CN118562727A - Method for purifying exosomes and exosome quality control method - Google Patents
Method for purifying exosomes and exosome quality control method Download PDFInfo
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Abstract
The application relates to a method for purifying exosomes and an exosome quality control method, belonging to the technical field of biology. Wherein the method of purifying exosomes comprises: and sequentially performing first TFF tangential flow purification, anion multi-mode chromatographic purification and gel filtration chromatographic purification treatment on the supernatant of the exosome to be purified to obtain the exosome. The exosomes with high purity, high activity and high safety can be obtained based on the purification method.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a method for purifying exosomes and an exosome quality control method.
Background
Exosomes (exosomes) are membrane vesicles released into the extracellular matrix after fusion of intracellular multivesicular bodies (multivesicular body, MVB) with cell membranes, the diameters of the membrane vesicles are about 30-150nm, substances such as proteins, lipids, polysaccharides, RNA and the like are contained in the membrane vesicles, the exosomes naturally exist in body fluids including blood, saliva, urine, breast milk and the like, and the secreted exosomes enter the body fluids such as blood, saliva, urine, breast milk and the like, reach other cells and tissues through the circulatory system, and generate remote regulation and control effects.
When exosome studies are performed, a major problem to be solved first is to obtain a sufficient amount of high purity exosomes. However, it is known that in the process of extracting exosomes, the purity and yield of exosomes are often two relatively contradictory problems, and some yield may be lost when trying to increase the purity, but only the yield is pursued, which may be more fatal, because it directly leads to inaccuracy of our marker screening or functional verification related data, influence of dose judgment, low repeatability of the experiment, or even no repeatability. Purity is a critical issue for drug delivery systems, low purity exosomes may affect drug delivery efficiency, and increased immunogenicity may result in unexpected toxicity. With the increasing depth of research on the field of exosomes by more scientific institutions and scientific researchers, the requirements on related data are more and more strict, so that the demands of the vast scientific researchers on how to obtain high-purity exosomes are more and more urgent.
Disclosure of Invention
The present application aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, an object of the present application is to provide a means for efficiently obtaining high purity exosomes.
Specifically, the application provides the following technical scheme:
In a first aspect of the application, the application provides a method of purifying an exosome. According to an embodiment of the application, the method comprises: and sequentially performing first TFF tangential flow purification, anion multi-mode chromatographic purification and gel filtration chromatographic purification treatment on the supernatant of the exosome to be purified to obtain the exosome. The exosomes with high purity, high activity and high safety can be obtained based on the purification method.
In a second aspect of the application, the application provides a method of exosome quality control. According to an embodiment of the application, the method comprises: purifying the exosomes using the method of the first aspect; and performing at least one of 1) -3) the detection of the obtained exosomes: 1) Western blot analysis; 2) Scanning by a transmission electron microscope; 3) SEC-HPLC purity detection. Based on the exosome quality control method, accurate characterization of exosomes can be realized, so that quality control of each stage of exosome production process is realized, and a large number of high-purity pollution-free exosomes are obtained.
Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.
Drawings
The foregoing and/or additional aspects and advantages of the application will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic diagram showing the results of SEC (exclusion chromatography) purity detection of buffers provided in the examples of the present application (control);
FIG. 2 is a schematic diagram showing the SEC purity detection results of exosomes purified by anion multimode chromatography according to the embodiment of the application;
FIG. 3 is a schematic diagram showing the SEC purity measurement results of the exosomes after the second concentration according to the embodiment of the present application;
FIG. 4 is a schematic diagram showing the SEC purity detection results of exosomes purified by gel filtration chromatography according to the embodiment of the present application;
FIG. 5 is a schematic diagram of the result of the electron microscopy of the exosome in different steps according to the embodiment of the present application; wherein, (a) anion multi-mode chromatography, (B) second concentration, (C) gel filtration chromatography;
FIG. 6 is a graph showing the comparison of the total recovery rate and the result of each embodiment provided by the embodiment of the application;
FIG. 7 is a comparative graph of ratio purity results for various embodiments of the present application.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
In this document, the terms "first," "second," and "first," are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated unless otherwise specified. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
In this document, the terms "comprise" or "comprise" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, and not to exclude other aspects, unless otherwise noted.
