An exosome purification method and quality control method suitable for industrialization level

Abstract

The application belongs to the technical field of exosome purification and characterization, and particularly relates to an exosome purification method and a quality control method suitable for industrial level. The application establishes an exosome purification process combining TFF and whole strong anion exchange chromatography, and selects a nuclease to remove chromatin contamination, aiming to provide an alternative reference for industrial level large-scale preparation of natural exosomes or engineered exosomes for clinical purification. Meanwhile, the application also establishes a whole exosome quality control system combining NTA, WB, TEM, ExoView and FFF-MALS-DLS. By using the exosome purification method and the quality control method, the physical properties and biological attributes of exosomes in the sample can be linked, the most accurate exosome characterization under the current technological conditions can be realized, the quality control of exosome samples at each stage of the process can be realized, and exosomes with almost no chromatin contamination can be obtained.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of exosome purification and characterization, and particularly relates to an exosome purification method and quality control method suitable for industrial level. BACKGROUND

[0002] Exosomes are a rapidly growing direction in the therapeutic field of the biopharmaceutical industry, and the source cells of exosomes include mesenchymal stem cells, which is the reason why exosomes are particularly concerned in the field of regenerative medicine. Recent studies have recorded the ability of mesenchymal stem cell exosomes to reverse the effects of severe stroke, highlighting their therapeutic potential. At the same time, it also emphasizes the need for scalable purification technology to drive these products through clinical trials and obtain permission for production.

[0003] The ultimate goal of exosomes is to be applied to the clinic as a biological agent, so it must meet the existing safety standards of biological agents, and host proteins, DNA, viruses and endotoxins must be reduced to a safe level. At the same time, the exosome product must maintain activity and be able to be concentrated to maintain dose control. Industrial exosome purification must consider the scalability of the process, as well as the protection of the structure and biological integrity of the exosomes, reducing the aggregation of exosomes, non-exosome particles and other protein contamination, improving the yield and purity of exosomes, and at the same time, this process must be economical and scalable, but there is no available solution for industrial large-scale production and purification of exosomes, and there is a lack of a unified quality control system. SUMMARY

[0004] The purpose of the present application is to provide an exosome purification method and quality control method suitable for industrial level, to provide an alternative reference for the industrial level large-scale preparation of natural exosomes or engineered exosomes that can be used for clinical purification and quality control.

[0005] The present application provides an exosome purification method suitable for industrial level, comprising the following steps:

[0006] (1) filtering the supernatant of exosome donor cells through a double-layer glass fiber filter to obtain an exosome clear liquid;

[0007] (2) passing the exosome clear liquid obtained in step (1) through a polyether sulfone liquid filter to obtain a sterile clear liquid;

[0008] (3) performing tangential flow filtration on the sterile clear liquid obtained in step (2) through a cellulose-based membrane to obtain a tangential flow filtrate;

[0009] (4) incubating the tangential flow filtrate obtained in step (3) with a nuclease after hollow fiber tangential flow filtration to obtain a chromatin-free exosome liquid;

[0010] (5) The achromatin-free exosome liquid obtained in step (4) is subjected to whole strong anion exchange chromatography column, and the purified peak is collected to obtain purified exosomes.

[0011] Preferably, the Sartorius The GF pre-filter is used for constant flow filtration of the supernatant of the collected exosome donor cells.

[0012] The Sartorius The filter diameter of the GF is 0.65 μm.

[0013] Preferably, the Sartorius The 2XLG filter is used for sterilization filtration of the exosome clarified liquid.

[0014] The Sartorius The pore size of the 2XLG filter is 0.8 / 0.2 μm.

[0015] Preferably, the Sartorius The 300 kDa 0.1 m 2 The ultrafiltration membrane pack is used for tangential flow filtration of the sterile clarified liquid.

[0016] Preferably, the Sartorius Single-use hollow fiber TFF is used in step (4) to incubate the Kryptonase and the tangential flow filtrate after solution short cycle for 1 h.

[0017] The pass particle size of the Sartorius Single-use hollow fiber TFF is not greater than 750 kDa.

[0018] Preferably, after the incubation in step (4), the obtained achromatin-free exosome liquid is further subjected to Sartorius Single-use hollow fiber TFF.

[0019] Preferably, the Sartorius BIA Separations CIMmultus TM The EV 1 ml 2 μm whole column is used for exosome fine purification of the achromatin-free exosome liquid.

