A plant-derived exosome stabilization and purification system and its process

Abstract

This invention discloses a plant-derived exosome stabilization and purification system and its process, belonging to the field of plant-derived exosome purification technology. The plant-derived exosome stabilization and purification system and its process include a sequentially linked suspension cell culture module, a multi-stage primary purification module, a stabilization treatment module, and a quality detection module. The suspension cell culture module uses MS basal medium supplemented with a hormone combination of 6-BA, NAA, meta-topolin, and 2,4-D, and cultures plant cells in suspension for 7-14 days under conditions of 25°C, 12h light / 12h dark, and 110rpm to form a source for exosome production. This plant-derived exosome stabilization and purification system and its process exhibit particle size fluctuation <10% and activity retention >85%, meeting the requirements for long-term industrial storage; ensuring consistency: modular design and online detection ensure that the coefficient of variation of particle size, potential, and activity between batches is <5%, complying with GMP production standards.

Description

Technical Field

[0001] This invention relates to the field of plant-derived exosome purification technology, and in particular to a plant-derived exosome stabilization and purification system and process. Background Technology

[0002] Plant-derived extracellular vesicles (PEVs) are natural vesicle structures secreted by plant cells. They possess excellent biocompatibility, low immunogenicity, and the ability to load natural active ingredients, and have shown great application potential in various fields in recent years. Currently, the production of plant exosomes largely relies on plant suspension cell culture systems. The extraction and purification process typically includes steps such as centrifugation, filtration, polyethylene glycol precipitation, and size exclusion chromatography. However, three core challenges exist in industrial-scale production:

[0003] Particle size aggregation problem: During the purification, concentration, cryopreservation and thawing stages, exosomes are prone to aggregation due to membrane surface charge imbalance, hydrogen bonding or hydrophobic interactions, resulting in abnormally large particle size (more than 300 nm), which seriously affects product uniformity and downstream application effects.

[0004] Insufficient stability: Existing technologies mostly use a single stabilizer (such as trehalose) or simple low-temperature freezing methods, lacking systematic control over key parameters such as buffer ionic strength, pH value, and shear stress, resulting in reduced activity and structural damage of exosomes during long-term storage.

[0005] Poor industrial adaptability: Existing processes are mostly fragmented unit operations with poor inter-module connectivity, making continuous production impossible. Furthermore, batch-to-batch parameter fluctuations are large, making it difficult to meet GMP (Good Manufacturing Practice) mass production requirements.

[0006] To address the aforementioned issues, some studies have attempted to optimize individual process parameters, such as adjusting centrifugation speed or changing the type of stabilizer. However, these efforts have failed to construct a stable purification system at the system level, thus failing to completely resolve the problems of exosome aggregation and stability. Therefore, developing an integrated, modular, and parameter-controllable plant-derived exosome stabilization and purification system is crucial for promoting its industrial application. Summary of the Invention

[0007] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a plant-derived exosome stabilization and purification system and process, which can solve the above-mentioned problems.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a plant-derived exosome stabilization and purification system, comprising a suspension cell culture module, a multi-stage primary purification module, a stabilization treatment module, and a quality detection module linked sequentially.

[0009] The multi-stage primary purification module includes, in process order:

[0010] Pre-centrifugation unit: Centrifuge at 3,000×g for 30 minutes to remove cell debris and large particulate impurities;

[0011] Ultracentrifugation unit: Centrifuge at 100,000×g for 60-90 minutes to recover exosome precipitates;

[0012] Polyethylene glycol precipitation unit: Adding 5-10% polyethylene glycol to achieve impurity concentration and auxiliary removal;

[0013] Size exclusion chromatography unit: using Sephadex CL-2B or G-100 packing material to remove residual small molecule impurities and protein debris;

[0014] The stabilization processing module includes:

[0015] Stabilization buffer unit: Use PBS, Tris or Hepes buffer, and control the ionic strength to 10-100mM and the pH value to 6.5-7.5.

[0016] Stabilizer compounding unit: Step-by-step addition of 1-5% w / v trehalose, 0.1-2.0 mM EDTA and 0.001-0.1% Tween 80;

[0017] Shear buffer unit: Equipped with a buffer and decompression device to control the concentration ratio of 10-50 times and prevent damage to the exosome membrane structure;

[0018] Temperature control cooling unit: Equipped with a programmable cooling controller, the cooling rate can be adjusted from -1 to -3°C / min, and the final freezing temperature is below -80°C;

[0019] Remelting unit: Equipped with an automatic compensation module for the concentration of compounded stabilizer, it slowly heats up to remelt while maintaining a constant stabilizer concentration;

[0020] The quality detection module is equipped with a nanoparticle tracking analyzer and a Zeta potential online monitoring device to detect the exosome particle size distribution and surface charge stability in real time.

[0021] As a preferred embodiment of the plant-derived exosome stabilization and purification system of the present invention, the suspension cell culture module uses MS basal medium supplemented with a hormone combination of 6-BA, NAA, meta-topolin, and 2,4-D to suspend plant cells for 7-14 days under conditions of 25°C, 12h light / 12h dark, and 110rpm to form a source of exosome production.

[0022] As a preferred embodiment of the plant-derived exosome stabilization and purification system of the present invention, the plant source is one or more combinations of Dendrobium officinale, Anoectochilus roxburghii, Lycium barbarum, and Ganoderma lucidum.

[0023] As a preferred embodiment of the plant-derived exosome stabilization and purification system of the present invention, the pre-centrifugation unit, ultracentrifugation unit, polyethylene glycol precipitation unit and size exclusion chromatography unit are connected in series through fluid pipelines to form a continuous purification flow path.

[0024] As a preferred embodiment of the plant-derived exosome stabilization and purification system of the present invention, the temperature control cooling unit is further provided with a cryopreservation termination temperature control module, which automatically stops the cooling program when the system temperature reaches below -80°C.

[0025] As a preferred embodiment of the plant-derived exosome stabilization and purification system of the present invention, wherein: in the stabilization buffer unit, the buffer replacement frequency is linked to the processing capacity of the multi-stage primary purification module, and the buffer is replaced once for every 10L of plant suspension cell culture supernatant processed.

[0026] A process for stabilizing and purifying plant-derived exosomes includes the following steps:

[0027] S1: Suspension cell culture: Plant cells were inoculated into MS medium containing hormone combinations and cultured in suspension at 25°C, 12h light / 12h dark, and 110rpm for 7-14 days. The culture supernatant was then collected.

[0028] S2: Multi-stage primary purification:

[0029] S21: Pre-centrifugation: Transfer the culture supernatant into the pre-centrifugation unit, centrifuge at 3,000×g for 30 minutes, and collect the supernatant;

[0030] S22: Ultracentrifugation: Transfer the supernatant collected in S21 into the ultracentrifugation unit, centrifuge at 100,000×g for 60-90 minutes, and collect the exosome precipitate;

[0031] S23: PEG precipitation: Add 5-10% polyethylene glycol solution to the exosome precipitate collected in S22, let stand at room temperature for 30 minutes, then centrifuge at 3,000×g for 15 minutes and collect the precipitate;

[0032] S24: Size exclusion chromatography: Resuspend the precipitate collected in S23 in stabilization buffer, load it onto a Sephadex CL-2B or G-100 column, and elute to collect the exosome components;

[0033] S3: Stabilization treatment:

[0034] S31: Buffer Adjustment: Introduce the exosome components collected in S24 into the stabilization buffer unit and adjust the ionic strength to 10-100 mM and the pH to 6.5-7.5;

[0035] S32: Stabilizer addition: Add 1-5% w / v trehalose, 0.1-2.0 mM EDTA and 0.001-0.1% Tween 80 to the system through the stabilizer compounding unit, and stir until homogeneous;

[0036] S33: Concentration control: The system is concentrated 10-50 times through a shear buffer unit, and the shear force is monitored in real time during the process to avoid exceeding 500Pa;

[0037] S34: Programmed cryopreservation: The concentrated system is introduced into a temperature-controlled cooling unit and cooled to below -80°C at a rate of -1 to -3°C / min for cryopreservation;

[0038] S35: Slow remelting: When needed, the remelting unit heats up at a rate of 0.5-1°C / min, while the concentration is maintained by the automatic concentration compensation module;

[0039] S4: Quality Inspection: After steps S24, S32, and S35, the exosome particle size and zeta potential are detected by the quality inspection module to ensure product quality.

[0040] As a preferred embodiment of the plant-derived exosome stabilization and purification process of the present invention, wherein the mass ratio of the amount of polyethylene glycol solution added in step S23 to the mass ratio of the exosome precipitate is 5:1-10:1.