In this document, the terms "optionally," "optional," or "optionally" mean, unless specified otherwise, that the subsequently described event or condition may, but need not, occur, and that the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
In order to solve the contradiction between purity and yield in the exosome extraction process, the purity and yield of exosome are improved. The application provides a method for purifying exosomes, which comprises the following steps: and sequentially performing first TFF tangential flow purification, anion multi-mode chromatographic purification and gel filtration chromatographic purification treatment on the supernatant of the exosome to be purified to obtain the exosome.
In some examples of the application, the supernatant is from a cell supernatant collected from cells under 2D culture conditions or 3D suspension culture conditions, the cells having the ability to secrete exosomes.
In some examples of the application, the cells are mesenchymal stem cells.
In some preferred examples of the application, the supernatant is from a cell supernatant of mesenchymal stem cells collected under 3D suspension culture conditions, the 3D suspension culture being performed by: comprises mixing the mesenchymal stem cells with the passage density of 3000-10000 cells/cm 2 with microcarriers; and inoculating the mixed treatment product to a bioreactor system, the bioreactor system comprising the culture medium.
In some examples of the application, the culture Medium is selected from at least one of mesenchymal stem cell serum-Free Medium, hapCult MSC, xeno-Free, SFM, MSC Xeno-Free SFM, MSC NutriStem, and MesenCult TM- ACF Plus Medium. In some preferred examples of the application, the medium is selected from mesenchymal stem cell serum-free medium. The serum-free culture medium of the mesenchymal stem cells is selected to be cultured, so that the influence of exogenous factors on the cell growth and phenotype can be reduced, the variables in the experiment are reduced, and the result is more reliable. Secondly, the serum-free culture medium can avoid the possible pollutants such as virus, bacteria, hormone and the like in serum, and improve the safety of biological products.
In some examples of the application, the microcarriers are present in the bioreactor system at a concentration of 1g/L to 4g/L after inoculation. The aforementioned concentration is optionally 1g/L, 2g/L, 3g/L or 4g/L.
In some examples of the application, the foregoing microcarrier is selected from at least one of Cytodex1, cytodex3, 3D TableTrix, plastics Plus, plastic, collagen, and Hillex II. In some preferred examples of the application, the microcarrier is selected from Cytodex1 and/or Collagen. Cytodex1 and/or Collagen microcarriers can better simulate the growth conditions of similar in vivo environments, facilitate the exchange of mesenchymal stem cells with nutrients and growth factors in the culture medium, and promote the growth and metabolic activity of cells.
It should be noted that the microcarrier can be reused.
In some examples of the application, the bioreactor system pH is selected from 7.0 to 7.8. The aforementioned pH is optionally 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8.
In some examples of the application, the inoculation is performed at a speed of 30rpm to 50rpm, alternatively 30rpm、31rpm、32rpm、33rpm、34rpm、35rpm、36rpm、37rpm、38rpm、39rpm、40rpm、41rpm、42rpm、43rpm、44rpm、45rpm、46rpm、47rpm、48rpm、49rpm or 50rpm.
In some examples of the application, the first day of the culture treatment is performed at a rotational speed of 30rpm to 50rpm, and after 24 hours at a rotational speed of 70rpm to 100rpm, and after 10rpm increase per day, up to 150 rpm. The culture strategy with the step-by-step rotation speed increase can effectively balance the shearing force of culture and the adaptability of cells, and is beneficial to improving the yield and quality of exosomes.
In some examples of the application, no or no fluid changes are made during the 3D suspension culture process.
In some examples of the application, the exchange is a 60% medium exchange at day 4 of culture.
In some examples of the application, the exchange is a 60% medium exchange at day 5 and day 7 of culture.
The stability of the culture environment can be maintained by periodically changing the liquid midway, the growth of cells and the production of exosomes are promoted, and the culture efficiency and the yield of exosomes are improved. The selective liquid exchange of 60% can effectively remove metabolites while keeping the culture environment stable, thereby meeting the nutrition requirements of cells and saving the culture cost.
In some examples of the application, the TFF tangential flow purification process comprises a first filtration process. The impurities of large particles are removed by the first filtration treatment, and the higher permeability and flux are maintained.
In some examples of the application, the first filtration treatment is performed by a filter made of a double layer PVDF membrane.
