Preparation method of high-purity exosome

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method of a high-purity exosome. According to the preparation method, a sample solution containing the exosome is sequentially subjected to clarification treatment, concentration and ultrafiltration treatment, composite chromatography and cation chromatography. By optimizing a multi-mode chromatography mode and improving a buffer solution system, the cost is effectively reduced, the purity and yield of the exosome are improved, and the industrial application requirements are met.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for preparing high-purity exosomes. Background Art

[0002] Exosomes are small vesicles with a diameter of 30-150nm and a double-layer lipid membrane structure secreted by living cells. They are widely present in the body, such as peripheral blood, ascites, urine, saliva, cerebrospinal fluid and other body fluids. They contain rich bioactive components such as proteins, lipids and nucleic acids, and play an important role in various physiological processes such as immune response, antigen presentation and signal transduction. With the increasing clinical application potential of exosomes, it has become imperative to optimize their isolation methods to obtain maximum yield, highest purity and best reproducibility.

[0003] Existing exosome separation technologies include centrifugation, ultrafiltration and chromatography, which have the problems of small processing volume, long separation time, high cost, low purity, unsatisfactory yield, selective restriction of exosome size by chromatography and ultrafiltration, susceptibility to centrifugal parameters (centrifugal force and centrifugal time), poor reproducibility, etc. In order to solve the above problems and meet the needs of large-scale industrial applications, the prior art provides some solutions, such as CN117511863A, a method for sequential separation of exosomes and its application, which discloses that the supernatant containing exosomes is treated by hollow fiber and then subjected to composite chromatography to purify the exosomes. CN116769692A, a method for separation and preparation of exosomes, discloses that a sample containing exosomes is subjected to double centrifugation, composite chromatography, and finally purified by hydroxyapatite column to extract exosomes. CN115449505A discloses a purification method for isolating exosomes and an exosome kit, which discloses that a sample containing exosomes is first centrifuged to obtain a primary extract, the primary extract is then subjected to composite chromatography to obtain a flow-through, and at least one of the primary extract or the flow-through is ultrafiltered using a hollow fiber or ultrafiltration membrane.

[0004] The existing methods for extracting exosomes also have the problem that the phosphate buffer used in ultrafiltration and chromatography is easily reacted with common calcium ions Ca 2+ Mg 2+ The defects of some heavy metal ions forming precipitation and inhibiting certain biochemical reaction processes have not been effectively solved. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for preparing high-purity exosomes. The method can effectively reduce costs, improve the purity and yield of exosomes, eliminate the influence of factors such as centrifugation that cause unstable quality of exosomes, improve the stability of exosome preparation, and meet the needs of industrial application through optimized multimodal chromatography and improved buffer system.

[0006] The technical solution of the present invention is a method for preparing high-purity exosomes, comprising the following steps:

[0007] (1) Clarification: The sample solution containing exosomes is clarified by a depth filter to obtain a clarified solution;

[0008] (2) Ultrafiltration and concentration: The clarified solution is subjected to ultrafiltration and concentration treatment under the action of tangential flow to obtain a concentrated solution;

[0009] (3) Cation chromatography: The concentrated solution is subjected to cation chromatography to obtain a cation chromatography flow-through.

[0010] The present invention provides a method for preparing high-purity exosomes, which further comprises the following steps: before the concentrated solution is subjected to cationic chromatography, composite chromatography is first performed to obtain a composite chromatography flow-through liquid; and the composite chromatography flow-through liquid is then subjected to cationic chromatography to obtain a cationic chromatography flow-through liquid.

[0011] In step (1), the pore size of the deep filter is 1.0-5.0 um, and the filtration pressure is less than 10 PSI.

[0012] In step (1), the sample liquid containing exosomes includes any one or a combination of cell supernatant, blood, tears, urine, saliva, milk, ascites, etc.

[0013] In step (2), the parameters of the tangential flow are: shear force is 1500-2000S -1 , TMP is 3-3.5PSI, flow rate is 120-150ml / min; preferably shear force is 2000S -1 , TMP is 3.5PSI and flow rate is 120ml / min.