[0041] As a preferred embodiment of the plant-derived exosome stabilization and purification process of the present invention, wherein the concentration process in step S33 adopts ultrafiltration membrane concentration method, and the molecular weight cutoff of the ultrafiltration membrane is 100kDa.

[0042] As a preferred embodiment of the plant-derived exosome stabilization and purification process described in this invention, in step S4, if the exosome particle size is detected to be greater than 150 nm or the zeta potential is higher than -10 mV, the process returns to step S31 to readjust the buffer solution.

[0043] Compared with the prior art, the beneficial effects of the present invention are:

[0044] 1. The plant-derived exosome stabilization and purification system and its process solve the aggregation problem: Through the multi-parameter synergy of the stabilization module, the exosome particle size can be stably controlled at 100-150nm, with an aggregation rate of <5% during storage, which is significantly better than the traditional process (aggregation rate >30%), improving stability: After storage at -80°C for 3 months, the particle size fluctuation of exosomes is <10%, and the activity retention rate is >85%, meeting the requirements for long-term industrial storage; ensuring consistency: Modular design and online detection ensure that the coefficient of variation of particle size, potential and activity between batches is <5%, which complies with GMP production standards.

[0045] 2. The plant-derived exosome stabilization and purification system and its process are applicable to multiple fields: The exosomes prepared by this system can be directly used in health foods (such as oral anti-aging preparations), beauty repair products (such as serums and masks), drug delivery carriers (such as loading small molecule drugs), and regenerative medicine (such as wound repair). The application scenarios are wide-ranging and the production capacity is high: The system modules can be scaled up (such as replacing the 5L bioreactor with a 100L one) to achieve kilogram-level exosome production, and the parameters do not need to be significantly adjusted, making it highly adaptable to industrialization. Attached Figure Description

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0047] Figure 1 This is a graph showing the test results of the screening experiment for the pre-centrifugation step of this invention;

[0048] Figure 2 The figure shows the test results of the screening experiment for the ultracentrifugation step of this invention;

[0049] Figure 3 This is a graph showing the results of a fine screening test for the concentration of the precipitant in this invention;

[0050] Figure 4 This is a graph showing the results of the fine-tuned synergistic optimization experiment of ionic strength and pH value in this invention;

[0051] Figure 5 This is a flowchart of the process for the prevention, control, and stabilization of plant-derived exosomes in this invention.

[0052] Figure 6 This is a detailed process flow diagram of the preparation of plant-derived exosomes for aggregation control and stabilization according to the present invention;

[0053] Figure 7 This is a particle size distribution analysis diagram (NTA detection) of the exosome samples before and after stabilization treatment in this invention. Detailed Implementation

[0054] This section will describe in detail specific embodiments of the present invention. Preferred embodiments of the present invention are shown in the accompanying drawings. The purpose of the drawings is to supplement the textual description with graphics, so that people can intuitively and vividly understand each technical feature and overall technical solution of the present invention, but they should not be construed as limiting the scope of protection of the present invention.

[0055] In the description of this invention, it should be understood that the orientation descriptions, such as up, down, front, back, left, right, etc., are based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0056] In the description of this invention, terms such as greater than, less than, and exceeding are understood to exclude the stated number, while terms such as above, below, and within are understood to include the stated number. The use of terms like "first" and "second" is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0057] In the description of this invention, unless otherwise explicitly defined, terms such as "set up," "install," and "connect" should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0058] Example 1

[0059] Reference Figure 1 and Figure 2 This is the first embodiment of the present invention. This embodiment provides an experiment for screening and optimizing ultracentrifugation parameters for plant exosomes. In a multi-stage purification process, ultracentrifugation is a key step in achieving the initial concentration and enrichment of exosomes from a large amount of culture medium. The magnitude of the centrifugal force and the reaction time not only directly determine the sedimentation efficiency, but are also closely related to the integrity of the exosome membrane structure. If the parameters are set too low, small-diameter exosomes cannot settle effectively, resulting in a significant decrease in yield; if the parameters are set too high, a prolonged high-overload environment will produce a strong shearing and squeezing effect, leading to exosome membrane rupture, irreversible aggregation, and co-precipitation of non-specific impurity proteins. Unless otherwise specified, this experiment and subsequent experiments all use tobacco BY-2 cell centrifugation supernatant as the same raw material.

[0060] 1.1 Pre-centrifugation step for screening

[0061] Pre-centrifugation, as the first step in a multi-stage purification module, has the core function of removing turbidity and impurities. Utilizing the sedimentation force generated by low-to-medium speed centrifugation, it rapidly separates and removes large cell debris, dead cells, and undegraded tissue particles from suspended cell culture media. Insufficient centrifugation intensity can leave large particles that clog subsequent ultrafiltration membranes or contaminate the chromatographic column; excessive centrifugation may lead to premature sedimentation and loss of some large exosomes or trigger non-specific aggregation of biomolecules.

[0062] The test range was set with centrifugal force from 1000g to 8000g, with a dimensional gradient set every 1000g; a dimensional gradient was set every 10 minutes from 10 to 60 minutes, and a cross-dimensional experimental group was set up. The centrifugation was carried out at 4℃.

[0063] After centrifugation, the supernatant of each group was carefully aspirated, and the absorbance (OD600) of the supernatant at 600 nm was measured. The percentage decrease in turbidity compared to the original culture medium was calculated to characterize the removal effect on cell debris and large particulate impurities. At the same time, an equal volume of supernatant was taken for Western blotting to detect and quantify the common marker proteins of plant exosomes (such as TET8 or HSP70) by gray-scale scanning. The retention rate was calculated with the uncentrifuged original liquid as 100% baseline to assess the loss of exosomes due to sedimentation during centrifugation.

[0064] Test results are as follows Figure 1 As shown, when the centrifugal force is low (<2000g), even if the centrifugation time is significantly extended to 60 minutes, the turbidity removal effect is still limited, indicating that the low gravity field cannot effectively settle small cell debris; while as the centrifugal force is increased to above 6000g, although the turbidity drops rapidly, the protein retention rate drops sharply, indicating that the excessive shear force has begun to cause non-specific sedimentation or structural damage of exosomes.

[0065] In terms of centrifugation time, a typical diminishing marginal effect is observed: the removal effect is significantly improved within the first 30 minutes, but after 40 minutes, the increase in the overall score tends to slow down or even declines due to increased exosome loss. Furthermore, excessively long centrifugation times not only reduce efficiency in industrial production but also increase the risk of sample heating and degradation.

[0066] Extreme value analysis was performed on the response surface model, and the theoretical peak coordinates of the comprehensive score were located at (3235.67g, 33.82min). A comprehensive evaluation of the accuracy of equipment parameter settings and production cycle control was conducted. It was necessary to avoid uncontrollable time due to excessively high centrifugal force (such as uneven instantaneous settling) and to prevent a surge in time costs due to excessively low centrifugal force. Ultimately, the parameters (3000g, 30min) that were closest to the theoretical peak and easiest to operate were selected as the pre-centrifugation process parameters.

[0067] 1.2 Screening and Optimization of Ultracentrifugation Parameters

[0068] As a core enrichment step in a multi-stage purification process, ultracentrifugation directly determines the final yield and purity of exosomes. Insufficient centrifugation intensity can cause small-diameter exosomes to suspend in the supernatant due to Brownian motion, resulting in yield loss. Conversely, excessively high centrifugation intensity or prolonged centrifugation, while increasing sedimentation, can easily lead to mechanical damage to the exosome membrane structure, irreversible aggregation, and non-specific co-precipitation of cytoplasmic proteins.

[0069] The test range was set with centrifugal force from 80,000g to 150,000g, with a dimensional gradient of 10,000g per 10000g. A cross-dimensional experimental group was also set up with a dimensional gradient of 30 to 120 minutes per 10 minutes. Centrifugation was performed at 4°C. After centrifugation, the supernatant was discarded, the exosome precipitate at the bottom was collected, and resuspended with an equal volume of buffer solution.

[0070] Nanoparticle tracking analysis was used to determine the particle concentration (Particles / mL) and average particle size in the resuspension. The particle concentration directly reflects the physical sedimentation yield of exosomes, while the average particle size and its distribution coefficient are used to monitor for membrane rupture fragments or abnormal aggregation caused by excessive centrifugation. The total protein concentration (μg / mL) in the sample was determined by the BCA method. The purity of exosomes was quantitatively evaluated by calculating the ratio of particle number to total protein (P / Pratio). A higher ratio means that there are more exosome particles in a unit of protein, i.e., fewer non-specific coprecipitated impurities.

