Device and method for continuously separating target object by using magnetic beads

Abstract

The invention discloses a device and method for continuously separating a target object through magnetic beads, the device comprises a magnetic attraction pool, a driving part, a leaching part and a double-roller structure, the double-roller structure comprises a magnetic insulation outer cylinder and a magnetic inner cylinder, the diameter of the magnetic inner cylinder is smaller than the size of the magnetic insulation outer cylinder, the magnetic inner cylinder is inserted into the magnetic insulation outer cylinder, and a rotating shaft is located above the axis of the magnetic inner cylinder; grooves extending in the axial direction are evenly distributed in the peripheral face of the magnetism-insulating outer cylinder, and the end covers are packaged at the two axial ends of the magnetism-insulating outer cylinder. When target impurity liquid and magnetic bead liquid are mixed in proportion, magnetic beads can be combined with target objects in the target impurity liquid and the magnetic bead liquid to form magnetic bead conjugates, the magnetic bead conjugates are separated out through the impurity liquid washing-out unit, after the separated magnetic bead conjugates are eluted, the target objects and the magnetic beads can be separated, the magnetic beads are recycled through the target liquid separation unit, and the target liquid is collected. According to the invention, automatic raw material adding and automatic target object and magnetic bead recovery can be realized, and continuous and industrial production of purification and separation of the target object by a magnetic bead method can be realized.

Description

Technical Field

[0001] The invention belongs to the field of separation, and in particular relates to a device and method for continuously separating target objects using magnetic beads. Background Art

[0002] Purification of biological substances is used to separate and purify the desired target molecules in a complex biological mixture matrix to obtain biological substances with high purity, stable properties and high recovery rate. Purification of biological substances generally requires centrifugation, membrane separation, salting out and chromatography steps. Chromatographic methods commonly used in the prior art include: gel filtration chromatography, ion exchange chromatography, hydrophobic layer plates, affinity / immunoprecipitation chromatography, multimodal chromatography, reversed phase chromatography, ion exchange chromatography, hydrophobic layer plates, affinity / immunoprecipitation chromatography, multimodal chromatography and reversed phase chromatography are all adsorption chromatography technologies.

[0003] The principles of affinity chromatography and immunoprecipitation chromatography are the same, both of which are to make the specific site of the target object bind to the ligand on the surface of the microsphere. Based on the characteristics of this type of chromatography technology, magnetic bead technology was developed, that is, adding metal substances to the affinity chromatography microspheres, so that the target substance can be quickly separated without column loading. By improving the microspheres, all the adsorption chromatography technologies introduced above can be replaced in the research and development stage.

[0004] Compared with traditional adsorption chromatography technology that requires column packing, the advantages of magnetic bead technology are: 1) It is easy to use and has low requirements for sample pretreatment; 2) It saves time. When purifying target molecules, it is only necessary to wash the magnetic beads 3 to 5 times with equilibrium buffer and add them to the sample. This process usually takes less than 5 minutes; 3) It has low requirements for equipment and only requires a magnetic stand; 4) It can achieve high-throughput purification. When purifying multiple samples, the magnetic bead purification method can be carried out on several samples in parallel at the same time.

[0005] However, the existing magnetic bead technology still has some disadvantages: 1) Sample processing capacity is limited. Since the magnetic bead purification instrument is based on the principle of magnetic field for separation, the effect of the magnetic field on the target is limited by distance. The existing magnetic frames and magnetic rods with limited size can only work at the milliliter level and upgrade level. This limits the magnetic bead technology to mainly serve scientific research circles such as universities and research institutes or corporate R&D. 2) Scale-up is hindered. Due to the limitation of sample processing capacity, the purification method currently developed by the magnetic bead method ultimately relies on the filler method for industrial production, which will lead to discontinuous scale-up of the purification process. Summary of the invention

[0006] The purpose of the present invention is to provide a device that utilizes magnetic beads to continuously separate target objects so as to solve the technical problem in the prior art that magnetic bead technology cannot be used to replace adsorption chromatography to quickly extract target objects from large-volume (ton-level) raw materials; the purpose of the present invention is also to provide a method for continuously separating target objects using the above-mentioned device.

[0007] To achieve the above object, a device for continuously separating target objects using magnetic beads of the present invention adopts the following technical solution:

[0008] A device for continuously separating a target object by using magnetic beads, comprising a foreign liquid washing unit and a target object separation unit, wherein the foreign liquid washing unit is used to separate a magnetic bead conjugate from foreign liquid, and the target object separation unit is used to separate the magnetic beads and the target object in the magnetic bead conjugate, wherein the foreign liquid washing unit and the target object separation unit are connected through a pipeline and both comprise a magnetic suction pool, a receiving pool, a driving part, a washing part and a double drum structure;

[0009] The upper end of the magnetic absorption pool is open to allow the double-drum structure to absorb the magnetic beads and / or magnetic bead conjugates therein;

[0010] The double-drum structure comprises an outer magnetic-insulating cylinder, a magnetic inner cylinder, a rotating shaft, an end cover and a driving part, wherein the driving part is used to drive the outer magnetic-insulating cylinder to rotate around the rotating shaft, the diameter of the magnetic inner cylinder is smaller than the size of the outer magnetic-insulating cylinder and is inserted therein, the rotating shaft is located above the axis of the magnetic inner cylinder so that when a certain position on the outer circumference of the outer magnetic-insulating cylinder rotates from bottom to top, the distance between the position and the magnetic inner cylinder gradually increases; the outer circumference of the outer magnetic-insulating cylinder is uniformly provided with grooves extending in the axial direction, the end covers are encapsulated at the axial ends of the outer magnetic-insulating cylinder, the grooves are inclined so that one end thereof tilts downward when the outer magnetic-insulating cylinder rotates to the top;

[0011] The rinsing part includes a rinsing head for spraying a cleaning liquid;

[0012] The position of the receiving pool corresponds to the position of the double-drum structure to receive the magnetic beads and / or magnetic bead combinations that roll down along the grooves under the action of the cleaning liquid.

