CN101745316A - Novel ultrafiltration method and ultrafiltration device for using gravitational field to assist to concentrate large biological molecules - Google Patents
Novel ultrafiltration method and ultrafiltration device for using gravitational field to assist to concentrate large biological molecules Download PDFInfo
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- CN101745316A CN101745316A CN201010100497A CN201010100497A CN101745316A CN 101745316 A CN101745316 A CN 101745316A CN 201010100497 A CN201010100497 A CN 201010100497A CN 201010100497 A CN201010100497 A CN 201010100497A CN 101745316 A CN101745316 A CN 101745316A
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Abstract
The invention relates to an ultrafiltration method and an ultrafiltration device for using gravitational field to assist to concentrate large biological molecules for realizing the aim of carrying out ultrafiltration concentration on the large biological molecules simply, rapidly, continuously and effectively. An ultrafiltration membrane assembly is arranged in a mode that the direction of a penetration liquid of the membrane is in parallel with the horizontal plane. The ultrafiltration concentration of solution of the large biological molecules is carried out in an end-filtration mode. A high concentration material liquid in a concentration polarization layer formed on the surface of the membrane is stripped and falls down continuously from the surface of the membrane under the action of gravity. The falling high concentration material liquid is directly extracted and collected so as to obtain concentrated solution of the target product. As the falling high concentration liquid, i.e. potential pollutants of the surface of the membrane, is discharged in time, the ultrafiltration concentration process of the invention can be stably operated for a long time in the end-filtration mode. Therefore, the method not only has the characteristics of low shearing, low energy consumption and self-cleaning, but also has the outstanding advantages of simple equipment, convenient operation, strong controllability, easy scale amplification and the like.
Description
Invention field
The ultrafiltration that the present invention relates to large biological molecule concentrates, particularly a kind of auxiliary macromolecular hyperfiltration process of concentrated biological of gravitational field and device thereof of utilizing.
Background technology
Ultrafiltration is a kind of in the membrane separation technique, and as the outstanding representative of green isolation technics, its main separating ranges is the material of molecular weight 1000～500000, has been widely used in fields such as food, biology, medicine, chemical industry, environmental protection.But do not have advantages such as phase transformation, the amplification of operating condition gentleness scale because of having process, ultrafiltration especially is fit to concentrating or desalination of bioactive macromolecule such as protein, enzyme, polypeptide etc.Yet in the big molecular process of ultrafiltration concentrated biological, concentration polarization and film pollute and cause the decline of permeation flux or the rising of transmembrane pressure, have reduced the thickening efficiency and the economy of this process.Usually select for use methods such as cross-flow filtration or dynamic membrane filtering to slow down the adverse effect of concentration polarization as much as possible in the prior art, but no matter take which kind of method to go to slow down concentration polarization, still belong to the power consumption operation.Therefore even to this day, concentration polarization still is considered as unfavorable factor by most people and is treated.
National inventing patent CN101269298 discloses a kind of macromolecular membrane filtering method of concentration polarization concentrated biological that utilizes first, form fast, layer interior high characteristic of solute concentration of speed according to the concentration polarization layer, proposition is provided with hollow type concentrate drawing device in the position near the film surface, by the concentrated liquid specially drawing device high concentration feed liquid in the film surface concentration polarization layer is drawn out, when obtaining the high concentration product solution, reduced face concentration polarization degree.Yet although this invention effect is remarkable, the concrete key of implementing depends on the making and the cost thereof of system core parts concentrate drawing device, has limited the substance of this method and has used; If there is not core component concentrate drawing device, this working of an invention then can't be guaranteed, and this has also influenced further applying of this invention simultaneously.
The present invention proposes a kind of novel ultrafiltration method for concentration and device that the concentrated liquid specially drawing device just can be derived highly concentrated solution in the face concentration polarization layer that need not, and promptly utilizes auxiliary macromolecular hyperfiltration process of concentrated biological of gravitational field and device.Up to now, still not having correlative study and patent both at home and abroad announces.
Summary of the invention
The object of the present invention is to provide macromolecular method of a kind of ultrafiltration concentrated biological and device, specifically provide a kind of macromolecular method of concentration polarization principle ultrafiltration concentrated biological and device of utilizing, more particularly provide a kind of auxiliary macromolecular hyperfiltration process of concentrated biological of gravitational field and device of utilizing, pollute to realize effective controlling diaphragm of macromolecular while of efficient concentrated biological.
