodiimide
HOBt: 1-hydroxybenzotriazoles
tBu: tert-butyl radical
Trt: trityl radical
Cys: cysteine
Glu: glutamic acid
Tyr: tyrosine
Asn: asparagine
Pro: proline
Ala: alanine
Thr: threonine
Gly: glycine
DMF: n, N' -dimethylformamide
MeOH: methanol
DCM: methylene dichloride
TFA: trifluoroacetic acid
EDT (electro-thermal transfer coating): ethanedithiol
PhSMe: phenylmethyl sulfide
Piperidine: hexahydropyridine
DIEA: n, N' -diisopropylethylamine
Therefore, the invention provides a method for purifying linaclotide, which is characterized by comprising the following steps:
(1) regulating the pH value of the crude linaclotide solution to 3.5 +/-0.2 by using trifluoroacetic acid;
(2) setting gradient according to volume fraction, washing the reversed phase packed column with 50% mobile phase B for 10min, and isocratically eluting with 5% mobile phase B for 10 min;
(3) loading the solution (1) into a reverse phase filler;
(4) setting a gradient according to volume fraction, keeping the mobile phase B at 5% in an initial state of an elution gradient for 5 minutes, increasing the proportion of the mobile phase B to 16% in 1 minute, increasing the proportion of the mobile phase B to 26% in 80 minutes, and collecting elution fraction;
(5) transferring salt, concentrating and freeze-drying the eluted fraction to obtain linaclotide refined peptide;
wherein, the reversed phase packed column is: (10 μm, 200-300A, 50mm × 250 mm);
mobile phase A: 0.3% aqueous acetic acid;
mobile phase B: acetonitrile;
the gradient program was: the initial state of the elution gradient was 5% mobile phase B, held for 5 minutes, then the mobile phase B proportion was increased to 16% in 1 minute, then the mobile phase B proportion was increased to 26% in 80 minutes;
the flow rate is: 80 mL/min;
the detection wavelength is as follows: 220 nm.
Through a large number of tests and screens, the method provided by the invention obtains more ideal experimental conditions as follows:
1. selection of the pore size of the packing
The aperture of the filler is as follows: 120A, 200A, 300A
2. Mobile phase a type selection
0.1% aqueous TFA, 0.3% aqueous acetic acid
3. Filler type selection
Silica gel C18 reversed phase packing, polymer UniPS
10 reverse phase packing
We also performed the following 5 experimental conditions for the purification of linaclotide:
experimental conditions 1: (1) sample treatment: adjusting the pH of the crude peptide solution of linaclotide to 3.5 +/-0.5 by using TFA; (2) column punching and balancing: washing the reverse phase packed column with 50% mobile phase A for 10 min; then isocratically eluting with 5% mobile phase A for balancing for 10 min; wherein a silica gel C18 reversed phase packed column is (10 μm, 120A, 50 mm. times.250 mm); mobile phase A: 0.1% aqueous TFA; mobile phase B: acetonitrile; (3) loading: loading the sample solution into a preparative column; (4) and (3) elution: the gradient program was: the initial state of the elution gradient is that the mobile phase B is 5 percent, the mobile phase B is kept for 5min, then the proportion of the mobile phase B is increased to 16 percent within 1min, and then the proportion of the mobile phase B is increased to 26 percent within 80 min; the flow rate is: 80 mL/min; the detection wavelength is 220 nm; and (3) central control detection: using an Agilent ZORBAX Bonus RP 5um 4.6 x 250mm column; adjusting pH to 2.1 with 10mM potassium dihydrogen phosphate, and using acetonitrile as mobile phase A and B; the flow rate is 1.0 ml/min; the column temperature is 30 ℃; the detection wavelength is 210 nm; the initial state of the elution gradient is 20% of mobile phase B, the mobile phase B is increased to 40% within 40 min, then the mobile phase B is increased to 70% within 5min, the initial state is returned within 2min, and the elution gradient is kept for 13 min; (5) transferring salt, concentrating and freeze-drying to obtain the linaclotide refined peptide.
the above results show that the purification effect of the filler with the pore diameter of 200A-300A is good, and the purification effect of 300A is optimal.
