Quality detection method for protein polymer

Abstract

The present invention relates to the field of biopharmaceuticals, and discloses a quality detection method for a protein polymer, comprising at least one of reverse-phase HPLC detection and SDS-PAGE analysis, mass spectrometry detection, particle size detection, exosome surface marker detection, sterile detection, and endotoxin detection. In some embodiments of the present invention, the quality detection method effectively achieves separation of components in a protein polymer sample by optimizing HPLC conditions, thereby obtaining better detection results, and establishing a quality control standard for the protein polymer.

Description

A method for quality detection of protein polymers

[0001] This disclosure claims priority to Chinese Patent Application No. 202410944797.4, filed on July 15, 2024, entitled "A Method for Quality Testing of Protein Polymers", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biopharmaceuticals, specifically relating to a method for quality detection of protein polymers. Background Technology

[0003] Mesenchymal stem cells (MSCs) possess self-replication and multi-lineage differentiation potential, and are widely found in tissues such as bone marrow, adipose tissue, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amnion, peripheral blood, muscle, and urine. They are characterized by their wide availability, lack of matching requirements, low infection rate, strong differentiation potential, strong proliferation capacity, and convenient collection. They can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), and interferon (IFN), which participate in regulating cell growth, apoptosis, cell differentiation, antiviral activity, and immune maturation. They can be used for immune regulation, tissue repair, and the treatment of diseases such as acute lung injury, severe pneumonia, and acute respiratory distress syndrome.

[0004] The inventors have discovered that by stimulating and then lysing MSCs, a protein polymer with neurorepair activity can be isolated and purified from intracellular proteins. This protein polymer has shown significant effects in completed animal and clinical trials, especially in treating ALS, demonstrating clear potential as a drug and is now in the clinical trial stage.

[0005] Currently, protein polymers are obtained by stimulating MSC expression and then isolating and purifying them, which makes quality control relatively difficult. Developing a quality detection method for protein polymers is of great significance for stabilizing their quality. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one deficiency of the prior art and provide a method for quality detection of protein polymers.

[0007] The technical solution adopted in this invention is:

[0008] A method for quality detection of protein polymers includes reversed-phase HPLC detection and at least one of the following methods:

[0009] SDS-PAGE analysis

[0010] Mass spectrometry detection

[0011] Particle size detection

[0012] Detection of exosome surface markers

[0013] Aseptic testing

[0014] Endotoxin testing;

[0015] The reversed-phase HPLC detection conditions were as follows: mobile phase A was 0.1% TFA aqueous solution, mobile phase B was 0.1% TFA acetonitrile solution, and the gradient elution conditions were as follows:

[0016] Alternatively, the conditions for reversed-phase HPLC detection are:

[0017] Mobile phase A was an aqueous solution of 0.1% TFA, and mobile phase B was a 71.4% acetonitrile solution of 0.075% TFA. Chromatographic conditions:

[0018] for:

[0019] Or the chromatographic conditions are:

[0020] In some examples of quality testing methods, the chromatographic column used for reversed-phase HPLC detection is a 300C4-T 4.6x150, 5μm column manufactured by Nanomicro Corporation. Preferably, the column temperature is 30℃, the flow rate is 0.8mL / min, the detection wavelength is 280nm, and the sample loading volume is 25μL.

[0021] In some examples of quality control methods, reversed-phase HPLC detection uses an XBridge Protein BEH C4 column. 3.5μm, 4.6mm*150mm, detection wavelength 220nm, flow rate 1.0mL / min.

[0022] In some examples of quality detection methods, the elution time of the sample after separation by reversed-phase HPLC is between 2 min and 20 min. The elution time of the main sample component group 1 is between 12 min and 20 min, the elution time of characteristic peak 1 is about 15 min, the elution time of sample component group 2 is between 2 min and 5 min, and the elution time of characteristic peak 2 is about 3 min.

