A method for assessing non-target protein residues in small extracellular vesicles

Abstract

This invention discloses a method for assessing non-target protein residues in small extracellular vesicles, belonging to the field of biodetection technology. The assessment method includes: determining the protein concentration of small extracellular vesicles, denoted as C0. TP The small extracellular vesicle product is filtered using an ultrafiltration tube or filter to obtain the filtrate. The protein concentration in the filtrate is then measured to obtain the concentration of the non-target protein, denoted as C. NP Vesicle protein concentration is expressed as C. VP And satisfy C TP =C VP +C NP Therefore, the proportion of non-target proteins is C. NP / C TP ×100%, vesicle protein percentage is C VP / C TP ×100%=1-C NP / C TP ×100%, representing purity. The evaluation method of this invention not only solves the problem that there is no way to evaluate the non-target protein residues in small extracellular vesicles in the prior art, but also indirectly provides a new method for evaluating the purity of small extracellular vesicles, realizing quality detection and control of small extracellular vesicles.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for evaluating non-target protein residues in small extracellular vesicles. Background Technology

[0002] Extracellular vesicles (EVs) are particles released by cells, enclosed in a lipid bilayer, and unable to self-replicate (lacking a functional nucleus). In describing EV subsets, there is a class of terms based on hypothetical biogenesis, such as exosomes and ectosomes. Exosomes refer to extracellular vesicles originating from within the cell, released via multivesicular bodies (MVBs), while ectosomes refer to extracellular vesicles formed by cell membrane budding.

[0003] In recent years, research and applications related to EVs have received widespread attention. Among EV products, stem cell-derived EVs carry the unique biological information of stem cells, possessing potential tissue regeneration and immunomodulatory functions. Moreover, compared to traditional stem cell therapies, they exhibit lower immunogenicity and better biocompatibility, thus being considered a safer and more universal cell-free treatment option. Currently, research on stem cell-derived EV drugs is extensive, but many challenges remain in quality research and pharmaceutical evaluation. Current isolation techniques struggle to enrich EVs generated by different mechanisms, and characterizing corresponding EV subpopulations is also difficult.

[0004] Small extracellular vesicles, generally less than 200 nm in diameter, typically ranging from 30 to 200 nm, are mainly composed of lipids, nucleic acids, and proteins. During cell culture, in addition to secreting extracellular vesicles into the culture supernatant, cells also secrete proteins into it. During the separation and purification of small extracellular vesicles using cell culture supernatant, some non-target proteins (free proteins) inevitably remain. Furthermore, different small extracellular vesicle production processes or parameters have varying effects on the residual non-target proteins.

[0005] Typically, EV purity analysis involves detecting the target vesicle particles present in the product, while impurity analysis includes detecting product-related impurities and process-related impurities. Currently, EV purity is commonly expressed as the particle-to-protein ratio or the percentage of target vesicle particles. However, particle protein content is an indirect indicator of purity; the percentage of target vesicle particles does not reflect the degree of removal of non-target proteins; and existing methods struggle to simultaneously assess both the overall purity of EVs and the level of non-target protein impurities, resulting in a technical limitation of relying on a single dimension for quality evaluation. Summary of the Invention

[0006] To address the above problems, the purpose of this invention is to propose an evaluation method for non-target protein residues in small extracellular vesicles. This method can evaluate the purity and impurity proteins to a certain extent, thereby enabling quality detection and control of small extracellular vesicles.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention proposes a method for assessing non-target protein residues in small extracellular vesicles, comprising the following steps:

[0009] S1: Determine the protein concentration of extracellular vesicle products from small cells, denoted as C. TP ;

[0010] S2: Filter the small extracellular vesicle product using an ultrafiltration tube or filter to obtain the filtrate. Measure the protein concentration in the filtrate; this is the concentration of the non-target protein, denoted as C. NP ;

[0011] S3: Vesicle protein concentration is expressed as C VP And satisfy C TP =C VP +C NP ;

[0012] Therefore, the proportion of non-target proteins is C. NP / C TP ×100%, vesicle protein percentage is C VP / C TP ×100%=1-C NP / C TP ×100%, representing purity.

[0013] Preferably, the small extracellular vesicle product is derived from neural stem cell conditioned medium (i.e., neural stem cell culture supernatant, provided by Shanghai Anji Xiekang Biotechnology Co., Ltd.).