Comparative experiments were performed on the same crude linaclotide peptide, and the purification process in prior art CN103848893A and the preferred method of the present invention are shown in example 4 and example 5, respectively, which have significant advantages, and the results of the comparative experiments are as follows:
the invention has the beneficial effects that: a method for purifying linaclotide with high purity, high yield and low cost is provided, in which multimeric impurities can be controlled particularly well.
Drawings
FIG. 1 shows HPLC chromatogram of linaclotide fine peptide.
FIG. 2 shows the detection spectrum of linaclotide fine peptide multimer.
FIG. 3 linaclotide mass spectrum.
Detailed Description
The invention is further illustrated by the following examples.
Specifically, with respect to each of the commercially available amino acids and amino acid fragments, and each of the commercially available resins, referred to in the following examples, the manufacturers and commercial models thereof are as follows:
the Fmoc protecting group amino acid raw material and the 2-CTC resin are conventional commercial reagents (manufacturer: Gill Biochemical (Shanghai) Co., Ltd.; chemical purity).
The organic solvent and other raw materials are all commercially available products (manufacturer: national drug group chemical reagents limited company; chemical purity).
Purifying the filler: daisogel SP-200-10-P, Daisogel SP-300-10-P, Nami UniPS, Suzhou
10。
In addition, the conditions and the equipment types and manufacturers used for the "rotary evaporation concentration" and "lyophilization" and the HPLC and mass spectrometry measurements mentioned in the following examples are as follows:
preparation HPLC: innovation general, Waters 2545;
and (3) rotary evaporation concentration equipment: rotary evaporator R-200/205 (Buchi, switzerland);
and (3) rotary evaporation concentration conditions: concentrating by rotary evaporation at 30 deg.C under vacuum (-0.1 Mpa), wherein the volume of concentrated solution is less than 75% of the total volume before rotary evaporation.
A freeze-drying device: lyophilizer FD-3 (Beijing Bo Yi kang laboratory instruments, Inc.);
freeze-drying conditions: the freeze-dried tray was placed in a freezer compartment of a refrigerator (-20 ℃) and pre-frozen for 6 h. Starting a freeze dryer, starting refrigeration, precooling for more than 30 min, and setting a freeze-drying curve as follows:
a first stage: running for 16 h at-27 ℃; and a second stage: running for 4 h at-5 ℃; a third stage: running for 2 h at 5 ℃; a fourth stage: run at 30 ℃ for 16 h.
HPLC: agilent ZORBAX Bonus RP 5um 4.6 x 250mm chromatography column; 10mM potassium dihydrogen phosphate (pH 2.1 adjusted by phosphoric acid) is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate is 1.0 ml/min; the column temperature is 30 ℃; the detection wavelength is 210 nm; the initial state of the elution gradient was 20% mobile phase B, which was raised to 40% within 40 min, then raised to 70% within 5min, and returned to the initial state within 2min for 13 min.
Gel chromatography (multimer detection): and (3) chromatographic column: TSK GEL 2000 SWXL 5 μm 7.8 × 300 mm; mobile phase: TFA acetonitrile to water = 0.05: 35: 65; column temperature: 30 ℃; flow rate: 0.8 ml/min; detection wavelength: 215 nm; gradient: isocratic elution for 30 min.