[0020] The application also provides a quality control method in the process of exosome production and purification, comprising (1) nanoparticle tracking analysis, (2) Western Blot analysis, (3) transmission electron microscopy scanning, (4) exosome separation and identification based on 18-angle laser light scattering-field flow separation detection system, and (5) exosome subpopulation analysis based on ExoView in the process of exosome production and purification.

[0021] Preferably, the Western Blot analysis comprises detection of exosome-specific markers such as Alix, TSG101, CD63, CD9, CD81, Syntenin-1, etc.

[0022] Preferably, the exosome subpopulation analysis comprises exosome subpopulation analysis based on ExoView, or analysis of exosome intact vesicles and exosome vesicle fragments by detecting the expression level of Syntenin-1 in the sample.

[0023] Beneficial effects: the application provides an exosome purification method suitable for industrial level, establishes an exosome purification process combining tangential flow filtering (TFF) and whole strong anion exchange chromatography, and selects a nuclease to remove chromatin contamination, aiming to provide an alternative reference for industrial level large-scale preparation of natural exosomes or engineered exosomes for clinical purification. Meanwhile, the application also establishes a whole exosome quality control system combining nanoparticle tracking analysis (NTA), Western Blot (WB), transmission electron microscopy (TEM), automatic exosome fluorescence detection system ExoView, and field flow separation-18-angle light scattering detection (FFF-MALS).

[0024] By using the exosome purification method and quality control method provided by the application, the physical properties and biological attributes of exosomes in the sample can be linked, the most accurate exosome characterization under current technological conditions can be achieved, the quality control of exosome samples at each stage of the process can be realized, and exosomes with almost no chromatin contamination can be obtained. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0026] Figure 1 Figure for an industrial level large-scale exosome purification process (A) and quality control system (B) overview;

[0027] Figure 2 Figure for clarification of filter rate pressure difference carrier change in the filtration process;

[0028] Figure 3 Figure for exosome sample specific protein expression detection results;

[0029] Figure 4 Figure for filter rate pressure difference carrier change in the sterilization filtration process;

[0030] Figure 5 Figure for TFF process filter rate TMP time change curve;

[0031] Figure 6 Figure for E, F exosome structure under electron microscope;

[0032] Figure 7 Figure for chromatogram;

[0033] Figure 8 Figure for G, H, I exosome structure under electron microscope;

[0034] Figure 9 Figure for normalized sample ultraviolet signal elution comparison results;

[0035] Figure 10 Figure for normalized sample light scattering signal elution graph comparison results;

[0036] Figure 11 Figure for elution particle size concentration analysis results;

[0037] Figure 12 Figure for G sample exosome CD63, CD81, CD9 subgroup analysis results;

[0038] Figure 13 Figure for H sample exosome CD63, CD81, CD9 subgroup analysis results;

[0039] Figure 14 Figure for I sample exosome CD63, CD81, CD9 subgroup analysis results;

[0040] Figure 15Figure for I sample CD63 capture channel exosome subpopulation analysis result graph;

[0041] Figure 16 Figure for I sample CD81 capture channel exosome subpopulation analysis result graph;

[0042] Figure 17 Figure for I sample CD9 capture channel exosome subpopulation analysis result graph. DETAILED DESCRIPTION

[0043] The present application provides an exosome purification method suitable for industrial level, the purification process flow thereof is shown in Figure A, comprising the following steps: Figure 1

[0044] (1) passing the supernatant of exosome donor cells through a double-layer glass fiber filter to obtain exosome clear liquid;

[0045] (2) passing the exosome clear liquid obtained in step (1) through a polyether sulfone liquid filter to obtain sterile clear liquid;

[0046] (3) passing the sterile clear liquid obtained in step (2) through a cellulose-based membrane for tangential flow filtration to obtain tangential flow filtrate;

[0047] (4) incubating the tangential flow filtrate obtained in step (3) with a nuclease after hollow fiber tangential flow filtration to obtain achromatin exosome liquid;

[0048] (5) passing the achromatin exosome liquid obtained in step (4) through a whole strong anion exchange chromatography column, collecting the purified peak to obtain purified exosomes.