[0023] In some examples of quality testing methods, the chromatographic column used for reversed-phase HPLC detection is a 300C4-T 4.6x150, 5μm column manufactured by Nanomicro Corporation. Preferably, the column temperature is 30℃, the flow rate is 0.8mL / min, the detection wavelength is 280nm, and the sample loading volume is 25μL. After sample separation by reversed-phase HPLC, the peak elution time is between 2min and 20min. The peak elution time of the main sample component group 1 is between 12min and 20min, the peak elution time of characteristic peak 1 is about 15min, the peak elution time of sample component group 2 is 2min to 5min, and the peak elution time of characteristic peak 2 is about 3min.

[0024] In some examples of quality testing methods, SDS-PAGE analysis uses 4-20% pre-mixed gel for sample separation and detection. The sample bands are mainly distributed in the range of 11KD to 100KD, with the molecular weight decreasing from large to small. The first band is located between 75KD and 100KD, and the second band is located between 63KD and 75KD.

[0025] In some quality testing methods, when measuring particle size, the particle size of the protein polymer sample should be between 30 nm and 150 nm.

[0026] In some examples of quality testing methods, particle size analysis is performed on protein polymer samples with particle sizes ranging from 40 nm to 90 nm.

[0027] In some quality control methods, particle size analysis is performed on protein polymer samples with particle sizes ranging from 50 nm to 80 nm. In other quality control methods, exosome surface marker detection is performed by assessing the expression of the exosome positive markers TSG101 / CD9 / HSP70 and the negative marker Calnexin; no exosome markers should be expressed. Preferably, the method used for exosome surface marker detection is Western blotting.

[0028] In some examples of quality control methods, the detection of exosome surface markers includes the following procedures:

[0029] Antibodies are used to specifically bind to protein polymer samples processed by gel electrophoresis. The detection signal is amplified by a labeled secondary antibody cascade, and then luminescence is emitted by a substrate chemiluminescent reagent to detect the expression of exosome markers.

[0030] In some examples of quality testing methods, the direct inoculation method is used for sterility testing.

[0031] In some examples of quality testing methods, the Limulus Amebocyte Lysate (LAL) assay or gel electrophoresis is used for endotoxin detection; the endotoxin content should be less than 10 EU / mL; preferably, the endotoxin content should be less than 2 EU / mL.

[0032] In some examples of quality control methods, mass spectrometry has been used to identify protein polymers containing the following two proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0033] Preferably, it further includes at least one of the following proteins: sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens; sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens; sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens; sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens; sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens; sp|P60709|ACTB_HUMAN Actin,cytoplasmic 1 OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0034] The beneficial effects of this invention are:

[0035] The quality detection methods of some examples of this invention can effectively separate components in protein polymer samples by optimizing HPLC conditions, thereby achieving better detection results.

[0036] The quality detection methods of some examples of this invention can achieve better separation of substances in samples and obtain better detection results by optimizing SDS-PAGE conditions.

[0037] The quality detection methods of some examples of the present invention can conveniently determine the expression of exosomes in a sample by selecting specific exosome surface markers. In particular, by optimizing the detection conditions, a better detection limit is obtained, and more accurate detection results can be obtained. Attached Figure Description

[0038] Figure 1 shows the reversed-phase HPLC chromatograms of two batches of protein polymer samples.

[0039] Figure 2 shows the gradient screening results.

[0040] Figure 3 shows the SDS-PAGE detection results of the protein polymer sample.

[0041] Figure 4 shows the protein concentration detected by BCA in the protein polymer sample.

[0042] Figure 5 shows the sterility test results of the protein polymer sample. Detailed Implementation

[0043] The technical solution of the present invention will be further explained below with reference to experiments.