[0014] In step (2), ultrafiltration and concentration are performed using a hollow fiber column, such as a 300KD hollow fiber column.

[0015] In step (2), the product is concentrated 5 times by ultrafiltration and concentration to obtain a concentrated solution.

[0016] In step (2), the concentrated solution obtained by ultrafiltration and concentration treatment is then replaced with a solution, and the solution used for the replacement is a sodium chloride solution or a phosphate buffer containing sodium chloride. The concentration of the sodium chloride solution is 300 mM, the pH of the phosphate buffer containing sodium chloride is 7.5-8.0, and the concentration of sodium chloride is 300 mM.

[0017] In step (3), the balancing solution for cationic chromatography is 3-5 column volumes, and the balancing solution is a sodium chloride solution or a phosphate buffer containing sodium chloride. The concentration of the sodium chloride solution is 300 mM, the pH of the phosphate buffer containing sodium chloride is 7.5-8.0, and the concentration of sodium chloride is 300 mM.

[0018] In step (3), the cationic chromatography column used for cationic chromatography is a Capto Mustang chromatography column, which is first treated with alkali and then rinsed to a neutral pH.

[0019] In step (3), the cation chromatography loading flow rate is 1 ml / min.

[0020] The balancing solution for the composite chromatography is 3-5 times the column volume, and the balancing solution is a sodium chloride solution or a phosphate buffer containing sodium chloride. The concentration of the sodium chloride solution is 300mM, the pH of the phosphate buffer containing sodium chloride is 7.5-8.0, and the concentration of sodium chloride is 300mM. The composite chromatography column for the composite chromatography is a Capto Core 700 chromatography column, which is first treated with alkali and then rinsed to a neutral pH.

[0021] The composite chromatography loading flow rate is 1 ml / min.

[0022] The advantages of the present invention are:

[0023] 1. Compared with traditional clarification methods such as ultracentrifugation, capsule filters have faster processing efficiency and higher loading capacity, are suitable for industrial scale-up production, and can greatly reduce the time used for clarification.

[0024] 2. The purity of exosomes can be further improved by optimizing and improving the combined chromatography method and buffer system, and purification of exosomes of different volumes can be achieved to meet the needs of industrial production.

[0025] 3. The combination of TFF tangential flow filtration and composite membrane chromatography can not only prepare high-purity exosomes that meet clinical requirements, but also greatly improve efficiency, yield, speed and robustness.

[0026] 4. For the traditional size exclusion chromatography method, an optimized composite mode combined with a cationic chromatography method is used, and a Capto Mustang weak cationic chromatography step is added. The flow-through liquid is collected by flow-through, and the impurities are combined with the chromatography medium to further remove residual proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the process for preparing high-purity exosomes of the present invention.

[0028] Figure 2 The recovery rate of exosomes obtained in each process step during the preparation of high-purity exosomes of the present invention.

[0029] Figure 3 The protein removal rates of each process step in the preparation of high-purity exosomes of the present invention are shown in Table 1. The sites and trend lines with the same or similar protein removal rates in the 300 mM sodium chloride buffer group and the phosphate buffer group containing 300 mM sodium chloride are shown only in the 300 mM sodium chloride buffer group. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0031] (1) Clarification and filtration: The cell supernatant containing exosomes is clarified using a 1.0-5.0 μm depth filter, during which the filtration pressure is controlled to be less than 10 PSI. The supernatant after filtration is tested for particle count and BCA. This step can remove residual cell debris, most apoptotic bodies and microvesicles to obtain a clarified filtrate.

[0032] (2) Ultrafiltration, concentration and fluid exchange: A 300KD hollow fiber column was used, and equal volumes of the clarified filtrate obtained were divided into control groups A and B. The buffer system used was 300 mM Nacl buffer (Group A) and phosphate buffer containing 300 mM Nacl (pH 7.5-8.0) (Group B).

