Preparation method of high-purity exosome bulk drug

Abstract

The invention discloses a preparation method of a high-purity exosome bulk drug, which comprises the following steps: S1, taking fresh plasma, placing in a collection tube, adding 20 [mu] L of a solution A into every 5 mL of plasma, slightly reversing, uniformly mixing, and standing for a period of time; s2, separating the solution obtained in S1, taking the supernatant as serum, putting the supernatant into a centrifugal tube, repeatedly centrifuging once, and sucking the supernatant for later use; and S3, sucking the centrifuged serum through an injector, filtering through a filter, and transferring to a new centrifugal tube for later use. The streptag II is marked on the aptamer through dehydration condensation of carboxyl of the streptag II and amino on the aptamer, then the exosome capturing tool CD63 aptamer-magnetic bead conjugate is generated through the specific binding effect of the streptag II and streptavidin on the surface of the magnetic bead, and compared with a tedious three-step magnetic bead modification process in an existing immunomagnetic bead method, the time consumption is about 10 h, and the efficiency is high. Only two steps are needed for magnetic bead modification, about 2 h is consumed, the process is simple, and consumed time is short.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome API preparation, and specifically to a method for preparing a high-purity exosome API. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] With the development of regenerative medicine, the application of exosomes has received more and more attention. Exosomes are round extracellular membrane vesicles secreted by different types of cells, with specific lipids and proteins, and a size between 30 and 120 nm. Almost all mammalian cells can secrete vesicles. They have a lipid bilayer membrane structure and a typical cup-shaped morphology. They usually contain functional molecules such as proteins, lipids, mRNA, and microRNA. They are widely present in various body fluids, such as blood, saliva, urine, cerebrospinal fluid, and breast milk. Exosomes participate in intercellular material exchange and information transmission, regulate immune response, tumor invasion, cell proliferation and other biological behaviors, and have natural advantages and great potential to become drug carriers and biological raw materials for the treatment of related diseases. However, due to the complexity of the composition of the body fluid environment in which the exosomes are located and the nanoscale size of the exosomes themselves, it is still very challenging to separate and obtain exosomes with high purity and functional structural integrity from various body fluids.

[0004] At present, the commonly used methods for separation and purification of exosomes include ultracentrifugation (UC), immunoaffinity, polymer precipitation, nanomembrane filtration, size exclusion chromatography, etc. Among them, ultracentrifugation (UC) is the gold standard for separation and purification of exosomes, but this method is time-consuming, low-yield, and relies on expensive precision instruments and equipment. At the same time, multiple ultracentrifugation concentrations may damage the structural integrity of exosomes, which is not conducive to downstream analysis and application of exosomes. Other separation technologies based on physical properties such as exosome size, density, and solubility also have problems such as low yield, low purity, and long time consumption. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a high-purity exosome API to solve the problems in the above-mentioned background technology that the separation and purification of exosomes is costly and time-consuming, and that the morphological structure and physiological function of the exosomes are easily destroyed, the integrity of the exosome structure is destroyed, and the downstream analysis of the exosomes is not conducive. A method for preparing a high-purity exosome API proposed in the present invention comprises the following steps: S1: Take fresh plasma, place it in a collection tube, add 20 μL of solution A to every 5 mL of plasma, gently invert to mix, and leave it for a while; S2: Separate the solution obtained in S1, take the supernatant as serum, put it in a centrifuge tube, repeat centrifugation once, and take out the supernatant for later use; S3: The serum after centrifugation is drawn up through a syringe, filtered, and then transferred to a new centrifuge tube for later use; S4: Take the activated Strep-tagII protein purification magnetic beads, add the saturated coupling amount of anti-CD63, and mix for a period of time at room temperature; S5: washing the solution obtained in S4 with a separation buffer, and after washing, adding amino acids for dehydration condensation; S6: add an appropriate amount of separation buffer to suspend the solution obtained in S5 to finally form a magnetic bead suspension; S7: Add 5 times the solution obtained in S3 to the magnetic bead suspension obtained in S6 and incubate; S8: After the incubation is completed, the system is washed with solution B, and the magnetic beads are separated by a magnetic rack to obtain a complex adsorbed with exosomes; S9: Mix the complex obtained in S8 with 400 μL of the eluent, shake at room temperature, and then separate and take the supernatant, which is the high-purity exosome API.

