Method for detecting the quality of a protein polymer

Abstract

The application belongs to the field of biological drugs, and discloses a quality detection method of protein polymer, which comprises at least one of reverse phase HPLC detection, SDS-PAGE analysis, mass spectrometry detection, particle size detection, exosome surface marker detection, sterility detection and endotoxin detection. The quality detection method of some examples of the application can effectively realize the separation of components in the protein polymer sample by optimizing the HPLC condition, achieve better detection results, and establish the quality control standard of the protein polymer.

Description

[0001] This application is a divisional application of Chinese patent application filed on July 15, 2024, with application number 2024109447974 and invention title "A method for quality testing of protein polymers". 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: A method for quality detection of protein polymers includes reversed-phase HPLC detection and at least one of the following methods: SDS-PAGE analysis Mass spectrometry detection Particle size detection Exosome surface marker detection Aseptic testing Endotoxin testing; 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:

[0008] Alternatively, the conditions for reversed-phase HPLC detection are: 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. The chromatographic conditions were as follows:

[0009] Or the chromatographic conditions are: .

[0010] In some examples of quality testing methods, the chromatographic column used for reversed-phase HPLC detection is a 300 C4-T 4.6x150, 5µm column manufactured by Nanomicro Corporation. Preferably, the column temperature is 30℃, the flow rate is 0.8 mL / min, the detection wavelength is 280 nm, and the sample loading volume is 25 µL.

[0011] In some examples of quality control methods, reversed-phase HPLC uses an XBridge Protein BEH C4 column, 300 Å, 3.5 μm, 4.6 mm * 150 mm, with a detection wavelength of 220 nm and a flow rate of 1.0 mL / min.

[0012] In some examples of quality detection methods, the elution time of the sample after separation by reversed-phase HPLC is between 2 and 20 min. The elution time of the main sample component group 1 is between 12 and 20 min, the elution time of characteristic peak 1 is about 15 min, the elution time of sample component group 2 is 2 to 5 min, and the elution time of characteristic peak 2 is about 3 min.

[0013] In some examples of quality testing methods, the chromatographic column used for reversed-phase HPLC detection is a 300 C4-T 4.6x150, 5µm column manufactured by Nanomicro Corporation. Preferably, the column temperature is 30℃, the flow rate is 0.8 mL / min, the detection wavelength is 280 nm, and the sample loading volume is 25 µL. After sample separation by reversed-phase HPLC, the peak elution time is between 2 and 20 min. The peak elution time of the main sample component group 1 is between 12 and 20 min, the peak elution time of characteristic peak 1 is about 15 min, the peak elution time of sample component group 2 is 2 to 5 min, and the peak elution time of characteristic peak 2 is about 3 min.

[0014] 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 11-100 kD, with the molecular weight decreasing from large to small. The first band is located between 75 kD and 100 kD, and the second band is located between 63 kD and 75 kD.

[0015] In some examples of quality testing methods, when measuring particle size, the particle size of the particles in the protein polymer sample should be 30-150 nm, preferably 50-80 nm.

[0016] In some quality control methods, exosome surface marker detection is determined by detecting the expression of exosome positive markers TSG101 / CD9 / HSP70 and negative marker Calnexin, and there should be no exosome marker expression; preferably, the method used for exosome surface marker detection is Western blotting.

[0017] In some examples of quality control methods, the detection of exosome surface markers includes the following procedures: 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.

[0018] In some examples of quality testing methods, the direct inoculation method is used for sterility testing.

[0019] In some examples of quality testing methods, the Limulus amebocyte lysate (LAL) reagent method 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.

[0020] 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; 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=Homosapiens; 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.

[0021] The beneficial effects of this invention are: 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.

[0022] 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.

[0023] 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

[0024] Figure 1 This is a reversed-phase HPLC chromatogram of a protein polymer sample.

[0025] Figure 2 This is the result of gradient filtering.

[0026] Figure 3 This is the SDS-PAGE detection result of the protein polymer sample.

[0027] Figure 4 These are the results of exosome surface marker detection in protein polymer samples.

[0028] Figures 5-8 It involves the analysis of particle size distribution and particle concentration of different protein polymer samples.

[0029] Figure 9 This is a BCA assay for protein concentration in protein polymer samples.

