Separation and purification reagent for detecting purity and particle number of biological nano active component, purity and particle number detection method and kit

Abstract

The invention provides a separation and purification reagent for detecting the purity and the particle number of nano-scale bioactive components such as vesicles and viruses, a method and a kit for detecting the purity and the particle number of biological nano-active substances, and an exclusion chromatographic column filled with a polymethacrylate filler and an agarose gel filler which are mixed in proportion. The separation degree between nano active ingredients and impurities is good, the separation degree between the active ingredients and impure protein and nucleic acid is ensured, and accurate quantitative detection of the nano active ingredients is facilitated; the chromatographic columns are used on HPLC (High Performance Liquid Chromatography) instruments of different manufacturers, and various detectors are used for detecting; the method can be used for more stable, more accurate and more operable quantitative detection of nano active components from different sources, can be used for accurately detecting the purity and content of the active components in a sample, and has important application value in quality control of biological nano active components. The kit disclosed by the invention can be used for rapidly and accurately detecting the purity and the concentration of the active ingredients, and a rapid and accurate detection way is provided.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological nano active ingredient analysis and detection, and particularly relates to a separation and purification reagent for biological nano active ingredient purity and particle number detection, a biological nano active ingredient purity and particle number detection method and a kit. BACKGROUND

[0002] Biological nano active ingredients refer to a group of microparticles with a particle size range of 1-1000 nanometers, including viruses, extracellular vesicles and exosomes. Biological nanoparticles are unevenly dispersed in solution and are in an aggregated state. Traditional fluorescence quantitative PCR and nanoparticle tracking analysis have small sample volume and small sampling amount. The sample is diluted multiple times before being put on the machine, which makes it difficult to truly reflect the concentration and purity of nanoparticles in the original sample. The detection results have poor reproducibility and wide range, and it is difficult to use biological nanoparticles as a precise quantitative detection method for drug application. Therefore, new methods and kits need to be developed for the development of biological nano drugs.

[0003] Vesicles (extracellular vesicles, EVs) are nanoscale lipid bilayer vesicles released by cells, including plant vesicles, microvesicles and apoptotic bodies; these cells include almost all cell types. EVs are widely present in various body fluids and cell supernatants, and stably carry important biological molecules such as nucleic acids, proteins and lipids, participate in the regulation of pathological and physiological processes such as cell proliferation and differentiation, angiogenesis and immune response, have various biological activities, and are important carriers of information exchange.

[0004] Plant vesicles differ greatly from mammalian EVs in morphology, composition and function, but play an important role in a non-cell autonomous manner due to the presence of cell walls, and can even exchange biological information between species. Plant vesicles are typically in the form of a tea tray with a diameter of 30-400 nm. The general separation and purification methods are differential centrifugation, density gradient centrifugation, polymer precipitation and size exclusion chromatography. Jae Young You et al. used size exclusion chromatography to separate nanovesicles from cabbage and purple cabbage, and verified their functions in cell proliferation, anti-inflammatory, anti-apoptotic and drug delivery. At the same time, three plant-derived nanovesicle separation methods were compared: size exclusion chromatography (SEC), PEG precipitation and ultracentrifugation (UC), and it was pointed out that size exclusion chromatography has significant advantages in yield, particle size uniformity (NTA method detection), protein impurity residue and function retention.

[0005] In terms of purity analysis, compared to the uniformity of animal cell vesicles, plant vesicles are more diverse and therefore more difficult to analyze. High-performance liquid chromatography (HPLC) requires small sample loading and has low vesicle content. Plant extracts contain macromolecules such as cellulose and pectin, and the uniform particle size and small pore size of the chromatographic packing material result in poor response of traditional SEC columns to the target plant vesicle detection signal. For particle count and content detection, current methods utilize dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS) to detect particle size, concentration, and potential. The bicinchoninicacid assay (BCA) is used to detect protein content in the sample, and the purity of plant vesicles is assessed by comparing it with the particle count, expressed as the number of particles per unit of protein. The Triton X-100 membrane rupture assay is used to break down membrane-bound particles (such as EVs), and the change in particle concentration before and after treatment is detected to calculate the proportion of membrane-bound particles, thus indirectly reflecting the purity of plant vesicles. The existing detection methods cannot eliminate the interference of nucleic acid and protein impurities in the sample on purity detection, while high performance liquid chromatography can effectively separate vesicles, proteins, nucleic acids, etc., to eliminate the interference of nucleic acid and protein impurities on purity detection.