[0006] Preferably, in S1, solution A is a blood coagulant, and the standing time is 30 minutes.

[0007] Preferably, in S2, the separation temperature is 4°C, the separation factor is 3000g, and the separation time is 5 minutes.

[0008] Preferably, in S4, the mass ratio between the anti-CD63 antibody and the Strep-tag II protein purification magnetic beads is 1:5.

[0009] Preferably, in S5, the separation buffer is a phosphate buffered saline solution containing 0.1% bovine serum albumin; preferably, the separation buffer is sterilized by filtering with a 0.22 micron filter membrane in advance.

[0010] Preferably, in S6, the amount of the separation buffer is 50-500 times the volume of the magnetic bead suspension.

[0011] Preferably, in S7, the incubation temperature is 37°C, the incubator parameters are 90° upside down rotation, 5 seconds tilt, 5° vibration for 1 second, and incubation time for 2 hours.

[0012] Preferably, in S8, the solution B is a phosphate buffered saline solution.

[0013] Preferably, in S9, the eluent is a glycine solution with a pH of 2.8 to 3.5, a glycine concentration of 0.1 to 0.2 mol / L, an oscillation time of 30 min, a separation factor of 3000 g, and a separation time of 5 min.

[0014] Preferably, in S1-S9, each result obtained is tested, recorded and uploaded to the traceability system.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention marks streptag II onto the aptamer through dehydration condensation between the carboxyl group of strep tag II and the amino group on the aptamer, and then generates an exosome capture tool, a CD63 aptamer-magnetic bead conjugate, through the specific binding of strep tag II and streptavidin on the surface of the magnetic beads. Compared with the cumbersome three-step magnetic bead modification process in the existing immunomagnetic bead method, which takes about 10 hours, the magnetic bead modification in the present invention only requires two steps and takes about 2 hours, so the process is simple and time-saving; The CD63 aptamer used in the present invention can specifically recognize the CD63 characteristic protein on the surface of exosomes, effectively overcoming the defect of co-separation of impurities such as lipoproteins in conventional exosome enrichment technology, greatly improving the purity of enriched exosomes, and ensuring the integrity of separated exosomes; The present invention uses deoxyribonuclease Dnase to destroy the CD63 aptamer structure, or based on the competitive binding between biotin solution, Strep-tag II and Strep-Tactin, to achieve the purpose of lossless release and recovery of exosomes adsorbed on the surface of magnetic beads, and the recovery rate of exosomes can reach more than 90%; The present invention uses CD63 aptamers to replace the CD63 antibodies used in the traditional immunomagnetic bead method. Compared with CD63 antibodies, CD63 aptamers can be rapidly synthesized in vitro in batches at low cost; and aptamers are not easily affected by external environmental factors such as temperature and pH, and their performance is more stable and reliable. Therefore, the invention can greatly reduce the economic cost of exosome separation and purification operations, while improving the stability and repeatability of experimental results. The present invention applies the biological raw material drug production quality control system and combines it with the traceability system to ensure the safe, effective and controllable production of exosome raw material drug preparations. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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.

[0017] The present invention provides a technical solution: a method for preparing a high-purity exosome raw material drug, comprising the following steps: S1: Take fresh plasma, place it in a collection tube, add 20 μL of solution A to every 5 mL of plasma, gently invert to mix, and leave it for a while; S2: Separate the solution obtained in S1, take the supernatant as serum, put it in a centrifuge tube, repeat centrifugation once, and take out the supernatant for later use; S3: The serum after centrifugation is drawn up through a syringe, filtered, and then transferred to a new centrifuge tube for later use; S4: Take the activated Strep-tagII protein purification magnetic beads, add the saturated coupling amount of anti-CD63, and mix for a period of time at room temperature; S5: washing the solution obtained in S4 with a separation buffer, and after washing, adding amino acids for dehydration condensation; S6: add an appropriate amount of separation buffer to suspend the solution obtained in S5 to finally form a magnetic bead suspension; S7: Add 5 times the solution obtained in S3 to the magnetic bead suspension obtained in S6 and incubate; S8: After the incubation is completed, the system is washed with solution B, and the magnetic beads are separated by a magnetic rack to obtain a complex adsorbed with exosomes; S9: Mix the complex obtained in S8 with 400 μL of the eluent, shake at room temperature, and then separate and take the supernatant, which is the high-purity exosome API.