[0013] Furthermore, the impurity liquid washing out unit and the target object separation unit also include a recovery pool, the bottom of which is open to allow the lower half of the double-drum structure to pass through, the bottom wall of the recovery pool is high in the middle and low around so that excess cleaning liquid flowing down the magnetically insulating outer cylinder can be gathered around, and an outlet is opened on the bottom wall for the accumulated liquid to flow out.

[0014] Furthermore, the rinsing part also includes a rinsing rack, the rinsing heads are fixed on the rinsing rack and are located above the double-drum structure, and the rinsing heads are arranged in multiple groups, and the multiple groups of rinsing heads are distributed at intervals along the axial direction of the double-drum structure.

[0015] Furthermore, it also includes a liquid preparation unit for storing raw materials, an incubation pool for mixing magnetic bead liquid and target impurity liquid, and an elution pool for mixing magnetic bead conjugate and elution liquid. The discharge end of the incubation pool is connected to the magnetic suction pool in the impurity liquid elution unit, and the discharge end of the elution pool is connected to the magnetic suction pool in the target separation unit. A part of the discharge ports of the liquid preparation unit are connected to the incubation pool through pipelines to transfer target impurity liquid and magnetic bead liquid to it, the corresponding discharge ports of the liquid preparation unit are connected to the elution pool through pipelines to transfer eluate to it, the corresponding discharge ports of the liquid preparation unit or the impurity liquid elution unit are connected to the elution pool through pipelines to transfer magnetic bead conjugate to it, and the incubation pools are arranged in series in multiple groups to allow the magnetic bead liquid to fully combine with the target impurity liquid, and the elution pools are arranged in series in multiple groups to allow the eluate to fully combine with the magnetic bead conjugate.

[0016] Furthermore, the magnetic inner cylinder is naturally placed in the magnetically insulated outer cylinder, and both ends of the magnetic inner cylinder are open; the outer circumferential surface of the magnetically insulated outer cylinder is a conical surface, and the end cover at the small diameter end is concave to form a recessed portion, and the receiving pool is partially inserted into the recessed portion; the inner circumferential surface of the magnetically insulated outer cylinder, the inner circumferential surface of the magnetic inner cylinder, and the outer circumferential surface of the magnetically insulated outer cylinder are all conical surfaces, and the tapers of the three are the same as the taper of the outer circumferential surface of the magnetically insulated outer cylinder.

[0017] Furthermore, along the flow direction of the fluid, a secondary recovery unit is provided after the impurity washing out unit and the target separation unit, and the secondary recovery unit includes a secondary recovery pool and an electromagnet, and the electromagnet is located below the secondary recovery pool, and an inlet is provided at one end of the secondary recovery pool, and a first outlet and a second outlet are provided at the other end, a first solenoid valve is provided at the first outlet, and a second solenoid valve is provided at the second outlet, the outlet of the magnetic suction pool and the outlet of the recovery pool are connected with the inlet of the corresponding secondary recovery pool through a pipeline, and the first outlet is connected with the inlet end of the corresponding incubation pool / elution pool through a pipeline.

[0018] Furthermore, it also includes a collecting unit, and the second discharge port of the secondary recovery unit is connected to the collecting unit through a pipeline; the distance between the bottom of the double-drum structure and the bottom of the magnetic suction pool is adjustable;

[0019] It also includes a similar molecule separation unit and a similar molecule elution pool arranged in front of the similar molecule separation unit, the similar molecule separation unit also includes the magnetic attraction pool, the receiving pool, the driving part, the elution part and the double-drum structure, the similar molecule separation unit is arranged between the impurity liquid elution unit and the target separation unit, the corresponding discharge port of the liquid preparation unit is connected to the similar molecule elution pool through a pipeline alone or together with the corresponding discharge port of the impurity liquid elution unit, the discharge end of the similar molecule elution pool is connected to the magnetic attraction pool of the similar molecule separation unit through a pipeline, and the outlet of the receiving pool in the similar molecule separation unit is used to transfer materials to the elution pool.

[0020] A method for continuously separating a target object using magnetic beads of the present invention adopts the following technical solution:

[0021] A method for continuously separating a target object using magnetic beads comprises the following steps: a first step of pre-treating a raw material containing the target object to be separated into a target impurity liquid, and pre-treating the magnetic beads into a magnetic bead liquid;

[0022] In the second step, the target impurity liquid and the magnetic bead liquid are continuously added in proportion, and after being mixed evenly, they are sent to the impurity liquid washing unit to separate the magnetic bead conjugate and the impurity liquid;

[0023] The third step is to continuously add the magnetic bead conjugate separated in the impurity washing unit and the elution liquid in proportion, and after being mixed evenly, send them to the target separation unit to separate the magnetic beads and the target;

[0024] This method is mainly used to replace affinity / immunoprecipitation chromatography technology to achieve rapid and continuous extraction of target substances from large volumes of raw materials.

[0025] Furthermore, the method for continuously separating the target object using magnetic beads also includes a fourth step: preparing the magnetic beads separated in the target object separation unit into a standard magnetic bead solution to be re-sent to the first step for use;

[0026] A secondary recovery unit is provided after the impurity liquid washing out unit and the target separation unit; in the second step, the impurity liquid separated in the impurity liquid washing out unit is sent to the subsequent secondary recovery unit, the magnetic beads and magnetic bead conjugates therein are recovered and the recovered products are mixed with the target impurity liquid again; in the third step, the target separated in the target separation unit is sent to the subsequent secondary recovery unit, the magnetic beads and magnetic bead conjugates therein are recovered and the recovered products are mixed with the eluent again.