[0006] Therefore, developing a rapid, accurate, and highly sensitive high-performance liquid chromatography method for detecting the purity and particle number of viruses, animal and plant vesicles has significant application value. Summary of the Invention

[0007] This application provides a separation and purification reagent, a method for detecting the purity and particle number of bioactive nanoparticles, and a kit for this purpose, in order to solve the problems existing in related technologies. The technical solution is as follows: In a first aspect, embodiments of this application provide a separation and purification reagent for detecting the purity and particle number of biological nano-components, the separation and purification reagent comprising a gel filter packing; the gel filter packing is composed of a mixture of polymethyl methacrylate packing and agarose gel packing; The volume ratio of polymethyl methacrylate filler to agarose gel filler is 1:(1~10); The particle size range of the gel filter media is 20~165μm.

[0008] In one embodiment, the polymethyl methacrylate filler has a particle size range of 9-70 μm and a pore size of 100-2000 Å.

[0009] In one embodiment, the agarose gel filler has a particle size range of 25~170μm.

[0010] In one embodiment, the mobile phase is further included; the mobile phase is a sodium chloride phosphate buffer; the concentration of the phosphate buffer is 5-50 mM; and the concentration of sodium chloride is 100-500 mM. The pH of the mobile phase is 7.0~8.0.

[0011] In one embodiment, a reference product is also included, which is a liquid reference product or a lyophilized product of a bioactive ingredient.

[0012] In one embodiment, the reference material is adenovirus, adeno-associated virus, MSC cell exosomes, 293 cell exosomes, bean sprout vesicles, and jujube seed vesicles.

[0013] Secondly, embodiments of this application provide a method for detecting the purity and particle number of bioactive components, which involves separating and purifying the sample using a size exclusion chromatography column filled with the aforementioned gel filter packing material; detecting the sample using a detector; and quantitatively detecting the bioactive components using a reference standard. The detector is any one or a combination of two or more of the following: an ultraviolet detector, a fluorescence detector, or a differential refractive index detector.

[0014] In one embodiment, the sample is separated, purified, and detected by a detector using a liquid chromatograph.

[0015] In one embodiment, when using an ultraviolet detector for detection, the detection wavelengths are set to UV260nm and UV280nm. When using a differential refractive index detector, the signal polarity is set to negative and the temperature is 35℃. When using a fluorescence detector for detection, the fluorescence detector is set to a wavelength of ex260 nm / em500 nm.

[0016] In one embodiment, the conditions for separating and purifying the sample using a size exclusion chromatography column are as follows: The mobile phase flow rate was 0.1–1.0 mL / min; the elution process was isocratic elution; and the column temperature was 25–35 °C.

[0017] In one embodiment, the sample is a virus, plant vesicle, or animal vesicle.

[0018] In one embodiment, the sample is an adenovirus, bean sprout, rice, jujube seed vesicle sample, or exosomes of mammalian origin.

[0019] Thirdly, embodiments of this application provide a kit for detecting the purity and particle number of bioactive nanoparticles, including the separation and purification reagents described above.

[0020] The advantages or beneficial effects of the above technical solutions include at least the following: The separation and purification reagent for detecting the purity and particle number of bioactive nanoparticles in this application is separated and purified by a gel filtration packing material including polymethyl methacrylate packing material and agarose gel packing material. The separation degree between bioactive nanoparticles and impurities is good, ensuring the separation degree between bioactive nanoparticles and impurities such as proteins and nucleic acids, which is beneficial to the accurate quantitative detection of bioactive nanoparticles.

[0021] The detection method for the purity and particle number of bioactive nanoparticles in this application uses gel filtration packing to prepare size exclusion chromatographic columns of different types, which can be used on HPLC instruments from different manufacturers with various detectors. It can more stably, accurately and operably quantify bioactive nanoparticles from different sources, and accurately detect the purity and content of active ingredients in samples. It has important application value in the quality control of bioactive nanoparticles.