[0018] In this embodiment, in S1, solution A is a blood coagulant, and the standing time is 30 minutes.

[0019] In this embodiment, in S2, the separation temperature is 4°C, the separation factor is 3000g, and the separation time is 5 minutes.

[0020] In this embodiment, in S4, the mass ratio between the anti-CD63 antibody and the Strep-tag II protein purification magnetic beads is 1:5.

[0021] In this embodiment, S5, the separation buffer is a phosphate buffered saline solution containing 0.1% bovine serum albumin; preferably, the separation buffer is sterilized by filtering with a 0.22 micron filter membrane in advance.

[0022] In this embodiment, in S6, the amount of the separation buffer is 50-500 times the volume of the magnetic bead suspension.

[0023] In this embodiment, in S7, the incubation temperature is 37°C, the incubator parameters are 90° inverted rotation, 5 seconds tilt, 5° vibration for 1 second, and incubation time for 2 hours.

[0024] In this embodiment, in S8, the solution B is a phosphate buffered saline solution.

[0025] In this embodiment, in S9, the eluent is a glycine solution with a pH of 2.8 to 3.5, a glycine concentration of 0.1 to 0.2 mol / L, an oscillation time of 30 min, a separation factor of 3000 g, and a separation time of 5 min.

[0026] In this embodiment, in S1-S9, each result obtained is tested, recorded and uploaded to the traceability system.

[0027] Embodiment 2: The difference from the first embodiment is that: S1: Take fresh plasma, place it in a collection tube, add 20 μL of solution A to every 5 mL of plasma, gently invert to mix, and leave it for a while; S2: Separate the solution obtained in S1, take the supernatant as serum, put it in a centrifuge tube, repeat centrifugation once, and take out the supernatant for later use; S3: The serum after centrifugation is drawn up through a syringe, filtered, and then transferred to a new centrifuge tube for later use; S4: Take the activated Strep-tagII protein purification magnetic beads, add the saturated coupling amount of anti-CD63, and mix for a period of time at room temperature; S5: washing the solution obtained in S4 with a separation buffer, and after washing, adding amino acids for dehydration condensation; S6: add an appropriate amount of separation buffer to suspend the solution obtained in S5 to finally form a magnetic bead suspension; S7: Add 5 times the solution obtained in S3 to the magnetic bead suspension obtained in S6 and incubate; S8: After the incubation is completed, the system is washed with solution B, and the magnetic beads are separated by a magnetic rack to obtain a complex adsorbed with exosomes; S9: Mix the complex obtained in S8 with 400 μL of the eluent, shake at room temperature, and then separate and take the supernatant, which is the high-purity exosome API.

[0028] In this embodiment, in S1, solution A is a blood coagulant, and the standing time is 30 minutes.

[0029] In this embodiment, in S2, the separation temperature is 4°C, the separation factor is 3000g, and the separation time is 5 minutes.

[0030] In this embodiment, in S4, the mass ratio between the anti-CD63 antibody and the Strep-tag II protein purification magnetic beads is 1:5.

[0031] In this embodiment, S5, the separation buffer is a phosphate buffered saline solution containing 0.1% bovine serum albumin; preferably, the separation buffer is sterilized by filtering with a 0.22 micron filter membrane in advance.

[0032] In this embodiment, in S6, the amount of the separation buffer is 50-500 times the volume of the magnetic bead suspension.

[0033] In this embodiment, in S7, the incubation temperature is 37°C, the incubator parameters are 90° inverted rotation, 5 seconds tilt, 5° vibration for 1 second, and incubation time of 4 hours.

[0034] In this embodiment, in S8, the solution B is a phosphate buffered saline solution.