[0027] Another method of the present invention for continuously separating a target object using magnetic beads adopts the following technical solution:

[0028] A method for continuously separating a target object using magnetic beads comprises the following steps: a first step of pre-treating a raw material containing a target object to be separated into a target impurity liquid, and pre-treating magnetic beads into a magnetic bead liquid;

[0029] In the second step, the target impurity liquid and the magnetic bead liquid are continuously added in proportion, and after being evenly mixed, they are sent to the impurity liquid washing unit to separate the magnetic bead conjugate and the impurity liquid;

[0030] The third step is to continuously add the magnetic bead conjugate separated in the impurity washing unit and the elution liquid in proportion, and after being mixed evenly, send them to the similar molecule separation unit to separate the similar molecules in the magnetic bead conjugate;

[0031] In the fourth step, the mixture of the magnetic bead conjugate separated in the similar molecule separation unit and the magnetic beads and the eluent 2 are continuously added in proportion, and after being mixed evenly, are sent to the target separation unit to separate the magnetic beads and the target;

[0032] In the fifth step, the magnetic beads separated in the target separation unit are prepared into a standard magnetic bead solution and then sent back to the first step for use.

[0033] This method is mainly used to replace ion exchange chromatography, hydrophobic chromatography, multimodal chromatography and reverse phase chromatography to achieve rapid and continuous extraction of target substances from large volumes of raw materials.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The magnetic inner cylinder will absorb the magnetic bead conjugate in the magnetic pool through the magnetic-insulating outer cylinder. However, due to the existence of the magnetic-insulating outer cylinder, the adsorbed magnetic bead conjugate will stay on the outer wall surface of the bottom of the magnetic-insulating outer cylinder and gather at the bottom of the groove as much as possible. When the magnetic-insulating outer cylinder rotates, the magnetic beads at the bottom will rotate upwards. When the outer wall surface of the magnetic-insulating outer cylinder to which the magnetic beads are attached rotates to the top, the adsorption force of the magnetic inner cylinder on the magnetic bead conjugate is significantly reduced to the point of demagnetization. The water source sprayed from the elution head can wash the magnetic beads down, and the water flow carries the magnetic beads downward along the groove and falls into the receiving pool.

[0036] 2. The liquid preparation unit puts the fermentation liquid and magnetic beads to be processed as needed. After the fermentation liquid and the magnetic beads are mixed in the three-stage incubation pool, the magnetic beads will separate the target substance in the fermentation liquid and adsorb it. After the mixed liquid is sent to the magnetic absorption pool, the magnetic bead conjugate in the mixed liquid can be separated with the continuous operation of the magnetic-free outer cylinder and the elution part. After being treated with the eluent, the magnetic beads are separated from the target, and the target separation unit is used to continuously separate the magnetic beads to obtain the target. The present invention can realize the automatic addition of raw materials, the automatic recovery of the target and the automatic recovery of the magnetic beads, can process tons of or even larger volumes of fermentation liquid, and can realize the continuous production of the magnetic bead method for purifying and separating the target.

[0037] 3. The axis of the rotating shaft coincides with the axis of the magnetically insulated outer cylinder, so it has the advantages of stable rotation, low noise and low malfunction.

[0038] 4. The present invention can replace ion exchange chromatography to extract amino acids, polypeptides, proteins, nucleotides, nucleic acids, plasmids, viruses, and lipids; it can also replace hydrophobic chromatography to extract proteins, enzymes, polysaccharides, nucleic acids, plasmids, viruses, ammonium sulfate protein samples (containing high salt), and protein macromolecules; it can replace affinity / immunoprecipitation chromatography to extract recombinant proteins, enzyme preparations, antibodies, antigens, hormones, viruses, lectins, cell membrane components, and cells; it can also replace multimodal chromatography to extract proteins, enzymes, polysaccharides, nucleic acids, and viruses; and it can replace reverse phase chromatography to extract peptides and oligonucleotides.

[0039] The parts not involved in the device are the same as the existing technology or can be implemented by using the existing technology. The structure of the invention is scientific and reasonable, and it is safe and convenient to use, which provides great help to people. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0041] Figure 1 It is a structural schematic diagram of an embodiment of a device for continuously separating target objects using magnetic beads according to the present invention;

[0042] Figure 2 It is a structural schematic diagram of the impurity washing unit;

[0043] Figure 3 It is a structural schematic diagram of a double drum structure;

[0044] Figure 4 It is a schematic diagram of the structure of the secondary recovery unit;

[0045] Figure 5 It is a structural schematic diagram of the impurity washing unit from another perspective;

[0046] Figure 6 is a system block diagram of a device that continuously separates target objects using magnetic beads;

[0047] Figure 7 is a process flow chart of a method 1 for continuously separating a target substance according to the present invention;

[0048] Figure 8 is a process flow chart of a second method for continuously separating a target substance according to the present invention;

[0049] In the figure: 1 liquid preparation unit, 2 incubation tank, 3 impurity washing unit, 4 target separation unit, 5 pipeline, 6 collection unit, 7 secondary recovery unit, 8 elution tank, 9 magnetic bead regeneration system, 11 magnetic bead bin, 12 raw material bin, 13 cleaning liquid bin, 14 magnetic bead conjugate bin, 15 elution liquid bin, 31 magnetic suction tank, 32 double drum structure, 33 driving part, 34 elution part, 35 recovery tank, 36 receiving tank, 321 magnetic inner cylinder, 322 magnetically insulated outer cylinder, 323 rotating shaft, 324 end cover, 3221 groove, 3241 recessed part, 61 impurity recovery bin, 62 target recovery bin, 63 magnetic bead conjugate recovery bin, 64 magnetic bead recovery bin, 65 similar molecule recovery bin, 71 secondary recovery tank, 72 feeding port, 73 first discharge port, 74 second discharge port, 75 electromagnet. DETAILED DESCRIPTION

[0050] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.