[0022] The reagent kit of this application can quickly and accurately detect the purity and concentration of active ingredients, providing a rapid and accurate detection method.

[0023] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0024] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0025] Figure 1 To detect the ultraviolet spectra of plant vesicles using different size exclusion chromatography columns; Figure 2 This is a diagram showing the results of detecting plant vesicles using a differential refractive index detector. Figure 3 This is a graph showing the results of detecting plant vesicles using a fluorescence detector; Figure 4 Spectra of plant vesicles and their strongly degraded samples used in methodological specificity validation; Figure 5 Spectra of plant vesicles at different concentrations; Figure 6 The standard curve for methodological linearity verification; Figure 7 This is a comparison chart of the concentration results measured by the method of this application with those measured by NTA and the nano-Coultre method; Figure 8 A spectrum for methodological repeatability verification; Figure 9 Spectra of different columns used in method robustness validation; Figure 10 Spectra of different high-performance liquid chromatographs used in method tolerance validation; Figure 11 The analysis results are for plant vesicle samples from different processing steps. Figure 12 These are the analytical results for different samples. Detailed Implementation

[0026] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0027] Existing methods for vesicle particle number detection, such as nanoparticle size tracking (NTA) and nanocoulter methods, suffer from narrow linear ranges, low detection upper limits, and require sample dilution before detection. NTA, in particular, requires 100-10000-fold absorption of the sample. The heterogeneity of vesicle solutions and uneven sampling during dilution lead to poor reproducibility. Therefore, this application provides a separation and purification reagent, a method for detecting the purity and particle number of bioactive nanoparticles, and a kit for this purpose.

[0028] A separation and purification reagent for detecting the purity and particle number of bioactive nanoparticles, the separation and purification reagent comprising a gel filter media; the gel filter media is composed of a mixture of polymethyl methacrylate media and agarose gel media; The volume ratio of polymethyl methacrylate filler to agarose gel filler is 1:(1~10); The particle size range of the gel filter media is 20~165μm.

[0029] The gel filter packing material of this application exhibits high sensitivity for bioactive nanoparticles, especially nano-sized biological particles such as plant vesicles; as an analytical chromatographic packing mixture, it can be self-packed into analytical chromatographic columns; and it is suitable for various analytical instruments. Furthermore, the mixture of polymethyl methacrylate (PMMA) and agarose packing materials increases the pressure resistance of the agarose packing material, making it suitable for high-pressure HPLC analysis, while simultaneously reducing the adsorption of plant exosomes by the PMMA packing material, thus improving overall detection sensitivity.

[0030] In one embodiment, the polymethyl methacrylate (PMMA) filler has a particle size range of 9–70 μm and a pore size of 100–2000 Å. In this embodiment, the PMMA filler particle size range is 10.0 ± 1.0, 15.0 ± 1.5, 30.0 ± 5.0, 60.0 ± 10.0 μm; it can also be any value between 9 and 70 μm. The PMMA filler pore size range is 500 Å, 100 Å, 2000 Å; it can also be any value between 100 and 2000 Å.

[0031] In one embodiment, the agarose gel filler particle size ranges from 25 to 170 μm. In this embodiment, the agarose gel filler particle size ranges from 25, 45, 80, 85, 90, 95, 100, 125, 170 μm or any value between the two.

[0032] As one embodiment, the mobile phase is further included; the mobile phase is a sodium chloride phosphate buffer; the concentration of the phosphate buffer is 5%. 50mM; sodium chloride concentration is 100~500mM; The pH of the mobile phase is 7.0~8.0.

[0033] In this embodiment, the concentration of phosphate buffer in the mobile phase is 5 mM, 20 mM, or 50 mM; it can also be any value between 5 and 50 mM.

[0034] The concentration of sodium chloride in the mobile phase is 200 mM, 250 mM, 300 mM or 500 mM; it can also be any value between 100 and 500 mM.

[0035] The pH value of the mobile phase is 7.0, 7.2, 7.4, 7.6, 7.8 or 8.0; or it can be any value between 7.0 and 8.0.