[0049] The present application passes the supernatant of exosome donor cells through a double-layer glass fiber filter to obtain exosome clear liquid, and the type of the exosome donor cells is not particularly limited, and any cell capable of producing exosomes in the art can be used, and the method for producing exosomes by the donor cells is not particularly limited, preferably a scheme based on a Sartorius clarification filter, a sterilization filter and a tangential flow filter is used to evaluate the exosome particle yield and the process duration, because the platform provided by Sartorius can be scaled up, the production cost of exosomes is very high, and in the examples, the above process is executed and verified based on a 250 mL original exosome sample, and based on the obtained data, the filter and ultrafiltration membrane package corresponding to the processing volume of the sample can be selected to realize ultrafiltration of exosomes in pilot and production scale, and realize large-scale preliminary purification of exosomes; the exosome purification method of the present application can be directly scaled up, such as tens of liters or even hundreds of liters of exosome donor cell supernatant, which can complete the same degree of purification. The present application preferably uses Sartorius ​The GF pre-filter filters the collected supernatant of the exosome donor cells at a constant flow rate; the Sartorius The pore size of the GF is preferably 0.65 pm, the Sartorius The GF pre-filter has excellent overall permeability and particle retention capacity, providing the most efficient liquid purification and high overall permeability, which can significantly reduce the cost of filtration. The Sartorius GF (0.65 pm, 17.3 cm 2 ) pre-filter filters the collected initial cell supernatant sample at a constant flow rate of 285 LMH at 25°C.

[0050] After obtaining the exosome clear liquid, the present application filters the exosome clear liquid through a polyether sulfone liquid filter to obtain a sterile clear liquid. The Sartorius 2XLG filter for sterilization filtration of the exosome clear liquid; the Sartorius The pore size of the 2XLG filter is preferably 0.8 / 0.2 pm. The Sartorius 2 is a high-performance polyether sulfone (PES) liquid filter, and different pore size combinations ensure high economic and safe filtration performance. The Sartorius 2XLG (0.8 / 0.2 pm, 17.3 cm 2 ) filter for sterilization filtration of the collected exosome clear liquid sample at a flow rate of 313 LMH at 25°C.

[0051] After obtaining the sterile clear liquid, the present application filters the sterile clear liquid through a cellulose-based membrane for tangential flow filtration to obtain a tangential flow filtrate, preferably using a Sartorius 300kDa 0.1m 2 Ultrafiltration membrane package TMP 0.2 bar, 25°C for tangential flow filtration of the sterile clear liquid. In the present application, High-performance membrane is a stable cellulose-based membrane that is stable in a wide pH range and is very hydrophilic, so it does not bind proteins and is almost free of contamination.

[0052] After obtaining the tangential flow filtrate, the tangential flow filtrate is incubated with nuclease after hollow fiber tangential flow filtration to obtain achromatin exosome fluid. The present application preferably uses Sartorius Single-use hollow fiber TFF to incubate the nuclease Kryptonase and the tangential flow filtrate after solution short cycle for 1 h; the Sartorius Single-use hollow fiber TFF has a particle size of no more than 750 kDa. The Sartorius hollow fiber tangential flow filtration (TFF) module in the present application provides high-performance separation in various upstream and downstream biological treatment unit operations, and Kryptonase is a comprehensive treatment enzyme that can reduce host cell DNA and help remove host cell protein contaminants. Using Sartorius Single-use hollow fiber TFF, 750 kDa injection of Kryptonase for solution short cycle incubation for 1 h removes chromatin-based vesicle contamination in the sample. In the present application, after the incubation, the obtained achromatin exosome fluid is preferably further subjected to Sartorius Single-use hollow fiber TFF again under the condition of TMP 0.2 bar and 25°C.

[0053] After obtaining the achromatin exosome fluid, the achromatin exosome fluid is subjected to a whole strong anion exchange chromatography column, and the purified peak is collected to obtain purified exosomes. One of the great difficulties in the art of separating exosomes from culture supernatant is the presence of non-vesicular macromolecular structures in different fluids. The present application uses tangential flow filtration (TFF) to remove macromolecular contaminants based on particle size or molecular weight changes. However, there is particle size heterogeneity, and further optimization is needed to separate exosomes from contaminated proteins; in the present application, the low-shear strong anion chromatography based on CIMmultus TM EV monolithic column is used for downstream exosome fine purification. CIMmultus TM EV monolithic column has a unique pore structure and corresponding anion ligand, which facilitates the flow and binding of exosomes in the chromatography column, and has a separation effect on exosomes and impurities, and exosomes can be separated from smaller protein and nucleic acid contaminants. Therefore, TFF samples can be significantly enriched in exosomes of different sample sources after passing through CIMmultus TM EV monolithic column. In the examples of the present application, Sartorius BIA Separations CIMmultus TM EV 1ml 2μm monolithic column is used for exosome fine purification of secondary tangential flow filtration samples, and the chromatography conditions are shown in Table 5.

