Continuous separation device and method for recombinant collagen

Abstract

The invention discloses a continuous separation device and method for recombinant collagen, and belongs to the field of recombinant collagen separation.The continuous separation device comprises a liquid storage tank, a separation mechanism of a separation barrel is arranged at the processing output end of the liquid storage tank, the separation barrel is installed on one side of the liquid storage tank, a centrifugal net is installed in the separation barrel, and a stirring assembly is arranged at the bottom end of the separation barrel; the stirring assembly comprises an annular plate fixedly installed at the bottom end of the separation cylinder, the annular plate is communicated with an inner cavity of the separation cylinder, and stirring blades used for stirring and precipitating eluent are rotatably installed in the annular plate. The continuous separation and purification of the recombinant collagen are realized, the centrifugal impeller and the stirring assembly in the separation cylinder are driven by the motor, so that a solution is uniformly mixed, the separation efficiency is improved, meanwhile, the resin column arranged in the filter cylinder can further adsorb impurities, and the purity of a final product is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of recombinant collagen separation, and in particular to a device and method for continuous separation of recombinant collagen. Background Art

[0002] As an important bioactive substance, recombinant collagen has broad application prospects in the fields of medicine, cosmetics, food, etc. In its production process, separation and purification are key steps to ensure product quality and purity.

[0003] Traditional recombinant collagen separation methods often rely on a single chromatography technology. A single chromatography technology cannot completely remove all impurity proteins, resulting in low purity of the final product. Especially when processing complex biological samples, the samples may contain multiple impurities with properties similar to the target protein. These impurities are difficult to effectively separate using a single chromatography technology. Secondly, traditional chromatography equipment often lacks an effective mixing and stirring mechanism during the separation process, resulting in uneven distribution of the solution in the chromatography column, affecting the separation effect and efficiency. At the same time, the eluate after a single separation will contain incompletely separated impurities or aggregates of the target protein, resulting in insufficient separation effect. Summary of the Invention

[0004] The object of the present invention is to provide a continuous separation device and method for recombinant collagen to solve the problem mentioned in the background art that there is a lack of effective mixing and stirring mechanism during the separation process, resulting in uneven distribution of the solution in the chromatography column.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solutions: a continuous separation device for recombinant collagen, comprising a liquid storage tank, a separation mechanism of a separation cylinder being provided at the processing output end of the liquid storage tank, the separation cylinder being installed on one side of the liquid storage tank, a centrifugal screen being installed inside the separation cylinder, a stirring assembly being provided at the bottom end of the separation cylinder, the stirring assembly comprising an annular plate fixedly installed at the bottom end of the separation cylinder, and the annular plate being connected to the inner cavity of the separation cylinder, a stirring blade for stirring and precipitating the eluate being rotatably installed inside the annular plate, a connecting plate being fixedly installed on one side of the stirring blade, a motor being fixedly installed on one side of the liquid storage tank, and the output shaft of the motor and the connecting plate being fixedly connected via a support frame.

[0006] As a preferred technical solution of the present invention, the bottom end of the annular plate is connected to a filter cartridge, and a resin column for adsorbing impurities is provided inside the filter cartridge.

[0007] As a preferred technical solution of the present invention, a rotating rod is rotatably installed inside the separation cylinder, and a plurality of centrifugal impellers for mixing the liquid inside the separation cylinder are fixedly installed on the outer surface of the rotating rod. The output shaft of the motor is fixedly connected to the rotating rod, and a circular ring is installed on the outer surface of the separation cylinder, and a third adding cylinder is installed on the outer surface of the circular ring.

[0008] As a preferred technical solution of the present invention, an anti-blocking component is provided inside the separation cylinder, and the anti-blocking component includes a scraper rotatably mounted on the outer surface of the rotating rod, and the scraper slides on the inner wall of the centrifugal screen, and a rotating plate is fixedly mounted on one side of the connecting plate, and a top plate is fixedly mounted on the outer surface of the rotating plate, and the top plate is fixedly connected to one end of the scraper.

[0009] As a preferred technical solution of the present invention, the scraper is provided with air holes on its surface close to the centrifugal screen, the scraper is provided with a cavity inside, and a plurality of air-permeable films are installed inside the cavity.

[0010] As a preferred technical solution of the present invention, a limit rod is slidably installed inside the cavity, a plurality of resistance springs are arranged between the limit rod and the breathable film, and a sticky plate for adhering impurities is installed inside the cavity.

