Purification method of urokinase and chromatographic column type adsorption device for extraction

Abstract

The invention relates to the technical field of urokinase, and discloses a urokinase purification method and a chromatographic column type adsorption device for extraction, the purification method comprises the following steps: S1, taking a urokinase raw material, carrying out microfiltration, adding a chitosan quaternary ammonium salt solution, carrying out directional flocculation, centrifuging, and collecting a supernatant; s2, adding the supernate in the step S1 into a weak anion exchange chromatographic column, eluting, collecting eluent with active components, and desalting the eluent; s3, adding the eluent desalted in the step S2 into a gel filtration chromatography column, eluting, and collecting the eluent; s4, the eluent in the step S3 is added to a Ni-IMAC metal chelating chromatographic column, and purified urokinase is obtained after elution. According to the method, multiple chromatographic columns are used for chromatography, and cost and operation complexity are reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of urokinase, in particular to a purification method of urokinase and a chromatographic column type adsorption device for extraction. BACKGROUND

[0002] The principle of the chromatographic column is that the migration speed of each component in the column is different due to the different distribution coefficients of each component in the sample mixture in the stationary phase and the mobile phase, thereby realizing separation. The chromatographic column is usually made of glass, organic glass or stainless steel and has good pressure resistance and corrosion resistance, and is used to contain the stationary phase and the mobile phase. The stationary phase is a material filled in the column and is usually composed of silica gel, resin or gel. The filling of the stationary phase is usually filled manually, and there is a lack of related equipment, resulting in a relatively cumbersome filling process.

[0003] Meanwhile, the existing urokinase purification technology takes "pre-treatment-chromatography combined use" as the core, and is divided into three types of salt precipitation-chromatography, membrane separation-chromatography and affinity chromatography assisted, but there are problems such as low impurity removal efficiency of pre-treatment (salt precipitation only removes 40%-50% impurities), large activity loss, difficult to balance chromatographic purity and recovery rate (final purity is usually <95%), high cost, and single activity protection, which cannot adapt to large-scale production, and new efficient and economical purification technology needs to be developed.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] To solve the above technical problems, the present application provides a purification method of urokinase and a chromatographic column type adsorption device for extraction. The stationary phase is filled by setting a discharge pipe, and the stationary phase can be fully filled in the chromatographic column body by the flow channel along the side wall of the chromatographic column body and the vibration of the eccentric wheel. Compared with the traditional manual filling, the filling effect is better and more convenient. The stationary phase is washed by setting a washing assembly, the through pipe is clamped by setting the first clamp rod and the second clamp rod, the control of the through pipe passage state is realized, and then the control of the flow of the mobile phase is realized, thereby avoiding the waste caused by the continuous flow of the mobile phase.

[0006] The technical scheme adopted by the present application to solve the technical problems is as follows:

[0007] A purification method of urokinase, comprising the following steps:

[0008] S1, taking urokinase raw materials, adding chitosan quaternary ammonium salt solution for directional flocculation after microfiltration, and collecting the supernatant by centrifugation;

[0009] S2, adding the supernatant in step S1 to a weak anion exchange chromatographic column, collecting the eluate with active components after elution, and desalting the eluate;

[0010] S3. Add the desalted eluent from step S2 to the gel filtration chromatography column, and collect the eluent after elution.

[0011] S4. Add the eluent from step S3 to a Ni²⁺-IMAC metal chelate chromatography column, and obtain purified urokinase after elution.

[0012] A chromatographic column adsorption device for urokinase extraction is used to fill a gel filtration chromatography column. The device includes a chromatography column body and a filling unit inside the chromatography column body. The filling unit includes a feeding module, a vibration module and a flushing module.

[0013] The feeding module includes a feeding pipe, and a discharge component is rotatably arranged at the bottom of the feeding pipe. The discharge component is used for feeding materials. A control component is also arranged on one side of the feeding pipe. The control component is used to control the discharge component to discharge materials.