[0036] One embodiment includes a reference material, which is a liquid or lyophilized reference material of a bioactive nanoparticle. Preferably, the reference material is adenovirus, adeno-associated virus, MSC cell exosomes, 293 cell exosomes, bean sprout vesicles, or jujube seed vesicles. More preferably, the reference material is a lipid bilayer membrane structure vesicle extracted from a plant and purified by one or more steps of chromatography. Preferably, the reference material is a liquid or lyophilized powder composed of a buffer system.

[0037] This application also provides a method for detecting the purity and particle number of bioactive nanoparticles, which involves separating and purifying the sample using a size exclusion chromatography column filled with the above-mentioned gel filtration packing; detecting the sample using a detector; and quantitatively detecting the bioactive nanoparticles using a reference standard. The detector is any one or a combination of two or more of the following: an ultraviolet detector, a fluorescence detector, or a differential refractive index detector.

[0038] The gel filtration packing material of this application is used to prepare a size exclusion chromatographic column for sample separation and purification. It exhibits good separation between bioactive nanoparticles and impurities, which is beneficial for the accurate quantitative detection of bioactive nanoparticles. Subsequent detection using a detector further enables accurate quantitative analysis of the bioactive nanoparticles. As one implementation method, the sample is separated, purified, and detected using a liquid chromatograph.

[0039] The gel filtration packing material of this application is used to prepare size exclusion chromatographic columns, which can be used in liquid chromatography (HPLC) to separate bioactive nanoparticles; and the use of detectors, especially ultraviolet detectors, can simultaneously achieve sample separation and purification and ultraviolet detection in a single liquid chromatograph, simplifying the testing process.

[0040] As one embodiment, the size exclusion column used is a 4.6 x 200 mm or other size column.

[0041] As one implementation method, when using an ultraviolet detector for detection, the detection wavelength is set to UV260nm and UV280nm.

[0042] As one implementation method, when using a differential refractive index detector for detection, the signal polarity is set to negative and the temperature is 35°C.

[0043] As one implementation method, when using a fluorescence detector for detection, the fluorescence detector is set to a wavelength of ex260 nm / em500 nm.

[0044] As one implementation method, the conditions for separating and purifying samples using a size exclusion chromatography column are as follows: The flow rate of the mobile phase is 0.1~1.0 mL / min; it can be 0.1 mL / min, 0.15 mL / min, 0.5 mL / min or 1.0 mL / min.

[0045] The elution process is isocratic elution; the column temperature is 25~35℃.

[0046] As one embodiment, the sample is a virus, plant vesicle, or animal vesicle.

[0047] As one embodiment, the sample is an adenovirus, bean sprout, rice, jujube seed vesicle sample, or exosomes derived from mammalian MSCs.

[0048] This application also provides a kit for detecting vesicle purity and particle number, including the separation and purification reagents described above.

[0049] The following is a further explanation using specific embodiments.

[0050] Example 1: Preparation of chromatographic column and corresponding tests The chromatographic column was prepared as follows: Liquid chromatograph: AKTA pure 25; SEC-HPLC column: self-made, with dimensions of 4.6 mm in diameter × 200 mm, packed with polymethyl methacrylate and agarose packing materials in different proportions and types; The filling ratios include: 2:1, 1:1, 1:2, and 1:5; Reference material: Plant vesicle liquid reference material or lyophilized product with 1E+11 particles / mL, which can be used for qualitative analysis or external standard method for quantification; Mobile phase: 10mM PBS + 300mM NaCl, pH 7.2~7.4; Elution process: isocratic elution Method parameters: The flow rate was adjusted from 0.15 mL / min to 20 mL / min, and the column back pressure was observed. If the flow rate and pressure remained linearly related when the pressure rose to 35 bar, and remained stable at 30 bar for 5 minutes, the flow rate was stopped. The column that passed the maximum pressure test was used for injection testing. The detection wavelengths were UV280 nm and UV260 nm, and the injection volume was 500 μL. The chromatograms were compared with those of the preparative chromatography to observe the sample separation effect. The results are shown in Table 1.

