Quality inspection method for protein aggregates

Abstract

The present invention belongs to the biopharmaceutical field and discloses a method for quality testing protein aggregates, comprising reverse-phase HPLC analysis and at least one of the following: SDS-PAGE analysis, mass spectrometry analysis, particle size analysis, exosome surface marker analysis, sterility testing, and endotoxin testing. Several examples of the quality testing method of the present invention can effectively achieve separation of components in protein aggregate samples, obtain better test results, and establish quality control standards for protein aggregates by optimizing HPLC conditions.

Description

Technical Field

[0001] The present disclosure claims the priority of the Chinese patent application filed with the China National Intellectual Property Administration on July 15, 2024, with the title of "Quality Inspection Method for Protein Aggregates" and application number 202410944797.4, and the entire content of the above application is incorporated into the present disclosure by reference.

[0002] <Technical Field> The present invention belongs to the field of biopharmaceuticals, and specifically relates to a quality inspection method for protein aggregates. Background Art

[0003] Mesenchymal stem cells (MSCs) have the capacity for self-renewal and multi-directional differentiation, and are widely present 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 feature a wide range of sources, no requirement for matching, low infection rate, strong differentiation potential, strong proliferation capacity, and convenient collection. MSCs can produce active factors including stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), interferon (IFN), and are involved in regulating processes such as cell growth, apoptosis, cell differentiation, antiviral activity, and immune maturation. They can be used for immunoregulation, tissue repair, and the treatment of diseases such as acute lung injury, severe pneumonia, and acute respiratory distress syndrome.

[0004] The inventors have found that by stimulating MSCs and then lysing the stimulated MSCs, protein aggregates with nerve repair activity can be obtained through separation and purification from intracellular proteins. The protein aggregates have shown remarkable effects in completed animal experiments and clinical trials, particularly exhibit significant therapeutic effects on ALS, have clear potential for development into a pharmaceutical drug, and have already entered the clinical trial stage.

[0005] At present, protein aggregates are obtained by stimulating the expression of MSCs and then separating and purifying them. This makes quality control relatively difficult, and developing methods for quality control of protein aggregates is of great importance in order to stabilize their quality. [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to overcome at least one drawback of the prior art and provide a method for quality inspection of protein aggregates. [Means for solving the problem]

[0007] The technical approach adopted by this invention is as follows: A method for inspecting the quality of protein aggregates, comprising reverse-phase HPLC testing and at least one of the following methods: SDS-PAGE analysis, Mass spectrometry, Particle size inspection, Exosome surface marker testing, Sterility testing, and Endotoxin test; The conditions for the reversed-phase HPLC analysis were as follows: Mobile phase A was a 0.1% TFA aqueous solution, mobile phase B was a 0.1% TFA acetonitrile solution, and the gradient elution conditions were as follows: [Table 1] It is said that, Alternatively, the conditions for reverse-phase HPLC testing are: Mobile phase A is an aqueous solution of 0.1% TFA, and mobile phase B is a 71.4% acetonitrile solution of 0.075% TFA. The chromatography conditions are as follows: [Table 2] It is said that, Alternatively, the chromatography conditions are: [Table 3] A quality inspection method that is considered to be the standard.

[0008] In some examples of quality inspection methods, the chromatography column used for reversed-phase HPLC testing is a NanoMicro 300 C4-T 4.6x150, 5μm column, preferably with a column temperature of 30°C, a flow rate of 0.8 mL / min, a test wavelength of 280 nm, and an injection volume of 25 μL.

[0009] In some examples of quality control methods, the chromatography column used for reverse-phase HPLC testing is an XBridge Protein BEH C4 chromatography column, 300 Å, 3.5 μm, 4.6 mm x 150 mm, with a test wavelength of 220 nm and a flow rate of 1.0 mL / min.

[0010] In some examples of quality inspection methods, the peak time after reverse-phase HPLC sample separation is between 2 min and 20 min, the peak time for major sample component group 1 is between 12 min and 20 min, the peak time for characteristic peak 1 is approximately 15 min, the peak time for sample component group 2 is between 2 min and 5 min, and the peak time for characteristic peak 2 is approximately 3 min.