[0014] The vibration module includes an eccentric wheel, which generates vibration when rotated;

[0015] The flushing module includes a flushing component, which includes a guide pipe. An adjustment component is provided on one side of the guide pipe to control the interval water output of the flushing component.

[0016] In a preferred embodiment of the present invention, the discharge assembly includes an upper connecting ring rotatably connected to the bottom of the discharge tube, a lower connecting ring fixedly installed at the bottom of the upper connecting ring, a flow channel installed on the side of the lower connecting ring, one end of the flow channel being in contact with the inner wall of the chromatography column body, and a sealing assembly provided on the lower connecting ring for sealing the discharge assembly.

[0017] In a preferred embodiment of the present invention, the sealing assembly includes a sealing ring, the bottom of which has an outlet, the top of which has a placement groove, the sealing ring being movably connected in the placement groove, the side wall of which has a sliding groove, the side of which is fixedly mounted with a first stop bar, the first stop bar being movably connected through the sliding groove, the side wall of which is fixedly mounted with a second stop bar, and an elastic rope being fixedly mounted between the first stop bar and the second stop bar.

[0018] In a preferred embodiment of the present invention, the control component includes a mounting plate that is movably sleeved on the feed tube. A toothed ring is rotatably connected to the bottom of the mounting plate. A connecting component is provided at the bottom of the toothed ring. The connecting component includes a first abrasive pad and a second abrasive pad. The first abrasive pad is fixedly installed on the top of the sealing ring. A connecting ring is fixedly installed on the top of the second abrasive pad. The connecting ring is fixedly installed at the bottom of the toothed ring.

[0019] In a preferred embodiment of the present invention, the control component further includes a gear, which is rotatably mounted on the bottom of the mounting plate. The gear meshes with a gear ring. A rotating column is fixedly mounted on the top of the gear through a connecting shaft passing through the mounting plate. A rotating shaft is movably inserted into the rotating column. A threaded section is provided at the bottom of the rotating shaft. The rotating column is threadedly connected to the bottom of the rotating shaft through the threaded section.

[0020] In a preferred embodiment of the present invention, the control component further includes a card plate and a card slot. The card slot is formed on the rotating column. A support block is rotatably mounted on the top of the card plate. A first telescopic rod is fixedly mounted on one end of the support block. A fixing block is fixedly mounted on the other end of the first telescopic rod. The fixing block is fixedly mounted on the top of the mounting plate. A first spring is movably sleeved on the first telescopic rod. An elastic plate is fixedly mounted on the top of the mounting plate. The elastic plate is located on both sides of the card plate and is used to push the card plate to conform to the rotating column.

[0021] In a preferred embodiment of the present invention, the vibration module further includes a first support frame, the eccentric wheel is rotatably connected to the first support frame, a second support frame is provided on the top of the first support frame, both the first and second support frames are fixedly connected to the feed pipe, a support platform is fixedly installed on one side of the feed pipe, a power source is fixedly installed on one side of the support platform, the rotating shaft movably passes through the second support frame and is connected to the power source, and a pulley mechanism is installed between the rotating shaft and the eccentric wheel.

[0022] In a preferred embodiment of the present invention, the rinsing assembly further includes a delivery pipe and a connecting pipe, both of which are connected to one end of the guide pipe. A spray nozzle mechanism is installed at the end of the connecting pipe. The spray nozzle mechanism is used to spray water onto the inner wall of the chromatography column. A support member is fixedly installed on the side of the feed pipe. The support member abuts against the inner wall of the chromatography column. The spray nozzle mechanism is fixedly inserted into the support member.

[0023] In a preferred embodiment of the present invention, the adjusting component includes a fixing frame, which is fixedly installed at one end of the feeding pipe. A first clamping rod is installed at one end of the fixing frame, and a groove is formed at one end of the fixing frame. A second clamping rod is slidably connected to the groove. The guiding pipe is located between the first clamping rod and the second clamping rod. Fixing frames are fixedly installed at both ends of the guiding pipe, and the fixing frames are fixedly connected to the fixing frame.