[0044] The manufacturing processes of the protein polymer test samples used in the following experiments include:

[0045] Culture mesenchymal stem cells and create a stress environment using ultraviolet irradiation;

[0046] Mesenchymal stem cells were lysed and purified using size exclusion chromatography to obtain protein polymers. Under size exclusion chromatography conditions with a flow rate of 0.2 mL / min, the elution volumes of the first fraction peak were 12 mL to 13.2 mL, the second fraction peak peak was 15.2 mL to 17 mL, the third fraction peak peak was 17 mL to 20 mL, the fourth fraction peak peak was 29 mL to 31 mL, and the fifth fraction peak peak was 31 mL to 34 mL.

[0047] Rapid purity analysis of protein polymer samples was performed using reversed-phase chromatography.

[0048] Based on the difference in hydrophobicity, reversed-phase chromatography can be used to separate the stimulated protein polymers obtained in this invention. Under initial conditions, the concentration of organic components in the mobile phase is low, and the complex protein (protein polymer) exhibits strong hydrophobic interaction with the stationary phase, resulting in almost complete adsorption. When the concentration of organic components in the mobile phase reaches a specific level, the complex protein (protein polymer) is completely eluted from the stationary phase and no longer interacts with it. Therefore, even minute changes in the organic components of the mobile phase can significantly affect the reversed-phase retention behavior of the complex protein (protein polymer).

[0049] Main instruments and equipment

[0050] Sample processing and testing conditions:

[0051] The collected sample stock solution was loaded into the container at a volume of 25 μL. The detection conditions were: column temperature 30℃, flow rate 0.8 mL / min, and wavelength 280 nm. Mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.1% TFA acetonitrile solution.

[0052] Washing conditions:

[0053] The detection results are shown in Figure 1. As can be seen from the figure, the separation effect is good, with no overlap between peaks, and the peak shapes of the HPLC curves for the two different samples are consistent. After separation by reversed-phase HPLC, the elution times of the samples range from 2 min to 20 min. The elution times of the main sample component group 1 are from 12 min to 20 min, and the elution time of characteristic peak 1 is approximately 15 min. The elution times of sample component group 2 are from 2 min to 5 min, and the elution time of characteristic peak 2 is approximately 3 min.

[0054] Protein polymer purity was determined by HPLC and subjected to gradient screening.

[0055] In some embodiments, higher injection volumes are required for more detailed purity analysis, thus the detection gradients of the HPLC method were screened.

[0056] Sample information: Protein polymer sample to be tested.

[0057] 1. Solution preparation

[0058] Mobile phase A (0.1% TFA aqueous solution): Take 1000 mL of ultrapure water, add 1 mL of trifluoroacetic acid, mix well and sonicate to obtain the mobile phase A.

[0059] Mobile phase B (71.4% acetonitrile solution of 0.075% TFA): Take 286 mL of ultrapure water, add 714 mL of acetonitrile and 0.75 mL of trifluoroacetic acid, mix well and sonicate to obtain the mobile phase B.

[0060] Protein polymer test sample: Weigh the test sample, dilute with PBS pH 7.2 buffer, mix well, and prepare a concentration of 1 mg / mL.

[0061] 2. Chromatographic conditions

[0062] Chromatographic conditions: For ease of proportion, the chromatographic column used in chromatographic conditions 1 to 6 is an XBridge Protein BEH C4 column. 3.5μm, 4.6mm*150mm, column temperature 40℃, mobile phase A: 0.1% TFA aqueous solution; mobile phase B: 0.075% TFA in 71.4% acetonitrile solution, detector emission wavelength is 220nm.

[0063] Chromatographic conditions 1

[0064] Chromatographic conditions 2

[0065] Chromatographic conditions 3

[0066] Chromatographic conditions 4

[0067] Chromatographic conditions 5

[0068] Chromatographic conditions 6

[0069] 3. Analysis Results

[0070] The detection was performed using chromatographic conditions 1 to 6, and the results are shown in Figure 2 and Tables 1 and 2.

[0071] Table 1 Purity Statistics of Six Gradient Screening Results

[0072] Table 2. Peak Area Statistics of Six Gradient Screening Results

[0073] As can be seen from Figure 2, Table 1 and Table 2, the peak areas and purity of the four parts of the sample peaks in the six gradient screening results are not significantly different.