[0011] As a preferred technical solution of the present invention, a circulation component is provided on the outside of the separation cylinder, and the circulation component includes a connecting pipe installed between the top and bottom ends of the separation cylinder, and a suction pump is provided on the connecting pipe for driving the liquid inside the connecting pipe.

[0012] As a preferred technical solution of the present invention, a fermentation mechanism is provided at the top of the liquid storage tank, and the fermentation mechanism includes a fermentation liquid barrel installed at the top of the liquid storage tank. A water pump is fixedly installed at the top of the liquid storage tank, and the output end of the water pump is connected to the fermentation liquid barrel through a feed pipe. A first adding cylinder is provided on the side wall of the fermentation liquid barrel, and a sedimentation tank is provided at the bottom end of the fermentation liquid barrel.

[0013] As a preferred technical solution of the present invention, the bottom end of the fermentation liquid barrel is connected to and installed with a chromatography cartridge, a second addition cartridge is provided on one side of the chromatography cartridge, the bottom end of the chromatography cartridge is connected to and installed with an elution cartridge, the bottom end of the elution cartridge is connected to and installed with a separation cartridge, and the bottom end of the separation cartridge is connected to and installed with a collection cartridge via a discharge pipe.

[0014] A continuous separation method for recombinant collagen, the preparation steps are as follows:

[0015] Step 1: Collect the fermentation broth or cell lysate to ensure that it contains recombinant collagen;

[0016] Step 2: Use phosphate buffer to equilibrate the affinity chromatography column to allow the filler in the column to reach a stable state. During the equilibration process, monitor the UV absorbance to ensure a stable baseline.

[0017] Step 3: Slowly add the treated sample to the affinity chromatography column, control the flow rate to allow the recombinant collagen to fully bind to the affinity ligand on the filler, and collect the flow-through for subsequent analysis;

[0018] Step 4: Wash the affinity column with binding buffer to remove unbound impurity proteins and monitor the UV absorbance until the absorbance drops to the baseline level, indicating that the impurities have been basically removed;

[0019] Step 5: Change the elution conditions to dissociate the recombinant collagen from the affinity ligand, and collect the eluate, which is the initially purified recombinant collagen;

[0020] Step 6: appropriately dilute or concentrate the eluate obtained from the affinity chromatography, and adjust its ionic strength and pH value to suit the conditions of ion exchange chromatography;

[0021] Step 7: Slowly add the treated sample to the ion exchange chromatography column, controlling the flow rate to allow the recombinant collagen to interact with the ionic groups on the filler;

[0022] Step 8: Gradually increase the salt concentration in the elution buffer to elute protein molecules with different charge properties in sequence, collect the eluate containing recombinant collagen, use elution buffers with different salt concentrations to perform elution in stages, and collect the eluate from each stage, which is the purified recombinant collagen.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention achieves continuous separation and purification of recombinant collagen by integrating multiple technologies such as fermentation, affinity chromatography, ion exchange chromatography and centrifugal separation. The centrifugal impeller and stirring assembly in the separation cartridge are driven by a motor to mix the solution evenly, thereby improving the separation efficiency. At the same time, the resin column arranged inside the filter cartridge can further adsorb impurities, significantly improving the purity of the final product.

[0025] During the separation process, the present invention can effectively remove large particle impurities and unbound proteins in the solution through the synergistic effect of the centrifugal mesh and the scraper. The scraper slides on the inner wall of the centrifugal mesh, preventing the accumulation of impurities on the mesh and further reducing impurity residues. In addition, the resin column in the filter cartridge has a high adsorption capacity for small molecular impurities, ensuring the purity of the eluent.

[0026] The anti-blocking components arranged inside the separation cylinder of the present invention include scrapers and air holes, which effectively prevent the centrifugal net from being blocked during the separation process. When the scraper slides on the inner wall of the centrifugal net, the air holes and the air-permeable film in the cavity can balance the air pressure, reduce the friction between the scraper and the centrifugal net, and improve the operating stability of the equipment. At the same time, the sticky plate in the cavity can adhere to some impurities, further reducing the risk of blockage.

[0027] The present invention realizes the recycling of eluent through the circulation component. The setting of the connecting pipe and the suction pump allows the eluent to circulate between the top and bottom ends of the separation cylinder, thereby improving the elution efficiency and reducing the amount of eluent used. This not only reduces production costs, but also reduces the discharge of waste liquid, meeting environmental protection requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the overall structure of the present invention;

[0029] Figure 2 It is a side structural schematic diagram of the present invention;

[0030] Figure 3 It is a schematic diagram of the structure of the circulation component of the present invention;

[0031] Figure 4 It is a schematic diagram of the centrifugal network structure of the present invention;

[0032] Figure 5 Schematic diagram of the internal structure of the separation cylinder of the present invention;

[0033] Figure 6 Schematic diagram of the stirring blade structure of the present invention;

[0034] Figure 7 Schematic diagram of the centrifugal impeller structure of the present invention;

[0035] Figure 8 It is a schematic diagram of the internal structure of the scraper of the present invention.