[0024] In a preferred embodiment of the present invention, the adjusting assembly further includes a movable frame, which is fixedly connected to a second clamping rod. A second telescopic rod is fixedly installed at one end of the movable frame, and one end of the second telescopic rod is fixedly connected to a feeding pipe. A second spring is movably sleeved on the second telescopic rod. The adjusting assembly also includes a cam, which is rotatably connected to a rotating shaft. The cam is aligned with the movable frame, and a ratchet mechanism is installed between the cam and the rotating shaft.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] This invention uses a feed pipe to fill the stationary phase, which enters along the side wall of the chromatography column through a flow channel. The vibration of the eccentric wheel ensures that the stationary phase fully fills the chromatography column. Compared with traditional manual filling, this invention has a better filling effect and is more convenient.

[0027] This invention uses a flushing assembly to flush the stationary phase and a first and second clamp to hold the conduit, thereby controlling the state of the conduit passage and controlling the outflow of the mobile phase, thus avoiding waste caused by continuous outflow of the mobile phase. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the overall structure of a chromatographic column adsorption device for urokinase extraction according to the present invention;

[0029] Figure 2 This is a schematic diagram of the internal structure of a chromatography column adsorption device for urokinase extraction according to the present invention;

[0030] Figure 3 This is a schematic diagram of the structure at the lower connecting ring of the present invention;

[0031] Figure 4 This is a schematic diagram of the elastic rope structure of the present invention;

[0032] Figure 5 This is a schematic diagram of the structure of the sealed ring in the detached state of the present invention;

[0033] Figure 6 This is a schematic diagram of the control component structure of the present invention;

[0034] Figure 7 This is a schematic diagram of the structure of the card plate of the present invention;

[0035] Figure 8 This is a schematic diagram of the structure at the threaded section of the present invention;

[0036] Figure 9 This is a schematic diagram of the structure at the eccentric wheel of the present invention;

[0037] Figure 10 This is a schematic diagram of the structure of the conductive tube of the present invention.

[0038] Figure label:

[0039] 100. Chromatography column body;

[0040] 200. Feed pipe; 201. Upper connecting ring; 202. Lower connecting ring; 203. Flow channel; 204. Placement groove; 205. Sealing ring; 206. Flow outlet; 207. Slide groove; 208. First stop bar; 209. Second stop bar; 210. Elastic rope; 211. First abrasive pad; 212. Support component;

[0041] 300. Mounting plate; 301. Gear ring; 302. Connecting ring; 303. Second frosted pad; 304. Gear; 305. Rotating column; 306. Slot; 307. Locking plate; 308. Elastic plate; 309. Support block; 310. First telescopic rod; 311. First spring; 312. Fixing block;

[0042] 400. Power source; 401. Rotating shaft; 402. Threaded section; 403. Belt pulley mechanism; 404. First support frame; 405. Eccentric wheel; 406. Second support frame; 407. Support platform;

[0043] 500, Fixed frame; 501, First clamping rod; 502, Groove; 503, Second telescopic rod; 504, Second spring; 505, Moving frame; 506, Second clamping rod; 507, Cam; 508, Ratchet mechanism; 509, Guide tube; 510, Fixed frame; 511, Delivery tube; 512, Connecting tube; 513, Nozzle mechanism. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention.

[0045] Example 1

[0046] A method for purifying urokinase, comprising the following steps:

[0047] S1. Take urokinase raw material, microfilter it, add chitosan quaternary ammonium salt solution for directional flocculation, and collect the supernatant by centrifugation.

[0048] S2. Add the supernatant from step S1 to a weak anion exchange chromatography column, collect the eluent containing the active component after elution, and desalt the eluent.

[0049] S3. Add the desalted eluent from step S2 to the gel filtration chromatography column, and collect the eluent after elution.