[0035] In this embodiment, in S9, the eluent is a glycine solution with a pH of 2.8 to 3.5, a glycine concentration of 0.1 to 0.2 mol / L, an oscillation time of 30 min, a separation factor of 3000 g, and a separation time of 5 min.

[0036] In this embodiment, in S1-S9, each result obtained is tested, recorded and uploaded to the traceability system.

[0037] Embodiment three: The difference between the first embodiment and the second embodiment is that: S1: Take fresh plasma, place it in a collection tube, add 20 μL of solution A to every 5 mL of plasma, gently invert to mix, and leave it for a while; S2: Separate the solution obtained in S1, take the supernatant as serum, put it in a centrifuge tube, repeat centrifugation once, and take out the supernatant for later use; S3: The serum after centrifugation is drawn up through a syringe, filtered, and then transferred to a new centrifuge tube for later use; S4: Take the activated Strep-tagII protein purification magnetic beads, add the saturated coupling amount of anti-CD63, and mix for a period of time at room temperature; S5: washing the solution obtained in S4 with a separation buffer, and after washing, adding amino acids for dehydration condensation; S6: add an appropriate amount of separation buffer to suspend the solution obtained in S5 to finally form a magnetic bead suspension; S7: Add 5 times the solution obtained in S3 to the magnetic bead suspension obtained in S6 and incubate; S8: After the incubation is completed, the system is washed with solution B, and the magnetic beads are separated by a magnetic rack to obtain a complex adsorbed with exosomes; S9: Mix the complex obtained in S8 with 400 μL of the eluent, shake at room temperature, and then separate and take the supernatant, which is the high-purity exosome API.

[0038] In this embodiment, in S1, solution A is a blood coagulant, and the standing time is 30 minutes.

[0039] In this embodiment, in S2, the separation temperature is 4°C, the separation factor is 3000g, and the separation time is 5 minutes.

[0040] In this embodiment, in S4, the mass ratio between the anti-CD63 antibody and the Strep-tag II protein purification magnetic beads is 1:5.

[0041] In this embodiment, S5, the separation buffer is a phosphate buffered saline solution containing 0.1% bovine serum albumin; preferably, the separation buffer is sterilized by filtering with a 0.22 micron filter membrane in advance.

[0042] In this embodiment, in S6, the amount of the separation buffer is 50-500 times the volume of the magnetic bead suspension.

[0043] In this embodiment, in S7, the incubation temperature is 37°C, the incubator parameters are 90° inverted rotation, 5 seconds tilt, 5° vibration for 1 second, and incubation time for 1 hour.

[0044] In this embodiment, in S8, the solution B is a phosphate buffered saline solution.

[0045] In this embodiment, in S9, the eluent is a glycine solution with a pH of 2.8 to 3.5, a glycine concentration of 0.1 to 0.2 mol / L, an oscillation time of 30 min, a separation factor of 3000 g, and a separation time of 5 min.

[0046] In this embodiment, in S1-S9, each result obtained is tested, recorded and uploaded to the traceability system.

[0047] Embodiment 4: The difference between the first embodiment, the second embodiment and the third embodiment is that: S1: Take fresh plasma, place it in a collection tube, add 20 μL of solution A to every 5 mL of plasma, gently invert to mix, and leave it for a while; S2: Separate the solution obtained in S1, take the supernatant as serum, put it in a centrifuge tube, repeat centrifugation once, and take out the supernatant for later use; S3: The serum after centrifugation is drawn up through a syringe, filtered, and then transferred to a new centrifuge tube for later use; S4: Take the activated Strep-tagII protein purification magnetic beads, add the saturated coupling amount of anti-CD63, and mix for a period of time at room temperature; S5: washing the solution obtained in S4 with a separation buffer, and after washing, adding amino acids for dehydration condensation; S6: add an appropriate amount of separation buffer to suspend the solution obtained in S5 to finally form a magnetic bead suspension; S7: add 10 times the solution obtained in S3 to the magnetic bead suspension obtained in S6 and incubate; S8: After the incubation is completed, the system is washed with solution B, and the magnetic beads are separated by a magnetic rack to obtain a complex adsorbed with exosomes; S9: Mix the complex obtained in S8 with 400 μL of the eluent, shake at room temperature, and then separate and take the supernatant, which is the high-purity exosome API.