Example 1: solid phase synthesis of linaclotide
Weighing 66.67g of 2-CTC resin with the substitution degree of 1.50mmol/g, adding the weighed resin into a solid phase reaction column, adding the resin into the solid phase reaction column, washing the solid phase reaction column for 1 time by DCM, swelling the resin by DCM for 30 minutes, dissolving 229.76g of Fmoc-Tyr (tBu) -OH (500mmol) in DMF, adding 124ml of DIEA (750mmol) in ice water bath for activation, adding the activated resin into the reaction column filled with the resin, reacting for 2 hours, adding 1000ml of anhydrous methanol and sealing for 1 hour. Washing with DMF 3 times gave Fmoc-Tyr (tBu) -CTC resin. Removing Fmoc protection by using a mixed solution of DMF and pyridine with the volume ratio of 4:1, washing the mixture for 6 times by using DMF, weighing 175.72g of Fmoc-Cys (Trt) -OH (300 mmol) and 40.52g of HOBt (300 mmol), adding the mixture into a mixed solution of DCM and DMF with the volume ratio of 1:1, adding the mixture into a reaction column filled with resin after adding 46ml of DIC (300 mmol) for activation in an ice-water bath, reacting for 2 hours at room temperature, detecting by an indetrione method to judge the reaction end point, and indicating that the reaction is complete if the resin is colorless and transparent; and if the resin is developed, the reaction is not complete, the reaction needs to be carried out for 1 hour, and the judgment standard is suitable for judging the reaction end point by the detection of an indetrione method in the subsequent amino acid coupling. Repeating the above steps of removing Fmoc protection and adding the corresponding amino acid for coupling, according to the linaclotide backbone peptide sequence, sequentially performing Fmoc-Gly-OH, Fmoc-Thr (tBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Trt) -OH, coupling, washing linaclotide CTC resin with DMF 3 times, removing Fmoc protection with a mixed solution of DMF: pyridine volume ratio of 4:1, then washing with DMF 6 times, DCM washing 3 times, MeOH 3 times, DCM washing 3 times, MeOH washing 3 times, the resin was dried by suction to obtain 330.87g of linaclotide linear peptide resin.
Example 2: cleavage of linaclotide
33.09g of linaclotide linear peptide resin is weighed and added into a 1000mL three-neck round-bottom flask, and EDT, PhSMe and H are added according to the volume ratio of 2.5: 5: 87.52Preparing 330ml of lysis solution by using O and TFA, adding the lysis solution into the resin, and reacting for 2 hours at the temperature of 20-30 ℃. The reaction solution was added to glacial ethyl ether for precipitation for 1 hour, centrifuged, washed with anhydrous ethyl ether for 6 times, and dried under vacuum to obtain 14.56 g of linaclotide linear peptide.
Example 3: cyclization of linaclotide
120g (1 mol) of sodium dihydrogen phosphate and 1.91g (20 mmol) of guanidine hydrochloride were added to 10L of a solution (water: acetonitrile =85: 15), and after stirring and dissolving, 10.2g of linaclotide linear peptide was added and reacted with stirring at 25-30 ℃ for 24 hours for standby. The purity of the crude linaclotide product is 64.5 percent (detected by HPLC), the content of polymer impurities is 2.96 percent (detected by gel chromatography), and the synthesis yield is 45.2 percent.
Example 4: purification by CN103848893A
The sample solution obtained in example 3 was purified by the method of CN103848893A as follows:
(1) the pH of 10L of the crude linaclotide solution obtained in example 3 was adjusted to 3.5. + -. 0.2 using trifluoroacetic acid, and 3.8L of the crude linaclotide solution was filtered, and the filtrate was collected for use.
(2) And (3) purification process: setting gradient according to volume fraction, wherein the initial state of elution gradient is that the mobile phase B is 10%, balancing the chromatographic column for 5 minutes, then loading, increasing the proportion of the mobile phase B to 40% within 35min, monitoring and collecting target peak eluent in sections. Performing purity detection on the collected eluent, and removing most acetonitrile from the eluent with the purity of more than or equal to 99.0% for salt conversion; removing most of acetonitrile from the eluent with the purity of more than or equal to 80% and less than 99.0%, recovering and repeating the purification process, collecting the eluent with the purity of more than or equal to 99.0% again, removing most of acetonitrile, and then using the eluent for salt transfer; the eluent with the purity of less than 80 percent is treated according to waste liquid. Injecting samples for two times, and repeating the operations.