In some examples of the application, the filter has an outer pore size of 1.0 μm and an inner pore size of 0.45 μm. The pore size of 1.0 μm can block large-particle impurities such as cells, microcarriers, cell metabolites and the like, but can keep higher permeability, so that the filtration is not easy to block, the pore size of 0.45 μm can enable large vesicles not to easily pass through, and exosomes with the diameter of 30-150 nm can easily pass through, thus improving the permeability and flux of the filtration, and intercepting cell vesicles with larger diameters and no need.
In some examples of the application, the flux of the first filtration treatment is selected from 220L/m 2-225 L/m2. In some preferred examples of the application, the first filtration treatment has a flux of 222L/m 2. Based on the flux, the filtering effect is better.
In some examples of the application, the flow rate of the first filtration treatment is selected from 10L/m 2/min-15L/m2/min. The flow rate can ensure that impurities can not be rapidly accumulated to form a filter cake in the filtering process due to the fact that the flow rate is too high, and the filter is blocked to influence the filtering flux.
In some examples of the application, the first filtering process further comprises: and (5) performing top washing treatment on the filter. The exosomes trapped in the pore size and adsorbed on the filter membrane are washed down by a top wash step to reduce the loss of particle count.
In some examples of the application, the top wash treatment is a 10L/m 2 buffer (e.g., 40mM PB, 125mM NaCl,pH7.8;PBS,pH7.4) wash of the filter in the first filtration treatment.
In some examples of the application, the first filtering process further comprises, after: and concentrating and ultrafiltering the first filtrate after the first filtering treatment. Impurities in the filtrate are removed through concentration and ultrafiltration, so that the purity of exosomes is improved.
In some examples of the application, the concentrated ultrafiltration treatment is by a hollow fiber module. Wherein the pore diameter of the hollow fiber in the hollow fiber module is 30nm-50nm, which is equivalent to 500KD. In some examples of the application, the use of hollow fiber modules produces less shear forces, which is beneficial to protecting the integrity of exosomes; secondly, the hollow fiber module can efficiently concentrate a large volume of fermentation supernatant, which is beneficial to the operation of the following steps such as anion composite chromatography and the like; in addition, the pore size of the hollow fiber module can effectively filter most of impurities such as cell fragments, proteins and nucleic acids, and exosomes are reserved, so that the exosomes are efficiently separated and concentrated; more importantly, compared with other concentration technologies, the hollow fiber module is simple to operate, easy to control and maintain, and convenient for experimental operation.
In some examples of the application, the transmembrane pressure difference of the concentration ultrafiltration treatment is less than or equal to 5Psi, so that the stability of exosomes in the concentration ultrafiltration process can be ensured and the process time can be shortened.
In some examples of the application, the flux of the concentrated ultrafiltration treatment is selected from 50L/m 2-60 L/m2. The flux in the above range can ensure the high efficiency and stability of the concentration ultrafiltration treatment process.
In some examples of the application, the time of the concentrated ultrafiltration treatment is selected from 6-8 hours. In some preferred examples of the present application, the concentration ultrafiltration treatment time is 7 hours, and during this treatment time, the integrity of the exosomes can be ensured, avoiding the exosomes from being broken due to the concentration ultrafiltration treatment for a long time.
In some examples of the application, after the concentrated ultrafiltration treatment, further comprising subjecting the concentrated ultrafiltration treated ultrafiltrate to a second filtration treatment. Based on the second filtering treatment, the concentrated and ultrafiltered feed liquid is clarified, so that agglomeration and blockage of anion composite chromatography media are prevented, and the use and impurity removal effects are influenced.
In some examples of the application, the anionic multimode chromatographic purification employs a chromatographic column for a third filtration treatment. Impurities in the solution are further removed through anion multimode chromatography, and the purity of exosomes is improved.
In some examples of the application, the chromatographic column packing particle size is selected from 85 μm to 95 μm. In some preferred examples of the application, the chromatographic column packing particle size is selected from 90 μm.