Proposition of the present invention is based on following principle: utilize that concentration polarization phenomenon intrinsic in the ultra-filtration process and concentration polarization layer form that speed is fast, the high characteristics of solute concentration in the layer, according to existing big concentration difference promptly to have certain density gradient between the middle mutually solution of face concentration polarization intrastratal solution and main body, in the concentration polarization layer high concentrations of solutes molecule under certain condition can be under the gravity effect from the film sur-face peeling and fall, under terminal ultrafiltration pattern, thereby the high concentration feed liquid that will fall is directly collected acquisition high concentration target product solution from membrane module bottom.
The ultrafiltration that the method that utilization the present invention proposes is carried out large biological molecule concentrates, the concentrated liquid specially drawing device need not be set just highly concentrated solution in the concentration polarization layer can be derived collection, hardware requirement is low, applied widely, overcome traditional big molecular method of concentration polarization principle concentrated biological that utilizes and to have selected for use suitable concentrated liquid specially drawing device could realize the technology barriers that concentrate is derived, thereby can realize continuous, the macromolecular purpose of high-performance ultrafiltration concentrated biological easily; Simultaneously, the high concentrations of solutes molecule that cause is fallen is that the potential pollutant of face is in time derived membrane module, the ultrafiltration concentration process can long-term stability move under the end-filtration pattern, thereby reduced of the height dependence of conventional cross-flow ultrafiltration concentration process, realized that easily feed liquid concentrates and film pollutes the dual purpose of control the feed liquid circular flow.
Therefore, though the method that the present invention proposes is to be based upon to utilize on the macromolecular basis of concentration polarization principle ultrafiltration concentrated biological, but utilize the macromolecular method of concentration polarization principle ultrafiltration concentrated biological different with traditional to a great extent, to how deriving in the concentration polarization layer this key core technology of highly concentrated solution carried out the essence quality and change more precisely, concrete method is as follows:
The mode that is parallel to horizontal plane according to film penetrating fluid direction is placed hyperfiltration membrane assembly, the ultrafiltration that the control system operational factor is carried out biological macromolecule solns under the end-filtration pattern concentrates, be formed at high concentration feed liquid in the concentration polarization layer on film surface under the gravity effect constantly from the film sur-face peeling and fall, collect the concentrate that obtains target product thereby the high concentration feed liquid that will fall directly extracts.
Wherein, the transmembrane pressure of ultra-filtration process is controlled at-100～300KPa between, the film permeation flux is controlled at 5～30L/m
2Between the h, the large biological molecule concentration of raw material extracts flow-control 2.5 * 10 between 0.1～1.5g/L
-3～5.0L/h, concentrate concentration is between 10～180g/L, and the time of extraction for the first time is between the initial 0.5～3.5h afterwards of ultrafiltration.
Described hyperfiltration membrane assembly pattern is doughnut formula, tubular type, plain film formula or rolling, and the film effective length is in 3000mm; Filter membrane is selected organic milipore filter of macromolecule or inorganic milipore filter for use, and selected milipore filter to the rejection of large biological molecule between 60.0-99.9%.
The organic milipore filter of described macromolecule comprises: cellulose family, polyether sulfone, polysulfones, Kynoar class, polyvinyl alcohol, polyvinyl acetaldehyde, polyvinyl chloride milipore filter or the blend film that may exist between them.
Described inorganic milipore filter comprises: ceramic membrane or metal film.
Described ultrafiltration concentration process adopts the end-filtration pattern, and wherein, the film penetrating fluid is capable of circulation back into material liquid tank, also can directly efflux collection.In order to increase the rate of recovery of large biological molecule in concentration process, adopt penetrating fluid to efflux earlier usually, treat that feeding liquid almost completely enters concentration systems after, switch to the penetrating fluid circulation pattern again.In addition,, also can adopt the penetrating fluid circulation pattern to concentrate, to improve the cycles of concentration and the rate of recovery of target product if the milipore filter of selecting for use is lower to the macromolecular rejection of target organism.
Described large biological molecule is protein, polypeptide, enzyme.