[0074] As shown in Figure 2, Table 1 and Table 2, the peak area and purity of peak 1 and peak 4 are basically the same, and the peak shape is basically the same. However, because these two parts account for a relatively low percentage of the overall purity, the RSD value is relatively large. The peak area and purity of peak 2 are basically the same. The different elution gradient times result in significant differences in peak shape. Overall, the peak shapes under chromatographic conditions 1 to 3 are acceptable, while the peak shapes under chromatographic conditions 4 to 6 are poor.

[0075] As shown in Figure 2, Table 1 and Table 2, the peak area and purity of peak 3 are basically consistent. The different elution gradient times lead to significant differences in peak shape. Overall, the peak separation of chromatographic conditions 2, 4 to 6 is poor and unstable. The peak shapes of chromatographic conditions 1 and 3 are acceptable. Moreover, the elution time of chromatographic condition 1 is longer than that of chromatographic condition 3. Therefore, chromatographic condition 3 is preferred.

[0076] The purity and apparent molecular weight of the protein polymer samples were analyzed by SDS-PAGE.

[0077] Main reagents

[0078] Solution preparation:

[0079] Electrophoresis buffer: Add one packet of denatured protein gel pre-prepared buffer to 1.5L of ultrapure water, dissolve thoroughly, and then bring the volume up to 2L.

[0080] Staining solution: Pour 8 mL of Coomassie Brilliant Blue rapid staining solution into a 1 L volumetric flask, rinse the 8 mL flask several times with ultrapure water, and finally bring the volume to 1 L. Destaining solution: 100 mL methanol, 100 mL glacial acetic acid, and bring the volume to 1 L with ultrapure water.

[0081] Sample testing and result processing

[0082] Sample preparation and loading: Mix 10 μL of protein polymer sample with 2 μL of 5x SDS-PAGE protein loading buffer, incubate at 100℃ for 3 min, then centrifuge rapidly for 30 s. Add the prepared sample and standard protein marker to the loading wells.

[0083] Electrophoresis: Cover the electrophoresis tank and connect the electrophoresis apparatus. Adjust the electrophoresis voltage to 150V and electrophoresis for about 40 minutes. Stop electrophoresis when the front edge of the loading buffer is 1cm away from the bottom edge of the gel frame and turn off the power.

[0084] Gel plate removal: After electrophoresis, use a tool to open the gel plate, remove the gel, and place the gel in 50 mL of Coomassie Brilliant Blue rapid staining solution.

[0085] Staining and destaining: Place the staining solution in a microwave oven and heat on high for 1 minute. Remove the staining box and place it on a horizontal shaker. Shake at medium speed for 3 to 5 minutes, then discard the staining solution. Add 50 mL of fresh staining solution and heat on high for 1 minute, then heat on low for 3 to 8 minutes. Discard the staining solution and destain with destaining solution until the residual faint background is completely removed.

[0086] Photo analysis: Place the decolorized gel on a white plate, take a picture, and use the protein marker to provide molecular weight information for protein identification.

[0087] The experimental results are shown in Figure 3. As can be seen from Figure 3, the sample bands are mainly distributed in the range of 11KD to 100KD. Among them, the molecular weight decreases from large to small. The first band is located between 75KD and 100KD, and the second band is located between 63KD and 75KD.

[0088] Detection of exosome surface markers

[0089] By performing Western blotting on exosomes, the expression levels of exosome positive markers TSG101 / CD9 / HSP70 and negative marker Calnexin were obtained, thereby determining the relationship between the protein polymer sample and exosomes.

[0090] Antibodies specifically bind to protein polymer samples processed by gel electrophoresis, and the detection signal is amplified by an HRP-labeled secondary antibody cascade. The sample is then luminescent using a substrate chemiluminescent reagent (such as ECL) to detect the protein components expressing the specific target gene separated by electrophoresis. Information on the expression of specific proteins is obtained by analyzing the position and depth of the bands. TSG101 / CD9 / HSP70 are positive protein markers for exosomes, and Calnexin is a negative protein marker for exosomes.