[0048] In this embodiment, in S1, solution A is a blood coagulant, and the standing time is 30 minutes.

[0049] In this embodiment, in S2, the separation temperature is 4°C, the separation factor is 3000g, and the separation time is 5 minutes.

[0050] In this embodiment, in S4, the mass ratio between the anti-CD63 antibody and the Strep-tag II protein purification magnetic beads is 1:5.

[0051] In this embodiment, S5, the separation buffer is a phosphate buffered saline solution containing 0.1% bovine serum albumin; preferably, the separation buffer is sterilized by filtering with a 0.22 micron filter membrane in advance.

[0052] In this embodiment, in S6, the amount of the separation buffer is 50-500 times the volume of the magnetic bead suspension.

[0053] In this embodiment, in S7, the incubation temperature is 37°C, the incubator parameters are 90° inverted rotation, 5 seconds tilt, 5° vibration for 1 second, and incubation time for 2 hours.

[0054] In this embodiment, in S8, the solution B is a phosphate buffered saline solution.

[0055] In this embodiment, in S9, the eluent is a glycine solution with a pH of 2.8 to 3.5, a glycine concentration of 0.1 to 0.2 mol / L, an oscillation time of 30 min, a separation factor of 3000 g, and a separation time of 5 min.

[0056] In this embodiment, in S1-S9, each result obtained is tested, recorded and uploaded to the traceability system.

[0057] Embodiment five: The difference between the first embodiment, the second embodiment, the third embodiment and the fourth embodiment is that: S1: Take fresh plasma, place it in a collection tube, add 20 μL of solution A to every 5 mL of plasma, gently invert to mix, and leave it for a while; S2: Separate the solution obtained in S1, take the supernatant as serum, put it in a centrifuge tube, repeat centrifugation once, and take out the supernatant for later use; S3: The serum after centrifugation is drawn up through a syringe, filtered, and then transferred to a new centrifuge tube for later use; S4: Take the activated Strep-tagII protein purification magnetic beads, add the saturated coupling amount of anti-CD63, and mix for a period of time at room temperature; S5: washing the solution obtained in S4 with a separation buffer, and after washing, adding amino acids for dehydration condensation; S6: add an appropriate amount of separation buffer to suspend the solution obtained in S5 to finally form a magnetic bead suspension; S7: Add 2 times the solution obtained in S3 to the magnetic bead suspension obtained in S6 and incubate; S8: After the incubation is completed, the system is washed with solution B, and the magnetic beads are separated by a magnetic rack to obtain a complex adsorbed with exosomes; S9: Mix the complex obtained in S8 with 400 μL of the eluent, shake at room temperature, and then separate and take the supernatant, which is the high-purity exosome API.

[0058] In this embodiment, in S1, solution A is a blood coagulant, and the standing time is 30 minutes.

[0059] In this embodiment, in S2, the separation temperature is 4°C, the separation factor is 3000g, and the separation time is 5 minutes.

[0060] In this embodiment, in S4, the mass ratio between the anti-CD63 antibody and the Strep-tag II protein purification magnetic beads is 1:5.

[0061] In this embodiment, S5, the separation buffer is a phosphate buffered saline solution containing 0.1% bovine serum albumin; preferably, the separation buffer is sterilized by filtering with a 0.22 micron filter membrane in advance.

[0062] In this embodiment, in S6, the amount of the separation buffer is 50-500 times the volume of the magnetic bead suspension.

[0063] In this embodiment, in S7, the incubation temperature is 37°C, the incubator parameters are 90° inverted rotation, 5 seconds tilt, 5° vibration for 1 second, and incubation time for 2 hours.

[0064] In this embodiment, in S8, the solution B is a phosphate buffered saline solution.

[0065] In this embodiment, in S9, the eluent is a glycine solution with a pH of 2.8 to 3.5, a glycine concentration of 0.1 to 0.2 mol / L, an oscillation time of 30 min, a separation factor of 3000 g, and a separation time of 5 min.

[0066] In this embodiment, in S1-S9, each result obtained is tested, recorded and uploaded to the traceability system.