Purification chromatographic conditions: the type of the high performance liquid chromatograph: waters 2545; a chromatographic column: 50 x 250mm, porous graphitized carbon as fixed phase stuffing of 7 micron size; flow rate: 80 mL/min; detection wavelength: 280 nm.
Mobile phase a phase: 0.2% (g/mL, W/V) of gamma-cyclodextrin in 30mM sodium dihydrogen phosphate in water was adjusted to pH 2.0 with phosphoric acid. The preparation process comprises the following steps: weighing 20g of gamma-cyclodextrin and 36g of sodium dihydrogen phosphate, dissolving with appropriate amount of purified water, filtering with 0.45 μm filter membrane, collecting the filtrate, adding purified water to 10L scale, and adjusting pH to 2.0 with phosphoric acid.
Mobile phase B phase: chromatographically pure acetonitrile.
Sample loading amount: 3.8L.
(3) A salt conversion process: setting gradient according to volume fraction, keeping the initial state of elution gradient at 5% of mobile phase B for 5min, balancing the chromatographic column for 5min, then loading, isocratically eluting with 5% of mobile phase B for 20min, then increasing the proportion of mobile phase B to 30% within 2min, isocratically eluting with 30% of mobile phase B for 10min, monitoring and collecting target peak eluent.
And (3) transferring salt chromatographic conditions: the high performance liquid chromatograph model: waters 2545; a chromatographic column: 50X 250mm, filled with reversed phase C18 chromatographic packing, the grain size of the packing being 10 μm; flow rate: 80 mL/min; detection wavelength: 280 nm.
Mobile phase a phase: 0.10% glacial acetic acid (v/v) solution.
Mobile phase B phase: chromatographically pure acetonitrile.
Sample loading volume: 200 mL.
(4) Concentrating the target peak eluent under reduced pressure by rotary evaporation to 20mL, and freeze-drying. And freeze-drying to obtain 1.63g of white powdery solid refined peptide. The purity of the purified linaclotide refined peptide is 99.45 percent (detected by HPLC), the content of polymer impurities is 0.34 percent (detected by gel chromatography), the purification yield is 80.7 percent (calculated by the linaclotide content in the crude product), and the total yield is 36.5 percent.
Example 5: purification of linaclotide
(1) The pH of 10L of the crude linaclotide solution obtained in example 3 was adjusted to 3.5. + -. 0.2 using trifluoroacetic acid, and 3.8L of the crude linaclotide solution was filtered, and the filtrate was collected for use.
(2) Setting a gradient according to volume fraction, and flushing the reversed-phase packed column for 10min by using 50% of mobile phase B; then isocratically eluting with 5% mobile phase B for 10 min; wherein, UniPS
The 10 reversed phase packing column is: 10 μm, 300A, 50mm × 250 mm; mobile phase A: 0.3% aqueous acetic acid; mobile phase B: acetonitrile; the flow rate is: 80 mL/min; the detection wavelength is as follows: 220 nm.
(3) Loading the filtered filtrate obtained in the step (1) into a preparation column completely;
(4) and (3) elution: the gradient program was: setting gradient according to volume fraction, keeping the initial state of elution gradient at 5% of mobile phase B for 5min, increasing the proportion of mobile phase B to 16% within 1min, and increasing the proportion of mobile phase B to 26% within 80 min; the flow rate is: 80 mL/min; the detection wavelength is 220 nm; collecting the elution fraction with the target peak purity of more than 99.0%, and performing center control detection: using an Agilent ZORBAX Bonus RP 5um 4.6 x 250mm column; adjusting pH to 2.1 with 10mM potassium dihydrogen phosphate, and using acetonitrile as mobile phase A and B; the flow rate is 1.0 ml/min; the column temperature is 30 ℃; the detection wavelength is 210 nm; the initial state of the elution gradient is 20% of mobile phase B, the mobile phase B is increased to 40% within 40 min, then the mobile phase B is increased to 70% within 5min, the initial state is returned within 2min, and the elution gradient is kept for 13 min; performing purity detection on the collected eluent, and removing most acetonitrile from the eluent with the purity of more than or equal to 99.0% for salt conversion; removing most of acetonitrile from the eluent with the purity of more than or equal to 80% and less than 99.0%, recovering and repeating the purification process, collecting the eluent with the purity of more than or equal to 99.0% again, removing most of acetonitrile, and then using the eluent for salt transfer; the eluent with the purity of less than 80 percent is treated according to waste liquid. Injecting samples for two times, and repeating the operations.