In some examples of the application, the chromatographic column packing is selected from Capto Core 700, sun Core 700, smac Core 700, maxtar Coll 700, monomix Core 1000XXX, preferably Capto Core 700. The outermost layer of the Capto Core 700 filler is wrapped with a passivation layer of 5 mu m, the passivation layer cannot be combined with exosomes, small holes with the diameter of 700KD (40-60 nm) are formed in the passivation layer, the small holes enter the inside of the filler, the inside of the filler contains multimode octylamine groups, substances such as HCP, BSA, endotoxin and nucleic acid can be combined, most exosomes cannot enter the inside of the filler through the small holes in the passivation layer due to the fact that the particle size distribution of the exosomes is 30-150 nm, and can only flow downwards through gaps between spheres, and most substances such as HCP, endotoxin and nucleic acid can enter the inside of the filler through the small holes in the passivation layer to be combined with the octylamine groups due to the fact that the particle size of the exosomes is small (generally within 500 KD).
In some examples of the application, the anionic multimodal chromatographic purification is performed in a buffer having a pH selected from 7.5-8, preferably 7.8. Most host proteins (Host Cell Protein, HCP), nucleic acids, endotoxins and the like have isoelectric points of less than 6.0, have negative charges at pH7.8, and the octyl amine groups in the filler have positive charges, so that impurities such as HCP, nucleic acids, endotoxins and the like can be effectively adsorbed.
In some examples of the application, the buffer comprises: 37mmol/L-42mmol/L sodium hydrogen phosphate and 120mmol/L-130mmol/L sodium chloride; preferably 40mmol/L sodium hydrogen phosphate and 125mmol/L sodium chloride. While 20mmol/L sodium phosphate is mostly used in a common buffer system, the filler pH can be more quickly washed to a preset pH value by using 40mmol/L sodium hydrogen phosphate; in addition, the octyl amine group in the filler contains a hydrophobic group, and 125mmol/L sodium chloride is added into the buffer solution, so that the hydrophobic effect of the octyl amine group can be better exerted.
In some examples of the application, the flow rate of the anionic multimodal chromatographic purification is no greater than 1cm/min. The smaller flow rate is favorable for substances such as HCP, nucleic acid, endotoxin and the like to smoothly enter the filler through the pores to be combined with the octylamine group, and the slow linear flow rate provides enough combination time, so that the impurities can be removed better.
In some examples of the application, the chromatography column packing loading is 11mg/ml or less. Since the exosome supernatant contains nucleic acid, endotoxin, pigment and other substances, binding sites on the octyl amine group are occupied, so that the binding capacity to HCP is reduced, and a filler with higher binding capacity needs to be selected in the experiment.
In some examples of the application, the third filtering process further comprises concentrating the third filtrate after the third filtering process and a fourth filtering process. The volume of the feed liquid is reduced by concentration, and the concentration of exosomes in the feed liquid is improved; based on the fourth filtering treatment, the concentrated feed liquid is clarified, so that caking and blockage of gel filtration chromatography media are prevented, and the use and impurity removal effects are affected.
In some examples of the application, the gel column is used for a fifth filtration treatment in the gel filtration chromatography purification. Through gel filtration chromatography, the purity of the exosome sample can be effectively improved, and impurities with smaller molecular size, such as proteins, nucleic acids, small molecular metabolites and the like, are removed, so that a purer exosome sample is obtained.
In some examples of the application, the filler particle size of the gel column is selected from 45 μm to 165 μm.
In some examples of the application, the packing material of the gel column is selected from Seplife FF, superose 6Increase, siperdex 200 include, TA-6FFXXX, preferably Seplife 6FF, which may further remove HCP, further reducing total protein content and thereby increasing ratio purity and SEC purity.
In some examples of the application, the gel column is loaded at 5% -15% column volume, alternatively 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%, preferably 5% -10% column volume. The recovery rate of exosome particles can be influenced by different loading volumes, and the inventor finds that through single factor experiments, the relation between the column volume and the recovery rate of exosome particles is researched: the larger the loading volume is, the higher the recovery rate is, and in the common gel filtration chromatography, the loading capacity is generally 1% -5% of the column volume, and the inventor finds that the loading volume of the exosome can reach 10% of the column volume through research, and the separation effect of the gel filtration chromatography is not influenced, so that the economy is greatly improved.