[0036] Figure: 1, liquid storage tank; 2, fermentation mechanism; 21, water pump; 22, feed pipe; 23, sedimentation tank; 24, fermentation liquid barrel; 25, first addition cylinder; 3, chromatography cylinder; 4, elution cylinder; 5, separation mechanism; 51, separation cylinder; 52, centrifugal screen; 53, stirring assembly; 531, motor; 532, rotating rod; 533, centrifugal impeller; 534, annular plate; 535, filter cartridge; 536, stirring blade; 537, support frame ; 538. Connecting plate; 539. Anti-blocking component; 5391. Rotating plate; 5392. Top plate; 5393. Scraper; 5394. Air vent; 5395. Breathable film; 5396. Limit rod; 5397. Resistance spring; 5398. Sticky plate; 5399. Cavity; 54. Ring; 55. Circulation component; 551. Connecting pipe; 552. Suction pump; 56. Third adding cylinder; 6. Discharge pipe; 7. Collection cylinder. DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0038] See also Figure 1-8 The present invention provides a continuous separation device for recombinant collagen, comprising a liquid storage tank 1, a separation mechanism 5 with a separation cylinder 51 provided at the processing output end of the liquid storage tank 1, the separation cylinder 51 being installed on one side of the liquid storage tank 1, a centrifugal net 52 being installed inside the separation cylinder 51, a stirring assembly 53 being provided at the bottom end of the separation cylinder 51, the stirring assembly 53 comprising an annular plate 534 fixedly installed at the bottom end of the separation cylinder 51, and the annular plate 534 being communicated with the inner cavity of the separation cylinder 51, a stirring blade 536 for stirring and precipitating the eluate being rotatably installed inside the annular plate 534, a connecting plate 538 being fixedly installed on one side of the stirring blade 536, a motor 531 being fixedly installed on one side of the liquid storage tank 1, and the output shaft of the motor 531 and the connecting plate 538 being fixedly connected via a support frame 537.

[0039] Among them, when separating the protein, multiple processing and separation are carried out through the separation cylinder 51, and after separation, the motor 531 is used to drive the stirring blade 536 to rotate in the annular plate 534 to stir and precipitate the eluate. At the same time, the annular plate 534 is connected to the inner cavity of the separation cylinder 51 to realize the circulation and sufficient mixing of the liquid, thereby realizing the continuous separation of recombinant collagen. The preliminary separation is carried out through the centrifugal net 52 in the separation cylinder 51, and the separation process is further optimized in combination with the stirring component 53, thereby improving the separation efficiency and purity.

[0040] In some embodiments, a filter cartridge 535 is installed in communication with the bottom end of the annular plate 534 , and a resin column for adsorbing impurities is provided inside the filter cartridge 535 .

[0041] The resin material in the resin column has the ability to absorb specific impurities. When the eluent flows through the filter cartridge 535, the impurities are absorbed by the resin column, thereby purifying the eluent and improving the purity of the eluent.

[0042] In some embodiments, a rotating rod 532 is rotatably installed inside the separation cylinder 51, and a plurality of centrifugal impellers 533 for mixing the liquid inside the separation cylinder 51 are fixedly installed on the outer surface of the rotating rod 532. The output shaft of the motor 531 is fixedly connected to the rotating rod 532. The outer surface of the separation cylinder 51 is connected to and installed with a ring 54, and the outer surface of the ring 54 is connected to and installed with a third adding cylinder 56.

[0043] The motor 531 drives the rotating rod 532 to rotate, thereby driving the centrifugal impeller 533 to rotate, generating centrifugal force to mix the liquid evenly and accelerate the separation process. At the same time, the third addition cylinder 56 is provided to facilitate the addition of elution buffer and the adjustment of separation conditions.

[0044] In some embodiments, an anti-blocking component 539 is provided inside the separation cylinder 51, and the anti-blocking component 539 includes a scraper 5393 rotatably mounted on the outer surface of the rotating rod 532, and the scraper 5393 slides on the inner wall of the centrifugal net 52. A rotating plate 5391 is fixedly mounted on one side of the connecting plate 538, and a top plate 5392 is fixedly mounted on the outer surface of the rotating plate 5391. The top plate 5392 is fixedly connected to one end of the scraper 5393.