[0030] Figure 10 This is the sterility test result for the protein polymer sample. Detailed Implementation

[0031] The technical solution of the present invention will be further explained below with reference to experiments.

[0032] The manufacturing processes of the protein polymer test samples used in the following experiments include: Culture mesenchymal stem cells and create a stress environment using ultraviolet irradiation; 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 12-13.2 ml, the second fraction peak peak was 15.2-17 ml, the third fraction peak peak was 17-20 ml, the fourth fraction peak peak was 29-31 ml, and the fifth fraction peak peak was 31-34 ml.

[0033] Rapid purity analysis of protein polymer samples was performed using reversed-phase chromatography. 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 exhibits strong hydrophobic interaction with the stationary phase, resulting in almost complete adsorption. When the organic component concentration in the mobile phase reaches a specific level, the complex protein is completely eluted from the stationary phase and no longer interacts with it. Therefore, even minute changes in the organic component of the mobile phase can significantly affect the reversed-phase retention behavior of the complex protein.

[0034] Main instruments and equipment

[0035] Sample processing and testing conditions: The collected sample was loaded at its original volume, 25 µL. Detection conditions were: column temperature 30℃, flow rate 0.8 mL / min, wavelength 280 nm. Mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.1% TFA acetonitrile solution.

[0036] Washing conditions:

[0037] Test results as follows Figure 1 As shown in the figure, the separation effect is good, with no overlap between peaks, and the peak shapes of the HPLC curves of the two different samples are consistent. After separation by reversed-phase HPLC, the elution time of the samples is between 2 and 20 min. The elution time of the main sample component group 1 is between 12 and 20 min, and the elution time of characteristic peak 1 is about 15 min. The elution time of sample component group 2 is between 2 and 5 min, and the elution time of characteristic peak 2 is about 3 min.

[0038] Protein polymer purity was determined by HPLC-SEC followed by gradient screening.

[0039] In some embodiments, higher injection volumes are required for more detailed purity analysis, thus the detection gradients of the HPLC-SEC method were screened.

[0040] Sample information: Protein polymer stock solution (ALEETO stock solution).

[0041] 1. Solution preparation

[0042] 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.

[0043] 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.

[0044] ALEETO sample: Weigh the ALEETO stock solution, dilute with PBS pH 7.2 buffer, mix well, and prepare a concentration of 1 mg / mL.

[0045] 2 Chromatographic conditions

[0046] Chromatographic conditions: For ease of comparison, in chromatographic conditions 1 to 6, the chromatographic column used was an XBridge Protein BEH C4, 300 Å, 3.5 μm, 4.6 mm * 150 mm, with a column temperature of 40℃. Mobile phase A was 0.1% aqueous solution of TFA; mobile phase B was 0.075% TFA in 71.4% acetonitrile solution, and the detector was 220 nm.

[0047] Chromatographic conditions 1

[0048] Chromatographic conditions 2

[0049] Chromatographic conditions 3

[0050] Chromatographic conditions 4

[0051] Chromatographic conditions 5

[0052] Chromatographic conditions 6

[0053] 3. Analysis Results The detection was performed using chromatographic conditions 1 through 6, and the results are as follows: Figure 2 As shown in Tables 1 and 2.

[0054] Table 1. Purity Statistics of Six Gradient Screening Results

[0055] Table 2. Peak Area Statistics of Six Gradient Screening Results

[0056] from Figure 2 As shown in Tables 1 and 2, the peak areas and purity of the four parts of the sample peaks in the six gradient screening results are not significantly different.

[0057] from Figure 2As shown in Tables 1 and 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 overall. The different elution gradient times lead to 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.

[0058] from Figure 2 As shown in Tables 1 and 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 gradient screening 1 is greater than that of chromatographic condition 3. Therefore, chromatographic condition 3 is preferred.

[0059] The purity and apparent molecular weight of the protein polymer samples were analyzed by SDS-PAGE.

[0060] Main reagents

[0061] Solution preparation: Electrophoresis buffer: Add one packet of denatured protein gel pre-prepared buffer to 1.5 L of ultrapure water, dissolve thoroughly, and then bring the volume up to 2 L.

[0062] 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.