[0051] Embodiment 1 of a device for continuously separating target objects using magnetic beads according to the present invention:

[0052] A device for continuously separating target objects using magnetic beads, the specific structure of which is as follows Figures 1 to 7 As shown, it includes a liquid preparation unit 1, an incubation pool 2, a foreign liquid washing unit 3, a target separation unit 4, a secondary recovery unit 7 and a collection unit 6, and each part is connected by a pipeline 5. This embodiment is used to replace affinity chromatography and immunoprecipitation chromatography technology, wherein the magnetic beads used are specific adsorption magnetic beads, such as affinity magnetic beads and immunoprecipitation magnetic beads.

[0053] The liquid preparation unit 1 is used to prepare various raw materials needed in the production process, including a magnetic bead bin 11 for storing magnetic beads, a raw material bin 12 for storing raw materials containing target materials, a cleaning liquid bin 13 for storing cleaning liquid, an eluent bin 15 for storing eluent, and a magnetic bead conjugate bin 14 for storing magnetic bead conjugates. The materials in the magnetic bead bin 11 and the raw material bin 12 are fed to the incubation pool 2 at a set flow rate, so that the magnetic beads and raw materials are continuously added in proportion.

[0054] In this embodiment, two cleaning liquid tanks 13 are arranged to supply cleaning liquid to the impurity washing unit 3 and the elution part 34 of the target separation unit 4 respectively.

[0055] The original magnetic bead protection solution is replaced by a buffer solution in the magnetic bead bin 11. The composition of the buffer solution is determined specifically according to the target object, and the pH range, imidazole concentration and salt ion concentration of the buffer solution need to be set. The target impurities are stored in the raw material bin 12. The target impurities are obtained by breaking the cell wall and lysing the substances in the cells under the protection of the buffer solution. The cleaning solution is used to be sent to the elution part, and the cleaning solution bin 13 is configured with a cleaning solution with a pH buffering capacity, imidazole concentration and salt ion concentration within a certain range. The concentration of the magnetic bead conjugate sent in is reconfigured using a buffer solution in the magnetic bead conjugate bin 14.

[0056] The collecting unit 6 is used to collect the target substance and impurities generated during the purification process, and includes an impurity recovery chamber 61 , a target substance recovery chamber 62 , a magnetic bead conjugate recovery chamber 63 and a magnetic bead recovery chamber 64 .

[0057] The magnetic beads and raw materials in the liquid preparation unit 1 are mixed through the incubation process and then sent to the impurity liquid washing unit 3, which performs the classification process one to obtain the magnetic bead conjugate and impurity liquid. The magnetic bead conjugate is sent to the target separation unit 4, which performs the separation operation two to obtain the magnetic beads and the target; the impurity liquid and the excess cleaning liquid flowing down from the outside of the double drum structure 32 are sent to the impurity liquid recovery bin 61 after the magnetic beads and magnetic bead conjugate therein are recovered by the first group of secondary recovery units 7, and the recovered magnetic beads and magnetic bead conjugate are sent to the incubation process again. The target is sent to the target recovery bin 62 after the magnetic beads and magnetic bead conjugate therein are recovered by the second group of secondary recovery units 7, and the recovered magnetic beads and magnetic bead conjugate are sent to the elution process again.

[0058] Incubation process: three groups of incubation pools 2 are arranged in series along the flow direction of the fluid. The material enters the incubation pool 2 at the first end through the feed port. After preliminary mixing in the incubation pool 2 at the first end, it is sent to the next two incubation pools 2 in sequence to ensure that the two are fully mixed. The discharge port of the incubation pool 2 at the tail end is used to send out the incubated mixture.

[0059] Separation operation 1: After the magnetic beads and the raw material containing the target (also called the target impurity liquid) are mixed in the incubation process, the magnetic beads will adsorb the target in the raw material to form a magnetic bead conjugate. Therefore, the mixture sent out in the incubation process contains magnetic bead conjugates, impurities and buffer components. These components enter the impurity washing unit 3 together and flow to the magnetic suction pool 31 in a stable and dilute posture. During the flow of the mixture in the magnetic suction pool 31, the magnetic bead conjugate and a small amount of magnetic beads are quickly adsorbed on the outside of the double-drum structure 32 under the magnetic attraction of the double-drum structure 32, and rotate with it; the elution part 34 rinses the outside of the double-drum structure 1, and the magnetic bead conjugate and a small amount of magnetic beads roll down along the conical surface of the magnetic-insulating outer cylinder 322 to the receiving pool 36 under the flushing action of the cleaning liquid. The impurities and buffer components in the magnetic pool 31 continue to flow along the magnetic pool 31 to the first group of secondary recovery pools 7; while the cleaning liquid rinses the outside of the double-drum structure 32, the excess cleaning liquid continues to flow to the first group of secondary recovery pools 7 through the recovery pool 35. The first group of secondary recovery pools 7 recover the magnetic bead conjugates and a small amount of magnetic beads and then send them back to the incubation pool 2. The remaining miscellaneous liquid in the first group of secondary recovery pools 7 enters the miscellaneous liquid recovery bin 61, and the magnetic bead conjugates collected in the receiving pool 36 enter the magnetic bead conjugate recovery bin 63.

[0060] Elution process: The magnetic bead conjugate and the elution liquid are mixed in a certain proportion, and the elution operation is performed to separate the magnetic beads from the target object bound thereto. The elution process also uses three-stage elution pools 8 connected in sequence to ensure that the magnetic beads and the conjugate are fully separated. The structure of the three-stage elution pool 8 is the same as that of the three-stage incubation pool 2, and its specific structure and principle will not be repeated here.