[0045] Among them, the scraper 5393 slides on the inner wall of the centrifugal net 52 driven by the rotating rod 532, scraping off impurities attached to the mesh, preventing blockage, and ensuring the continuity of the separation process. At the same time, the setting of the rotating plate 5391 and the top plate 5392 ensures that the scraper 5393 and the stirring blade 536 move synchronously, optimizing the anti-blocking effect.

[0046] In some embodiments, a surface of the scraper 5393 close to the centrifugal net 52 is provided with ventilation holes 5394 , an interior of the scraper 5393 is provided with a cavity 5399 , and a plurality of breathable films 5395 are installed inside the cavity 5399 .

[0047] Among them, the conical air holes 5394 and the breathable film 5395 can balance the air pressure between the scraper 5393 and the centrifugal net 52, reduce friction, and improve the operating stability of the anti-blocking component 539. At the same time, the through holes opened on the surface of the rotating plate 5391 can stir the air flow, and the air flow can also exhaust the conical air holes 5394 to further reduce the blockage of the centrifugal net 52 and increase the long-term stable separation effect of centrifugal separation.

[0048] In some embodiments, a limiting rod 5396 is slidably installed inside the cavity 5399, a plurality of resistance springs 5397 are arranged between the limiting rod 5396 and the breathable film 5395, and a sticky plate 5398 for adhering impurities is installed inside the cavity 5399.

[0049] Among them, the limiting rod 5396 limits the expansion range of the breathable film 5395, the resistance spring 5397 provides a buffering force to reduce the impact force between the scraper 5393 and the centrifugal net 52, and the sticky plate 5398 can adhere to some impurities to reduce the risk of clogging of the breathable film 5395.

[0050] In some embodiments, a circulation assembly 55 is provided on the outside of the separation cylinder 51. The circulation assembly 55 includes a connecting pipe 551 installed between the top and bottom ends of the separation cylinder 51. The connecting pipe 551 is provided with a suction pump 552 for driving the liquid inside the connecting pipe 551.

[0051] The arrangement of the connecting tube 551 and the suction pump 552 enables the eluent to circulate between the top and bottom ends of the separation cylinder 51, thereby improving the elution efficiency and reducing the amount of eluent used.

[0052] In some embodiments, a fermentation mechanism 2 is provided at the top of the liquid storage tank 1, and the fermentation mechanism 2 includes a fermentation liquid barrel 24 installed at the top of the liquid storage tank 1. A water pump 21 is fixedly installed at the top of the liquid storage tank 1, and the output end of the water pump 21 is connected to the fermentation liquid barrel 24 through a feed pipe 22. A first adding cylinder 25 is provided on the side wall of the fermentation liquid barrel 24, and a sedimentation tank 23 is provided at the bottom end of the fermentation liquid barrel 24.

[0053] Among them, the water pump 21 introduces the fermentation liquid from the liquid storage tank 1 into the fermentation liquid barrel 24, the first addition cylinder 25 is used to add phosphate buffer reagent, and the sedimentation tank 23 is used to preliminarily separate impurities in the fermentation liquid. The fermentation mechanism 2 can achieve continuous processing and preliminary separation of the fermentation liquid, providing high-quality raw materials for the subsequent separation process.

[0054] In some embodiments, the bottom end of the fermentation liquid barrel 24 is connected to and installed with a chromatography cylinder 3, a second addition cylinder 31 is provided on one side of the chromatography cylinder 3, the bottom end of the chromatography cylinder 3 is connected to and installed with an elution cylinder 4, the bottom end of the elution cylinder 4 is connected to and installed with a separation cylinder 51, and the bottom end of the separation cylinder 51 is connected to and installed with a collection cylinder 7 through a discharge pipe 6.

[0055] The fermentation broth tank 24, chromatography cartridge 3, elution cartridge 4, and separation cartridge 51 are sequentially connected. By adding various reagents and adjusting separation conditions, impurities are gradually removed and the recombinant collagen is purified. Finally, the separated recombinant collagen is stably introduced into the collection cartridge 7 through the discharge pipe 6. Through multiple separation processes and impurity removal, a continuous separation and purification process from fermentation broth to final product is achieved, improving production efficiency and product quality.