[0033] The samples of groups A and B were concentrated 5 times using hollow fiber columns. During the concentration process, the flow rate at the inlet end was controlled at 120 ml / min and the shear force was controlled at 2000 s. -1 , TMP was controlled within 3.5psi to reduce the damage of shear force to exosomes. After concentration, the two groups of samples were replaced 10 times with 300mM Nacl buffer (Group A) and phosphate buffer containing 300mM Nacl (pH 7.5-8.0) (Group B) with equal volumes.

[0034] (3) Multimode chromatography: After two 20 mL Capto Core 700 chromatography columns were treated with alkali, they were rinsed to a neutral pH and 300 mM Nacl buffer (Group A) and phosphate buffer containing 300 mM Nacl (pH 7.5-8.0) (Group B) were used as the buffer system. The columns were balanced for 3-5 column volumes. The sample flow rate was controlled at 1 ml / min, and the flow-through was collected and sampled for particle number and BCA detection.

[0035] (4) Cationic chromatography: Capto Mustang cationic filler was used. After two 1 mL Capto Mustang cationic chromatography columns were treated with alkali, they were rinsed to a neutral pH. 300 mM Nacl buffer (Group A) and phosphate buffer containing 300 mM Nacl (pH 7.5-8.0) (Group B) were used as buffer systems, 3-5 column volumes of the column were balanced, the sample flow rate was controlled at 1 ml / min, the flow-through liquid was collected, and samples were taken to detect the number of particles and BCA. This step can remove residual positively charged impurities, further improve the purity of exosomes, and compare the final results of the two buffer systems.

[0036]

[0037] It should be pointed out that the above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology of this project to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing high-purity exosomes, characterized in that the steps include: (1) Clarification: The sample solution containing exosomes is clarified by a depth filter to obtain a clarified solution; (2) Ultrafiltration and concentration: The clarified solution is subjected to ultrafiltration and concentration treatment under the action of tangential flow to obtain a concentrated solution; (3) Cation chromatography: The concentrated solution is subjected to cation chromatography to obtain a cation chromatography flow-through.

2. The preparation method according to claim 1, characterized in that: Before the concentrated solution is subjected to the cation chromatography, the concentrated solution is first subjected to the composite chromatography to obtain the composite chromatography flow-through solution; the composite chromatography flow-through solution is then subjected to the cation chromatography to obtain the cation chromatography flow-through solution.

3. The preparation method according to claim 1 or 2, characterized in that: In step (1), the pore size of the deep filter is 1.0-5.0 um, and the filtration pressure is less than 10 PSI.

4. The preparation method according to claim 1 or 2, characterized in that: In step (1), the sample liquid containing exosomes includes any one or a combination of cell supernatant, blood, tears, urine, saliva, milk, ascites, etc.

5. The preparation method according to claim 1 or 2, characterized in that: In step (2), the parameters of the tangential flow are: shear force is 1500-2000S -1 , TMP is 3-3.5PSI, and flow rate is 120-150ml / min.

6. The preparation method according to claim 1 or 2, characterized in that: In step (2), a hollow fiber column is used for concentration and ultrafiltration.

7. The preparation method according to claim 1 or 2, characterized in that: In step (2), the concentrated solution obtained by ultrafiltration and concentration is then replaced with a sodium chloride solution or a phosphate buffer containing sodium chloride.

8. The preparation method according to claim 7, characterized in that: In step (2), the liquid replacement process is to replace the liquid with an equal volume of the concentrated liquid 10 times.

9. The preparation method according to claim 1 or 2, characterized in that: In step (3), the cation chromatography equilibrium solution is 3-5 times the column volume, and the equilibrium solution is a sodium chloride solution or a phosphate buffer containing sodium chloride.

10. The preparation method according to claim 2, characterized in that: The balancing liquid used for the composite chromatography is 3-5 times of the column volume, and the balancing liquid is a sodium chloride solution or a phosphate buffer containing sodium chloride.

Images