[0061] Separation operation 2: The components of the mixture after the elution process include magnetic beads, a small amount of magnetic bead conjugates, target substances and buffer solutions. The mixture enters the target separation unit 4 and flows to the magnetic suction pool 31 in a stable and dilute state. During the flow in the magnetic suction pool 31, a small amount of magnetic bead conjugates and magnetic beads are quickly adsorbed on the outside of the double-drum structure 32 under the magnetic attraction of the double-drum structure 32, and rotate with it; the elution part 34 rinses the outside of the double-drum structure 32, and a small amount of magnetic bead conjugates and magnetic beads roll down along the cone of the magnetic-insulating outer cylinder 322 to the receiving pool 36 under the action of the washing liquid. The target substances and buffer solution components in the magnetic suction pool 31 continue to flow along the magnetic suction pool 31 to the second group of secondary recovery pools 7; while the washing liquid rinses the outside of the double-drum structure 32, the excess washing liquid will continue to flow to the second group of secondary recovery pools 7 through the recovery pool 35. The magnetic beads and a small amount of magnetic bead conjugates recovered by the second group of secondary recovery pools 7 are sent back to the elution pool 8, the target objects in the second group of secondary recovery pools 7 enter the target object recovery bin 2, and the magnetic beads collected by the receiving pool 36 enter the magnetic bead recovery bin 64.

[0062] Magnetic bead regeneration process: The magnetic beads recovered from the target separation unit 4 to the magnetic bead recovery bin 64 flow along the pipeline 5 to the magnetic bead regeneration system 9 for standardized configuration of the magnetic bead liquid and then enter the incubation pool 2 for recycling. This operation can use a small amount of magnetic beads to continuously achieve target extraction and reduce costs. The flow power of the fluid along the pipeline can be provided by a pump, which belongs to the prior art and will not be described in detail in this application.

[0063] The specific structure of the impurity washing unit 3 is as follows: Figure 2 and Figure 3 , including a magnetic suction pool 31, a double-drum structure 32, a driving part 33, a elution part 34, a recovery pool 35 and a receiving pool 36.

[0064] The double-drum structure 32 includes a magnetic inner cylinder 321, a magnetically insulated outer cylinder 322, a rotating shaft 323 and an end cap 324. The magnetically insulated outer cylinder 322 is fixed to the outer periphery of the rotating shaft 323 to rotate therewith. The magnetic inner cylinder 321 is inserted into the magnetically insulated outer cylinder 322. The rotating shaft 323 does not contact the magnetic inner cylinder 321. Therefore, when the rotating shaft 323 drives the magnetically insulated outer cylinder 322 to rotate, it does not drive the magnetic inner cylinder 321 to rotate. The magnetically insulated outer cylinder 322 is made of plastic, which does not isolate the magnetic field and is not magnetized. Since the diameters of the magnetic inner cylinder 321 and the magnetically insulated outer cylinder 322 are different, and the lower end of the magnetic inner cylinder 321 contacts the inner wall surface of the lower end of the magnetically insulated outer cylinder 322 under the action of gravity, the distance between any point on the outer circumference of the magnetically insulated outer cylinder 322 and the magnetic inner cylinder 321 gradually increases during the process of rotating from bottom to top. In other words, the magnitude of the magnetic force at different positions of the magnetically insulated outer cylinder 322 in the circumferential direction is different. Specifically, the distance between the lower end of the outer circumference of the magnetic inner cylinder 321 and the lower end of the outer circumference of the magnetically insulated outer cylinder 322 is the smallest (only the wall thickness of the magnetically insulated outer cylinder 322), and the distance between the upper end of the outer circumference of the magnetic inner cylinder 321 and the upper end of the outer circumference of the magnetically insulated outer cylinder 322 is the largest. Accordingly, the magnetic force at the lower end of the magnetically insulated outer cylinder 322 is the largest, and the magnetic force at the upper end is the smallest.

[0065] The recovery pool 35 is high in the middle and low around, and a hole is provided at the bottom thereof for the lower part of the double drum structure 32 to pass through. An outlet is provided at the lower end of the recovery pool 35 and is connected to the secondary recovery pool 7 through a pipeline 5.

[0066] The end caps 324 are located at both axial ends of the magnetically insulated outer cylinder 322, and their function is to seal both ends of the magnetically insulated outer cylinder 322. The outer circumferential surface of the magnetically insulated outer cylinder 322 is a frustum. The inner and outer wall surfaces of the magnetically insulated outer cylinder 322 and the inner and outer wall surfaces of the magnetic inner cylinder 321 have the same taper, so the magnetic forces on the outer circumferential surfaces of the magnetically insulated outer cylinder 322 that are in corresponding positions in the circumferential direction and located at different axial sections in the axial direction are the same. The outer circumferential surface of the magnetically insulated outer cylinder 322 is provided with a plurality of grooves 3221 extending in the axial direction, and the end cap 324 at the small diameter end is concave to form a recessed portion 3241.

[0067] The elution part 34 includes an elution rack 341, an elution pipe 342, an elution head 343 and an elution pool 344. The receiving pool 36 is fixed on the elution rack 341 and partially extends into the recessed portion 3241. The elution pool 344 is fixed on the elution rack 341. The elution heads 343 are arranged in multiple groups and extend along the axial direction of the magnetically insulated outer cylinder 322.

[0068] The working principle of the double-drum structure is as follows: the magnetic inner cylinder 321 will absorb the magnetic bead conjugate and magnetic beads in the magnetic pool 31 through the magnetic-insulating outer cylinder 322, but due to the existence of the magnetic-insulating outer cylinder 322, the adsorbed magnetic bead conjugate and magnetic beads will stay on the outer wall surface at the bottom of the magnetic-insulating outer cylinder 322 and gather as much as possible at the bottom of the groove 3221. When the magnetic-insulating outer cylinder 322 rotates, the magnetic beads and magnetic bead conjugate at the bottom are blocked by the groove wall of the groove 3221 and will rotate upward with the magnetic-insulating outer cylinder 322. When the outer wall surface of the magnetic-insulating outer cylinder 322 to which the magnetic beads and magnetic bead conjugate are attached rotates to the top, the adsorption force of the magnetic inner cylinder 321 on the magnetic beads and magnetic bead conjugate is significantly reduced to the point of demagnetization.