[0050] S4. Add the eluent from step S3 to a Ni²⁺-IMAC metal chelate chromatography column, and obtain purified urokinase after elution.

[0051] In step S3, the column volume of the gel filtration chromatography column is 1000 ml, and the ratio of column height to diameter is (10-12):1. When packing, fix the gel filtration chromatography column, inject 500 ml of buffer solution into the column to remove air bubbles, and slowly inject the packing medium from the top of the gel filtration chromatography column to avoid generating air bubbles. During the injection process, use buffer solution to rinse and compact the packing medium.

[0052] Example 2

[0053] like Figures 1 to 10 As shown, a chromatographic column adsorption device for urokinase extraction is used to fill a gel filtration chromatography column. It includes a chromatography column body 100, and a filling unit is provided inside the chromatography column body 100. The filling unit includes a feeding module, a vibration module and a water flushing module.

[0054] The feeding module includes a feeding pipe 200, a discharge component is rotatably installed at the bottom of the feeding pipe 200 for feeding, and a control component is also installed on one side of the feeding pipe 200 for controlling the discharge component to discharge.

[0055] The vibration module includes an eccentric wheel 405, which generates vibration when rotated;

[0056] The flushing module includes a flushing component, which includes a guide pipe 509. An adjustment component is provided on one side of the guide pipe 509 to control the intermittent water output of the flushing component.

[0057] like Figures 3 to 5 As shown, in a specific embodiment, the discharge assembly includes an upper connecting ring 201, which is rotatably connected to the bottom of the feed pipe 200. A lower connecting ring 202 is fixedly installed at the bottom of the upper connecting ring 201. A flow channel 203 is installed on the side of the lower connecting ring 202, and one end of the flow channel 203 is in contact with the inner wall of the chromatography column body 100. A sealing assembly is provided on the lower connecting ring 202 to seal the discharge assembly. In this configuration, one end of the flow channel 203 is provided with a rubber coating. When it contacts the inner wall of the chromatography column body 100, the rubber coating generates friction between the flow channel 203 and the chromatography column body 100, preventing it from easily rotating.

[0058] like Figures 3 to 5As shown, the sealing assembly further includes a sealing ring 205, with an outlet 206 at its bottom and a mounting groove 204 at the top of the lower connecting ring 202. The sealing ring 205 is movably connected within the mounting groove 204. A sliding groove 207 is formed on the side wall of the sealing ring 205. A first stop 208 is fixedly installed on the side of the upper connecting ring 201, and the first stop 208 movably passes through the sliding groove 207. A second stop 209 is fixedly installed on the side wall of the sealing ring 205. An elastic rope 210 is fixedly installed between the first stop 208 and the second stop 209. In this configuration, the tension of the elastic rope 210 acts on the first stop 208 and the second stop 209, causing the sealing ring 205 to rotate and reset.

[0059] Example 3

[0060] like Figure 4 , Figure 6 As shown, in a specific embodiment, the control component includes a mounting plate 300, which is movably sleeved on the feed tube 200. A toothed ring 301 is rotatably connected to the bottom of the mounting plate 300. A connecting component is provided at the bottom of the toothed ring 301. The connecting component includes a first abrasive pad 211 and a second abrasive pad 303. The first abrasive pad 211 is fixedly installed on the top of the sealing ring 205, and a connecting ring 302 is fixedly installed on the top of the second abrasive pad 303. The connecting ring 302 is fixedly installed at the bottom of the toothed ring 301. In this configuration, the friction generated by the mutual pressing contact between the first abrasive pad 211 and the second abrasive pad 303 keeps the toothed ring 301 and the sealing ring 205 rotating synchronously.