[0051] Table 1. Effects of different mixing ratios on plant vesicle separation efficiency

[0052] Table 2. Effects of different polymethacrylate fillers on plant vesicle separation efficiency when mixed at a 1:2 ratio.

[0053] Example 2: Detection using an ultraviolet detector Liquid chromatograph: Agilent 1260 Infinity II with UV detector.

[0054] SEC-HPLC column: self-made, with dimensions of 4.6 × 200 mm (diameter × length), composed of polymethyl methacrylate (PMMA) and agarose packing materials; the volume ratio of PMMA to agarose packing materials is 1:2; the PMMA packing material has a particle size of 30.0 ± 5.0 μm and a pore size of 2000 Å; the agarose packing material has an average particle size of 90 μm. Reference material: Plant vesicle liquid reference material or lyophilized product with 1E+11 particles / mL, which can be used for qualitative analysis or external standard method for quantification.

[0055] Mobile phase: 10mM PBS + 300mM NaCl, pH 7.2~7.4; Elution process: isocratic elution.

[0056] Method parameters: flow rate 0.15 mL / min, detection wavelengths UV280 nm and UV260 nm, elution time 35 min, injection volume 100 μL, sample chamber uncontrolled, column oven uncontrolled.

[0057] Example 3: Detection using a fluorescence detector Liquid chromatograph: Agilent 1260 Infinity II with fluorescence detector.

[0058] SEC-HPLC column: self-made; dimensions: diameter × length 4.6 × 200 mm; polymethyl methacrylate (PMMA) and agarose (AG) packing materials; volume ratio of PMMA to AAG: 1:2; PMMA particle size: 30.0 ± 5.0 μm, pore size: 2000 Å; AAG average particle size: 90 μm. Reference material: Plant vesicle liquid reference material or lyophilized product with 1E+11 particles / mL, which can be used for qualitative analysis or external standard method for quantification.

[0059] Mobile phase: 10mM PBS + 300mM NaCl, pH 7.2~7.4.

[0060] Elution process: isocratic elution.

[0061] Method parameters: flow rate 0.15 mL / min, detection wavelength ex260 nm / em500 nm, elution time 35 min, injection volume 100 μL, sample chamber uncontrolled, column oven uncontrolled.

[0062] Example 4: Detection results of the differential refractive index detector Liquid chromatograph: Agilent 1260 Infinity II with differential refractive index detector.

[0063] SEC-HPLC column: self-made; dimensions: diameter × length 4.6 × 200 mm; polymethyl methacrylate (PMMA) and agarose (AG) packing materials; volume ratio of PMMA to AAG: 1:2; PMMA particle size: 30.0 ± 5.0 μm, pore size: 2000 Å; AAG average particle size: 90 μm. Reference material: Plant vesicle liquid reference material or lyophilized product with 1E+11 particles / mL, which can be used for qualitative analysis or external standard method for quantification.

[0064] Mobile phase: 10mM PBS + 300mM NaCl, pH 7.2~7.4.

[0065] Elution process: isocratic elution.

[0066] Method parameters: flow rate 0.15 mL / min, differential refractive index detector signal polarity set to negative, temperature 35℃, elution time 35 min, injection volume 100 μL, sample chamber uncontrolled, column oven uncontrolled.

[0067] Performance testing results (1) Detector selection Following the methods in Examples 1-3, the same plant vesicle sample was analyzed using the same injection volume, and the results are as follows: Figures 1-3 As shown.

[0068] from Figure 1 and Figure 3 It can be seen that the differential refractive index detector has no response to the principal component; the fluorescence detector (ex260nm / em500nm) has a low response value (the main peak area is only about 42 Lu*s), while under the same sample volume of UV detection, the UV detector (UV280nm or UV260nm) has the highest response value, with a main peak area of ​​about 400 mAU*min. This indicates that the UV detector has a superior detection effect.

[0069] (2) Ultraviolet detector detection, selection of chromatographic column Commercially available size exclusion chromatography columns (TSKgel G4000SW, 7.8 mm × 300 mm, 13 μm, 450 Å), XBridge BEH 450 Å SEC, 5 μm, 7.8 mm × 300 mm, and self-made columns #1 and #2 were used to analyze the same plant vesicle samples according to the above-described detection method. When using commercially available columns, the flow rate was set to 0.75 mL / min, while other parameters remained consistent. The detection results are as follows: Figure 1 As shown.