[0014] Preferably, the concentration of the small extracellular vesicle protein is determined using at least one of the BCA method, the Bradford method, and the Lowry method.

[0015] Preferably, the extracellular vesicle protein concentration of the small cells is determined by lysis of the extracellular vesicles.

[0016] Preferably, a pinhole filter is used for filtering the small extracellular vesicles.

[0017] More preferably, the pinhole filter is a pinhole filter with a specification of 0.02um.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention proposes a method for assessing non-target protein residues in small extracellular vesicles, solving the problem of the lack of assessment methods for evaluating non-target protein residues in small extracellular vesicles. It also indirectly provides a new method for assessing the purity of small extracellular vesicles, thereby enabling quality detection and control of small extracellular vesicles. Attached Figure Description

[0020] Figure 1 The nanoparticle count results for the exosome product NE260127 in the examples are shown. Detailed Implementation

[0021] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0022] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0023] The following examples present a method for assessing non-target protein residues in small extracellular vesicles, comprising the following steps:

[0024] S1: Determine the concentration of extracellular vesicle proteins in small cells, denoted as C. TP ;

[0025] S2: Filter the small extracellular vesicle product using an ultrafiltration tube or filter to obtain the filtrate. Measure the protein concentration in the filtrate; this is the concentration of the non-target protein, denoted as C. NP ;

[0026] S3: Vesicle protein concentration is expressed as C VP And satisfy C TP =C VP +C NP ;

[0027] Therefore, the proportion of non-target proteins is C. NP / C TP ×100%, vesicle protein percentage is C VP / C TP ×100%=1-C NP / C TP ×100%, representing purity.

[0028] Example 1: Ultrafiltration centrifuge tube separation and purification of small extracellular vesicles

[0029] 1. Take 20 mL of neural stem cell conditioned medium (i.e. neural stem cell culture supernatant, provided by Shanghai Anji Xiekang Biotechnology Co., Ltd.) and divide it into two groups. Centrifuge at 3500g for 30 min using 50kD and 100kD ultrafiltration centrifuge tubes respectively. Collect the concentrated solution and dilute it to 1 mL.

[0030] 2. The concentration of the concentrate was determined using the Bradford Protein Concentration Assay Kit (Beyotime Biotechnology, catalog number: P0006C).

[0031] 3. Filter the concentrate using a 0.02µm pinhole filter (Cytiva, catalog number 6809-1102), and then determine the concentration of the filtrate using the Bradford Protein Assay Kit (Beyotime Biotechnology, catalog number: P0006C).

[0032] IV. Calculation of non-target protein concentration and vesicle protein concentration

[0033] The results are shown in Table 1. The proportion of non-target proteins in the small extracellular vesicle product separated and concentrated using a 50kd ultrafiltration tube was 71.34%, while the proportion of non-target proteins in the small extracellular vesicle product separated and concentrated using a 100kd ultrafiltration tube was 49.93%. Using a 50kd ultrafiltration centrifuge tube resulted in the retention of more non-target proteins compared to a 100kd ultrafiltration centrifuge tube, which is consistent with the expected trend.

[0034] Table 1: Evaluation of Non-Target Proteins in Products Separated by Different Ultrafiltration Tubes

[0035]

[0036] Example 2: Isolation and purification of small extracellular vesicles using an exosome harvesting system

[0037] 1. Take 10 L of neural stem cell conditioned medium (i.e., neural stem cell culture supernatant, provided by Shanghai Anji Xiekang Biotechnology Co., Ltd.), and use the 3D FloTrix® vivaEXO exosome harvesting system (Huakan Biotechnology) to isolate and purify small extracellular vesicles, obtaining approximately 500 mL of concentrated small extracellular vesicle solution. Detect the NTA nanoparticle count using PBS to obtain a standard dilution buffer of at least 2 × 10^9 particles / mL. Perform protein concentration analysis on the diluted small extracellular vesicle product according to the steps below, and simultaneously analyze the nanoparticle count (NTA count).

[0038] 2. The small extracellular vesicle products were lysed using Triton X-100 (Beyotime Biotechnology, catalog number: ST795), and the concentration was determined using a BCA protein concentration assay kit (Beyotime Biotechnology, catalog number: P0010).

[0039] 3. Small extracellular vesicle products were filtered using a 0.02µm pinhole filter (Cytiva, catalog number 6809-1102). The filtrate was lysed using a Triton X-100 (Beyotime Biotechnology, catalog number: ST795). The concentration of the filtrate was determined using a BCA protein concentration assay kit (Beyotime Biotechnology, catalog number: P0010).