[0071] Test results are as follows Figure 2 As shown, when the centrifugal force is below 80,000 g or the centrifugation time is less than 50 min, insufficient sedimentation driving force results in a large number of small-diameter exosomes remaining suspended in the supernatant, leading to low yield and purity. Conversely, when the centrifugal force exceeds 120,000 g or the time is extended to over 90 min, although the particle concentration reading continues to rise due to nonspecific sedimentation, the total protein concentration increases exponentially, causing a sharp drop in the P / P ratio, and the particle size deviation deteriorates drastically due to severe mechanical aggregation. This indicates that excessively high centrifugation intensity and excessively long reaction time, while increasing the quantity, severely sacrifice the quality of exosomes.

[0072] Extreme value analysis was performed on the response surface. The theoretical saturation point coordinates for particle concentration were (112, 450 g, 94.3 min), the theoretical peak coordinates for the P / P ratio were (92, 300 g, 72.8 min), and the theoretical minimum coordinates for particle size deviation were (88, 600 g, 63.5 min). Considering that yield is directly related to production cost in industrial production, yield and purity were given higher weights (40% each) in the comprehensive evaluation, while integrity was used as a basic constraint (20%). The theoretical comprehensive optimal point coordinates were obtained by weighted geometric center calculation (99, 620 g, 79.54 min). Considering the universality of parameter settings for industrial centrifuge equipment, the parameters were rounded to obtain (100000 g, 80 min).

[0073] Example 2

[0074] Reference Figure 3 This is the second embodiment of the present invention. This embodiment provides a screening experiment for a deep purification process of ultracentrifuged products. After treatment with the optimal centrifugation parameters determined in Example 1, although most exosomes could be recovered, the test data showed that a considerable proportion of soluble proteins and some nucleic acid impurities remained in the resuspension, resulting in the specific purity (P / PRatio) of the product not yet meeting the high purity standard of clinical-grade raw materials. In addition, the volume of the centrifuged resuspension is large, making direct fine separation inefficient. To resolve the contradiction between impurity residue and processing efficiency, this experiment aims to screen an efficient impurity precipitation and removal strategy.

[0075] 2.1 Screening and Optimization of Physical Purification Strategies

[0076] To further improve the purity of ultracentrifuged products and remove residual free proteins and small molecule metabolites, this experiment proposes a separation strategy based on differences in physical properties for fine purification. Physical purification typically utilizes significant differences in physical characteristics such as particle size and hydrodynamic radius between the target product and impurities to achieve separation. Its advantage lies in not introducing exogenous chemical reagents and preserving the natural structure and biological activity of exosomes to the greatest extent. However, different physical separation media have drastically different pore structures and fractionation ranges, directly determining their resolution and recovery efficiency for plant exosomes, which are nanoparticles of a specific size.

[0077] 2.1.1 Preliminary screening of physical separation media

[0078] Experimental groups: A0 control group; A1 dead-end microfiltration; A2 tangential flow filtration (TFF); A3 size exclusion chromatography (SEC); A4 density gradient centrifugation (DGC); A5 dialysis; A6 microfluidic separation.

[0079] The process effectiveness is comprehensively evaluated by detecting the purity enhancement factor (change in P / P ratio) and yield, combined with the throughput per unit time.

[0080] The test results are as follows:

[0081] Experimental Groups P / P ratio change Exosome yield (%) Processing capacity per unit time (L / h) A0 1.00 100.0 - A1 1.05 42.3 0.36 A2 1.85 88.6 2.58 A3 2.92 85.4 1.21 A4 4.15 65.2 0.02 A5 1.12 92.1 0.27 A6 3.2 55.8 0.74

[0082] Among these methods, density gradient centrifugation, as a laboratory standard, demonstrates a significant advantage in purity enhancement, but its yield and throughput make it unsuitable for large-scale industrial production. Microfluidic separation suffers from the same problems as density gradient centrifugation. Dialysis offers extremely high yields but does not improve purity. Tangential flow filtration has the largest throughput and excellent yields, but its purification effect is only average. Considering all factors, size exclusion chromatography was ultimately chosen as the optimal physical separation medium.

[0083] 2.1.2 Screening of chromatographic packing materials

[0084] After determining size exclusion chromatography (SUC) as the physical purification process, the selection of the packing material type becomes crucial to the separation effect. Different types of packing materials have different size exclusion limits and fractionation ranges. If the pore size of the packing material is too small, although most exosomes can flow out of the dead volume, slightly larger protein molecules may be misjudged as size exclusion and co-eluted; if the pore size is too large, some small-diameter exosomes may enter the pores, leading to peak broadening and dilution.

[0085] Experimental groups: Group B1: Sephadex G-50 (resistivity limit 3W), Group B2: Sephadex G-100 (resistivity limit 15W), Group B3: Sephadex G-200 (resistivity limit 60W), Group B4: Sephacryl S-400HR (resistivity limit 800W), Group B5: Sephacryl S-500HR (resistivity limit 2000W), Group B6: Sephacryl S-1000SF (resistivity limit over 100 millionW), Group B7: Sepharose 4B (resistivity limit 2000W), Group B8: Sephadex CL-4B (resistivity limit 2000W), Group B9: Sephadex CL-2B (resistivity limit 4000W).

[0086] The test results are as follows:

[0087] Separation degree (Rs) Exosome recovery rate (%) B1 0.45 98.5 B2 1.23 96.2 B3 1.85 92.4 B4 2.15 88.6 B5 2.68 85.3 B6 1.15 72.4 B7 3.12 89.5 B8 3.45 91.2 B9 4.28 93.5

[0088] Note: Resolution (Rs) is an indicator used in chromatographic analysis to measure the degree of separation between two adjacent chromatographic peaks (i.e., exosome peaks and impurity peaks). The larger the Rs value, the greater the distance between the two peaks and the less overlap. It is generally considered that when Rs>1.5, the two peaks have achieved baseline separation (i.e., complete separation).

[0089] Among them, G-50, due to its small pore size, resulted in the co-elution of most contaminating proteins and exosomes within the dead volume, leading to extremely low separation. Although G-100 had limited ability to distinguish large molecular weight proteins, it had extremely high recovery rates for exosomes and could remove small molecule impurities such as PEG and salt ions with great efficiency, making it very suitable as a primary desalting or solvent displacement step. CL-2B achieved complete separation of exosomes from most contaminating proteins (including large molecular complexes) while maintaining a high recovery rate, making it the best choice for deep purification.

[0090] Considering that crude plant exosomes contain both large amounts of PEG residues (requiring the removal of small molecules) and co-precipitated proteins (requiring the removal of large molecules), a single-dimensional filler cannot simultaneously address both needs. The optimal process route was not determined by a single choice, but rather by flexibly switching based on the actual impurity profile: for batches requiring only the removal of PEG and small molecules, Sephadex G-100 was preferred to ensure high yield; while for pharmaceutical-grade batches requiring ultimate purity, Sephadex CL-2B was used for deep purification.

[0091] 2.2 Screening and Optimization of Chemical Precipitation Parameters

[0092] To further improve exosome yield and effectively reduce sample volume, this experiment introduced chemical precipitation as an auxiliary purification method. The core principle of chemical precipitation is the exclusion volume effect formed by highly hydrophilic polymers in solution. Polymer molecules occupy a large amount of space and bind free water, causing a sharp decrease in solvent availability around proteins and membrane vesicle structures, thus forcing them to aggregate and settle. Compared to ultracentrifugation, chemical precipitation requires less equipment, allows for large batch throughput, and is gentler to operate, significantly improving the recovery rate of low-abundance exosomes. However, this method also has significant limitations: poor selectivity in precipitation. If parameters are not properly controlled, it can easily lead to the co-precipitation of large amounts of non-specific proteins or even the polymer itself with the exosomes, severely affecting the purity of the final product.

[0093] 2.2.1 Screening of precipitant types

[0094] Experimental groups: Group C0 (blank control) underwent only low-speed centrifugation under the same conditions;

[0095] Group C1 was supplemented with 8% (w / v) PEG8000 and 0.5M NaCl (PEG precipitation method).

[0096] Group C2 was supplemented with ammonium sulfate ((NH4)2SO4) at 50% saturation (salting-out method);

[0097] Group C3 was treated with 3 times the volume of cold acetone (organic solvent precipitation method).

[0098] Group C4 was supplemented with 0.5 mg / mL protamine sulfate (protamine precipitation method);

[0099] Group C5 was supplemented with 0.1M sodium acetate (calcium acetate / sodium precipitation method).

[0100] Group C6 was supplemented with 0.6 times its volume of isopropanol (isopropanol precipitation method).

[0101] In addition to testing the exosome recovery rate (characterizing yield) and P / P ratio (characterizing purity) established in the aforementioned experiments, it is also necessary to use the DPPH free radical scavenging experiment to determine the antioxidant activity after reconstitution of the precipitate, and calculate the retention rate with the original solution activity as a 100% baseline to assess the degree of damage to the product's functionality by the process; the clarity of the precipitate after reconstitution is observed by the naked eye and the aggregation is examined by microscopic examination, because precipitates that are difficult to reconstitute will seriously hinder subsequent formulation and filtration steps.