The present invention provides the ultrafiltration enrichment facility of realizing the method for the invention simultaneously.This device basic composition unit comprises ultrafiltration upgrading unit, concentrate collector unit, concentrate concentration online detection unit.Wherein, the material liquid import is arranged on the membrane module top, concentrate is collected mouth and is arranged on membrane module bottom, concentrate is collected mouth and is linked to each other with concentrate concentration online detection unit with the concentrate collector unit by threeway, fall collect via draw-off pump to the concentrate of membrane module bottom before, analyze by concentrate concentration online detection unit earlier, open draw-off pump when arriving preset value and collect with continuous or mode intermittently.
UV-detector is limit because of range, the high concentration concentrate can't directly carry out concentration analysis by on-line ultraviolet detector, therefore, device provided by the invention not only can be realized the macromolecular purpose of efficient concentrated biological, also provides the high concentration feed liquid online measuring ability simultaneously.The online measuring ability of high concentration feed liquid is by selecting for use serial dilution input mode or quantitative sample injection mode to be achieved.Therefore,
Described concentrate concentration online detection unit, necessary building block comprises sampling pump, dilution pumps, on-line ultraviolet detector and blender, and wherein sampling pump and dilution pumps are constant flow pump, and the working flow scope is 0.6 * 10
-4～0.6L/h.
Described serial dilution input mode is promptly to control online dilution ratio by the flow of regulating sampling pump and dilution pumps to be achieved, solution after the dilution enters on-line ultraviolet detector analysis again, dilute sampling pump flow when by adjustment, can obtain preferable relatively diluent concentration, by the anti-concentrate concentration that pushes away of thinner ratio, wherein the sample introduction flow of sampling pump should not be lower than 1.5 * 10 again
-3L/h.
Described quantitative sample injection mode is to inject the highly concentrated solution of fixed amount in moment by sampling pump, quantitative highly concentrated solution with enter on-line ultraviolet detector analysis after continuous dilution mixes by blender, by control injection rate and dilution pumps flow-control appearance time and peak area, and then calculate the actual concentration of concentrate, wherein the sample size of sampling pump should not be lower than 5 * 10
-6L, optimal flow dilution phase flow rates 1.8-15.0 * 10
-2L/h.
Described continuous drawing mode is: when the biological macromolecule solns concentration at concentrate collection mouthful place arrives the expection concentration value after testing, continuously extract the highly concentrated solution that concentrate is collected the mouth place.At this moment, the setting of concentrate extraction flow can be calculated according to permanent calculation of material.
Described extraction at intermittence mode is: when the biological macromolecule solns concentration at concentrate collection mouthful place arrives the expection concentration value after testing, extract concentrate and collect mouthful highly concentrated solution at place, after extracting a period of time, when the biological macromolecule solns concentration at concentrate collection mouthful place is lower than the expection concentration value, stop extraction operation; Solution to be concentrated is collected mouthful biological macromolecule solns concentration at place and is returned to when expecting concentration value, extracts once more, so repeatable operation.
The gravitational field that utilizes provided by the invention is assisted the macromolecular method and apparatus of concentrated biological, has following outstanding feature and advantage:
1. combine the field-effect of concentration polarization self-characteristic and gravitational field, the thickening efficiency height.
2. need not the concentrated liquid specially drawing device, method is simple, and is low to the hardware requirement of device, and easily scale is amplified, and application is strong.
3. concentration process is particularly suitable for concentrating shear sensitive large biological molecule not having shearing or hanging down under the condition of shearing and carry out.
4. concentration process moves under the end-filtration pattern, and energy consumption greatly reduces.
Description of drawings
Fig. 1. utilize gravitational field to assist the macromolecular ultrafiltration apparatus schematic flow sheet of concentrated biological.
Reference numeral
1. feed pump 2. concentrate draw-off pumps 3. sampling pumps 4. dilution pumps 5. blenders 6. on-line ultraviolet detector 7. membrane modules 8,9. flow sensor 10,11,12. valves 13,14. pressure sensors
The specific embodiment
The present invention will be further described below in conjunction with embodiment.Theme protection domain involved in the present invention is not limited only to these embodiment.
Embodiment 1.