[0011] In some examples of quality inspection methods, the chromatography column used for reversed-phase HPLC inspection is a NanoMicro 300 C4-T 4.6x150, 5μm column, preferably with a column temperature of 30°C, a flow rate of 0.8 mL / min, an inspection wavelength of 280 nm, an injection volume of 25 μL, a peak time after reversed-phase HPLC sample separation between 2 min and 20 min, a peak time of 12 min to 20 min for major sample component group 1, a peak time of approximately 15 min for characteristic peak 1, a peak time of 2 min to 5 min for sample component group 2, and a peak time of approximately 3 min for characteristic peak 2.

[0012] In some examples of quality inspection methods, during SDS-PAGE analysis, sample separation and inspection are performed using a 4% to 20% precast gel, and the bands of the sample are mainly distributed between 11 KD and 100 KD. Among them, in descending order of molecular weight, the first band is located between 75 KD and 100 KD, and the second band is located between 63 KD and 75 KD.

[0013] In some examples of quality inspection methods, during particle size inspection, the particle size of particles in the protein aggregate sample should be between 30 nm and 150 nm.

[0014] In some examples of quality inspection methods, during particle size inspection, the particle size of particles in the protein aggregate sample is between 40 nm and 90 nm.

[0015] In some examples of quality inspection methods, during particle size inspection, the particle size of particles in the protein aggregate sample is between 50 nm and 80 nm. In some examples of quality inspection methods, inspection of exosome surface markers is identified by detecting the expression status of exosome positive markers TSG101 / CD9 / HSP70 and negative marker Calnexin, and there should be no expression of exosome markers; preferably, the method used for inspection of exosome surface markers is Western blotting.

[0016] In some examples of quality inspection methods, the operation for inspection of exosome surface markers is: it comprises performing specific binding on the gel-electrophoresed protein aggregate sample with an antibody, cascaded amplification of an inspection signal by a labeled secondary antibody, and detecting the absence of expression of exosome markers based on the luminescence generated by the substrate chemiluminescent reagent.

[0017] In some examples of quality inspection methods, sterility testing is performed using the direct inoculation method.

[0018] In some examples of quality inspection methods, the limulus reagent method (LAL) or gel method is used to conduct endotoxin detection, the endotoxin content should be less than 10EU / mL, and preferably, the endotoxin content should be less than 2EU / mL.

[0019] In some examples of quality inspection methods, the mass spectrometry inspection shows that protein aggregates comprise the following two proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens, and sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens; preferably, further 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, and sp|P01024|C3 HUMAN Complement C3 OS = Identifies that it contains at least one protein from Homo sapiens.

[0020] The beneficial effects of this invention are as follows: Some examples of quality inspection methods of the present invention can effectively separate components in protein aggregate samples and achieve better inspection results by optimizing HPLC conditions. Some examples of quality inspection methods of the present invention can achieve good separation of substances in a sample and obtain better inspection results by optimizing SDS-PAGE conditions. Some examples of quality inspection methods of the present invention can easily identify the expression status of exosomes in a sample by selecting specific exosome surface markers, and in particular, by optimizing the inspection conditions, better detection limits can be obtained and more accurate inspection results can be obtained. [Brief explanation of the drawing]

[0021] [Figure 1] These are reverse-phase HPLC chromatograms of two batches of protein aggregate samples. [Figure 2] This is the result of the gradient selection. [Figure 3] This is the result of SDS-PAGE testing of a protein aggregate sample. [Figure 4]This is the protein concentration obtained from BCA testing of protein aggregate samples. [Figure 5] This is the result of the sterility test on the protein aggregate sample. [Modes for carrying out the invention]

[0022] The technical proposal of this invention will be further explained below in conjunction with the experiments.