[0069] The working principle of the elution part is as follows: the cleaning liquid in the cleaning liquid tank 13 is sent to the elution pool 344 through the pipeline, the elution pipe 342 is connected to the bottom of the elution pool 344 and guides the cleaning liquid to multiple groups of elution heads 343, and the cleaning liquid finally flows out from the water outlet provided at the lower end of the elution head 343. The cleaning liquid sprayed from the water outlet can flush down the magnetic beads and the magnetic bead complex, and the water flow carries the magnetic beads and the magnetic bead complex downward along the groove 3221 and falls into the receiving pool 36.

[0070] The function of the recovery pool 35 is as follows: the excess cleaning liquid flowing down the circumference of the magnetic-insulating outer cylinder 322 will enter the secondary recovery pool 7 through the recovery pool 35, preventing excessive cleaning liquid from entering the magnetic absorption pool 31 and affecting the concentration of the magnetic bead conjugate therein and slowing down the processing efficiency.

[0071] The outlet of the receiving pool 36 is connected to the magnetic bead conjugate recovery chamber 63 through the pipeline 5. The magnetic bead conjugate collected by the magnetic bead conjugate recovery chamber 63 is then sent to the magnetic bead conjugate chamber 14. After the concentration and state are adjusted in the magnetic bead conjugate chamber 14, the eluent in the eluent chamber 15 is incubated in proportion and then sent to the target separation unit 4. The structure of the target separation unit 4 is the same as that of the impurity elution unit 3, and the only difference is that the raw materials sent in and the materials sent out are different. For details, please refer to the separation operation 2, which will not be repeated here.

[0072] The secondary recovery unit 7 includes a rectangular secondary recovery pool 71, a feed port 72 is arranged at one end of the secondary recovery pool 71 in the length direction, a first discharge port 73 and a second discharge port 74 are arranged at the other end in the length direction, a first solenoid valve is arranged at the first discharge port 73, and a second solenoid valve is arranged at the second discharge port 74, and an electromagnet 75 and a switch for controlling the power-on and power-off state of the electromagnet 75 are arranged at the bottom of the secondary recovery pool 71. When the electromagnet 75 is energized, the bottom plate of the secondary recovery pool 71 is magnetized and adsorbs the magnetic beads. At this time, the first solenoid valve is closed and the second solenoid valve is opened, and the liquid in the secondary recovery pool 71 flows to the miscellaneous liquid recovery bin 61 or the target recovery bin 62 through the second discharge port 74. When the electromagnet 75 loses power, the bottom plate of the secondary recovery pool 71 loses magnetism and can no longer adsorb the magnetic beads. At this time, the first solenoid valve is opened and the second solenoid valve is closed, and the magnetic beads and the magnetic bead conjugate in the secondary recovery pool 71 flow to the incubation pool 2 through the pipeline 5.

[0073] The second group of secondary recovery units 7 has the same structure as the first group of secondary recovery units 7, except that the inlet and outlet are connected to different silos. The second outlet 74 in the second group of secondary recovery units 7 is connected to the target recovery silo 62, and the first outlet 73 is connected to the elution tank 8.

[0074] It should be emphasized that the greater the distance between the bottom of the double-drum structure 32 and the bottom of the magnetic pool 31, the longer the time it takes for the magnetic inner cylinder 321 to adsorb the magnetic bead conjugate. Therefore, in a more preferred embodiment, the distance between the double-drum structure 32 and the magnetic pool 31 can be adjusted, so that the distance between the two can be fine-tuned after the device is installed to ensure that the double-drum structure 32 meets the standard for the collection rate of the magnetic bead conjugate in the magnetic pool 31 at the set speed. In this embodiment, an electric push rod is used to push the magnetic pool 31 up and down to adjust the distance between the double-drum structure 32 and the magnetic pool 31.

[0075] Embodiment 2 of a device for continuously separating target objects using magnetic beads according to the present invention:

[0076] The difference from Example 1 is that this example is used to replace ion exchange chromatography, hydrophobic chromatography, multimodal chromatography and reverse phase chromatography, and the magnetic beads used are non-specific magnetic beads such as ion exchange magnetic beads, hydrophobic magnetic beads, reverse phase magnetic beads, multimodal magnetic beads, etc.

[0077] However, non-specific magnetic beads will bind to some molecules similar to the target while binding to the target, so it is necessary to add an additional separation step for separating similar molecules between separation operation one and separation operation two. Therefore, another group of similar molecule separation units needs to be added between the impurity elution unit 3 and the target separation unit 4, and a group of elution pools 8 (similar molecule elution pools) are added before the similar molecule separation units.

[0078] The structure of the similar molecule separation unit is the same as that of the impurity washing unit 3, and the only difference is that the raw materials sent in and the materials sent out are different. What are sent into the similar molecule separation unit are magnetic beads, a combination of magnetic beads and a target, a combination of magnetic beads and similar molecules, and a buffer solution. In the similar molecule separation unit, an eluent 1 (the specific components are determined according to the similar molecules and the target, and this part belongs to the prior art and will not be described in detail here) is used to separate the similar molecules. What the similar molecule separation unit sends into the target separation unit 4 are magnetic beads, a combination of magnetic beads and a target (i.e., a magnetic bead combination). The similar molecules separated by the similar molecule separation unit are collected by the similar molecule recovery bin 65. It should be emphasized that the eluent used in the target separation unit 4 is different from the eluent 1 used in the similar molecule separation unit, and is an eluent 2 determined according to the target (selecting a specific eluent according to the target is a prior art and will not be described in detail in this application).

[0079] A set of secondary recovery units 7 is also arranged after the similar molecule separation unit. The buffer solution, the conjugate of magnetic beads and target, the conjugate of magnetic beads and similar molecules, the magnetic beads, and the eluent in the similar molecule separation unit are recovered by the secondary recovery unit 7, and then the conjugate of magnetic beads and target, and the conjugate of magnetic beads and similar molecules are sent to the elution pool 8 in front of the similar molecule separation unit again for re-elution of similar molecules.