[0102] The test results are as follows:

[0103] Experimental Groups Exosome recovery rate (%) <![CDATA[P / P ratio (×10 7 ).]]> Activity retention rate (%) Reconstitution and Dispersibility C0 5.2 3.12 100 5 C1 86.5 2.85 83.4 4.2 C2 92.1 1.15 65.2 2.5 C3 95.4 1.88 32.1 3 C4 73.6 2.45 88.3 2 C5 49.2 2.92 95.8 4.8 C6 91.3 1.65 45.6 3.5

[0104] Ammonium sulfate, cold acetone, and isopropanol have extremely high recovery rates, but they cause severe damage to exosomes; sodium acetate is mild and has high purity, but its sedimentation driving force is insufficient, resulting in low recovery rates that cannot meet production capacity requirements. PEG precipitation has a high recovery rate and causes less damage to exosomes, thus it is used as the final chemical precipitant.

[0105] 2.2.2 Fine screening and optimization of precipitant concentration

[0106] PEG precipitation is a non-linear process based on concentration thresholds: at low concentrations, only a very small number of large particles aggregate; as the concentration increases, the exosome sedimentation rate rises rapidly in an S-shaped curve; however, exceeding a certain threshold, a large number of small molecule proteins will also be co-precipitated, and excessively high viscosity will hinder precipitation separation. Starting at 2% (w / v), a concentration gradient was set at 2% intervals up to the industrial operating limit of 20% (w / v), resulting in 10 experimental groups to examine the process performance at different final concentrations. The experiments continued to use exosome recovery rate, P / P ratio (purity), and reconstitution dispersibility score as the core evaluation indicators.

[0107] Test results are as follows Figure 3 As shown, in the low concentration area, the sedimentation driving force is insufficient and the recovery rate increases slowly; when the concentration exceeds 6%, the recovery rate shows an explosive growth, and reaches the growth inflection point (marginal effect is maximized) at around 10.4%, and then enters a plateau period. Even if the concentration continues to increase to 20%, the yield improvement is very weak.

[0108] At low concentrations (<6%), the purity remains high due to the sedimentation of only large particles; however, as the concentration exceeds 6.8%, a large number of soluble impurities begin to be nonspecifically co-precipitated, causing the specific purity to decline rapidly; when the concentration exceeds 12%, the high viscosity leads to an aggravated physical retention effect, and the reconstitution and dispersibility deteriorate sharply, seriously affecting the product appearance.

[0109] The theoretical optimal point for exosome recovery is 10.4%, while the optimal point for maintaining purity and dispersibility is 6.8%. To ensure high yield while maintaining high purity as much as possible, and to consider the flowability of subsequent operations, a weighted balance range between the two was selected. Taking into account the convenience of industrial solution preparation, the optimal PEG precipitation concentration was finally determined to be 8%-10%. This ensures a high recovery rate of over 85% while keeping the co-precipitation of impurities within an acceptable range.

[0110] 2.3 Validation of the synergistic effect of the purification process combination

[0111] After determining the dual purification strategy of physical chromatography followed by chemical precipitation, the tandem sequence of the process steps directly determines the final purification efficiency and product quality. Control groups were tested using either physical chromatography followed by chemical chromatography or chemical chromatography followed by physical chromatography.

[0112] Experimental results show that the precipitate-then-chemical process has significant logical flaws. Directly loading large volumes of centrifuged resuspension onto the chromatographic column results in extremely long separation cycles and severe peak tailing. Furthermore, the subsequent PEG precipitation step introduces small molecule impurities (such as buffer salts) that were just removed by chromatography, causing exosomes to aggregate in their final form. In contrast, the chemi-then-precipitate process, with PEG precipitation acting as a coarse capture step, reduces the sample volume by approximately 20 times and removes most non-exosome proteins, significantly improving the loading concentration and separation resolution of subsequent chromatographic steps. The subsequent SEC step acts as a refining step, thoroughly removing residual PEG molecules and small molecule impurities, and displacing exosomes into the final preservation buffer, achieving monodisperse elution. Therefore, the optimal tandem process sequence is determined to be PEG precipitation and concentration followed by SEC chromatographic purification.

[0113] Example 3

[0114] refer to Figure 4This is the third embodiment of the present invention. This embodiment provides an experiment on the construction and optimization of a plant exosome stabilization system. Plant exosomes, as nanoscale lipid bilayer vesicles, are inherently thermodynamically unstable. During extraction, purification, and subsequent storage and transportation, they are highly susceptible to environmental stresses such as ionic strength fluctuations, oxidative stress, shear forces, and freeze-thaw cycles, leading to membrane rupture, leakage of contents, or irreversible non-specific aggregation, thus losing their biological activity and targeting function. Simply relying on traditional cryopreservation often fails to maintain their long-term dispersion stability and membrane integrity. This experiment aims to construct a multi-dimensional stabilization and protection system to endow exosomes with excellent structural stability and activity retention under complex operating conditions.

[0115] 3.1 Buffer Selection and Optimization

[0116] As the microenvironmental matrix for exosomes, the physicochemical properties of the buffer solution directly determine the dispersion state and structural integrity of exosomes. A suitable pH environment and ionic strength can maintain the charge distribution on the surface of the exosome membrane, generate sufficient electrostatic repulsion to resist hydrophobic aggregation between particles, and at the same time avoid membrane rupture or shrinkage caused by osmotic pressure imbalance.

[0117] 3.1.1 Buffer Screening Experiment

[0118] This experiment consisted of 12 experimental groups, each formulated according to its optimal universal concentration and pH in the field of biopharmaceuticals:

[0119] Group C0 (blank control) used ultrapure water;

[0120] Group C1 used 20 mM MES buffer (2-(N-morpholine) ethanesulfonic acid), pH 6.0;

[0121] Group C2 used 10mM PBS buffer (phosphate), pH 7.4;

[0122] Group C3 used 50mM Acetate buffer (pH 5.0);

[0123] Group C4 used 20 mM Tris-HCl buffer (tris(hydroxymethyl)aminomethane), pH 7.5;

[0124] Group C5 used 50mM Citrate buffer (citrate), pH 6.0;

[0125] Group C6 used 20mM MMOPS buffer (3-morpholinopropanesulfonic acid), pH 7.2;

[0126] Group C7 used 50 mM Borate buffer (borate), pH 8.5;

[0127] Group C8 used 25 mM HEpes buffer (hydroxyethylpiperazine ethanesulfonic acid), pH 7.0;

[0128] Group C9 used 10 mM PPIES buffer (piperazine-N,N'-bis(2-ethanesulfonic acid)), pH 6.8;

[0129] Group C10 used 25mM Bicarbonate buffer (bicarbonate), pH 7.4;

[0130] Group C11 used 154mM physiological saline (0.9% NaCl).

[0131] During storage, particularly long-term storage of exosomes, factors such as precipitation from the container material, dissolution of CO2 from the air, or accumulation of trace degradation products can all lead to pH drift, thereby disrupting membrane protein activity. Each group of samples underwent three standard freeze-thaw cycles (-80℃ to 25℃), and the pH values ​​before and after the freeze-thaw cycles were measured using a high-precision pH meter. The absolute difference (ΔpH = |pH after - pH before|) was calculated. A smaller ΔpH indicates a more stable buffer system.

[0132] Ions in solution interact with the charge layer on the surface of exosomes. An unsuitable ionic environment can lead to compression of the electric double layer, weakening the electrostatic repulsion between particles and causing irreversible aggregation and precipitation. After storing the sample at 4°C for 7 days, it was diluted, and the surface potential was measured using a Zeta potential analyzer. The Zeta potential is a core parameter characterizing the strength of electrostatic repulsion on the particle surface. Simultaneously, the average particle size was measured using a nanoparticle tracking analyzer (NTA), and the percentage increase in particle size relative to the initial particle size was calculated. The particle size increase rate directly reflects the degree of aggregation.

[0133] During freezing, the crystallization of water molecules causes a sharp increase in the solute concentration in the remaining solution. This localized high osmotic pressure difference generates strong mechanical shear forces on the exosome membrane, leading to membrane rupture or leakage of contents. Calcein-AM fluorescence leakage rate (%) is commonly used as a detection indicator. Calcein-AM is a membrane-permeable non-fluorescent dye that, upon entering intact exosomes, is hydrolyzed by esterases into Calcein, which emits strong green fluorescence and is locked within the membrane. If the membrane ruptures, the fluorescent substance leaks into the external environment. Exosomes were pre-labeled with Calcein-AM, and after washing away the free dye, they were resuspended in various buffer solutions. After one freeze-thaw cycle, the mixture was centrifuged at high speed (to remove intact exosomes), and the fluorescence intensity in the supernatant was measured. Stronger fluorescence indicates more severe leakage and greater membrane damage. To address the challenge of temperature changes, temperature fluctuations that may occur during cold chain transportation were simulated. The recovery rate of exosomes after three freeze-thaw cycles was measured to evaluate the ability of different buffer solutions to maintain pH and ionic environment during low-temperature crystallization.