[0063] Sample testing and result processing Sample preparation and loading: Mix 10 µL of protein polymer sample with 2 µL of 5x SDS-PAGE protein loading buffer, incubate at 100°C for 3 min, then centrifuge rapidly for 30 s. Add the prepared sample and standard protein marker to the loading wells.

[0064] Electrophoresis: Cover the electrophoresis tank and connect the electrophoresis apparatus. Adjust the electrophoresis voltage to 150 V and electrophoresis for about 40 minutes. Stop electrophoresis when the leading edge of the loading buffer is 1 cm from the bottom edge of the gel frame and turn off the power.

[0065] 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.

[0066] 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 3-5 minutes and discard the staining solution. Add 50 mL of fresh staining solution and heat on high for 1 minute, then heat on low for 3-8 minutes. Discard the staining solution and destain with destaining solution until the residual faint background is completely removed.

[0067] 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.

[0068] Experimental results are as follows Figure 3 As shown, from Figure 3 It can be seen that the sample bands are mainly distributed in the range of 11 to 100 kD. Among them, the molecular weight decreases from large to small. The first band is located between 75 KD and 100 KD, and the second band is located between 63 KD and 75 KD.

[0069] Exosome surface marker detection

[0070] 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.

[0071] 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.

[0072] The experimental results are shown in Table 3 and Figure 4 As shown.

[0073] Table 3. Detection results of exosome surface markers in different samples

[0074] 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.

[0075] Particle size distribution and particle concentration of protein polymer samples were analyzed by nanoflow cytometry.

[0076] Nanoflow cytometry can perform label-free, individual analysis of extracellular vesicles 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 extracellular vesicles into particle size under the same sampling conditions.

[0077] 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, adjusting it to its optimal detection state (where the signals from both the scattering and fluorescence channels are at their strongest and most uniform). Then, calibrate the instrument's concentration testing state using concentration standards and its particle size distribution testing state using particle size distribution standards. Clean the sample inlet capillary with ultrapure water and cleaning solution sequentially, following 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. Exosome samples are pre-diluted with HES to an appropriate factor. Data acquisition is performed in the nanoflow cytometer to determine the particle concentration and particle size distribution.

[0078] The experimental results are shown in Table 4 and Figures 5-8 As shown. Among them, Figure 5 The image shows the detection result for sample AA590-001. Figure 6 This is the detection image for sample AA590-002. Figure 7 This is the detection image for sample AA590-003. Figure 8 This is the detection image for sample AA590-004.

[0079] Table 4. Particle size / particle size detection results for different protein polymer samples

[0080] Endotoxin analysis of protein polymer samples was performed using the horseshoe crab reagent method. Main reagents

[0081] Preparation of bacterial endotoxin standard solution Take one vial of bacterial endotoxin standard (10 EU / vial), add 1 mL of water for bacterial endotoxin testing, reconstitute, and 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).

[0082] Sample dilution Samples to be tested: high-purity sample (labeled DEW SEC19.5) and sample diluted 5 times with cell culture medium and filtered through a 0.22µm filter membrane (labeled 5x DEW SEC19.5).

[0083] Sample dilution: Since the remaining volume of the DEW 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.

[0084] For the 5xDEW SEC19.5 sample, take 100 µL of the 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.

[0085] Detection and Result Processing 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, making two replicates for each sample. Seal the tubes and gently mix. Place each reaction tube vertically in a 37°C water bath and incubate for 60 ± 2 min, avoiding vibration during incubation.

[0086] 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 only valid 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.

[0087] The experimental results are shown in Table 5.

[0088] Table 5. Endotoxin detection results of protein polymer samples

[0089] Protein content analysis of protein polymer samples was performed using the BCA method. Main reagents and kits

[0090] Solution preparation Preparation of BCA working solution 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.

[0091] Preparation of BSA Standards Prepare the BSA standard system according to the table below (microplate detection, linear range 20-2000 µg / mL).

[0092]

[0093] Sample testing and result processing Sample determination: Add 25 µL of standard and test sample to each well of a microplate. Add 200 µL of BCA working solution to each well and vortex for 30 s to mix thoroughly. Cover the microplate and incubate at 37°C for 30 min. After the microplate cools to room temperature, measure the absorbance at 562 nm using a microplate reader.