[0080] Embodiment 1 of the method for continuously separating a target object using magnetic beads of the present invention:

[0081] Methods for continuous separation of target substances using magnetic beads, such as Figure 6 and Figure 7 As shown, the specific steps include:

[0082] In the first step, the raw material containing the target to be separated is pretreated to become a target impurity liquid, and the magnetic beads are pretreated to become a magnetic bead liquid;

[0083] In the second step, the target impurity liquid and magnetic bead liquid are continuously added in proportion, and after being evenly mixed, they are sent to the impurity liquid washing unit to separate the magnetic bead conjugate and the impurity liquid;

[0084] The third step is to continuously add the magnetic bead conjugate separated in the impurity washing unit and the elution liquid in proportion, and after being mixed evenly, send them to the target separation unit to separate the magnetic beads and the target;

[0085] In the fourth step, the magnetic beads separated in the target separation unit are prepared into a standard magnetic bead solution to be sent back to the first step for use.

[0086] The impurities separated in the second step are sent to the first set of secondary recovery units 7. After the first set of secondary recovery units 7 recover the magnetic beads and magnetic bead conjugates therein, the recovered products are sent back to the second step to be mixed with the target impurities to increase the loading capacity of the magnetic beads.

[0087] The target separated in the third step is first sent to the second group of secondary recovery units 7 to recover the magnetic beads and magnetic bead conjugates therein and then sent to the target object warehouse for collection. The magnetic beads and magnetic bead conjugates recovered in the second group of secondary recovery units 7 are sent back to the third step and mixed with the eluent to improve the recovery rate of the target.

[0088] Embodiment 2 of the method for continuously separating target objects using magnetic beads of the present invention:

[0089] The flowchart of this embodiment is as follows Figure 8 As shown, the specific steps include:

[0090] In the first step, the raw material containing the target to be separated is pretreated into a target impurity liquid, and the magnetic beads are pretreated into a magnetic bead liquid;

[0091] In the second step, the target impurity liquid and the magnetic bead liquid are continuously added in proportion, and after being mixed evenly, they are sent to the impurity liquid washing unit to separate the magnetic bead conjugate and the impurity liquid;

[0092] The third step is to continuously add the magnetic bead conjugate separated in the impurity washing unit and the elution liquid in proportion, and after being mixed evenly, send them to the similar molecule separation unit to separate the magnetic bead conjugate and the similar molecules;

[0093] In the fourth step, the mixture of the magnetic bead conjugate separated in the similar molecule separation unit and the magnetic beads and the eluent 2 are continuously added in proportion, and after being mixed evenly, are sent to the target separation unit to separate the magnetic beads and the target;

[0094] In the fifth step, the magnetic beads separated in the target separation unit are prepared into a standard magnetic bead solution and then sent back to the first step for use.

[0095] In the second step, the separated impurities are sent to the first set of secondary recovery units, which recover the magnetic beads and magnetic bead conjugates therein and then send the recovered products back to the second step to mix with the target impurities to increase the loading capacity of the magnetic beads.

[0096] The impurities separated in the third step are sent to the second set of secondary recovery units 7. The second set of secondary recovery units 7 recover the magnetic beads and magnetic bead conjugates therein and then send the recovered products back to the third step to be mixed with the eluent.

[0097] In the fourth step, the target object is first sent to the third group of secondary recovery units 7 to recover the magnetic beads and magnetic bead conjugates therein and then sent to the target object warehouse for collection. The magnetic beads and magnetic bead conjugates recovered in the third group of secondary recovery units 7 are re-sent to the fourth step and mixed with the elution solution 2 to improve the recovery rate of the target object.

[0098] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A device for continuously separating a target object using magnetic beads, characterized in that: It includes a debris washing unit and a target separation unit, the debris washing unit is used to separate the magnetic bead conjugate and the debris, the target separation unit is used to separate the magnetic beads and the target in the magnetic bead conjugate, the debris washing unit and the target separation unit are connected through a pipeline and both include a magnetic suction pool, a receiving pool, a driving part, a washing part and a double drum structure; The upper end of the magnetic absorption pool is open to allow the double-drum structure to absorb the magnetic beads and / or magnetic bead conjugates therein; The double-drum structure comprises an outer magnetic-insulating cylinder, a magnetic inner cylinder, a rotating shaft, an end cover and a driving part, wherein the driving part is used to drive the outer magnetic-insulating cylinder to rotate around the rotating shaft, the diameter of the magnetic inner cylinder is smaller than the size of the outer magnetic-insulating cylinder and is inserted therein, the rotating shaft is located above the axis of the magnetic inner cylinder so that when a certain position on the outer circumference of the outer magnetic-insulating cylinder rotates from bottom to top, the distance between the position and the magnetic inner cylinder gradually increases; the outer circumference of the outer magnetic-insulating cylinder is uniformly provided with grooves extending in the axial direction, the end covers are encapsulated at the axial ends of the outer magnetic-insulating cylinder, the grooves are inclined so that one end thereof tilts downward when the outer magnetic-insulating cylinder rotates to the top; The rinsing part includes a rinsing head for spraying a cleaning liquid; The position of the receiving pool corresponds to the position of the double-drum structure to receive the magnetic beads and / or magnetic bead combinations that roll down along the grooves under the action of the cleaning liquid.

2. The device for continuously separating target objects using magnetic beads according to claim 1, characterized in that: The impurity liquid washing unit and the target separation unit also include a recovery pool, the bottom of which is open to allow the lower half of the double-drum structure to pass through, the bottom wall of the recovery pool is high in the middle and low around so that the excess cleaning liquid flowing down the magnetically insulated outer cylinder is gathered around, and an outlet is opened on the bottom wall for the accumulated liquid to flow out.

3. The device for continuously separating target objects using magnetic beads according to claim 1, characterized in that: The rinsing part also includes a rinsing frame, the rinsing heads are fixed on the rinsing frame and are located above the double-drum structure, and the rinsing heads are arranged in multiple groups, which are spaced apart and distributed along the axial direction of the double-drum structure.