In some examples of the application, the flow rate of the gel filtration chromatography treatment is less than or equal to 1cm/min. The gel filtration chromatography instruction recommended flow rate is less than or equal to 5cm/min, and the inventor finds that the flow rate is less than or equal to 1cm/min, so that the separation effect can be improved and the elution time can be reduced.
In some examples of the application, the gel filtration chromatography purification treatment further comprises subjecting the fifth ultrafiltrate after the fifth filtration treatment to a sterile filtration treatment. The limit of microorganisms can be effectively reduced through the sterilization and filtration treatment, and the safety of exosomes is ensured.
In some examples of the application, the sterile filtration is performed using a 0.2 μm size sterile filter.
In other examples of the application, the method of purifying an exosome further comprises: the method further comprises the step of performing a second TFF tangential flow purification treatment on the anion multi-mode chromatographic purification product after anion multi-mode chromatographic purification and before gel filtration chromatographic purification.
In some examples of the application, the second TFF tangential flow purification treatment and the first TFF tangential flow purification treatment are the same or partially optimized.
The method for purifying exosomes of the present application can be used for industrial applications with a greatly increased throughput compared to general ultracentrifugation and density gradient centrifugation. The granularity (particles) of the exosome after being treated and purified by the method is high (> 1E+11particles/ml), the protein concentration is low (< 200 mu g/ml), and the most purity index of the particles/protein can reach more than 1E+9particles/mu g which is far higher than the group standard proposed by China exosome society: > 1E+8 parts ics/. Mu.g. Through detection, the exosome SEC-HPLC purity is very high and can reach more than 98%. More importantly, the method is mild, and does not damage the appearance, morphology, markers and the like of the exosomes.
In another aspect of the present application, the present application provides a method for controlling exosome quality, the method comprising: performing at least one of 1) -3) the detection of the obtained exosomes: 1) Western blot analysis; 2) Scanning by a transmission electron microscope; 3) SEC-HPLC purity detection. By using the exosome quality control method, the accurate characterization of the exosome can be realized, so that the quality control of each stage of the exosome production process is realized, and a large number of high-purity pollution-free exosomes are obtained.
In some examples of the application, the western blot analysis comprises detecting at least one of exosome-specific markers Calnexin, alix, CD, CD9, and CD 81.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
Test example 1: methods of purifying exosomes (including first TFF tangential flow purification, anion multimode chromatographic purification and gel filtration chromatographic purification treatments)
1.1 First TFF tangential flow purification
1.1.1 Concentrating the feed liquid, placing the feed liquid end and the reflux end of the hollow fiber into a container, placing the permeation end into a waste liquid barrel, starting a peristaltic pump to begin concentrating, wherein the concentration is 10-20 times, and the TMP is less than or equal to 5psi.
1.1.2 Ultrafiltration: after the sample is concentrated, adding balance liquid A into the feed liquid, continuously concentrating while keeping the volume of the concentrated liquid basically unchanged, washing and filtering for 4-5 times until the pH reaches 7.7-7.9, and then emptying the pipeline to recycle the feed liquid.
1.2 Anion Multi-mode chromatography purification
1.2.1 Equilibrium: the balance liquid A is used for washing 15-20 CV, and the flow rate is less than or equal to 1cm/min.
1.2.2 Loading: and loading the concentrated and ultrafiltered sample with loading capacity less than or equal to 11mg/ml and flow rate less than or equal to 1cm/min.
1.2.3 Collection: during loading or elution, collection was started when uv absorbance (280 nm wavelength) increased and stopped when uv absorbance decreased to baseline.
1.3 Purification by gel filtration chromatography
1.3.1 Rinsing with equilibration liquid A at a flow rate of 1cm/min or less for 3-5 CV.
1.3.2 Loading: the loading volume is less than or equal to 10 percent of the column volume, and the flow rate is less than or equal to 1cm/min.
1.3.2 Collection: after the sample loading is finished, the balance liquid A is used for flushing the sample at a speed of 1-3 CV, the flow rate is less than or equal to 1cm/min, the collection is started when the ultraviolet absorption (280 nm wavelength) is increased, and the collection is stopped when the ultraviolet absorption is reduced to a base line.