[0094] 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 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.

[0095] The results of the BCA protein concentration detection experiment are as follows: Figure 9 As shown.

[0096] Aseptic detection and analysis of protein polymer samples

[0097] 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.

[0098] 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 FTM to TSB inoculation is 2:1, for a total of 6 vials. Divide the FTM into two groups of 2 vials each and incubate at 23℃ and 33℃ respectively. Incubate the TSB at 23℃. Culture for at least 14 days. Negative Control: Simultaneously, take 2 vials from each treatment as negative controls and co-culture them at the corresponding temperature. Positive Control: Simultaneously, take 2 vials from each treatment as positive controls, add no more than 100 CFU of Staphylococcus aureus to each tube, and incubate at the corresponding temperature. Positive controls should show good growth within 72 h.

[0099] Result interpretation: No turbidity was observed in the negative control, while turbidity was observed in the positive control, indicating that the test sample 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.

[0100] The results of the sterility test are shown in Table 6 and Figure 10 As shown.

[0101] Table 6. Sterility test results of protein polymer samples

[0102] Note: -: no sterile growth, +: bacterial growth, N / A: not applicable 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. DEW purified samples did not show any turbidity during the observation period.

[0103] Mass spectrometry analysis was performed on the sample, and the mass spectrometry data was matched with known proteins to confirm that the protein polymer contained the following two proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens; 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=Homosapiens; 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.

[0104] 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 quality detection of protein polymers, characterized in that, Including reversed-phase HPLC detection and at least one of the following methods: SDS-PAGE analysis, mass spectrometry detection, particle size analysis, exosome surface marker detection, sterility detection, or endotoxin detection; The conditions for the reversed-phase HPLC detection are as follows: 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. The chromatographic conditions were as follows: ； Or the chromatographic conditions are: ； The chromatographic column used for the reversed-phase HPLC detection was an XBridge Protein BEH C4 column, 300 Å, 3.5 μm, 4.6 mm * 150 mm, with a detection wavelength of 220 nm. The production process of the protein polymer includes: Culture mesenchymal stem cells and create a stress environment using ultraviolet irradiation; 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 12-13.2 ml, the second fraction peak peak was 15.2-17 ml, the third fraction peak peak was 17-20 ml, the fourth fraction peak peak was 29-31 ml, and the fifth fraction peak peak was 31-34 ml. The protein polymer was diluted with PBS pH 7.2 buffer and then detected by reversed-phase HPLC.

2. The quality inspection method according to claim 1, characterized in that, During SDS-PAGE analysis, 4%~20% precast gel was used for sample separation and detection. The sample bands were mainly distributed in the range of 11KD~100KD, with the molecular weight decreasing from large to small. The first band was located between 75KD~100KD, and the second band was located between 63KD~75KD.

3. The quality inspection method according to claim 1, characterized in that, When measuring particle size, the particle size of the protein polymer sample should be between 30 nm and 150 nm.

4. The quality inspection method according to claim 3, characterized in that, When measuring particle size, the particle size of the protein polymer sample should be between 50 nm and 80 nm.

5. The quality inspection method according to claim 1, characterized in that, Exosome surface marker detection is determined by detecting the expression of exosome positive markers TSG101 / CD9 / HSP70 and negative marker Calnexin; no exosome markers should be expressed.

6. The quality inspection method according to claim 5, characterized in that, The method used for detecting exosome surface markers was Western blotting.

7. The quality inspection method according to claim 6, characterized in that, The procedure for detecting exosome surface markers includes: 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.

8. The quality inspection method according to claim 1, characterized in that, Aseptic testing was performed using the direct inoculation method.

9. The quality inspection method according to claim 1, characterized in that, Endotoxin detection should be performed using the horseshoe crab reagent method or gel electrophoresis; the endotoxin concentration should be less than 10 EU / mL.

10. The quality detection method of claim 9, wherein, The endotoxin content should be less than 2 EU / mL.

11. The quality detection method of claim 1, wherein, Mass spectrometry analysis confirmed that the protein polymer contained the following two proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens.

12. The method of claim 11, wherein, Mass spectrometry analysis revealed that the protein polymer contained 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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