4. The device for continuously separating target objects using magnetic beads according to claim 1, characterized in that: It also includes a liquid preparation unit for storing raw materials, an incubation pool for mixing magnetic bead liquid and target impurities, and an elution pool for mixing magnetic bead conjugates and elution liquid. The discharge end of the incubation pool is connected to the magnetic suction pool in the impurity elution unit, and the discharge end of the elution pool is connected to the magnetic suction pool in the target separation unit. A part of the discharge ports of the liquid preparation unit are connected to the incubation pool through pipelines to transfer target impurities and magnetic bead liquid to the incubation pool, and the corresponding discharge ports of the liquid preparation unit are connected to the elution pool through pipelines to transfer eluate to the incubation pool, and the corresponding discharge ports of the liquid preparation unit or the impurity elution unit are connected to the elution pool through pipelines to transfer magnetic bead conjugates to the incubation pool. The incubation pools are arranged in series in multiple groups so that the magnetic bead liquid can be fully combined with the target impurities, and the elution pools are arranged in series in multiple groups so that the eluate can be fully combined with the magnetic bead conjugate.

5. The device for continuously separating target objects using magnetic beads according to claim 1, characterized in that: The magnetic inner cylinder is naturally placed in the magnetically insulated outer cylinder, and both ends of the magnetic inner cylinder are open; the outer circumferential surface of the magnetically insulated outer cylinder is a conical surface, and the end cover at the small diameter end is concave to form a recessed portion, and the receiving pool is partially inserted into the recessed portion; the inner circumferential surface of the magnetically insulated outer cylinder, the inner circumferential surface of the magnetic inner cylinder, and the outer circumferential surface of the magnetically insulated outer cylinder are all conical surfaces, and the tapers of the three are the same as the taper of the outer circumferential surface of the magnetically insulated outer cylinder.

6. The device for continuously separating target objects using magnetic beads according to claim 4, characterized in that: Along the flow direction of the fluid, a secondary recovery unit is arranged behind the impurity washing out unit and the target separation unit, and the secondary recovery unit includes a secondary recovery pool and an electromagnet, and the electromagnet is located below the secondary recovery pool, an inlet is arranged at one end of the secondary recovery pool, and a first outlet and a second outlet are arranged at the other end, a first solenoid valve is arranged at the first outlet, and a second solenoid valve is arranged at the second outlet, the outlet of the magnetic suction pool and the outlet of the recovery pool are connected with the inlet of the corresponding secondary recovery pool through a pipeline, and the first outlet is connected with the inlet end of the corresponding incubation pool / elution pool through a pipeline.

7. The device for continuously separating target objects using magnetic beads according to claim 6, characterized in that: It also includes a collecting unit, and the second discharge port of the secondary recovery unit is connected to the collecting unit through a pipeline; the distance between the bottom of the double-drum structure and the bottom of the magnetic suction pool is adjustable; It also includes a similar molecule separation unit and a similar molecule elution pool arranged in front of the similar molecule separation unit, the similar molecule separation unit also includes the magnetic attraction pool, the receiving pool, the driving part, the elution part and the double-drum structure, the similar molecule separation unit is arranged between the impurity liquid elution unit and the target separation unit, the corresponding discharge port of the liquid preparation unit is connected to the similar molecule elution pool through a pipeline alone or together with the corresponding discharge port of the impurity liquid elution unit, the discharge end of the similar molecule elution pool is connected to the magnetic attraction pool of the similar molecule separation unit through a pipeline, and the outlet of the receiving pool in the similar molecule separation unit is used to transfer materials to the elution pool.

8. A method for continuously separating a target object using the device for continuously separating a target object using magnetic beads according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: a first step of pre-treating a raw material containing a target object to be separated into a target impurity liquid, and pre-treating magnetic beads into a magnetic bead liquid; In the second step, the target impurity liquid and the magnetic bead liquid are continuously added in proportion, and after being evenly mixed, they are sent to the impurity liquid washing unit to separate the magnetic bead conjugate and the impurity liquid; In the third step, the magnetic bead conjugate separated in the impurity washing unit and the elution liquid are continuously added in proportion, and after being mixed evenly, they are sent to the target separation unit to separate the magnetic beads and the target.

9. A method for continuous separation of a target object according to claim 8, characterized in that: The fourth step is to prepare the magnetic beads separated in the target separation unit into a standard magnetic bead solution and then send it back to the first step for use; A secondary recovery unit is provided after the impurity liquid washing out unit and the target separation unit; in the second step, the impurity liquid separated in the impurity liquid washing out unit is sent to the subsequent secondary recovery unit, the magnetic beads and magnetic bead conjugates therein are recovered and the recovered products are mixed with the target impurity liquid again; in the third step, the target separated in the target separation unit is sent to the subsequent secondary recovery unit, the magnetic beads and magnetic bead conjugates therein are recovered and the recovered products are mixed with the eluent again.

10. A method for continuously separating a target object using the device for continuously separating a target object using magnetic beads according to claim 7, characterized in that: The method comprises the following steps: a first step of pre-treating a raw material containing a target object to be separated into a target impurity liquid, and pre-treating magnetic beads into a magnetic bead liquid; In the second step, the target impurity liquid and the magnetic bead liquid are continuously added in proportion, and after being evenly mixed, they are sent to the impurity liquid washing unit to separate the magnetic bead conjugate and the impurity liquid; The third step is to continuously add the magnetic bead conjugate separated in the impurity washing unit and the elution liquid in proportion, and after being mixed evenly, send them to the similar molecule separation unit to separate the magnetic bead conjugate and the similar molecules; In the fourth step, the mixture of the magnetic bead conjugate separated in the similar molecule separation unit and the magnetic beads and the eluent 2 are continuously added in proportion, and after being mixed evenly, are sent to the target separation unit to separate the magnetic beads and the target; In the fifth step, the magnetic beads separated in the target separation unit are prepared into a standard magnetic bead solution and then sent back to the first step for use.

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