[0091] The experimental results are shown in Table 3.

[0092] Table 3. Detection results of exosome surface markers in different samples

[0093] The positive control, sample AA659-017, and sample AA659-018 showed no expression of the positive protein marker CD9; the positive control showed expression of the positive protein marker TSG101 / HSP70, while the positive samples AA659-017 and AA659-018 showed no expression of the positive protein marker TSG101 / HSP70; the negative protein marker Calnexin was not expressed.

[0094] Particle size distribution and particle concentration of protein polymer samples were analyzed by nanoflow cytometry.

[0095] Nanoflow cytometers can perform label-free, individual analysis of protein polymers at the single-particle level. Using silica nanoparticles as a standard for particle size measurement, a standard working curve can be established using a mixture of silica nanoparticles with known particle sizes (characterized by TEM) to convert the scattered light intensity of protein polymers into particle size under the same sampling conditions.

[0096] Operating Procedures: First, dilute the concentration / particle size distribution standards to an appropriate factor using ultrapure water. Perform quality control of the nanoflow cytometer according to the operating procedures. After adjusting the nanoflow cytometer to its optimal detection state (where the signals of both the scattering and fluorescence channels are at their strongest and most uniform), calibrate the instrument's concentration testing state using concentration standards and the particle size distribution testing state using particle size distribution standards. Clean the sample inlet capillary with ultrapure water and cleaning solution sequentially according to the instrument's operating instructions. Use a self-made HES diluent to detect the number of HES particles in the Blabelled Exo sample measurement mode of the nanoflow cytometer as a blank control. HES is also used as a diluent for subsequent samples. Protein polymer samples are pre-diluted with HES to an appropriate factor. Data are collected in the nanoflow cytometer to determine the particle concentration and particle size distribution of the samples. Each sample is tested twice.

[0097] The experimental results are shown in Table 4.

[0098] Table 4. Particle size / size detection results for different protein polymer samples.

[0099] Endotoxin analysis of protein polymer samples was performed using the horseshoe crab reagent method.

[0100] Main reagents

[0101] Preparation of bacterial endotoxin standard solution

[0102] Take one vial of bacterial endotoxin standard (10 EU / vial), add 1 mL of water for bacterial endotoxin testing, reconstitute, and then use a pipette tip to thoroughly dissolve any residue on the tube wall. Transfer the solution to a test tube and vortex thoroughly to obtain a 10 EU / mL bacterial endotoxin standard solution. Pipette 200 μL of the 10 EU / mL standard solution into 1.8 mL of water for testing to obtain a 1 EU / mL bacterial endotoxin standard solution; pipette 500 μL of the 1 EU / mL standard solution into 1.5 mL of water for testing to obtain a 0.25 EU / mL bacterial endotoxin standard solution (positive control).

[0103] Sample dilution

[0104] Samples to be tested: high-purity samples (labeled SEC19.5) and samples diluted 5-fold with cell culture medium and filtered through a 0.22 μm membrane (labeled 5x SEC19.5).

[0105] Sample dilution: Since the remaining volume of the SEC19.5 sample was less than 100 μL, 50 μL of the sample was added to 200 μL of test water to obtain a 1:4 test solution. Then, 125 μL of the solution from the previous step was successively added to 125 μL of test water to obtain dilutions of 1:8, 1:16, and 1:32. This test was not repeated.

[0106] Take 100 μL of the 5xSEC19.5 sample and add it to 400 μL of test water to obtain a 1:4 test solution. Then, take 250 μL of the solution from the previous step and add it to 250 μL of test water to obtain dilution solutions of 1:8, 1:16, and 1:32 respectively.