[0023] The process for preparing the test samples of protein aggregates used in the following experiment is as follows: This involves culturing mesenchymal stem cells and creating a stress environment through UV irradiation. This method includes lysing mesenchymal stem cells, separating and purifying them by size exclusion chromatography, and obtaining protein aggregates. Under size exclusion chromatography conditions with a flow rate of 0.2 mL / min, the elution volume of the first component peak was 12 mL to 13.2 mL, the elution volume of the second component peak was 15.2 mL to 17 mL, the elution volume of the third component peak was 17 mL to 20 mL, the elution volume of the fourth component peak was 29 mL to 31 mL, and the elution volume of the fifth component peak was 31 mL to 34 mL.

[0024] Rapid purity analysis of protein aggregate test samples using reverse-phase chromatography column Based on differences in hydrophobicity, the protein aggregates obtained by stimulation in this invention can be separated by reverse-phase chromatography. Under initial conditions, the concentration of organic components in the mobile phase is low, and the complex protein (protein aggregate) exhibits strong hydrophobicity with the stationary phase, resulting in almost complete adsorption to the stationary phase. When the organic components in the mobile phase reach a specific concentration, the complex protein (protein aggregate) completely elutes from the stationary phase and ceases to interact with it. Therefore, even a slight change in the organic components of the mobile phase clearly affects the reverse-phase retention behavior of the complex protein (protein aggregate).

[0025] Major equipment and devices [Table 4]

[0026] Sample processing and testing conditions The collected stock sample was injected, with an injection volume of 25 μL. The test conditions were a column temperature of 30°C, a flow rate of 0.8 mL / min, and a wavelength of 280 nm. Mobile phase A was a 0.1% TFA aqueous solution, and mobile phase B was a 0.1% TFA acetonitrile solution.

[0027] Elution conditions [Table 5]

[0028] The test results are shown in Figure 1. As can be seen from the figure, the separation effect is good, there is no overlap between the peaks, and the peak shapes of the HPLC curves of the two different samples match. The peak times after reverse-phase HPLC sample separation are between 2 min and 20 min, the peak time for major sample component group 1 is between 12 min and 20 min, the peak time for characteristic peak 1 is approximately 15 min, the peak time for sample component group 2 is between 2 min and 5 min, and the peak time for characteristic peak 2 is approximately 3 min.

[0029] HPLC-based purity testing and gradient sorting of protein aggregates In some examples, a higher sample injection volume was required to perform a more detailed purity analysis, so the HPLC test gradient was selected accordingly. Test sample information: Protein aggregate test sample.

[0030] 1 Solution preparation Mobile phase A (aqueous solution of 0.1% TFA): Prepared by taking 1000 mL of ultrapure water, adding 1 mL of trifluoroacetic acid, mixing uniformly, and then sonicating. Mobile phase B (71.4% acetonitrile solution of 0.075% TFA): 286 mL of ultrapure water was taken, 714 mL of acetonitrile and 0.75 mL of trifluoroacetic acid were added, and after homogeneous mixing, the phase was prepared by sonication. Protein aggregate test sample: The test sample was weighed, diluted with PBS pH 7.2 buffer, and mixed uniformly to prepare a concentration of 1 mg / mL.

[0031] 2. Chromatography conditions Chromatography conditions: For the sake of ratios, the chromatography column used in Chromatography conditions 1 to 6 was XBridge Protein BEH C4, 300 Å, 3.5 μm, 4.6 mm * 150 mm, column temperature 40°C. 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 emission wavelength of the analyzer was 220 nm.

[0032] Alkylometry Condition 1 [Table 6]

[0033] chromatography conditions 2 [Table 7]

[0034] chromatography condition 3 [Table 8]

[0035] Chromatography conditions 4 [Table 9]

[0036] Chromatography condition 5 [Table 10]

[0037] Chromatography conditions 6 [Table 11]

[0038] 3 Analysis results The results measured under chromatography conditions 1 to 6 are shown in Figure 2 and Tables 1 and 2.

[0039] [Table 12]

[0040] [Table 13]

[0041] As can be seen from Figure 2, Table 1, and Table 2, when comparing the results of the six gradient selections, there was not a significant difference in the peak area and purity of the four parts of the sample peak.