[0134] The test results are as follows:

[0135] Experimental group number Zeta potential (mV) Particle size increase rate (%) Freeze-thaw pH drift (ΔpH) Fluorescence leakage rate (%) C0 -5.2 128.5 1.85 68.4 C1 -8.4 18.2 0.12 15.6 C2 -22.5 4.2 0.48 8.5 C3 -12.3 25.4 0.22 22.1 C4 -20.1 5.6 0.35 6.8 C5 -18.6 2.8 0.18 45.2 C6 -15.5 6.2 0.02 7.5 C7 -28.4 14.5 0.25 18.2 C8 -24.8 3.1 0.05 4.2 C9 -16.2 8.4 0.08 3.5 C10 -15.4 12.1 0.85 12.4 C11 -9.8 22.5 1.25 35.6

[0136] Among them, borates performed well in terms of zeta potential, providing extremely strong electrostatic repulsion, but their strong alkaline environment caused the membrane structure to swell, resulting in a high particle size increase rate (14.5%). Citrate achieved the best control over particle size increase and had excellent dispersibility, but its strong calcium ion chelation led to an extremely high fluorescence leakage rate, indicating that the membrane structure had undergone catastrophic rupture. 3-morpholinopropanesulfonic acid and piperazine-N,N'-bis(2-ethanesulfonic acid) both had low zeta potentials and insufficient electrostatic protection, making it difficult to cope with long-term charge shielding challenges.

[0137] In comparison, PBS (phosphate), Tris-HCl (tris(hydroxymethyl)aminomethane), and Hepes (hydroxyethylpiperazine ethanethioic acid) have good overall performance and were therefore chosen as the best buffers. The optimal choice can be made based on the specific circumstances.

[0138] 3.1.2 Fine-grained synergistic optimization of ionic strength and pH value

[0139] After determining PBS, Tris, and Hepes as the preferred buffer matrices, their specific ionic strength and pH parameters become the microenvironmental factors determining the stabilization effect. Ionic strength directly affects the thickness of the electric double layer and the range of electrostatic repulsion by adjusting the Debye length, while pH regulates the zeta potential and membrane rigidity by changing the protonation state of phospholipids and proteins on the membrane surface. There is a complex coupling effect between the two: excessively high ionic strength can compress the electric double layer, leading to aggregation; excessively low pH may cause membrane fusion; and excessively high pH may lead to lipid hydrolysis. Therefore, this experiment uses the Hepes buffer, which has the best overall performance in Example 3.1, as a benchmark and designs a full-factor crossover experiment covering the ionic strength range of 20 mM to 200 mM (with a gradient of 20 mM) and the pH range of 6.0 to 8.0 (with a gradient of 0.2). The focus is on examining two core indicators: zeta potential (electrostatic stability) and particle size polydispersity index (PDI, physical homogeneity), aiming to construct an accurate heatmap of stability parameters.

[0140] Test results are as follows Figure 4As shown, coupled analysis of the three-dimensional response surfaces of Zeta potential and PDI revealed a misalignment between the theoretical optimal region of Zeta potential (low salt, high alkalinity) and the theoretical optimal region of PDI (medium salt, neutral). Considering both electrostatic repulsion and physical homogeneity, the calculated theoretical equilibrium point coordinates (72.4 mM, pH 7.12) were determined. Based on the convenience and versatility of industrial solution preparation, the process parameters were ultimately standardized and corrected to an ionic strength of 75 mM and a pH of 7.2.

[0141] 3.2 Screening and Optimization of Stabilizer Formulation

[0142] To further enhance the resilience of exosomes during freeze-thaw cycles and long-term storage, and to address the physical and chemical degradation issues that cannot be completely avoided by simple buffer solutions, this study investigated three core failure modes of exosome storage: ice crystal damage, lipid oxidation, and interfacial adsorption. Various potential protective agents with different mechanisms (including sugars, polyols, metal chelators, surfactants, and large protein molecules) were selected. Their protective efficacy against membrane structural integrity and physicochemical properties was compared and examined to identify the advantages and applicable scenarios of each protective agent, providing experimental evidence for constructing high-performance stabilization formulations.

[0143] Experimental groups: Group D0 was a blank control; Group D1 was supplemented with 10% glycerol; Group D2 was supplemented with 5% dimethyl sulfoxide (DMSO); Group D3 was supplemented with 3% trehalose; Group D4 was supplemented with 5% sucrose; Group D5 was supplemented with 1.0 mM ethylenediaminetetraacetic acid (EDTA); Group D6 was supplemented with 2.0 mM ethylene glycol diaminoethyl ether tetraacetic acid (EGTA); Group D7 was supplemented with 0.05% Tween 80; Group D8 was supplemented with 0.02% sodium dodecyl sulfate (SDS); Group D9 was supplemented with 0.5% human serum albumin (HSA); Group D10 was supplemented with 4% mannitol; Group D11 was supplemented with 1% poloxamer 188.

[0144] The freeze-drying protectant prevents ice crystals from piercing the membrane by replacing water molecules to form a glassy state. The average particle size of the sample after three standard freeze-thaw cycles was measured, and the percentage recovery relative to the initial particle size was calculated; a value closer to 100% indicates better anti-ice crystallization performance. The malondialdehyde (MDA) content in the stored sample was determined using the thiobarbituric acid method (TBA method). MDA is a lipid peroxidation product; a lower MDA value indicates more stable membrane lipids. A quantitative exosome solution was allowed to stand in a standard storage tube for 24 hours before being transferred. The amount of residual protein eluted from the original tube wall was determined using the BCA method, and its percentage of the total protein was calculated to evaluate the anti-interfacial adsorption and shearing effects.

[0145] The test results are as follows:

[0146] Experimental group number Particle size recovery rate (%) MDA content (nmol / mg) Adsorption residue rate (%) D0 65.4 12.5 15.8 D1 88.5 10.2 14.2 D2 98.5 11.5 13.8 D3 96.2 8.5 8.2 D4 94.2 9.1 9.5 D5 78.5 2.8 12.5 D6 75.2 3.2 12.8 D7 82.3 7.8 2.5 D8 12.5 13.5 0.5 D9 91.5 1.5 5.2 D10 85.6 9.8 10.5 D11 88.2 7.2 3.5

[0147] While dimethyl sulfoxide (DMSO) performs well in preventing ice crystal growth, its residual cytotoxicity prevents its application in the biopharmaceutical field. Sodium dodecyl sulfate (SOS) has an extremely low adsorption residue rate, but it completely destroys the lipid bilayer of exosomes, leading to product failure. Human serum albumin can effectively reduce MDA content, but as a foreign protein, it contaminates the raw materials.

[0148] Trehalose offers excellent antifreeze properties and is safe and non-toxic; EDTA boasts excellent antioxidant capabilities and structural stability; and Tween 80 exhibits excellent anti-adsorption capabilities, while its non-ionic properties ensure the integrity of the membrane structure. These three components precisely target the three major needs of antifreeze, antioxidant, and anti-adsorption, respectively, and none of them have fatal safety or quality defects. Ultimately, it was decided to combine them to construct a multi-dimensional protective stabilizer system.

[0149] Example 4

[0150] This embodiment provides a pilot-scale production and stabilization process verification of plant exosomes based on the tobacco BY-2 suspension cell system, aiming to verify the applicability and scalability of the aforementioned optimized process in a general model plant system.

[0151] Activated tobacco BY-2 callus was inoculated into modified MS liquid medium supplemented with a hormone combination of 0.2 mg / L 2,4-D, 1.0 mg / L NAA, and 0.5 mg / L 6-BA to maintain rapid cell proliferation. Suspension culture was performed under constant temperature of 25°C, a photocycle of 12 h light / 12 h dark, and continuous stirring at 110 rpm. The cell density reached peak at day 10 (approximately 3.5 × 10⁻⁶). 6 (cells / mL), at this point, a total of 5L of culture supernatant was collected as the starting material for exosome production.