[0107] Detection and Result Processing

[0108] Take 16 Limulus amebocyte lysate (LAL) reagents, flick the powder to the bottom of each tube, and add 100 μL of test water to each tube to dissolve it. Add 100 μL of negative control (bacterial endotoxin test water), positive control solution (0.25 EU / ml), and the test solution at each dilution ratio to the dissolved LAL reagents, respectively. Perform duplicates for each sample. Seal the tubes and gently shake to mix. Place each reaction tube vertically in a 37°C water bath and incubate for 60 ± 2 min, avoiding vibration during incubation.

[0109] Gently remove the test tube from the thermostat and slowly invert it 180°. If the contents of the tube are a firm gel that does not deform or slip off the tube wall, it is positive and recorded as (+); if it does not form a gel or forms a gel but cannot remain intact and slip off the tube wall, it is negative and recorded as (-). The test is valid only if the negative control tube is negative and the positive control tube and the test sample positive control tube are positive; otherwise, it is invalid.

[0110] The experimental results are shown in Table 5.

[0111] Table 5. Endotoxin detection results of protein polymer samples.

[0112] Protein content analysis of protein polymer samples was performed using the BCA method.

[0113] Main reagents and kits

[0114] Solution preparation

[0115] Preparation of BCA working solution

[0116] Prepare the required amount by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (A:B = 50:1) and mixing thoroughly.

[0117] Preparation of BSA Standards

[0118] Prepare the BSA standard system according to the table below (microplate detection, linear range 20 μg / mL~2000 μg / mL).

[0119] Sample testing and result processing

[0120] Sample determination: Add 25 μL of standard and the sample to be tested to a microplate. Add 200 μL of BCA working solution to each well and shake for 30 seconds to mix thoroughly. Cover the microplate and incubate at 37°C for 30 minutes. After the microplate cools to room temperature, measure the absorbance at 562 nm using a microplate reader.

[0121] Results processing: Based on the absorbance of the BSA standard (subtracting the OD value of the blank well in the standard to obtain the final reading), a standard curve was plotted (X-axis represents protein concentration in μg / mL, Y-axis represents the final OD562 nm). The protein concentration of the sample was calculated based on the standard curve and the sample dilution factor.

[0122] The results of the BCA protein concentration assay are shown in Figure 4.

[0123] Aseptic detection and analysis of protein polymer samples

[0124] Culture medium preparation: Prepare a certain amount of culture medium according to the instructions for thioglycolate fluid medium and tryptic soy peptone liquid medium. After dissolving, adjust the pH of the two media to 7.1 and 7.3 respectively using a pH meter, and sterilize at 121℃ for 15 min. After sterilization, place the culture medium in a biosafety cabinet for 2 days to observe for contamination. The culture medium that tests sterile is used for sterility testing experiments.

[0125] Sample Culture: Inoculate 100 μL to 1 mL of thioglycolate fluid medium (FTM) or 100 μL to 1 mL of tryptic soy liquid medium (TSB) with the purified sample solution diluted 5-fold into the cell culture medium. The ratio of thioglycolate fluid medium to tryptic soy liquid medium inoculated is 2:1, for a total of 6 tubes. Divide the thioglycolate fluid medium into two groups of 2 tubes each and incubate at 23℃ and 33℃ respectively. Incubate the tryptic soy liquid medium at 23℃. Incubate for at least 14 days. Negative Control: Simultaneously, take 2 tubes from each treatment as negative controls and co-incubate them at the corresponding temperature. Positive Control: Simultaneously, take 2 tubes from each treatment as positive controls, add no more than 100 CFU of Staphylococcus aureus to the tubes, and incubate at the corresponding temperature. The positive controls should grow well within 72 hours.

[0126] Result interpretation: No turbidity was observed in the negative control, while turbidity was observed in the positive control, indicating that the test samples should show sterile growth. Due to the small volume of the sample, 5 mL of the corresponding culture medium was added to each tube during the photographing process.

[0127] The results of the sterility test are shown in Table 6 and Figure 5.

[0128] Table 6. Sterility test results of protein polymer samples.