[0054] The application also provides a quality control method in the process of exosome production and purification, the flow chart of which is shown in Figure B, including in the process of exosome production and purification, performing (1) nanoparticle tracking analysis, (2) Western Blot analysis, (3) transmission electron microscope scanning, (4) exosome separation and identification based on 18-angle laser light scattering-field flow fractionation detection system, and (5) exosome subpopulation analysis based on ExoView. Figure 1

[0055] The application establishes a quality control standard based on exosome total population and subpopulation analysis to monitor the process of exosome production and purification, which specifically includes: (1) Nanoparticle tracking analysis (NTA) for nanoparticle counting in the sample; (2) Western Blot (WB) test for expression of exosome-specific proteins; (3) Transmission electron microscope (TEM) for exosome morphology characterization; (4) exosome separation and identification based on 18-angle laser light scattering-field flow fractionation detection system (FFF-MALS-DLS), which realizes precise classification of different particle sizes of small exosomes (Exo-S, 60-80 nm), large exosomes (Exo-L, 90-120 nm), and exomeres (non-membrane nanoparticles, Avg: 35 nm) according to pure physical properties, and simultaneously combines UV / Vis absorption spectrum and Zeta potential to precisely detect the physical properties of exosomes; (5) exosome subpopulation analysis based on ExoView, which can detect up to 5 types of markers at the same time by using a labeled chip to capture exosomes expressing specific membrane proteins, can classify and analyze exosome subpopulations by detecting the fluorescence of different markers, and can also quantify the contents of exosomes by perforating the exosomes, thereby evaluating engineered exosomes; or (5) Flow Nano Analyzer can detect the particle size and specific markers of exosomes to achieve the functions similar to ExoView. In the application, the specific operation schemes of NTA, WB, TEM, FF-MALS-DLS, and ExoView are not particularly limited and can be operated according to the device instructions.

[0056] ​In the present application, when performing exosome subpopulation analysis, ExoView can detect natural samples without any treatment, reducing the interference of complex operations on exosomes, while the chip can realize data consistency analysis with control, and the chip capture count determines that the detected material is exosome or exosome fragment, and the proportion of fragments can be confirmed by reagents. Nanoflow has requirements for the purity of exosome samples, and the detected material is particles, which cannot completely represent the nature of exosomes and will interfere with quality control. If the purification process can ensure the purity of exosomes, the quality control system can supplement the nanoflow data for more comprehensive exosome evaluation.

[0057] In the present application, the FFF-MALS-DLS detection platform can separate and identify exosomes with high sensitivity, wherein the FFF system is based on Eclipse FFF instrument and combined with a pump and an automatic sampler. The separation occurs in a thin channel with a porous bottom wall, including an ultrafiltration membrane supported by a frit. The constriction of the channel outlet causes part of the flow to pass through the bottom, forming a cross-flow. During sample injection, the cross-flow concentrates the sample towards the membrane. Brownian motion acts as a counteracting force, preventing cross-flow and allowing particles to move upwards and away from the membrane. The balance between cross-flow and diffusion produces a cloud of particles whose concentration decreases exponentially with distance from the membrane. Smaller particles, with higher diffusion, will be above the channel, while larger particles will remain closer to the membrane. During elution, the laminar flow of the carrier liquid along the channel results in a velocity profile that depends on the height above the membrane. Smaller particles, which are in contact with the faster flow of the larger cloud, will be eluted faster than larger particles, mainly because they encounter slower flow. This results in very efficient separation of particles with little or no shear or membrane interaction.

[0058] The ExoView described in the present application performs precise subpopulation and content analysis of exosomes in different process steps, and the exosome sample can remain in the original biological state without complex processing procedures, and up to 16 samples can be automatically measured. 488nm, 555nm, 640nm and 750nm, 4 color channels, single molecule sensitive fluorescence means that even the smallest exosome can be detected. Exosome subpopulations are described by the expression of biomarkers. Up to 5 markers can be detected and characterized for each exosome, enabling specific detection and analysis of exosome contents.