[0152] 5 L of culture supernatant was centrifuged at 4,200 × g for 35 minutes (with fine-tuning based on the parameters optimized in Example 1) to thoroughly remove cell debris. The clear supernatant was collected for pre-centrifugation. The supernatant was then ultracentrifuged at 100,000 × g for 80 minutes (rounding down based on the parameters optimized in Example 1.2) to enrich the exosome precipitate. The precipitate was resuspended and PEG8000 was added to a final concentration of 10% (w / v) (based on the parameters optimized in Example 2.2). The precipitate was allowed to stand overnight at 4°C to achieve preliminary removal of impurities and volume reduction. After reconstitution of the PEG precipitate, it was loaded onto a chromatography column packed with Sephadex CL-2B (based on the medium optimized in Example 2.1), eluted with PBS buffer, and the characteristic peak components of the exosomes were collected to achieve deep desalting and purification.

[0153] An optimized stabilizing agent was added to the purified exosome fraction, and the final system parameters were controlled as follows:

[0154] Buffer environment: 75 mM Heps buffer, pH 7.2 (based on the parameters optimized in Example 3.1).

[0155] Compound stabilizer: containing 3% (w / v) trehalose, 1.0 mM EDTA and 0.05% Tween 80 (based on the optimized formulation of Example 3.2).

[0156] The prepared product was characterized in multiple dimensions, and the results are as follows:

[0157] Physical characterization: NTA analysis showed that tobacco exosomes had a highly uniform particle size, with the main peak concentrated in the 115-135 nm range, and aggregated particles larger than 200 nm accounted for only 1.5%; the zeta potential was stable at -24.2 mV.

[0158] Bioactivity: Cell scratch repair assay showed a repair rate of 18.5% and a DPPH free radical scavenging rate of 12.6% after 48 hours.

[0159] Long-term stability: After storing the finished product at -80°C for 3 months, the particle size fluctuation was 6.5%, the zeta potential change was <3%, the activity retention rate (relative to initial activity) was 92%, and no obvious aggregation was observed. These results confirm that even at low concentrations, the stabilization and purification process of this invention can still endow exosomes with excellent structural stability and activity retention.

[0160] Example 5

[0161] Please see Figure 5-7 This is the fifth embodiment of the present invention. The present invention provides a technical solution: a plant-derived exosome stabilization and purification system, comprising a suspension cell culture module, a multi-stage primary purification module, a stabilization treatment module, and a quality detection module that are sequentially linked; Suspension cell culture module: MS basal medium and optimized hormone combination (6-BA, NAA, meta-topolin, 2,4-D) are used to simulate the natural growth environment of plant cells and ensure the continuous and stable secretion of exosomes; at the same time, the culture temperature, light cycle and stirring rate are controlled to avoid interference from impurities caused by cell breakage.

[0162] Suspension cell culture module: MS basal medium and optimized hormone combination (6-BA, NAA, meta-topolin, 2,4-D) are used to simulate the natural growth environment of plant cells and ensure the continuous and stable secretion of exosomes; at the same time, the culture temperature, light cycle and stirring rate are controlled to avoid interference from impurities caused by cell breakage.

[0163] Multi-stage primary purification module: This module is connected in series in the order of pre-centrifugation, ultracentrifugation, PEG precipitation, and size exclusion chromatography to achieve stepwise purification from crude extract to high-purity exosomes.

[0164] Pre-centrifugation (3,000×g, 30 min) quickly removes cell debris and large particulate impurities, reducing the burden on subsequent purification;

[0165] Ultracentrifugation (100,000×g, 60-90 min) utilizes density differences to achieve preliminary enrichment of exosomes;

[0166] Polyethylene glycol precipitation (5-10% concentration) helps remove soluble impurities by changing osmotic pressure, thus improving exosome recovery rate;

[0167] Size exclusion chromatography (Sephadex CL-2B or G-100 packing material) separates residual proteins from small molecule impurities based on particle size differences, ensuring exosome purity >95%.

[0168] Stabilization module: As the core innovation of this invention, it solves the problem of exosome aggregation through multi-dimensional parameter control.

[0169] Buffer system: Use PBS, Tris or Hepes buffer, and control the ionic strength to 10-100mM (to avoid membrane structure compression caused by excessive ionic strength) and pH 6.5-7.5 (to match the isoelectric point of plant exosomes and maintain surface charge stability).

[0170] Compound stabilizers: Trehalose (1-5% w / v) protects the membrane structure through hydrogen bonding, EDTA (0.1-2.0 mM) chelates metal ions to prevent oxidation, and Tween 80 (0.001-0.1%) reduces interfacial tension and inhibits aggregation. The three work synergistically to improve stability.

[0171] When preparing the stabilizer, a magnetic stirrer, beaker, pH meter, and other equipment are needed. Deionized water is used as the solvent. Add approximately 80% of the total volume of deionized water to the beaker, and start stirring (200-300 rpm). Heat to 50-60°C, then slowly add trehalose, continuing to stir until completely dissolved (the solution is clear). Then turn off the heating and allow the solution to cool to 30-40°C. While maintaining stirring, add the weighed EDTA. If dissolution is slow, adjust the pH to 6.0-7.0 with 0.1 mol / L sodium hydroxide or 1% hydrochloric acid until the EDTA is completely dissolved. After the mixture has cooled to room temperature (20-25℃), slowly add Tween 80 dropwise along the beaker wall using a pipette, while simultaneously reducing the stirring speed to 150-200 rpm. Stir for 5-10 minutes until it is evenly dispersed (without layering or oil film). Finally, add deionized water to the set total volume and continue stirring at low speed for 2-3 minutes to mix thoroughly. Check if the solution is clear and transparent, without precipitation or foam. Retest the pH to ensure it is between 6.0 and 7.0. During operation, please note: the mixing order must not be reversed (to avoid Tween 80 deactivation at high temperatures or EDTA clumping); heating is only required when dissolving trehalose, and cooling is necessary afterwards; control the stirring speed when adding Tween 80 to prevent foaming; if a small amount of foam is generated, allow it to stand to dissipate.

[0172] Shear stress control: The concentration factor is limited to 10-50 times by a buffer pressure reduction device to avoid exosome membrane damage caused by excessive shear force during ultrafiltration or centrifugation;

[0173] Programmable temperature control: A slow cooling rate of -1 to -3°C / min is adopted to reduce the damage to the membrane structure caused by ice crystal formation during freezing. At the same time, the concentration is automatically compensated during the thawing stage to avoid secondary aggregation caused by stabilizer dilution.

[0174] Quality Inspection Module: Equipped with a nanoparticle tracking analyzer (NTA) and a Zeta potential online monitoring device to monitor exosome particle size (controlled at 100-150nm) and surface charge (controlled at -10 to -30mV) in real time, ensuring consistent quality for each batch of products;

[0175] The Zeta potential is controlled between -10 and -30 mV. This range ensures the stability of exosome suspension (avoiding aggregation) while matching the natural charge characteristics of their physiological environment. The control mechanism must cover the entire process from pretreatment to production, monitoring, and adjustment, as detailed below:

[0176] First, the rationality of the target range is clarified: -10 to -30mV is in the range of weak to moderate negative potential. On the one hand, it can prevent exosomes from aggregating through moderate electrostatic repulsion between particles (exosomes with an absolute potential value below 10mV are prone to aggregation, while those above 30mV may affect their biological activity due to excessive charge); on the other hand, it matches the natural Zeta potential range of exosomes in human body fluids (usually -15 to -25mV), which can reduce the risk of immune rejection in subsequent applications.

[0177] The control mechanism needs to be implemented in four steps: The first step is buffer regulation during the pretreatment stage. A low ionic strength buffer (such as 0.01M PBS) with a pH of 7.2-7.4 should be selected to avoid high concentrations of salt ions (such as >0.1M NaCl) compressing the electric double layer and causing a decrease in the absolute value of the potential. At the same time, the buffer should be filtered through a 0.22μm filter membrane to remove impurities that interfere with the charge. The second step is control of production process parameters. When centrifuging exosomes (such as differential centrifugation or ultracentrifugation), the rotation speed (10000-12000×g) and temperature (4℃) should be controlled to avoid excessive shear force damaging the exosome membrane structure (membrane structure damage will lead to uneven surface charge distribution). If a stabilizer is added (such as the previously compounded trehalose-EDTA system), its concentration (trehalose 1-3% w / v, EDTA 0.5-1.0mM) must be ensured not to change the exosome surface. The third step is real-time monitoring and linkage adjustment. The Zeta potential online monitoring equipment is linked with the production system, and automatic sampling and detection are set every 15 minutes. If the potential deviates from the target range (e.g., below -10mV), 0.01MPBS is immediately added through the automatic replenishment device to adjust the ionic strength, or the centrifugation temperature is finely adjusted (±1℃). The fourth step is abnormal handling. If the potential continues to deviate (e.g., two consecutive detections >-10mV or <-30mV), production is suspended. Particle size is detected simultaneously through NTA (to rule out potential changes caused by abnormal particle size), and the integrity of exosome membrane proteins is detected (e.g., Western blotting of CD63 protein). After confirming that there is no membrane damage, the parameters are readjusted to ensure that the potential and particle size of each batch are within the target range, ultimately achieving consistent quality.