[0061] like Figure 6 , Figure 8 As shown, the control assembly further includes a gear 304, which is rotatably mounted on the bottom of the mounting plate 300. The gear 304 meshes with the gear ring 301. A rotating column 305 is fixedly mounted on the top of the gear 304 through a connecting shaft passing through the mounting plate 300. A rotating shaft 401 is movably inserted into the rotating column 305. A threaded section 402 is provided at the bottom of the rotating shaft 401. The rotating column 305 is threadedly connected to the bottom of the rotating shaft 401 through the threaded section 402. In this configuration, the threaded length between the threaded section 402 and the rotating column 305 is less than the length of the threaded section 402 to provide space for the rotating column 305 to move on the rotating shaft 401. The difference between the two is slightly less than the distance between the first abrasive pad 211 and the second abrasive pad 303 when they are separated, so that the first abrasive pad 211 and the second abrasive pad 303 can make full contact.

[0062] like Figures 6 to 7As shown, the control assembly further includes a locking plate 307 and a locking slot 306. The locking slot 306 is formed on the rotating column 305. A support block 309 is rotatably mounted on the top of the locking plate 307. A first telescopic rod 310 is fixedly mounted on one end of the support block 309, and a fixing block 312 is fixedly mounted on the other end of the first telescopic rod 310. The fixing block 312 is fixedly mounted on the top of the mounting plate 300. A first spring 311 is movably sleeved on the first telescopic rod 310. An elastic plate 308 is fixedly mounted on the top of the mounting plate 300. The elastic plate 308 is located on both sides of the locking plate 307 and is used to push the locking plate 307 to fit against the rotating column 305. In this configuration, when the rotating column 305 rotates, the locking plate 307 is pushed through the locking slot 306, causing the locking plate 307 to move against the elastic force of the first spring 311 and move to the other side of the rotating column 305.

[0063] Example 4

[0064] like Figure 2 , Figure 9 As shown, in a specific embodiment, the vibration module further includes a first support frame 404, an eccentric wheel 405 rotatably connected to the first support frame 404, a second support frame 406 on the top of the first support frame 404, both the first support frame 404 and the second support frame 406 being fixedly connected to the feed pipe 200, a support platform 407 fixedly installed on one side of the feed pipe 200, a power source 400 fixedly installed on one side of the support platform 407, a rotating shaft 401 movably passing through the second support frame 406 and connected to the power source 400, and a pulley mechanism 403 installed between the rotating shaft 401 and the eccentric wheel 405. In this configuration, the rotating shaft 401 drives the eccentric wheel 405 to rotate through the pulley mechanism 403, and the eccentric wheel 405 vibrates when it rotates under its eccentric setting.

[0065] like Figure 2 , Figure 3 , Figure 9 , Figure 10 As shown, the rinsing assembly further includes a delivery pipe 511 and a connecting pipe 512. Both the delivery pipe 511 and the connecting pipe 512 are connected to one end of the guide pipe 509. A spray mechanism 513 is installed at the end of the connecting pipe 512. The spray mechanism 513 is used to spray water onto the inner wall of the chromatography column body 100. A support member 212 is fixedly installed on the side of the feed pipe 200, and the support member 212 abuts against the inner wall of the chromatography column body 100. The spray mechanism 513 is fixedly inserted into the support member 212. In this configuration, the rinsing assembly is used to deliver the mobile phase to rinse the stationary phase, preventing excess stationary phase from adhering to the side wall of the chromatography column body 100.

[0066] like Figures 9 to 10As shown, the adjustment assembly further includes a fixing frame 500, which is fixedly installed at one end of the feed pipe 200. A first clamping rod 501 is installed at one end of the fixing frame 500, and a groove 502 is formed at one end of the fixing frame 500. A second clamping rod 506 is slidably connected to the groove 502. A guide pipe 509 is located between the first clamping rod 501 and the second clamping rod 506. Fixing frames 510 are fixedly installed at both ends of the guide pipe 509, and the fixing frames 510 are fixedly connected to the fixing frame 500. In this configuration, the clamping action of the first clamping rod 501 and the second clamping rod 506 causes the guide pipe 509 to deform, thereby cutting off the passage and achieving control over the outflow of the mobile phase.