[0059] By combining the above detection means, the physical properties and biological attributes of exosomes in the sample can be linked to achieve the most accurate exosome characterization under current technological conditions, and thus realize quality control of exosome samples at each stage of the process.

[0060] In order to further illustrate the present application, an exosome purification method and quality control method suitable for industrialization level provided by the present application are described in detail below in combination with the drawings and examples, but they cannot be understood as limiting the protection scope of the present application.

[0061] Example 1

[0062] The fine purification sample is 300 mL of human umbilical cord-derived mesenchymal stem cell 2D culture supernatant, the cell is hUC-MSC-0214, the culture system is a T-175 culture flask, and the culture medium is RoosterNourish TM MSC-XF complete medium is used for mesenchymal stem cell expansion and RoosterCollection-EV Pro TM Complete medium is used for exosome production, and RoosterCollection-EV Pro is replaced after the cells grow to 90% confluence TM , and the cell supernatant after 4 days of culture is collected. NTA, WB, TEM, ExoView, FFF-MALS-DLS are used for quality control during the process.

[0063] (1) Exosome initial sample clarification filtration: use GF (0.65 μm, 17.3 cm 2 ) filter to filter and clarify the original cell supernatant (A) in a constant flow mode, 251 mL of sample (B) is filtered out in 30.5 min, the average flux is 285 LMH, the load is 145 L / m 2 , and the pressure difference during the filtration process rises from 0 to 0.6 bar. The filtration load and pressure difference change as shown in Figure 2 , which meets the requirements.

[0064] NTA detection of particle numbers in A and B before and after clarification, the clarification test results are shown in Table 1;

[0065] Table 1 Clarification test results

[0066]

[0067] WB detects exosome-specific markers TSG101, CD63 and β-actin internal reference in A and B, and all have expression of exosome-specific markers Figure 3 ). The above data show that after clarification filtration, the particle number of sample A is lost by 38.9%, both samples A and B have expression of exosome-specific markers, and the filtrate does not have co-expression of CD63 and TSG101 Figure 3 in the middle J), which shows that most of the particles lost by the sample after filtration clarification are not exosomes.

[0068] (2) Exosome sterilization filtration: using 2XLG (0.8 / 0.2μm, 17.3cm) 2 The filter used a constant flow method to sterilize the collected exosome clear fluid sample (B). 253 mL of sample (C) was filtered out in 28 minutes, with an average throughput of 313 LMH and a loading capacity of 146 L / m³. 2 The pressure differential during the filtration process increased from 0 to 0.3 bar. The changes in filter load and pressure differential are shown in Figure 4, which meet the requirements.

[0069] The results of the clarification test, based on the particle counts in B and C before and after NTA detection, are shown in Table 2.

[0070] Table 2 Clarification Test Results

[0071]

[0072] Western blot analysis revealed the expression of exosome-specific markers TSG101, CD63, and β-actin internal reference in exosomes B and C. Figure 3 ).

[0073] The above data shows that after sterilization filtration, almost no particle number is lost, and exosome-specific markers are expressed in both samples B and C.

[0074] (3) Exosome tangential flow filtration: at room temperature, using 300kDa 0.1m 2 260 mL of sterile filtered sample (C) was concentrated 2.6 times by tangential flow filtration using an ultrafiltration membrane pack, followed by 7-fold fluid replacement to obtain 100 mL of concentrate (D). The entire tangential flow process took 53.1 min, with an inlet pressure of 0.4 bar, a reflux pressure of 0 bar, a permeation pressure of 0 bar, a TMP of 0.2 bar, and an average filtration rate of 43 LMH. During concentration, the filtration rate decreased slightly with increasing feed concentration, while during filtration, the filtration rate tended to stabilize. The permeation velocity and TMP over time curves are shown below. Figure 5 As shown in Table 3, the results of the ultrafiltration experiment, combined with NTA detection of particle numbers in cells C and D before and after tangential flow filtration, are presented. Western blotting analysis of exosome-specific markers TSG101, CD63, and β-actin (internal reference) in cells C and D showed expression of these exosome-specific markers. Figure 3 ).