Test example 2 (this patent embodiment): methods of purifying exosomes (including first TFF tangential flow purification, anion multi-mode chromatographic purification, second TFF tangential flow purification, and gel filtration chromatographic purification treatments)
2.1 First TFF tangential flow purification
2.1.1 Concentrating the feed liquid, placing the feed liquid end and the reflux end of the hollow fiber into a container, placing the permeation end into a waste liquid barrel, starting a peristaltic pump to begin concentrating, wherein the concentration is 10-20 times, and the TMP is less than or equal to 5psi.
2.1.2 Ultrafiltration: after the sample is concentrated, adding balance liquid A into the feed liquid, continuously concentrating while keeping the volume of the concentrated liquid basically unchanged, washing and filtering for 4-5 times until the pH reaches 7.7-7.9, and then emptying the pipeline to recycle the feed liquid.
2.2 Anion Multi-mode chromatography purification
2.2.1 Equilibrium: the balance liquid A is used for washing 15-20 CV, and the flow rate is less than or equal to 1cm/min.
2.2.2 Loading: and loading the concentrated and ultrafiltered sample with loading capacity less than or equal to 11mg/ml and flow rate less than or equal to 1cm/min.
2.2.3 Collection: during loading or elution, collection was started when uv absorbance (280 nm wavelength) increased and stopped when uv absorbance decreased to baseline.
2.3 Second tangential flow purification
2.3.1 Concentrating the feed liquid, placing the feed liquid end and the reflux end of the hollow fiber into a container, placing the permeation end into a waste liquid barrel, starting a peristaltic pump to begin concentrating, concentrating by 4-5 times, wherein TMP is less than or equal to 5psi, and then emptying a pipeline to recycle the feed liquid.
2.4 Gel filtration chromatography purification
2.4.1 Flushing with balancing solution A at flow rate less than or equal to 1cm/min for 3-5 CV.
2.4.2 Loading: the loading volume is less than or equal to 10 percent of the column volume, and the flow rate is less than or equal to 1cm/min.
2.4.2 Collection: after the sample loading is finished, the balance liquid A is used for flushing the sample at a speed of 1-3 CV, the flow rate is less than or equal to 1cm/min, the collection is started when the ultraviolet absorption (280 nm wavelength) is increased, and the collection is stopped when the ultraviolet absorption is reduced to a base line.
As can be seen from FIGS. 1 to 4, the purity of the exosomes after anion multi-mode chromatography purification and gel filtration chromatography purification is greatly improved, and the purity after gel filtration is 99.6%. As can be seen from SEC results, the exosomes do not aggregate, and almost no host proteins and host DNA are present in the sample, and the purity meets the group standard. As can be seen from FIG. 5, the exosome has a particle size ranging from 30 to 150nm, a tea tray shape, round edges and no ridges, and shows that the exosome has good shape, no damage to the membrane and meets the group standard.
Comparative example
1. Comparative example 1 (tangential flow filtration + size exclusion chromatography)
1.1 Tangential flow filtration
1.1.1 Concentrating the feed liquid, placing the feed liquid end and the reflux end of the hollow fiber into a container, placing the permeation end into a waste liquid barrel, starting a peristaltic pump to begin concentrating, wherein the concentration is 10-20 times, and the TMP is less than or equal to 5psi.
1.1.2 Ultrafiltration: after the sample is concentrated, PBS is added into the feed liquid, the concentration is continued while the concentration liquid volume is kept basically unchanged, the washing and filtering are carried out for 4-5 times, and then the feed liquid is recovered by the emptying pipeline.
1.2 Size exclusion
1.2.1 Equilibrium: the column was rinsed with 10ml PBS.
1.2.2 Loading: the loading volume was 1ml.
1.2.3 Collection: the column was rinsed with 10ml PBS, collection was started when uv absorbance (280 nm wavelength) increased and stopped when uv absorbance decreased to baseline.
2. Comparative example 2 (tangential flow filtration + anion exchange)
2.1 Tangential flow filtration
2.1.1 Concentrating the feed liquid, placing the feed liquid end and the reflux end of the hollow fiber into a container, placing the permeation end into a waste liquid barrel, starting a peristaltic pump to begin concentrating, wherein the concentration is 10-20 times, and the TMP is less than or equal to 5psi.
2.1.2 Ultrafiltration: after the sample is concentrated, 20mM Tris pH8.0 is added into the feed liquid, the concentration is continued, the volume of the concentrated liquid is kept basically unchanged, the washing and filtering are carried out for 4-5 times, and then the feed liquid is recovered by a emptying pipeline.