[0067] like Figures 9 to 10 As shown, the adjusting assembly further includes a movable frame 505, which is fixedly connected to the second clamping rod 506. A second telescopic rod 503 is fixedly installed at one end of the movable frame 505, and one end of the second telescopic rod 503 is fixedly connected to the feed pipe 200. A second spring 504 is movably sleeved on the second telescopic rod 503. The adjusting assembly also includes a cam 507, which is rotatably connected to the rotating shaft 401. The cam 507 is aligned with the movable frame 505, and a ratchet mechanism 508 is installed between the cam 507 and the rotating shaft 401. In this configuration, the cam 507 controls the movement of the second clamping rod 506 to achieve intermittent flow of the flowing phase, avoiding waste. When the rotating shaft 401 reverses, the ratchet mechanism 508 prevents the cam 507 from being driven by the rotating shaft 401, thus keeping the second clamping rod 506 and the first clamping rod 501 in a clamping state.

[0068] The implementation principle of the chromatographic column adsorption device for urokinase extraction in this embodiment is as follows: During operation, the entire filling unit is first inserted into the chromatography column body 100, and the lower connecting ring 202 is brought close to the bottom of the chromatography column body 100. At this time, the power source 400 is started, and the power source 400 drives the rotating shaft 401 to rotate. At this time, the rotating column 305 is kept in a non-rotating state due to the abutment of the slot 306 and the clamping plate 307. At the same time, the elastic force of the first spring 311 holds the clamping plate 307 in place to prevent it from being pushed. When the rotating shaft 401 rotates, the threaded section 402 on the rotating shaft 401 rotates, which drives the rotating column 305 to descend, thereby causing the mounting plate 300 to descend. At this time, the mounting plate 300 drives the toothed ring 301 and the connecting ring 302 to descend, thereby causing the second abrasive pad 303 to descend and come into contact with the first abrasive pad 211. When the mounting plate 300 cannot descend further, the rotating column 305 cannot descend. At this time, the rotating shaft 401 rotates and drives the rotating column 305 to rotate. The rotating column 305 drives the gear 304 to rotate. The gear 304 drives the gear ring 301 to rotate through meshing. The gear ring 301 drives the sealing ring 205 to rotate through the friction with the first abrasive pad 211 and the second abrasive pad 303. At this time, one end of the flow channel 203 abuts against the inner wall of the chromatography column body 100, so that the lower connecting ring 202 and the upper connecting ring 201 will not rotate. When the sealing ring 205 rotates to the position where the outlet 206 is aligned with the flow channel 203, the sliding groove 207 on the sealing ring 205 abuts against the first stop bar 208, thereby driving the upper connecting ring 201 and the lower connecting ring 202 to rotate together. At this time, the fixed phase through the feed pipe 200 enters the chromatography column body 100 along the flow channel 203.

[0069] When the rotating shaft 401 rotates, it drives the eccentric wheel 405 to rotate through the belt pulley mechanism 403. The vibration generated when the eccentric wheel 405 rotates is transmitted to the chromatography column body 100 through the support member 212, so that the chromatography column body 100 is filled with the internal stationary phase after being vibrated.

[0070] When the rotating shaft 401 rotates, it drives the cam 507 to rotate through the ratchet mechanism 508. The cam 507 abuts against the moving frame 505 to make it reciprocate. The first clamping rod 501 and the second clamping rod 506 perform intermittent clamping movements under the action of the moving frame 505 to intermittently clamp the guide tube 509, thereby causing the nozzle mechanism 513 to intermittently spray the mobile phase to homogenize the stationary phase on the side wall of the chromatography column body 100.