[0075] The above data indicate that tangential flow filtration achieved concentration of the exosome solution and buffer replacement, resulting in a 12.3% loss of particle number. Both samples C and D showed expression of exosome-specific markers, and the filtrate did not show co-expression of CD63 and TSG101. Figure 3 The K value indicates that the vast majority of particles lost during tangential flow filtration were not exosomes.

[0076] Table 3 Ultrafiltration test results

[0077]

[0078] (4) Kryptonase enzyme treatment: under room temperature conditions, using Sartorius Single-use hollow fiber TFF, 750 kDa, 100 mL of exosome tangential flow filtration concentrate (D) and Kryptonase enzyme were injected into the solution for short cycle and low pressure incubation for 1 hour, obtaining 100 mL of Kryptonase enzyme treated exosome concentrate (E).

[0079] Combined with NTA detection of the particle number in D and E before and after tangential flow filtration, there was no particle loss. WB detection of exosome specific markers TSG101, CD63 and β-actin internal reference in D and E, all had expression of exosome specific markers Figure 3 ).

[0080] The above data show that after Kryptonase enzyme treatment, the chromatin treatment of exosome solution is realized, and there is no obvious particle loss, and there is expression of exosome specific markers in D and E samples.

[0081] Table 4 NTA detection of particle number after tangential flow filtration

[0082]

[0083] (5) Secondary tangential flow filtration: under room temperature conditions, using Sartorius Single-use hollow fiber TFF, 750 kDa, 100 mL of Kryptonase enzyme treated exosome concentrate (E) was subjected to secondary tangential flow filtration to obtain 100 mL of concentrate (F), the whole tangential flow process was 40 min, the inlet pressure was 0.4 bar, the back pressure was 0 bar, the permeate pressure was 0 bar, the TMP was 0.2 bar, the average filtration rate was 40 LMH, and the filtration rate was stable during the washing process.

[0084] WB detection of exosome specific markers TSG101, CD63 and β-actin internal reference in E and F, all had expression of exosome specific markers Figure 3 ). The filtrate did not have co-expression of CD63 and TSG101 Figure 3 , indicating that the sample was not damaged by enzyme treatment and secondary tangential flow filtration.

[0085] TEM detection of E and F, both can see obvious exosome structure Figure 6), the above data show that after twice tangential flow filtration, no obvious particle loss is caused, and the E and F samples have expression of exosome-specific markers, and the vesicles show exosomes under electron microscopy.

[0086] (6) Fine purification of exosomes based on Sartorius BIA Separations monolith strong anion exchange chromatography

[0087] Due to the CIMmultus TM EV monolith has a unique pore structure and corresponding anion ligand, which facilitates the flow and binding of exosomes in the chromatographic column, and has a good separation effect on exosomes and impurities, so the CIMmultus TM EV 1 mL is used for fine purification of exosomes, and the sample solution before purification is 100 mL of exosome solution F filtered twice by tangential flow filtration, and after purification, according to the chromatogram shown in Figure 7 , three purification peaks (G, H, and I) are collected.

[0088] Table 5 Chromatographic conditions

[0089]

[0090] NTA detects the particle number of F, G, H, and I, and the results are as follows: F: 1.3E+10 Particles / mL, G: 3.2E+9 Particles / mL, H: 1.5E+10 Particles / mL, and I: 1.0E+10 Particles / mL. NTA detects the particle size distribution of F, G, H, and I, and the results are as follows: F: 96.7 nm (97.4%), G: 98.7 nm (97.8%), H: 92 nm (97.4%), and I: 106.1 nm (98.3%).

[0091] WB detects exosome-specific markers TSG101, CD63, and β-actin internal reference of F, G, H, and I, and all have expression of exosome-specific markers Figure 3 ). The filtrate does not have co-expression of CD63 and TSG101 Figure 3 -M), indicating that most of the exosomes in the sample are enriched during the chromatography process and are not lost.

[0092] TEM detects G, H, and I, and all can see Figure 8 the obvious exosome structure shown. The above data show that after CIMmultus TM EV monolith, G, H, and I samples have expression of exosome-specific markers, and the vesicles under electron microscopy are typical exosome structures, but the exosome particle size distribution and marker expression level of the three peaks collected have differences, and more accurate means are needed for subpopulation analysis.