2.2 Anion exchange
2.2.1 Equilibrium: 100CV was rinsed with 20mM Tris pH8.0, flow rate 5CV/min.
2.2.2 Loading: the ultrafiltered samples were loaded and, after loading was completed, 60CV was rinsed with 20mM Tris.
2.2.3 Elution: 20mM PB,2M NaCl pH8.0 at 0-100%, eluting 30cv, and flowing speed 1cv/min.
3. Comparative example 3 (anion exchange chromatography + cation exchange chromatography + molecular sieve chromatography)
3.1 Anion exchange
3.1.1. The pH of the chromatographic column was equilibrated to 8.0 using a pH8.0,20mM phosphate buffer, the column volume was 80ml, and after equilibration, the UV detector of the chromatographic system was set to zero.
3.1.2 Using an AKTA150 chromatographic system loading pump to load samples, wherein the loading flow rate is 4ml/min, the sample starts to be collected after the ultraviolet absorption value is increased to 1mAU in the loading process, and the sample is finished to be collected after the ultraviolet absorption value is reduced to 1 mAU.
3.2 Cation exchange chromatography
3.2.1 The pH of the column was equilibrated to 7.4 using pH7.4,20mM phosphate buffer, column volume was 100ml, and after equilibration was set to zero by UV detector of the chromatography system.
3.2.2 Regulating the pH value of the sample after cation exchange chromatography to 7.4, loading the sample by using an AKTA150 chromatography system loading pump, wherein the loading flow rate is 20ml/min, starting to collect the sample after the ultraviolet absorption value is increased to 1mAU in the loading process, and ending to collect the sample after the ultraviolet absorption value is reduced to 1 mAU.
3.3 Molecular sieve chromatography
3.3.1 The pH of the column was equilibrated to 7.4 using pH7.4,20mM phosphate buffer, column volume 200ml, and after equilibration was set to zero by UV detector of the chromatography system.
3.3.2 Loading the ultrafiltered sample by using an AKTA150 chromatographic system loading pump, wherein the loading flow rate is 20cm/h, after loading, the sample starts to be collected when the ultraviolet absorption value of the chromatographic system rises, the first ultraviolet absorption value of the outer water peak is reduced to be stable, the collection is ended,
3.3.3 The first exotic peak sample collected was the exosome sample, which was stored at 80℃for low temperature.
As shown in fig. 6-7, after the exosomes are purified by the method of the application, the total recovery rate of the exosomes is obviously higher than that of comparative case 2 and comparative case 3, slightly higher than that of comparative case 1, and the ratio purity is higher than that of comparative cases 1, 2 and 3; the gel filtration chromatography (size exclusion chromatography) in comparative cases 1 and 3 was relatively low in volume loading, typically 1% to 10% of column volume, and low in single pass; the application firstly concentrates the sample and then carries out anion multi-mode chromatography, so that most of impurities are removed, and then the sample volume is reduced by concentration, so that the sample can be completely purified by single gel filtration chromatography, and the purity and the recovery rate are higher than those of the comparative example.
In conclusion, the exosomes with high purity and high quality can be obtained based on the technical scheme of the application.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. A method of purifying an exosome comprising:
and sequentially performing first TFF tangential flow purification, anion multi-mode chromatographic purification and gel filtration chromatographic purification treatment on the supernatant of the exosome to be purified to obtain the exosome.
2. The method of claim 1, wherein the supernatant is from a cell supernatant collected from cells under 2D culture conditions or 3D suspension culture conditions, the cells having the ability to secrete exosomes.
3. The method of claim 2, wherein the cells are mesenchymal stem cells.
4. The method of claim 1, wherein the TFF tangential flow purification process comprises a first filtration process;
Optionally, the flux of the first filtration treatment is selected from 220L/m 2-225 L/m2, preferably 222L/m 2;
Optionally, the flow rate of the first filtration treatment is selected from 10L/m 2/min-15L/m2/min;
optionally, the first filtering process further comprises: and (5) performing top washing treatment on the filter.