[0071] When the stationary phase is filled to a certain extent, it needs to be filled again. At this time, the filling of the stationary phase needs to be stopped, and the power source 400 is rotated in reverse so that the rotating shaft 401 is reversed. When the rotating shaft 401 is reversed, the rotating column 305 is unable to rotate again under the action of the clamping plate 307, the clamping groove 306 and the first spring 311, so that the rotating shaft 401 drives the rotating column 305 and the mounting plate 300 to rise through the threaded section 402. At this time, the mounting plate 300 drives the toothed ring 301 and the connecting ring 302 to rise, so that the first abrasive pad 211 and the second abrasive pad 303 are separated. At this time, the sealing ring 205 is rotated and reset under the action of the elastic rope 210, so that the outlet 206 and the flow channel 203 are misaligned, and the stationary phase cannot flow out through the flow channel 203.

[0072] When the rotating shaft 401 reverses, due to the action of the ratchet mechanism 508, the cam 507 no longer rotates. At this time, the moving frame 505 abuts against the cam 507, causing the cam 507 to rotate and drive the second clamping rod 506 to abut against the first clamping rod 501, so that the conduction tube 509 is in the cut-off state, pausing the output of the flowing phase.

[0073] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for purifying urokinase, characterized in that, Includes the following steps: S1. Take urokinase raw material, microfilter it, add chitosan quaternary ammonium salt solution for directional flocculation, and collect the supernatant by centrifugation. S2. Add the supernatant from step S1 to a weak anion exchange chromatography column, collect the eluent containing the active component after elution, and desalt the eluent. S3. Add the desalted eluent from step S2 to the gel filtration chromatography column, and collect the eluent after elution. S4. Add the eluent from step S3 to a Ni²⁺-IMAC metal chelate chromatography column, and obtain purified urokinase after elution.

2. A chromatographic column adsorption device for urokinase extraction, used for packing the gel filtration chromatography column of claim 1, comprising a chromatography column body (100), characterized in that, The chromatography column body (100) is provided with a packing unit inside, the packing unit including a feeding module, a vibration module and a flushing module; The feeding module includes a feeding pipe (200), and a discharge component is rotatably arranged at the bottom of the feeding pipe (200). The discharge component is used for feeding. A control component is also arranged on one side of the feeding pipe (200). The control component is used to control the discharge component to discharge. The vibration module includes an eccentric wheel (405) which generates vibration when rotated; The flushing module includes a flushing component, which includes a guide pipe (509). An adjustment component is provided on one side of the guide pipe (509) to control the intermittent water output of the flushing component.

3. The chromatographic column adsorption device for urokinase extraction according to claim 2, characterized in that, The discharge assembly includes an upper connecting ring (201), which is rotatably connected to the bottom of the discharge pipe (200). A lower connecting ring (202) is fixedly installed at the bottom of the upper connecting ring (201). A flow channel (203) is installed on the side of the lower connecting ring (202). One end of the flow channel (203) is attached to the inner wall of the chromatography column body (100). A sealing assembly is provided on the lower connecting ring (202). The sealing assembly includes a sealing ring (205). An outlet (206) is opened at the bottom of the sealing ring (205). The lower connecting ring (202) has a mounting groove (204) at its top. The sealing ring (205) is movably connected in the mounting groove (204). The sealing ring (205) has a sliding groove (207) on its side wall. The upper connecting ring (201) has a first stop bar (208) fixedly installed on its side. The first stop bar (208) movably passes through the sliding groove (207). The sealing ring (205) has a second stop bar (209) fixedly installed on its side wall. An elastic rope (210) is fixedly installed between the first stop bar (208) and the second stop bar (209).

4. The chromatographic column adsorption device for urokinase extraction according to claim 3, characterized in that, The control component includes a mounting plate (300), which is movably sleeved on the feed tube (200). A toothed ring (301) is rotatably connected to the bottom of the mounting plate (300). A connecting component is provided at the bottom of the toothed ring (301). The connecting component includes a first abrasive pad (211) and a second abrasive pad (303). The first abrasive pad (211) is fixedly installed on the top of the sealing ring (205). A connecting ring (302) is fixedly installed on the top of the second abrasive pad (303). The connecting ring (302) is fixedly installed on the bottom of the toothed ring (301).