[0093] (7)High sensitivity separation and identification of exosomes based on FFF-MALS-DLS detection platform

[0094] Based on the FFF-MALS-DLS detection platform, the samples at each stage of the process were analyzed, and the data results of the C and I samples were analyzed. The normalized sample ultraviolet signal elution comparison showed that compared with the C (blue) sample, the I (red) sample realized the field flow separation of exosome particles. Figure 9 )。

[0095] The normalized sample light scattering signal elution comparison showed that compared with the C (blue) sample, the I (red) sample realized the enrichment of exosome particles ( Figure 10 ), and the exosome peak and other particle peaks could be clearly distinguished.

[0096] Combined with the ASTRA analysis software, the separation and analysis of particles of different sizes could be realized, and the precise concentration and particle size distribution quality control of sEv and lEv in the exosome sample could be realized. Figure 11 )。

[0097] (8)ExoView for precise subpopulation and content analysis of exosomes

[0098] To more finely control the quality and subpopulation analysis of the harvested exosomes, based on the ExoView detection platform, the samples at each stage of the process were analyzed, and the data results of the G( Figure 12 ), H( Figure 13 ), and I( Figure 14 ) samples were analyzed. It could be known that each sample captured the co-expression analysis of exosome particles CD61, CD81, and CD9 in the corresponding fluorescence channel, and thus realized the precise analysis of exosome subpopulations. Since the exosome capture chip could be customized to express markers, the capture and quantitative analysis of exosome membrane specific expression could be realized through the customized chip, and the quality control and precise analysis of engineered exosomes could be realized. At the same time, the expression level of Syntenin in the sample could also be detected to realize the analysis of exosome intact vesicles and exosome vesicle fragments, which was an important exosome integrity data in biological preparation quality control.

[0099] Based on the ExoView analysis data, the relationship between the expression level of different markers in the corresponding capture channel and the particle size could be obtained. For the I sample, the expression levels of CD63( Figure 15 ), CD81( Figure 16 ), and CD9( Figure 17Data analysis captured by the FFF-MALS-DLS detection platform can clarify the relationship between the expression levels and particle size of exosome markers in each subpopulation. Combined with the exosome particle and concentration data obtained from the FFF-MALS-DLS platform, precise subpopulation analysis and quality control of exosomes can be achieved with current technological capabilities. This provides reliable, accurate, and detailed data support for the clinical application of natural exosomes and the development of engineered exosome-based biopharmaceuticals. This analytical and characterization technique is also applicable to the analysis and quality control of various engineered nanoparticles.

[0100] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for purifying exosomes suitable for industrial-scale production, characterized in that, Includes the following steps: (1) The supernatant of exosome donor cells was passed through a double-layer glass fiber filter to obtain a clear exosome solution; using Sartorius... The GF pre-filter performs constant flow filtration on the supernatant of the collected exosome donor cells; (2) Pass the exosome clarification solution obtained in step (1) through a polyethersulfone liquid filter to obtain a sterile clarification solution; use Sartorius... The 2XLG filter sterilizes the exosome clarification solution. (3) The sterile clarified liquid obtained in step (2) is subjected to tangential flow filtration through a cellulose-based membrane to obtain the tangential flow filtrate; using Sartorius... 300kDa 0.1m 2 The ultrafiltration membrane pack performs tangential flow filtration on the sterile clarified liquid; (4) The tangential flow filtrate obtained in step (3) is filtered through hollow fiber tangential flow and then incubated with nuclease to obtain chromatin-free exosome fluid; The nuclease Kryptonase and tangential flow filtrate were incubated for 1 hour after short-circuiting in Sartorius Single-use hollow fiber TFF; the incubation process also included passing the resulting achromatin-free exosome fluid through Sartorius Single-use hollow fiber TFF once more. (5) The chromatin-free exosome fluid obtained in step (4) is passed through a monolithic strong anion exchange chromatography column, and the purification peak is collected to obtain purified exosomes; Sartorius BIA Separations CIMmultus is used. TM The achromatin-free exosome fluid was finely purified using an EV 1ml 2μm monolithic column.

2. The exosome purification method according to claim 1, characterized in that, The Sartorius mentioned in step (1) The filter diameter of GF is 0.65 μm.

3. The exosome purification method according to claim 1, characterized in that, In step (2), Sartorius The 2XLG filter has a pore size of 0.8 / 0.2μm.

4. The exosome purification method according to claim 1, characterized in that, The passing particle size of the Sartorius Single-use hollow fiber TFF described in step (4) is no greater than 750 kDa.

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