5. The method of claim 4, further comprising, after the first filtering process: concentrating and ultrafiltering the first filtrate after the first filtering treatment;
Optionally, the concentrated ultrafiltration treatment is performed by a hollow fiber module;
optionally, the hollow fibers in the hollow fiber module have a pore size of 30nm to 50nm;
optionally, the concentrated ultrafiltration treatment has a transmembrane pressure differential selected from 5psi or less;
Optionally, the flux of the concentrated ultrafiltration treatment is selected from 50L/m 2-60 L/m2;
optionally, the time of the concentrated ultrafiltration treatment is selected from 6 to 8 hours, preferably 7 hours;
Optionally, after the concentrated ultrafiltration treatment, further comprising subjecting the ultrafiltrate after the concentrated ultrafiltration treatment to a second filtration treatment.
6. The method of claim 4 or 5, wherein the anionic multimode chromatographic purification employs a chromatographic column for a third filtration treatment;
optionally, the chromatographic column packing particle size is selected from 85 μm to 95 μm;
optionally, the chromatographic column packing is selected from Capto Core 700, sun Core 700, smac Core 700, maxtar Coll Core 700, monomix Core 1000XXX, preferably Capto Core 700;
optionally, the anionic multimodal chromatographic purification is performed in a buffer having a pH selected from 7.5-8, preferably 7.8;
Optionally, the buffer comprises: 37mmol/L-42mmol/L sodium hydrogen phosphate and 120mmol/L-130mmol/L sodium chloride; preferably 40mmol/L sodium hydrogen phosphate and 125mmol/L sodium chloride;
optionally, the flow rate of the anionic multimodal chromatographic purification is not greater than 1cm/min;
optionally, the third filtration treatment further comprises concentrating the third filtrate after the third filtration treatment and performing a fourth filtration treatment.
7. The method according to claim 6, wherein the gel column is used for fifth filtration treatment in the gel filtration chromatography purification;
Optionally, the filler particle size of the gel column is selected from 45-165 μm;
optionally, the packing of the gel column is selected from Seplife FF, superose 6Increase, siperdex 200 increment, TA-6FF, preferably Seplife FF;
optionally, the gel column loading is from 5% to 15% column volume, preferably from 5% to 10% column volume;
optionally, the flow rate of the gel filtration chromatography treatment is less than or equal to 1cm/min;
Optionally, the gel filtration chromatography purification treatment further comprises a sterilization filtration treatment of the fifth filtrate after the fifth filtration treatment.
8. The method according to claim 1, characterized in that it comprises: the method further comprises the step of performing a second TFF tangential flow purification treatment on the anion multi-mode chromatographic purification product after anion multi-mode chromatographic purification and before gel filtration chromatographic purification.
9. A method according to claim 2 or 3, wherein the supernatant is derived from a cell supernatant of mesenchymal stem cells collected under 3D suspension culture conditions, the 3D suspension culture being performed by:
Mixing the mesenchymal stem cells with the passage density of 3000-10000 cells/cm 2 with microcarriers;
inoculating the mixed treatment product to a bioreactor system, the bioreactor system comprising the culture medium;
Optionally, the culture Medium is selected from at least one of mesenchymal stem cell serum-Free Medium, hapCult MSC, xeno-Free, SFM, MSC Xeno-Free SFM, MSC NutriStem, and MesenCult TM- ACF Plus Medium;
preferably, the culture medium is a mesenchymal stem cell serum-free culture medium;
Optionally, after inoculation, the microcarrier is present in the bioreactor system at a concentration of 1-4 g/L;
Optionally, the microcarrier is selected from at least one of Cytodex1, cytodex3, 3D TableTrix, plastics Plus, plastic, collagen and Hillex II, preferably Cytodex1 and/or Collagen;
Optionally, the bioreactor system pH is selected from 7.0 to 7.8;
optionally, the inoculation is carried out at a rotation speed of 30rpm to 50 rpm.
10. A method for controlling exosome quality, comprising:
purifying exosomes by the method of any one of claims 1-8; and
Performing at least one of 1) -3) the detection of the obtained exosomes:
1) Western blot analysis;
2) Scanning by a transmission electron microscope;
3) SEC-HPLC purity detection;
optionally, the western blot analysis comprises detecting at least one of exosome-specific markers Calnexin, alix, CD, CD9, and CD 81.
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