[0042] As can be seen from Figure 2, Table 1, and Table 2, the peak area and purity of Peak 1 and Peak 4 are almost identical, and the peak shapes are almost identical. However, because the proportion of these two parts to the overall purity is low, the RSD value is large. The overall peak area and purity of Peak 2 are almost identical, and the difference in peak shape due to the difference in elution gradient time is clear. Overall, the peak shapes for chromatography conditions 1 to 3 are all good, while the peak shapes for chromatography conditions 4 to 6 are poor.

[0043] As can be seen from Figure 2, Table 1, and Table 2, the overall peak area and purity of the peak 3 portion are almost identical, and the difference in peak shape due to the difference in elution gradient time is clear. Overall, the separation of peak shapes for chromatography conditions 2 and 4 to 6 is poor and unstable, while the peak shapes for chromatography conditions 1 and 3 are both good, and the elution time for chromatography condition 1 is longer than that for chromatography condition 3, therefore chromatography condition 3 is preferred.

[0044] Purity and apparent molecular weight analysis of protein aggregate test samples using SDS-PAGE method Main reagents [Table 14]

[0045] Solution preparation Electrophoresis buffer: One packet of denatured protein gel precast buffer was added to 1.5 L of ultrapure water, thoroughly dissolved, and then the volume was adjusted to 2 L. Staining solution: 8 mL of Coomassie Brilliant Blue rapid staining solution was placed in a 1 L volumetric flask, and the 8 mL flask was washed several times with ultrapure water until the final volume was 1 L. Decolorizing solution: 100 mL methanol, 100 mL glacial acetic acid, and ultrapure water were added to a final volume of 1 L.

[0046] Sample testing and result processing Sample processing and sample addition: 10 μL of protein aggregate sample and 2 μL of 5x SDS-PAGE protein loading buffer were taken, homogeneously mixed, and then treated in a 100°C metal bath for 3 minutes. After removal, the mixture was rapidly centrifuged for 30 seconds. The treated sample and standard protein markers were added to the injection wells. Electrophoresis: Close the lid of the electrophoresis tank, connect the electrophoresis apparatus, adjust the electrophoresis voltage to 150V, perform electrophoresis for approximately 40 minutes, stop the electrophoresis when the front end of the loading buffer reaches 1 cm from the bottom edge of the gel frame, and turn off the power. Gel plate removal: After electrophoresis was complete, the gel preparation plate was opened with a tool, the gel was removed, and the gel was placed in 50 mL of Coomassie Brilliant Blue rapid staining solution. Staining and decolorization: Place the staining solution in a microwave oven and heat on high heat for 1 minute. Remove the gel staining box and place it in a horizontal shaker. Shake at medium speed for 3 to 5 minutes, then discard the staining solution. Add another 50 mL of fresh staining solution and heat on high heat for 1 minute, then heat on low heat for 3 to 8 minutes. Remove the staining solution and decolorize with decolorizing solution until any remaining faint background is completely gone. Photographic analysis: The decolorized gel was placed on a white plate and photographed, providing molecular weight information for protein identification based on protein markers. As can be seen from Figure 3, the experimental results show that the sample bands are mainly distributed between 11KD and 100KD. Of these, the first band is located between 75KD and 100KD, and the second band is located between 63KD and 75KD, in order of decreasing molecular weight.

[0047] Exosome surface marker testing Western blotting was performed on exosomes to obtain the expression status of the exosome-positive markers TSG101 / CD9 / HSP70 and the negative marker Calnexin, and the relationship between the protein aggregate test sample and the exosomes was determined. Protein aggregate samples treated with gel electrophoresis using antibodies were specifically bound to the antibody, and the test signal was cascade-amplified using an HRP-labeled secondary antibody. The protein component expressing the specific target gene after electrophoretic separation was examined by the emission of a substrate chemiluminescent reagent (e.g., ECL). Information on the expression status of specific proteins was obtained by analyzing the position and concentration of the bands. TSG101 / CD9 / HSP70 are exosome-positive protein markers, and Calnexin is an exosome-negative protein marker. The experimental results are shown in Table 3.