[0067] In summary: the method for preparing a high-purity exosome raw material drug of the present invention is not only simple in process and short in preparation time, but also greatly improves the purity of the enriched exosomes, and can ensure the integrity of the separated exosomes. The exosome raw material drug obtained by the ratio method of Example 2 is increased to 4 hours, which not only prolongs the preparation time, but also causes the purity of the exosomes to decrease. The exosome raw material drug obtained by the ratio method of Example 3 is shortened to 4 hours. Although the preparation time is delayed, the integrity of the exosomes is reduced. The exosome raw material drug obtained by the ratio method of Example 4 is reduced in the recovery rate of the exosomes by adding 10 times the solution obtained in S3 to the magnetic bead suspension obtained by S6. The exosome raw material drug obtained by the ratio method of Example 5 is increased in the recovery rate of the exosomes by adding 2 times the solution obtained in S3 to the magnetic bead suspension obtained by S6, but the economic cost of the exosome separation and purification operation is increased.

[0068] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0069] 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 appended claims and their equivalents.

Claims

1. A method for preparing a high-purity exosome raw material drug, characterized in that: The following steps are involved: S1: Take fresh plasma, place it in a collection tube, add 20 μL of solution A to every 5 mL of plasma, gently invert to mix, and leave it for a while; S2: Separate the solution obtained in S1, take the supernatant as serum, put it in a centrifuge tube, repeat centrifugation once, and take out the supernatant for later use; S3: The serum after centrifugation is drawn up through a syringe, filtered, and then transferred to a new centrifuge tube for later use; S4: Take the activated Strep-tagII protein purification magnetic beads, add the saturated coupling amount of anti-CD63, and mix for a period of time at room temperature; S5: washing the solution obtained in S4 with a separation buffer, and after washing, adding amino acids for dehydration condensation; S6: add an appropriate amount of separation buffer to suspend the solution obtained in S5 to finally form a magnetic bead suspension; S7: Add 5 times the solution obtained in S3 to the magnetic bead suspension obtained in S6 and incubate; S8: After the incubation is completed, the system is washed with solution B, and the magnetic beads are separated by a magnetic rack to obtain a complex adsorbed with exosomes; S9: Mix the complex obtained in S8 with 400 μL of the eluent, shake at room temperature, and then separate and take the supernatant, which is the high-purity exosome API.

2. The method for preparing a high-purity exosome API according to claim 1, characterized in that: In the S1, solution A is a blood coagulant and the standing time is 30 minutes.

3. The method for preparing a high-purity exosome API according to claim 1, characterized in that: In the S2, the separation temperature is 4°C, the separation factor is 3000g, and the separation time is 5 minutes.

4. The method for preparing a high-purity exosome API according to claim 1, characterized in that: In S4, the mass ratio between the anti-CD63 antibody and the Strep-tag II protein purification magnetic beads is 1:

5.

5. The method for preparing a high-purity exosome API according to claim 1, characterized in that: In S5, the separation buffer is a phosphate buffered saline solution containing 0.1% bovine serum albumin; preferably, the separation buffer is sterilized by filtering with a 0.22 micron filter membrane in advance.

6. The method for preparing a high-purity exosome API according to claim 1, characterized in that: In S6, the amount of the separation buffer is 50-500 times the volume of the magnetic bead suspension.

7. The method for preparing a high-purity exosome API according to claim 1, characterized in that: In the S7, the incubation temperature is 37°C, the incubator parameters are 90° rotation, 5 seconds tilt, 5° vibration for 1 second, and incubation time for 2 hours.

8. The method for preparing a high-purity exosome API according to claim 1, characterized in that: In S8, the solution B is a phosphate buffered saline solution.

9. The method for preparing a high-purity exosome API according to claim 1, characterized in that: In the S9, the eluent is a glycine solution with a pH of 2.8 to 3.5, a glycine concentration of 0.1 to 0.2 mol / L, an oscillation time of 30 min, a separation factor of 3000 g, and a separation time of 5 min.

10. The method for preparing a high-purity exosome API according to claim 1, characterized in that: In S1-S9, each result obtained is tested, recorded and uploaded to the traceability system.