(5) Salt conversion: and (3) transferring salt chromatographic conditions: preparation HPLC: innovation is constant, 50 mm; a chromatographic column: 50X 250mm, filled with reversed phase C18 chromatographic packing, the grain size of the packing being 10 μm; flow rate: 80 mL/min; detection wavelength: 220 nm.
Mobile phase a phase: 0.10% glacial acetic acid (v/v) solution.
Mobile phase B phase: chromatographically pure acetonitrile.
Sample loading volume: 200 mL.
A salt conversion process: setting gradient according to volume fraction, keeping the initial state of elution gradient at 5% of mobile phase B for 5min, balancing the chromatographic column for 5min, then loading, isocratically eluting with 5% of mobile phase B for 20min, then increasing the proportion of mobile phase B to 30% within 2min, isocratically eluting with 30% of mobile phase B for 10min, monitoring and collecting target peak eluent.
(6) Concentration and freeze-drying: concentrating the target peak eluent under reduced pressure by rotary evaporation to 20mL, and freeze-drying. After freeze-drying, 1.80 g of white powdery solid refined peptide is obtained, the purity of the purified linaclotide refined peptide is 99.74% (HPLC detection, figure 1), the content of polymer impurities (the polymer impurities are 9.907min and 10.500min in retention time) is 0.03% (gel chromatography detection, figure 2), the purification yield is 88.9% (calculated by the linaclotide content in the crude product), and the total yield is 40.2%. The mass spectrum is shown in FIG. 3, [ M + Na ]]+: 1548.318, the theoretical exact molecular weight of linaclotide: 1526.39, the mass spectrometry results of the samples correspond to the theoretical molecular weight.
The foregoing is a more detailed description of the present invention in connection with specific modified embodiments thereof, and it is not intended that the present invention be limited to the specific embodiments thereof. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (1)
1. A method for purifying linaclotide, which is characterized by comprising the following steps: the method comprises the following steps:
(1) regulating the pH value of the crude linaclotide solution to 3.5 +/-0.2 by using trifluoroacetic acid;
(2) setting gradient according to volume fraction, washing the reversed phase packed column with 50% mobile phase B for 10min, and isocratically eluting with 5% mobile phase B for 10 min;
(3) loading the solution in the step (1) into an exchange column;
(4) setting a gradient according to volume fraction, keeping the mobile phase B at 5% in an initial state of an elution gradient for 5 minutes, increasing the proportion of the mobile phase B to 16% in 1 minute, increasing the proportion of the mobile phase B to 26% in 80 minutes, and collecting elution fraction;
(5) transferring salt, concentrating and freeze-drying the eluted fraction to obtain pure linaclotide;
wherein,
the reversed phase packed column is: multimer
10 reversed-phase packed columns, 10 μm,
50mm×250mm；
mobile phase A: 0.3% aqueous acetic acid;
mobile phase B: acetonitrile;
the gradient procedure was to elute the initial state of the gradient as 5% mobile phase B, hold for 5 minutes, then increase the mobile phase B proportion to 16% in 1 minute, then increase the mobile phase B proportion to 26% in 80 minutes;
the flow rate is: 80 mL/min;
the detection wavelength is as follows: 220 nm.
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