[0040] IV. Calculation of non-target protein concentration and vesicle protein concentration

[0041] The results are shown in Table 2. Under this process, it can be seen that the lower the proportion of non-target proteins, the higher the particle-to-protein ratio; the higher the proportion of vesicle proteins, the higher the particle-to-protein ratio. Both trends are consistent in assessing the purity of small extracellular vesicles, but the proportion of vesicle proteins is more intuitive. The nanoparticle counting results of the exosome product NE260127 are shown in Table 2. Figure 1 As shown.

[0042] Table 2: Evaluation of Non-Target Proteins in Exosome Harvesting System Separation Products

[0043]

[0044] Example 3: Precision and accuracy assessment of a method for evaluating non-target protein residues in small extracellular vesicles

[0045] 1. Produce small extracellular vesicle products (batch NE260624) according to the method of Example 2.

[0046] 2. Small extracellular vesicle products were analyzed using the Bradford Protein Assay Kit. The products were filtered through a 0.02 μm pinhole filter (Cytiva, catalog number 6809-1102), and the concentration of the filtrate was determined using the Bradford Protein Assay Kit. The assay was repeated six times, and the coefficient of variation was calculated.

[0047] 3. Take one portion of the small extracellular vesicle product, add PBS at a volume ratio of 1:4 and mix well; add BSA at a volume ratio of 1:4 and mix well. The final BSA concentration should be 0.05 mg / mL. Measure the concentration using a Bradford protein assay kit. Filter the small extracellular vesicle product using a 0.02 μm pin filter (Cytiva, catalog number 6809-1102), and measure the concentration of the filtrate using a Bradford protein assay kit. Calculate the sample spike recovery rate, where the actual spiked value (i.e., the amount of standard added) is calibrated by the pre-filter spiked protein concentration and the unspecified protein concentration.

[0048] 4. Calculate the coefficient of variation and the sample spike recovery rate.

[0049] The coefficient of variation is calculated as (sample standard deviation / sample mean) × 100%. The test results are shown in Table 3, with a coefficient of variation of 10.24%.

[0050] The sample spike recovery rate is equal to [(spike measured value - sample base value) / amount of standard added)] × 100%, and the results are shown in Table 4. The spike recovery rate is 101.76%.

[0051] Table 3: Accuracy Evaluation Results

[0052]

[0053] Table 4: Accuracy Assessment Results

[0054]

[0055] In summary, this invention provides a practical technical solution for evaluating impurity proteins or non-target proteins during the development of small extracellular vesicle drugs, and also provides a more intuitive evaluation index for the purity of small extracellular vesicles from a protein perspective.

[0056] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for assessing non-target protein residues in small extracellular vesicles, characterized in that, Includes the following steps: S1: Determine the concentration of extracellular vesicle proteins in small cells, denoted as C. TP ; S2: Filter the small extracellular vesicle product using an ultrafiltration tube or filter to obtain the filtrate. Measure the protein concentration in the filtrate; this is the concentration of the non-target protein, denoted as C. NP ; S3: Vesicle protein concentration is expressed as C VP And satisfy C TP =C VP +C NP ; Therefore, the proportion of non-target proteins is C. NP / C TP ×100%, vesicle protein percentage is C VP / C TP ×100%=1-C NP / C TP ×100%, representing purity.

2. The method for evaluating non-target protein residues in small extracellular vesicles according to claim 1, characterized in that, The concentration of the small extracellular vesicle protein was determined using at least one of the BCA method, the Bradford method, and the Lowry method.

3. The method for assessing non-target protein residues in small extracellular vesicles according to claim 1, characterized in that, Before determining the concentration of the extracellular vesicle protein in the small cells, the extracellular vesicles were subjected to vesicle lysis.

4. The method for assessing non-target protein residues in small extracellular vesicles according to claim 1, characterized in that, A pinhole filter was used to filter the small extracellular vesicles.

5. The method for evaluating non-target protein residues in small extracellular vesicles according to claim 4, characterized in that, The pinhole filter is a 0.02µm pinhole filter.

6. The method for evaluating non-target protein residues in small extracellular vesicles according to claim 1, characterized in that, The small extracellular vesicle product is derived from neural stem cell conditioned medium, which is the supernatant of neural stem cell culture.

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