[0056] A continuous separation method for recombinant collagen, the preparation steps are as follows:

[0057] Step 1: Collect the fermentation broth or cell lysate to ensure that it contains recombinant collagen;

[0058] Step 2: Use phosphate buffer to equilibrate the affinity chromatography column to allow the filler in the column to reach a stable state. During the equilibration process, monitor the UV absorbance to ensure a stable baseline.

[0059] Step 3: Slowly add the treated sample to the affinity chromatography column, control the flow rate to allow the recombinant collagen to fully bind to the affinity ligand on the filler, and collect the flow-through for subsequent analysis;

[0060] Step 4: Wash the affinity column with binding buffer to remove unbound impurity proteins and monitor the UV absorbance until the absorbance drops to the baseline level, indicating that the impurities have been basically removed;

[0061] Step 5: Change the elution conditions and increase the imidazole concentration to dissociate the recombinant collagen from the affinity ligand. Collect the eluate, which is the preliminarily purified recombinant collagen.

[0062] Step 6: appropriately dilute or concentrate the eluate obtained from the affinity chromatography, and adjust its ionic strength and pH value to suit the conditions of ion exchange chromatography;

[0063] Step 7: Slowly add the treated sample to the ion exchange chromatography column, controlling the flow rate to allow the recombinant collagen to interact with the ionic groups on the filler;

[0064] Step 8: Gradually increase the salt concentration (sodium chloride concentration) in the elution buffer to elute protein molecules with different charge properties in sequence, collect the eluate containing recombinant collagen, use elution buffers with different salt concentrations to perform staged elution, and collect the eluate from each stage, which is the purified recombinant collagen.

[0065] The working principle is as follows: the fermentation liquid in the fermentation liquid barrel is added with phosphate buffer through the first addition cylinder 25, and after mixing, it enters the affinity chromatography column to react to obtain a flow-through liquid, which is combined with the buffer affinity column in the chromatography cylinder 3 to remove unbound impurity proteins, and then in the elution cylinder 4, the second addition cylinder 31 is used to increase the imidazole concentration to dissociate the recombinant collagen from the affinity ligand, and the eluate is collected, which is the preliminarily purified recombinant collagen. The eluate obtained by affinity chromatography is appropriately diluted or concentrated, and its ionic strength and pH value are adjusted. Further, it is introduced into the separation cylinder 51. In the electron exchange chromatography column, the sodium chloride concentration in the elution buffer is gradually increased through the third addition cylinder 56, and the centrifugal impeller 533 is driven by the motor 531 in the separation cylinder 51 to stir the inside of the separation cylinder 51, so that the solution is mixed evenly and the separation effect is improved. At the same time, after separation, it is introduced into the annular plate 534, and the stirring blade 536 is driven by the motor 531 to continuously stir the liquid inside the annular plate 534, and cooperate with the filter cylinder 535 to improve the stability of the separation, and then the separated recombinant collagen is stably introduced into the collection cylinder 7 through the discharge pipe 6 for collection.

[0066] The above are only specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions, or modifications based on the present invention to solve substantially the same technical problems and achieve substantially the same technical effects are all included in the scope of protection of the present invention.

Claims

1. A continuous separation device for recombinant collagen, comprising a liquid storage tank (1), characterized in that: The processing output end of the liquid storage tank (1) is provided with a separation mechanism (5) of a separation cylinder (51), the separation cylinder (51) is installed on one side of the liquid storage tank (1), a centrifugal net (52) is installed inside the separation cylinder (51), and a stirring assembly (53) is provided at the bottom end of the separation cylinder (51), the stirring assembly (53) comprises an annular plate (534) fixedly installed at the bottom end of the separation cylinder (51), and the annular plate (534) is communicated with the inner cavity of the separation cylinder (51), and a stirring blade (536) for stirring and precipitating the eluent is rotatably installed inside the annular plate (534), a connecting plate (538) is fixedly installed on one side of the stirring blade (536), and a motor (531) is fixedly installed on one side of the liquid storage tank (1), and the output shaft of the motor (531) and the connecting plate (538) are fixedly connected via a support frame (537).

2. The continuous separation device for recombinant collagen according to claim 1, characterized in that: The bottom end of the annular plate (534) is connected to a filter cartridge (535) and a resin column for adsorbing impurities is provided inside the filter cartridge (535).

3. The continuous separation device for recombinant collagen according to claim 1, characterized in that: A rotating rod (532) is rotatably mounted inside the separation cylinder (51), and a plurality of centrifugal impellers (533) for mixing the liquid inside the separation cylinder (51) are fixedly mounted on the outer surface of the rotating rod (532). The output shaft of the motor (531) is fixedly connected to the rotating rod (532), and a circular ring (54) is mounted on the outer surface of the separation cylinder (51), and a third adding cylinder (56) is mounted on the outer surface of the circular ring (54).