See also Fig. 1.A kind of device of the auxiliary big molecule hyperfiltration process of concentrated biological of gravitational field that utilizes comprises feed pump 1, concentrate draw-off pump 2, sampling pump 3, dilution pumps 4, blender 5, on-line ultraviolet detector 6, membrane module 7, flow sensor 8 and 9, valve 10,11 and 12, pressure sensor 13 and 14.
(Tianjin University of Technology provides filling polyether sulfone hollow-fibre membrane, and molecular cut off 10KDa, film effectively load area 0.30m in the membrane module 7
2, film silk effective length 315mm).Material liquid is bovine serum albumin(BSA) (purity is greater than 98% for BSA, the molecular weight 68KDa) solution of 2L concentration 0.5g/L.Valve-off 11,12 is opened valve 10.Material liquid injects the feeding liquid mouth that is positioned at membrane module 7 tops via feed pump 1 continuous constant current amount, and penetrating fluid is got back to (being the penetrating fluid circulation pattern) in the raw material flow container through behind the flow sensor 9.Flow sensor 8 and 9 shows that feed rate and permeate flow are 4.2L/h.Transmembrane pressure (TMP=(P1+P2)/2) progresses into plateau (about 65.6KPa) after through one period of short duration quick rise period.This film is 97.7% to BSA initial-abstraction retention rate.2.0h after, open valve 12, open sampling pump 3, dilution pumps 4, dynamic mixer 5 and on-line ultraviolet detector 6, sampling pump flow 0.003L/h, dilution pumps flow 0.297L/h, record this moment membrane module 7 bottom concentrates to collect mouthful place's concentration be 65.0g/L, be 130.0 times of feeding liquid concentration.At this moment, open valve 11, open concentrate draw-off pump 2, take intermittently decimation pattern collect (open 2 minutes/closed 30 minutes, concentrate draw-off pump 2 flow sets are 0.03L/h), shutdown system after 10.5 hours.System's run duration, TMP drops to 52.7KPa gradually by the highest 65.6KPa, collects high power concentrate 16ml altogether, includes BSA0.92g, 115.4 times of average cycles of concentration.And if adopt conventional terminal filtering and concentrating process (membrane module 7 is identical, does not draw operation), the cycles of concentration theory can only reach 12.5 times at most.
Other conditions are constant, if the material liquid volume is increased to 4L, TMP is equally through progressing into plateau (about 68.9KPa) after one period of short duration quick rise period.2.0h after record membrane module 7 bottom concentrates to collect mouthful BSA of place concentration be 125.55g/L, be 251.1 times of concentration of raw material.10.5 shutdown system after hour, system's run duration, TMP drops to 51.3KPa gradually by the highest 68.9KPa, collect high power concentrate 16ml altogether, include BSA1.88g, 235.0 times of average cycles of concentration are 18.8 times that conventional ultrafiltration concentrates the theoretical maximum cycles of concentration.Obviously, the film method for concentration that proposes of the present invention is simple to operate and concentrated effect is remarkable.
Adopt the membrane module identical with embodiment 1, the BSA solution of same concentrations, identical feeding liquid flow, the material liquid volume increases to 10L.The 10L material liquid is continuously pumped into carries out ultrafiltration in the membrane module 7 and concentrate, before ultrafiltration concentrated 2h, penetrating fluid effluxed, and switches back the penetrating fluid circulation pattern behind the 2h.TMP is equally through progressing into plateau (about 71.8KPa) after one period of short duration quick rise period.Recording membrane module 7 bottom concentrates during 2h, to collect mouthful BSA of place concentration be 161.89g/L, is 323.8 times of material liquid.21.0h back shutdown system, system's run duration, TMP drops to 61.6KPa gradually by the highest 71.8KPa, collect high power concentrate 32ml altogether, include BSA4.75g, 296.9 times of average cycles of concentration are 23.8 times that conventional ultrafiltration concentrates the theoretical maximum cycles of concentration.Present embodiment has confirmed that further method for concentration that the present invention proposes has the conventional method concentrated effect that is beyond one's reach.