[0070] Self-made chromatographic column #1: Dimensions are 4.6 × 200 mm (diameter × length), using polymethyl methacrylate (PMMA) and agarose packing materials; the volume ratio of PMMA to agarose packing materials is 1:2; the PMMA packing material has a particle size of 30.0 ± 5.0 μm and a pore size of 2000 Å; the agarose packing material has an average particle size of 90 μm. Self-made chromatographic column #2: Dimensions are 4.6 × 200 mm (diameter × length), using polymethyl methacrylate (PMMA) and agarose packing materials; the volume ratio of PMMA to agarose packing materials is 1:2; the PMMA packing material has a particle size of 30.0 ± 5.0 μm and a pore size of 2000 Å; the agarose packing material has an average particle size of 34 μm. from Figure 1 As can be seen, the self-made chromatographic column has a better separation effect on plant vesicles.

[0071] (3) Detection effect of the detection method in Example 2 Three bottles containing the same plant vesicle sample were used. One bottle contained 10% HCl, another contained 0.1% SDS and 0.1 M NaOH, and the third served as the original sample. The detection method described in Example 2 was used for each sample injection and analysis. The results are as follows: Figure 4 As shown.

[0072] Figure 4 As can be seen, the detection method in Example 2 has a good separation effect on the main component and impurities, and the resolution (USP) between the main peak and the impurity peak is 5.44.

[0073] (4) Linearity determination of the detection method in Example 2 Plant vesicle reference samples were serially diluted and analyzed using the detection method described in Example 2. Linear regression was performed with the plant vesicle concentration as the x-axis and the corresponding peak area as the y-axis to obtain the linear regression equation and correlation coefficient R². The experimental results regarding the linear range of this detection method are shown below. Figure 5 and Figure 6 .

[0074] The analysis results showed that plant vesicles were at 5E+9 particles / mL. The linearity R² for 1E+11 particles / mL is 0.9999, which meets the method validation requirements.

[0075] (5) Accuracy of quantitative analysis of the detection method in Example 2 Based on the NTA concentration test results, plant vesicle reference standards with concentrations of 1E+11 Particles / mL, 5E+10 Particles / mL, 2E+10 Particles / mL, 1E+10 Particles / mL, and 5E+9 Particles / mL were prepared by dilution. The concentrations were detected using nanoparticle tracking analysis (NTA), pulse resistive detection (RPS), and the detection method described in Example 2, respectively. Specific test results are shown in Table 3. Linear regression was performed with the nominal concentration of plant vesicles on the x-axis and the corresponding measured concentration on the y-axis to obtain the linear regression equation and correlation coefficient R². The experimental results are shown in Table 3. Figure 7 .

[0076] Table 3

[0077] Analysis Table 3 and Figure 7 The results showed that the NTA, RPS, and Example 2 detection methods all had good linearity, with corresponding correlation coefficients R2 of 0.9852, 0.9976, and 0.9999, respectively. The measured concentration of the Example 2 method was between that of the NTA and RPS detection methods, indicating that the Example 2 method can be used for the concentration detection of plant vesicles.

[0078] (6) Repeatability of the method in Example 2 Using the detection method of Example 2 with the lowest linear concentration sample of 5E+9 particles / mL, the sample was injected six times repeatedly. The peak area of ​​the main peak was recorded for each injection, and the RSD value was calculated. The results are shown in Table 4, and the chromatogram is shown below. Figure 8 As shown.

[0079] Table 4

[0080] The RSD was 3.7%, and the repeatability met the method validation requirements.

[0081] (7) Method tolerance test in Example 2 Using the detection method described in Example 2, the effects of two self-made columns, Column #1 (agarose gel packing) and Column #2 (agarose and dextran composite gel packing), on the separation performance were investigated on a Thermo UltiMate 3000 liquid chromatograph to evaluate the tolerance of this detection method to different columns. The test results are shown in [Figure 2]. Figure 9 The separation performance of different chromatographic columns can meet the requirements.