5. The chromatographic column adsorption device for urokinase extraction according to claim 4, characterized in that, The control component also includes a gear (304), which is rotatably mounted on the bottom of the mounting plate (300). The gear (304) meshes with a gear ring (301). The top of the gear (304) passes through the mounting plate (300) via a connecting shaft and is fixedly mounted with a rotating column (305). A rotating shaft (401) is movably inserted into the rotating column (305). A threaded section (402) is provided at the bottom of the rotating shaft (401). The rotating column (305) is threadedly connected to the bottom of the rotating shaft (401) via the threaded section (402).

6. The chromatographic column adsorption device for urokinase extraction according to claim 5, characterized in that, The control assembly also includes a card plate (307) and a card slot (306). The card slot (306) is opened on the rotating column (305). A support block (309) is rotatably installed on the top of the card plate (307). A first telescopic rod (310) is fixedly installed on one end of the support block (309). A fixing block (312) is fixedly installed on the other end of the first telescopic rod (310). The fixing block (312) is fixedly installed on the top of the mounting plate (300). A first spring (311) is movably sleeved on the first telescopic rod (310). An elastic plate (308) is fixedly installed on the top of the mounting plate (300). The elastic plate (308) is located on both sides of the card plate (307). The elastic plate (308) is used to push the card plate (307) to make it fit against the rotating column (305).

7. The chromatographic column adsorption device for urokinase extraction according to claim 6, characterized in that, The vibration module also includes a first support frame (404), the eccentric wheel (405) is rotatably connected to the first support frame (404), a second support frame (406) is provided on the top of the first support frame (404), the first support frame (404) and the second support frame (406) are both fixedly connected to the feed pipe (200), a support platform (407) is fixedly installed on one side of the feed pipe (200), a power source (400) is fixedly installed on one side of the support platform (407), the rotating shaft (401) movably passes through the second support frame (406) and is connected to the power source (400), and a belt pulley mechanism (403) is installed between the rotating shaft (401) and the eccentric wheel (405).

8. The chromatographic column adsorption device for urokinase extraction according to claim 7, characterized in that, The rinsing assembly also includes a delivery pipe (511) and a connecting pipe (512). The delivery pipe (511) and the connecting pipe (512) are both connected to one end of the guide pipe (509). A spray mechanism (513) is installed at the end of the connecting pipe (512). The spray mechanism (513) is used to spray water onto the inner wall of the chromatography column body (100). A support member (212) is fixedly installed on the side of the feed pipe (200). The support member (212) abuts against the inner wall of the chromatography column body (100). The spray mechanism (513) is fixedly inserted into the support member (212).

9. The chromatographic column adsorption device for urokinase extraction according to claim 8, characterized in that, The adjustment assembly includes a fixing frame (500), which is fixedly installed at one end of the feed tube (200). A first clamping rod (501) is installed at one end of the fixing frame (500), and a groove (502) is opened at one end of the fixing frame (500). A second clamping rod (506) is slidably connected to the groove (502). The guide tube (509) is located between the first clamping rod (501) and the second clamping rod (506). Fixing frames (510) are fixedly installed at both ends of the guide tube (509), and the fixing frames (510) are fixedly connected to the fixing frame (500).

10. A chromatographic column adsorption device for urokinase extraction according to claim 9, characterized in that, The adjustment assembly further includes a movable frame (505), which is fixedly connected to a second clamping rod (506). A second telescopic rod (503) is fixedly installed at one end of the movable frame (505). One end of the second telescopic rod (503) is fixedly connected to a feed pipe (200). A second spring (504) is movably sleeved on the second telescopic rod (503). The adjustment assembly also includes a cam (507), which is rotatably connected to a rotating shaft (401). The cam (507) is aligned with the movable frame (505). A ratchet mechanism (508) is installed between the cam (507) and the rotating shaft (401).

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