[0178] Through the multi-parameter synergy of the stabilization module, the exosome particle size can be stably controlled within 100-150 nm, with an aggregation rate of <5% during storage, significantly better than traditional processes (aggregation rate >30%), thus improving stability: after storage at -80°C for 3 months, the particle size fluctuation of exosomes is <10%, and the activity retention rate is >85%, meeting the requirements for long-term industrial storage; ensuring consistency: modular design and online detection ensure that the coefficient of variation of particle size, potential and activity between batches is <5%, which complies with GMP production specifications;

[0179] The method for determining the activity retention rate is as follows: exosomes before storage (baseline group) and after 3 months of storage (storage group) are taken and diluted with serum-free culture medium to the same concentration (e.g., 100 μg / mL).

[0180] Target cells were seeded into 96-well plates (density 5×10³ cells / well), and after incubation for 24 h, exosomes from the baseline group, storage group and blank culture medium (control group) were added respectively, with 3 replicates for each group.

[0181] Continue incubation for 48 hours, add 20 μL MTT solution (5 mg / mL) to each well, incubate at 37°C for 4 hours, discard the supernatant, add 150 μL LDMSO to dissolve the crystals, and measure the absorbance (OD value) at 490 nm using a microplate reader.

[0182] Activity retention rate calculation: (OD value of storage group - OD value of control group) / (OD value of baseline group - OD value of control group) × 100%, the result must be > 85%;

[0183] The activity retention rate was tested once before storage (day 0) to obtain baseline data (particle size, zeta potential, activity value) as a benchmark for subsequent comparison, ensuring that the initial state met the quality requirements (particle size 100-150nm, potential -10 to -30mV).

[0184] Mid-term storage (1 month, 2 months): Test once each to monitor the trend of activity changes. If the activity decreases (e.g., <90%), adjust the storage conditions in advance (e.g., check whether the -80°C refrigerator is frequently opened and closed, and whether the exosomes are aliquoted and sealed) to avoid the activity not meeting the standard in the later stage.

[0185] End of storage (3 months): Test once as the final activity determination basis. Particle size (confirmed fluctuation <10%) and activity (confirmed >85%) must be tested simultaneously. If both indicators meet the standards, the batch can be judged to be qualified for storage.

[0186] Post-reconstitution validation (optional): Exosomes are removed at the end of storage and their activity is tested at 0h, 2h and 4h after reconstitution at 4℃ to assess short-term stability after reconstitution and ensure that the activity still meets the requirements for subsequent use.

[0187] A process for stabilizing and purifying plant-derived exosomes, the specific steps of which are as follows:

[0188] Suspension cell culture S1: Plant cells (such as Dendrobium officinale callus) are inoculated into MS medium containing a hormone combination and cultured for 7-14 days in a shake flask or bioreactor at 25°C, 12h light / 12h dark, and 110rpm until the cell density reaches 1×10⁻⁶ cells / year. 6 -5×10 6 When the cell count is 10 ...

[0189] Multi-stage primary purification S2:

[0190] S21 Pre-centrifugation: Transfer the crude extract into the pre-centrifugation unit, centrifuge at 3,000×g for 30 minutes, discard the precipitate (cell debris and large particulate impurities), and collect the supernatant;

[0191] S22 Ultracentrifugation: Transfer the supernatant of S21 to an ultracentrifuge tube and centrifuge at 100,000×g for 60-90 minutes (adjust according to the plant species, such as Ganoderma lucidum exosomes, which require centrifugation for 90 minutes). Discard the supernatant and collect the exosome precipitate at the bottom.

[0192] S23 PEG precipitation: Add 5-10% polyethylene glycol (PEG8000) solution to the S22 precipitate, let stand at room temperature for 30 minutes, then centrifuge at 3,000×g for 15 minutes and collect the precipitate (to further remove soluble proteins).

[0193] S24 size exclusion chromatography: Resuspend the S23 precipitate in 1-2 mL of stabilization buffer (such as PBS, pH 7.0, ionic strength 50 mM), load it onto a pre-equilibrated Sephadex CL-2B column, elute with the same buffer, and collect the main peak component of exosomes using a UV detector (280 nm).

[0194] Stabilization process S3:

[0195] S31 Buffer Adjustment: The exosome components collected in S24 are introduced into the stabilization buffer unit, and the ionic strength is adjusted to 10-100 mM and the pH to 6.5-7.5 using an online pH meter and conductivity meter.

[0196] S32 Stabilizer Addition: Start the stabilizer compounding unit, add trehalose, EDTA and Tween 80 in proportion, and magnetically stir (50-100 rpm) for 10 minutes to ensure uniform mixing;

[0197] S33 Concentration Control: The system after S32 treatment is introduced into the shear buffer unit and concentrated using a 100kDa ultrafiltration membrane. The concentration factor is controlled at 10-50 times, and the shear force is monitored by a pressure sensor during the process (not exceeding 500Pa).

[0198] S34 program cryopreservation: Transfer the concentrate to cryopreservation tubes, place them in a temperature-controlled cooling unit, and cool at a rate of -1 to -3°C / min. When the temperature reaches below -80°C, transfer it to a liquid nitrogen tank or a -80°C freezer for long-term storage.

[0199] S35 Slow Remelting: Before use, place the cryovial into the thawing unit and heat it to room temperature at a rate of 0.5-1°C / min. At the same time, the volatile stabilizer is replenished through the automatic concentration compensation module to maintain a constant concentration.

[0200] Quality Inspection S4: Samples were taken and tested at three key points: S24 (after chromatographic elution), S32 (after stabilizer addition), and S35 (after remelting).

[0201] Particle size analysis: The particle size distribution of exosomes was determined by NTA, requiring the main peak to be concentrated in the 100-150 nm range, and the proportion of particles with a diameter >200 nm to be <5%.

[0202] Zeta potential detection: Surface charge is measured using a Zeta potential meter, with the potential value required to be between -10 and -30mV to ensure dispersion stability;

[0203] Activity assay: The functional activity of exosomes was verified by the scratch assay, the antioxidant free radical scavenging assay (DPPH), or the MTT assay (e.g., repair rate > 80%, DPPH scavenging rate > 50%).

[0204] If the test results are not up to standard (e.g., particle size exceeds 150nm), return to the previous critical step for reprocessing (e.g., return to S31 to readjust the buffer solution) to ensure that the final product quality meets industrialization requirements.

[0205] Example 6

[0206] This embodiment provides a process for stabilizing and purifying exosomes derived from Dendrobium officinale.

[0207] Suspension cell culture: Dendrobium officinale callus tissue was taken and inoculated into MS medium containing 0.5 mg / L 6-BA, 0.1 mg / L NAA, 0.2 mg / L meta-topolin, and 0.05 mg / L 2,4-D. The culture was carried out in a 5L bioreactor at 25°C, 12h light / 12h dark, and 110 rpm for 10 days, and 3L of culture supernatant was collected.

[0208] Multi-stage primary purification:

[0209] Pre-centrifugation: Centrifuge at 3,000×g for 30 minutes and collect 2.8L of supernatant;

[0210] Ultracentrifugation: Centrifuge at 100,000×g for 75 minutes and collect approximately 0.5 mL of exosome precipitate;

[0211] PEG precipitation: Add 5 mL of 8% PEG8000 solution, let stand at room temperature for 30 minutes, then centrifuge at 3,000×g for 15 minutes and collect the precipitate;

[0212] Size exclusion chromatography: The precipitate was resuspended in 2 mL PBS (pH 7.2, ionic strength 50 mM), loaded onto a Sephadex CL-2B column (column volume 20 mL), eluted at a flow rate of 1 mL / min, and 8-12 mL of the eluent (exosome main peak) was collected.

[0213] Stabilization treatment:

[0214] Buffer conditioning: The eluted fraction is introduced into the stabilization buffer unit, and the pH is adjusted to 7.0 and the ionic strength to 50 mM.

[0215] Stabilizer addition: Add 2% w / v trehalose, 1.0 mM EDTA, and 0.01% Tween 80, and stir for 10 minutes;

[0216] Concentration control: Concentrate to 0.5 mL using a 100 kDa ultrafiltration membrane (concentration factor 10 times), with the shear force maintained at 300 Pa during the process;

[0217] Programmatic freezing: Cool to -85°C at a rate of -2°C / min, then store in a -80°C freezer;

[0218] Remelting: Heat to room temperature at a rate of 1°C / min, and automatically replenish with 0.002% Tween 80 through concentration compensation.