Adopt the membrane module identical with embodiment 1, the BSA solution of same concentrations, identical feeding liquid flow, the material liquid volume increases to 45L.The 45L material liquid is continuously pumped into carries out ultrafiltration in the membrane module 7 and concentrate, penetrating fluid effluxes.Adopt the continuous drawing mode, concentrate draw-off pump flow set is 0.02L/h, begins continuous drawing during 1.5h, this moment membrane module 7 bottom concentrates to collect mouthful BSA of place concentration be 130.29g/L, be 260.6 times of concentration of raw material.10.7h back shutdown system is collected out high power concentrate 214ml altogether, includes BSA22.09g, 206.5 times of average cycles of concentration.System operation beginning during the 1.5h in, TMP rapidly increases to 73.4KPa, TMP enters slow growth district behind the 1.5h, when system was out of service behind the 10.7h, TMP was 80.2KPa, the pressure climbing speed that slowly increases the district only is 0.74KPa/h.Obviously, can prove that the method for concentration that the present invention proposes not only has the efficient outstanding advantage that concentrates, and has also possessed " automatically cleaning " effect simultaneously, can realize the concentration operation of long-time continuous fully by present embodiment.
Embodiment 4
(German Memos company, molecular cut off 100KDa, film effectively load area 0.80m to filling Kynoar tubular membrane in the membrane module 7
2, film pipe effective length 950mm).Material liquid is gamma globulin (molecular weight 156kD, purity is greater than the 98%) solution of 20L concentration 0.5g/L.Valve-off 11,12 is opened valve 10.Material liquid injects the feeding liquid mouth that is positioned at membrane module 7 tops continuously via feed pump 1, and penetrating fluid is got back to (being the penetrating fluid circulation pattern) in the raw material flow container through behind the flow sensor 9.Flow sensor 8 and 9 shows that feed rate and permeate flow are 9.6L/h.TMP progresses into plateau (about 86.6KPa) after through one period of short duration quick rise period.This film is 93.5% to BSA initial-abstraction retention rate.1.5h after open valve 12, open sampling pump 3, dilution pumps 4, dynamic mixer 5 and on-line ultraviolet detector 6, record this moment membrane module 7 bottom concentrates to collect mouthful place's concentration be 42.35g/L, be 84.7 times of feeding liquid concentration.Open valve 11 this moment, open concentrate draw-off pump 2, take the continuous drawing pattern to collect (concentrate draw-off pump 2 flow sets are 0.05L/h), shutdown system after 6.5 hours.System's run duration, TMP drops to 74.5KPa gradually by the highest 86.6KPa, collects high power concentrate 250ml altogether, includes BSA9.37g, 75.0 times of average cycles of concentration.
(Jinsui River, Jiangyin film company, to the average rejection 63.5% of BSA, single periosteum effectively loads membrane area 0.83m to filling Kynoar tubular membrane in the membrane module 7
2, single film pipe effective length 1150mm).Membrane module 7 is composed in parallel by four tubular membrane and vertically places.Material liquid is the BSA solution of 60L concentration 0.5g/L.Valve-off 11,12 is opened valve 10.Material liquid injects the feeding liquid mouth that is positioned at membrane module 7 tops continuously via feed pump 1, and flow sensor 8 and 9 shows that feed rate and permeate flow are 21.0L/h.Before ultrafiltration concentrated 2.5h, penetrating fluid effluxed, and switches back the penetrating fluid circulation pattern behind the 2.5h.TMP progresses into plateau (about 256.4KPa) after through one period of short duration quick rise period.Open valve 12 during 3h, open sampling pump 3, dilution pumps 4, dynamic mixer 5 and on-line ultraviolet detector 6, record this moment membrane module 7 bottom concentrates to collect mouthful place's concentration be 28.52g/L, be 57.0 times of material liquid.Open valve 11 this moment, open concentrate draw-off pump 2, take the continuous drawing pattern to collect (concentrate draw-off pump 2 flow sets are 0.15L/h).13.5h back shutdown system, system's run duration, TMP drops to 157.8KPa gradually by the highest 256.4KPa, collects high power concentrate 950ml altogether, includes BSA15.05g, 31.7 times of average cycles of concentration.Even method for concentration that the present invention proposes is described under low rejection condition, has the conventional method concentrated effect that is beyond one's reach equally, and can long-term stability move.