4. The continuous separation device for recombinant collagen according to claim 1, characterized in that: An anti-blocking component (539) is provided inside the separation cylinder (51), and the anti-blocking component (539) includes a scraper (5393) rotatably mounted on the outer surface of the rotating rod (532), and the scraper (5393) slides on the inner wall of the centrifugal net (52). A rotating plate (5391) is fixedly mounted on one side of the connecting plate (538), and a top plate (5392) is fixedly mounted on the outer surface of the rotating plate (5391), and the top plate (5392) is fixedly connected to one end of the scraper (5393).

5. The continuous separation device for recombinant collagen according to claim 4, characterized in that: The scraper (5393) is provided with ventilation holes (5394) on the surface close to the centrifugal net (52), and a cavity (5399) is provided inside the scraper (5393), and a plurality of breathable films (5395) are installed inside the cavity (5399).

6. The continuous separation device for recombinant collagen according to claim 5, characterized in that: A limiting rod (5396) is slidably installed inside the cavity (5399), and a plurality of resistance springs (5397) are arranged between the limiting rod (5396) and the breathable film (5395). A sticky plate (5398) for adhering impurities is installed inside the cavity (5399).

7. The continuous separation device for recombinant collagen according to claim 1, characterized in that: A circulation assembly (55) is provided on the outside of the separation cylinder (51), and the circulation assembly (55) includes a connecting pipe (551) connected and installed between the top end and the bottom end of the separation cylinder (51), and a suction pump (552) is provided on the connecting pipe (551) for driving the liquid inside the connecting pipe (551).

8. The continuous separation device for recombinant collagen according to claim 1, characterized in that: A fermentation mechanism (2) is provided at the top of the liquid storage tank (1), and the fermentation mechanism (2) includes a fermentation liquid barrel (24) installed at the top of the liquid storage tank (1). A water pump (21) is fixedly installed at the top of the liquid storage tank (1), and the output end of the water pump (21) is connected to the fermentation liquid barrel (24) through a feed pipe (22). A first addition cylinder (25) is provided on the side wall of the fermentation liquid barrel (24), and a sedimentation tank (23) is provided at the bottom end of the fermentation liquid barrel (24).

9. The continuous separation device for recombinant collagen according to claim 8, characterized in that: The bottom end of the fermentation liquid barrel (24) is connected to a chromatography cylinder (3) and installed thereon. A second addition cylinder is provided on one side of the chromatography cylinder (3). The bottom end of the chromatography cylinder (3) is connected to an elution cylinder (4) and installed thereon. The bottom end of the elution cylinder (4) is connected to a separation cylinder (51) and installed thereon. The bottom end of the separation cylinder (51) is connected to a collection cylinder (7) and installed thereon via a discharge pipe (6).

10. A method for continuous separation of recombinant collagen, comprising the following steps: Step 1: Collect the fermentation broth or cell lysate to ensure that it contains recombinant collagen; Step 2: Use phosphate buffer to equilibrate the affinity chromatography column to allow the filler in the column to reach a stable state. During the equilibration process, monitor the UV absorbance to ensure a stable baseline. Step 3: Slowly add the treated sample to the affinity chromatography column, control the flow rate to allow the recombinant collagen to fully bind to the affinity ligand on the filler, and collect the flow-through for subsequent analysis; Step 4: Wash the affinity column with binding buffer to remove unbound impurity proteins and monitor the UV absorbance until the absorbance drops to the baseline level, indicating that the impurities have been basically removed; Step 5: Change the elution conditions (increase the imidazole concentration) to dissociate the recombinant collagen from the affinity ligand, and collect the eluate, which is the preliminarily purified recombinant collagen; Step 6: appropriately dilute or concentrate the eluate obtained from the affinity chromatography, and adjust its ionic strength and pH value to suit the conditions of ion exchange chromatography; Step 7: Slowly add the treated sample to the ion exchange chromatography column, controlling the flow rate to allow the recombinant collagen to interact with the ionic groups on the filler; Step 8: Gradually increase the salt concentration (sodium chloride concentration) in the elution buffer to elute protein molecules with different charge properties in sequence, collect the eluate containing recombinant collagen, use elution buffers with different salt concentrations to perform staged elution, and collect the eluate from each stage, which is the purified recombinant collagen.

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