[0219] Quality Inspection:

[0220] Particle size: NTA analysis showed that the particle size was concentrated in the 120-140nm range, with particles >200nm accounting for 2.3%;

[0221] Zeta potential: -22.5mV;

[0222] Activity: Cell scratch repair assay showed a repair rate of 85% and a DPPH clearance rate of 58% after 48 hours.

[0223] After being stored at -80°C for 3 months, this batch of exosomes showed a particle size fluctuation of 8%, a zeta potential change of <5%, an activity retention rate of >90%, and no obvious aggregation.

[0224] Example 7

[0225] This embodiment provides a process for stabilizing and purifying exosomes derived from Anoectochilus roxburghii.

[0226] Suspension cell culture: Anoectochilus roxburghii callus tissue was taken and inoculated into MS medium containing 0.3 mg / L 6-BA, 0.2 mg / L NAA, 0.1 mg / L meta-topolin, and 0.1 mg / L 2,4-D. The culture was carried out in a 5L bioreactor at 25°C, 12h light / 12h dark, and 110 rpm for 12 days, and 2.5L of culture supernatant was collected.

[0227] Multi-stage primary purification:

[0228] Pre-centrifugation: Centrifuge at 3,000×g for 30 minutes and collect 2.3L of supernatant;

[0229] Ultracentrifugation: Centrifuge at 100,000×g for 90 minutes and collect approximately 0.4 mL of exosome precipitate;

[0230] PEG precipitation: Add 4 mL of 10% PEG8000 solution, let stand at room temperature for 30 minutes, then centrifuge at 3,000×g for 15 minutes and collect the precipitate;

[0231] Size exclusion chromatography: Resuspend the precipitate in 2 mL Tris buffer (pH 7.4, ionic strength 80 mM), load the sample onto a Sephadex G-100 column, and collect 7-11 mL of the eluent.

[0232] Stabilization treatment:

[0233] Buffer adjustment: Adjust pH to 7.2 and ionic strength to 80 mM;

[0234] Stabilizer addition: Add 3% w / v trehalose, 0.5 mM EDTA, and 0.05% Tween 80, and stir for 10 minutes;

[0235] Concentration control: Concentrate to 0.4 mL (concentration factor 12 times), and maintain shear force at 400 Pa;

[0236] Programmed cryopreservation: Cool to -82°C at a rate of -1.5°C / min;

[0237] Remelting: Increase the temperature at a rate of 0.8°C / min, compensating with 0.01% Tween 80.

[0238] Quality Inspection:

[0239] Particle size: 110-130nm, with particles >200nm accounting for 1.8%;

[0240] Zeta potential: -18.7mV;

[0241] Activity: MTT assay showed a 75% increase in cell viability and a 62% DPPH clearance rate.

[0242] After 3 months of storage, the particle size of this batch of exosomes fluctuated by 7%, and the activity retention rate was >88%, meeting the raw material requirements for beauty repair products.

[0243] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A plant-derived exosome stabilization purification system, characterized by, It includes a sequentially linked suspension cell culture module, a multi-stage primary purification module, a stabilization treatment module, and a quality testing module; The multi-stage primary purification module includes, in process order: Pre-centrifugation unit: Centrifuge at 3,000×g for 30 minutes to remove cell debris and large particulate impurities; Ultracentrifugation unit: Centrifuge at 100,000×g for 60-90 minutes to recover exosome precipitates; Polyethylene glycol precipitation unit: Adding 5-10% polyethylene glycol to achieve impurity concentration and auxiliary removal; Size exclusion chromatography unit: using Sephadex CL-2B or G-100 packing material to remove residual small molecule impurities and protein debris; The stabilization processing module includes: Stabilization buffer unit: Use PBS, Tris or Hepes buffer, and control the ionic strength to 10-100mM and the pH value to 6.5-7.

5. Stabilizer compound unit: 1-5% w / v trehalose, 0.1-2.0 mM EDTA and 0.001-0.1% Tween 80 added; Shear buffer unit: Equipped with a buffer and decompression device to control the concentration ratio of 10-50 times and prevent damage to the exosome membrane structure; Temperature control cooling unit: Equipped with a programmable cooling controller, the cooling rate can be adjusted from -1 to -3°C / min, and the final freezing temperature is below -80°C; Remelting unit: Equipped with an automatic compensation module for the concentration of compounded stabilizer, it slowly heats up to remelt while maintaining a constant stabilizer concentration; The quality detection module is equipped with a nanoparticle tracking analyzer and a Zeta potential online monitoring device to detect the exosome particle size distribution and surface charge stability in real time.

2. A plant-derived exosome stabilization and purification system according to claim 1, characterized in that: The suspension cell culture module: plant cells are cultured in MS basal medium supplemented with a hormone combination of 6-BA, NAA, meta-topolin, and 2,4-D for 7-14 days under the conditions of 25°C, 12h light / 12h dark, and 110rpm to form a source for exosome production.

3. The plant-derived exosome stabilization and purification system of claim 1, wherein: The plant source is one or more combinations of Dendrobium officinale, Anoectochilus roxburghii, Lycium barbarum, and Ganoderma lucidum.

4. The plant-derived exosome stabilization and purification system of claim 1, wherein: The pre-centrifugation unit, ultracentrifugation unit, polyethylene glycol precipitation unit, and size exclusion chromatography unit are connected in series via fluid pipelines to form a continuous purification flow path.

5. The plant-derived exosome stabilization and purification system according to claim 1, characterized in that: The temperature control and cooling unit is also equipped with a freezing termination temperature control module, which automatically stops the cooling program when the system temperature reaches below -80°C.

6. The plant-derived exosome stabilization and purification system according to claim 1, characterized in that: In the stabilization buffer unit, the buffer replacement frequency is linked to the processing capacity of the multi-stage primary purification module, and the buffer is replaced once for every 10L of plant suspension cell culture supernatant processed.

7. A plant-derived exosome stabilization and purification process based on the system described in any one of claims 1-6, characterized in that, Includes the following steps: S1: Suspension cell culture: Plant cells were inoculated into MS medium containing hormone combinations and cultured in suspension at 25°C, 12h light / 12h dark, and 110rpm for 7-14 days. The culture supernatant was then collected. S2: Multi-stage primary purification: S21: Pre-centrifugation: Transfer the culture supernatant into the pre-centrifugation unit, centrifuge at 3,000×g for 30 minutes, and collect the supernatant; S22: Ultracentrifugation: Transfer the supernatant collected in S21 into the ultracentrifugation unit, centrifuge at 100,000×g for 60-90 minutes, and collect the exosome precipitate; S23: PEG precipitation: Add 5-10% polyethylene glycol solution to the exosome precipitate collected in S22, let stand at room temperature for 30 minutes, then centrifuge at 3,000×g for 15 minutes and collect the precipitate; S24: Size exclusion chromatography: Resuspend the precipitate collected in S23 in stabilization buffer, load it onto a Sephadex CL-2B or G-100 column, and elute to collect the exosome components; S3: Stabilization treatment: S31: Buffer Adjustment: Introduce the exosome components collected in S24 into the stabilization buffer unit and adjust the ionic strength to 10-100 mM and the pH to 6.5-7.5; S32: Stabilizer addition: Add 1-5% w / v trehalose, 0.1-2.0 mM EDTA and 0.001-0.1% Tween 80 to the system through the stabilizer compounding unit, and stir until homogeneous; S33: Concentration control: The system is concentrated 10-50 times through a shear buffer unit, and the shear force is monitored in real time during the process to avoid exceeding 500Pa; S34: Programmed cryopreservation: The concentrated system is introduced into a temperature-controlled cooling unit and cooled to below -80°C at a rate of -1 to -3°C / min for cryopreservation; S35: Slow remelting: When needed, the remelting unit heats up at a rate of 0.5-1°C / min, while the concentration is maintained by the automatic concentration compensation module. S4: Quality Inspection: After steps S24, S32, and S35, the exosome particle size and zeta potential are detected by the quality inspection module to ensure product quality.

8. The plant-derived exosome stabilization and purification system and process according to claim 6, characterized in that: In step S23, the mass ratio of the amount of polyethylene glycol solution added to the exosome precipitate is 5:1-10:

1.

9. The plant-derived exosome stabilization and purification system and process according to claim 6, characterized in that: The concentration process described in step S33 uses ultrafiltration membrane concentration, with the ultrafiltration membrane having a molecular weight cutoff of 100 kDa.

10. The plant-derived exosome stabilization and purification system and process according to claim 6, characterized in that: If, as described in step S4, an exosome particle size exceeding 150 nm or a zeta potential higher than -10 mV is detected, return to step S31 to readjust the buffer solution.

Images