[0129] Note: -: no sterile growth, +: bacterial growth, N / A: not applicable

[0130] During the incubation period, all negative controls showed no bacterial growth, and all inoculated positive controls showed no turbidity within 72 hours, indicating the experiment was valid. The purified samples did not show any turbidity during the observation period.

[0131] Mass spectrometry analysis was performed on the sample. Based on the mass spectrometry data, known proteins were matched, and the protein polymer was confirmed to contain the following two proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0132] Furthermore, the protein polymer of this application includes at least one of the following proteins: sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens; sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens; sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens; sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens; sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens; sp|P60709|ACTB_HUMAN Actin,cytoplasmic 1OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0133] Serum albumin protein content is at least 38% of the total protein polymer content, and Serotransferrin protein content is at least 2% of the total protein polymer content.

[0134] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A method for detecting the quality of a protein polymer, characterized by, The reverse phase HPLC detection and at least one of the following methods: SDS-PAGE analysis Mass spectrometry detection Particle size detection Exosome surface marker detection Sterile detection Endotoxin detection The conditions for the reverse phase HPLC detection are as follows: mobile phase A is 0.1% TFA in water, mobile phase B is 0.1% TFA in acetonitrile, and the gradient elution conditions are as follows: The conditions of the reverse phase HPLC detection are as follows: The mobile phase A is 0.1% TFA in water, the mobile phase B is 0.075% TFA in 71.4% acetonitrile, and the chromatographic conditions are as follows: or chromatographic conditions are:

2. The quality detection method according to claim 1, characterized in that, The chromatographic column used in the reverse phase HPLC detection is a 300C4-T 4.6x150, 5μm chromatographic column produced by Nanomicro Corporation, preferably, the column temperature is 30℃, the flow rate is 0.8mL / min, the detection wavelength is 280nm, and the sample volume is 25μL; or The chromatographic column used for the reverse phase HPLC detection was the column XBridge Protein BEH C4, 3.5μm, 4.6mm*150mm, detection wavelength 220nm, flow rate 1.0mL / min.

3. The quality detection method according to claim 2, characterized in that, The peak time of sample separation after reverse phase HPLC is between 2min and 20min, the peak time of main sample component group 1 is between 12min and 20min, the peak time of characteristic peak 1 is about 15min, the peak time of sample component group 2 is between 2min and 5min, and the peak time of characteristic peak 2 is about 3min.

4. The quality detection method according to claim 1, wherein in the SDS-PAGE analysis, 4%-20% precast gel is used for sample separation detection, and the sample bands are mainly distributed in 11KD-100KD, wherein the first band is located between 75KD and 100KD, and the second band is located between 63KD and 75KD.

5. The quality detection method of claim 1, wherein, In the particle size detection, the particle size of the particles in the protein polymer sample should be between 30nm and 150nm, preferably between 50nm and 80nm.

6. The quality detection method of claim 1, wherein The exosome surface marker detection is determined by detecting the expression of exosome positive markers TSG101 / CD9 / HSP70 and negative markers Calnexin, and there should be no expression of exosome markers; preferably, the method used for exosome surface marker detection is Western Blot; Further, the operation of exosome surface marker detection includes: Using antibodies to specifically bind to the protein polymer sample treated by gel electrophoresis, detecting the signal through labeled secondary antibody cascade amplification, and detecting the expression of no exosome markers through substrate chemiluminescence reagent luminescence.

7. The quality detection method of claim 1, wherein, The sterile detection is performed using the direct inoculation method.

8. The quality detection method of claim 1, wherein, The endotoxin detection is performed using the Limulus reagent method or the gel method; the endotoxin content should be less than 10EU / mL; preferably, the endotoxin content should be less than 2EU / mL.

9. The quality detection method of claim 1, wherein, Mass spectrometry detection determines that the protein polymer contains the following 2 proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens; Preferably, further comprising at least one of the following proteins: sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens; sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens; sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens; sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens; sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens; sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

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