[0082] Using the same chromatographic column from Example 2, detection was performed on both a Thermo UltiMate 3000 and an Agilent 1260 Infinity II liquid chromatograph to investigate the effect of different instruments on the separation performance, thereby evaluating the tolerance of this detection method to different instruments. The test results are shown in [Figure 1]. Figure 10 The separation effect of different devices can meet the requirements.

[0083] (8) Detection of plant vesicle samples from different sources, exosomes from mammalian MSCs, adenoviruses, and plant vesicle samples from different processing steps: The detection method of Example 2 was used to detect the bean sprout vesicles before purification, after the first purification, and after the second purification. The detection results are shown in [Figure 2]. Figure 11 ; The detection method of Example 2 was used to detect vesicle samples from bean sprouts, rice, and jujube seeds, as well as exosomes and adenoviruses derived from mammalian MSCs. The detection results are shown in [Figure 2]. Figure 12 ; from Figure 11 and Figure 12 It can be seen that the methods in Example 2 can effectively separate the main peak from the impurity peak.

[0084] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0085] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0086] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A separation and purification reagent for detecting the purity and particle number of bioactive nanoparticles, characterized in that, The separation and purification reagent includes a gel filtration packing material; the gel filtration packing material is a mixture of polymethyl methacrylate packing material and agarose gel packing material; The volume ratio of polymethyl methacrylate filler to agarose gel filler is 1:(1~10); The particle size range of the gel filter media is 20~165μm.

2. The separation and purification reagent for detecting the purity and particle number of bioactive nanoparticles according to claim 1, characterized in that, The particle size range of polymethyl methacrylate fillers is 9~70 μm; the pore size is 100~2000 Å. The particle size range of agarose gel filler is 25~170μm.

3. The separation and purification reagent for detecting the purity and particle number of bioactive nanoparticles according to claim 1, characterized in that, It also includes a mobile phase; the mobile phase is a sodium chloride phosphate buffer; the concentration of the phosphate buffer is 5~50mM; the concentration of sodium chloride is 100~500mM; The pH of the mobile phase is 7.0~8.

0.

4. The separation and purification reagent for detecting the purity and particle number of bioactive nanoparticles according to claim 1, characterized in that, It also includes reference materials, which are liquid or lyophilized bio-nanoactive ingredients; preferably, the reference materials are adenovirus, adeno-associated virus, MSC cell exosomes, 293 cell exosomes, bean sprout vesicles, and jujube seed vesicles.

5. A method for detecting the purity and particle number of bioactive nanoparticles, characterized in that, The sample was separated and purified using a size exclusion chromatography column filled with the gel filtration packing material as described in any one of claims 1-4; Detection was performed using a detector; quantitative detection of bioactive nanoparticles was conducted using reference standards. The detector is any one or a combination of two or more of the following: an ultraviolet detector, a fluorescence detector, or a differential refractive index detector.

6. The method for detecting the purity and particle number of bioactive nanoparticles according to claim 5, characterized in that, The samples were separated, purified, and detected by a detector using a liquid chromatograph.

7. The method for detecting the purity and particle number of bioactive nanoparticles according to claim 5, characterized in that, When using an ultraviolet detector, the detection wavelengths are set to UV260nm and UV280nm. When using a differential refractive index detector, the signal polarity is set to negative and the temperature is 35℃. When using a fluorescence detector for detection, the fluorescence detector is set to a wavelength of ex260 nm / em500 nm.

8. The method for detecting the purity and particle number of bioactive nanoparticles according to claim 5, characterized in that, The conditions for separating and purifying samples using size exclusion chromatography columns are as follows: The mobile phase flow rate was 0.1–1.0 mL / min; the elution process was isocratic elution; and the column temperature was 25–35 °C.

9. The method for detecting the purity and particle number of bioactive nanoparticles according to claim 4, characterized in that, The sample is a plant vesicle, an animal vesicle, or a virus; preferably, the sample is a vesicle sample of bean sprouts, rice, or jujube seed, an exosome of mammalian origin, an adenovirus, or an adeno-associated virus.

10. A kit for detecting the purity and particle number of bioactive nanoparticles, characterized in that, Includes the separation and purification reagents according to any one of claims 1-4.

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