Claims (9)
1. a gravitational field is assisted the macromolecular hyperfiltration process of concentrated biological, it is characterized in that: the mode that is parallel to horizontal plane according to film penetrating fluid direction is placed hyperfiltration membrane assembly, the ultrafiltration that the control system operational factor is carried out biological macromolecule solns under the end-filtration pattern concentrates, be formed at high concentration feed liquid in the concentration polarization layer on film surface under the gravity effect constantly from the film sur-face peeling and fall, collect the concentrate that obtains target product thereby the high concentration feed liquid that will fall directly extracts.
2. method according to claim 1 is characterized in that: the hyperfiltration membrane assembly pattern is doughnut formula, tubular type, plain film formula or rolling, and the film effective length is in 3000mm; Filter membrane is selected organic milipore filter of macromolecule or inorganic milipore filter for use, and selected milipore filter to the rejection of large biological molecule between 60.0-99.9%.
3. the organic milipore filter of macromolecule according to claim 2 comprises: cellulose family, polyether sulfone, polysulfones, Kynoar class, polyvinyl alcohol, polyvinyl acetaldehyde, polyvinyl chloride milipore filter or the blend film that may exist between them.
4. inorganic milipore filter according to claim 2 comprises: ceramic membrane or metal film.
5. method according to claim 1 is characterized in that: the transmembrane pressure of ultra-filtration process is controlled at-100～300KPa between, the film permeation flux is controlled at 5～30L/m
2Between the h, the large biological molecule concentration of raw material extracts flow-control 2.5 * 10 between 0.1～1.5g/L
-3～5.0L/h, concentrate concentration is between 10～180g/L, and the time of extraction for the first time is between the initial 0.5～3.5h afterwards of ultrafiltration.
6. method according to claim 1 is characterized in that: described large biological molecule is protein, polypeptide or enzyme.
7. ultrafiltration apparatus of realizing the described method of claim 1, it is characterized in that: this device basic composition unit comprises ultrafiltration upgrading unit, concentrate collector unit, concentrate concentration online detection unit.
8. device according to claim 7, it is characterized in that: the material liquid import is arranged on the membrane module top, concentrate is collected mouth and is arranged on membrane module bottom, concentrate is collected mouth and is linked to each other with concentrate concentration online detection unit with the concentrate collector unit by threeway, fall collect via draw-off pump to the concentrate of membrane module bottom before, analyze by concentrate concentration online detection unit earlier, open draw-off pump when arriving preset value and collect with continuous or mode intermittently.
9. according to claim 7 or 8 described concentrate concentration online detection unit, necessary building block comprises sampling pump, dilution pumps, on-line ultraviolet detector and blender, and wherein sampling pump and dilution pumps are constant flow pump, and the working flow scope is 0.6 * 10
-4～0.6L/h.
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| CN2010101004976A CN101745316B (en) | 2010-01-22 | 2010-01-22 | Novel ultrafiltration method and ultrafiltration device for using gravitational field to assist to concentrate large biological molecules |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102814121A (en) * | 2012-08-06 | 2012-12-12 | 中国科学院过程工程研究所 | Membrane filtration method and device for concentrating sugar solution by adopting concentration polarization principle |
| CN106914137A (en) * | 2015-12-24 | 2017-07-04 | 国家开发投资公司 | A kind of film concentration systems and method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5256294A (en) * | 1990-09-17 | 1993-10-26 | Genentech, Inc. | Tangential flow filtration process and apparatus |
| CN101269298B (en) * | 2007-03-23 | 2011-06-01 | 中国科学院过程工程研究所 | Membrane filtration method and device for polarization of concentration biomacromolecule with concentration |
| CN201244450Y (en) * | 2008-08-22 | 2009-05-27 | 周志杰 | Film component working under hypergravity condition |
-
2010
- 2010-01-22 CN CN2010101004976A patent/CN101745316B/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102814121A (en) * | 2012-08-06 | 2012-12-12 | 中国科学院过程工程研究所 | Membrane filtration method and device for concentrating sugar solution by adopting concentration polarization principle |
| CN106914137A (en) * | 2015-12-24 | 2017-07-04 | 国家开发投资公司 | A kind of film concentration systems and method |
| CN106914137B (en) * | 2015-12-24 | 2019-11-22 | 国投生物科技投资有限公司 | A kind of film concentration systems and method |
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| CN101745316B (en) | 2011-12-28 |
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