[0048] [Table 15]

[0049] In the positive control, sample AA659-017, and sample AA659-018, the positive protein marker CD9 was not expressed. In the positive control, the positive protein marker TSG101 / HSP70 was expressed. In sample AA659-017 and sample AA659-018, the positive protein marker TSG101 / HSP70 was not expressed. The negative protein marker Calnexin was not expressed.

[0050] Particle size distribution and particle concentration analysis of protein aggregate samples using nanoflow cytometry. Nanoflow cytometers enable the individual, unlabeled analysis of protein aggregates at the single-particle level. By using silicon dioxide nanoparticles as a standard for particle size measurement and utilizing a mixture of silicon dioxide nanoparticles with known particle sizes (characterized by TEM), a standard calibration curve of scattered light intensity and particle size can be created, allowing the scattered light intensity of protein aggregates under equivalent sampling conditions to be converted to particle size. Operating procedure: First, the concentration / particle size distribution standard was diluted to an appropriate ratio with ultrapure water. Following the operating procedure of the nanoflow cytometer, quality control of the nanoflow cytometer was performed and the nanoflow cytometer was adjusted to the optimal testing state (the scattering and fluorescence channel signals were both the strongest and most uniform). Then, the concentration standard was used to calibrate the instrument's concentration measurement state, and the particle size distribution standard was used to calibrate the instrument's particle size distribution measurement state. The sample injection capillary was then sequentially washed with ultrapure water and washing solution according to the instrument's operating instructions. Using a homemade HES diluent, the number of HES particles was checked in the Labeled Exo sample measurement mode of the nanoflow cytometer and used as a blank control. The HES was also used as the dilution solvent for subsequent samples. Protein aggregate samples were pre-diluted to an appropriate ratio with HES, sample data was collected using the nanoflow cytometer, and the particle concentration and particle size distribution of the samples were measured. The test was repeated twice for each sample. The experimental results are shown in Table 4.

[0051] [Table 16]

[0052] Endotoxin analysis of protein aggregate test samples using the Limulus reagent method Main reagents [Table 17]

[0053] Preparation of bacterial endotoxin standard solutions One 10 EU / mL bacterial endotoxin standard was taken, 1 mL of sterile water was added, and after redissolution, any deposits on the tube wall were thoroughly dissolved using a pipette tip. The mixture was then transferred to a test tube and thoroughly mixed uniformly using a vortex mixer to obtain a 10 EU / mL bacterial endotoxin standard solution. 200 μL of the 10 EU / mL standard solution was drawn into 1.8 mL of sterile water to obtain a 1 EU / mL bacterial endotoxin standard solution. 500 μL of the 1 EU / mL standard solution was drawn into 1.5 mL of sterile water to obtain a 0.25 EU / mL bacterial endotoxin standard solution (positive control).

[0054] Sample Dilution Test samples: High-purity sample (labeled SEC19.5) and a sample diluted 5-fold with cell culture medium and passed through a 0.22 μm filtration membrane (labeled 5x SEC19.5). Dilution of test samples: 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 each solution were sequentially taken from the previous step and added to 125 μL of test water to obtain 1:8, 1:16, and 1:32 dilutions, respectively. This test should not be repeated. For the 5xSEC19.5 sample, 100 μL of the sample was taken and added to 400 μL of test water to obtain a 1:4 test solution. Then, 250 μL of each solution was taken from the previous step and added to 250 μL of test water to obtain dilutions of 1:8, 1:16, and 1:32, respectively.

[0055] Inspection and result processing Sixteen tubes of Limulus reagent were taken, and after dropping the powder to the bottom of each tube, 100 μL of test water was added to each tube to dissolve the reagent. To each dissolved Limulus reagent, 100 μL of negative control (bacterial endotoxin test water), positive control solution (0.25 EU / ml), and the test solution at each dilution ratio were added. Two of each sample were placed parallel to each other, the tube openings were sealed, and the tubes were gently and uniformly shaken. Each reaction tube was placed vertically in a 37°C water bath and incubated at constant temperature for 60 ± 2 minutes, avoiding vibration during incubation. The test tube was gently removed from the incubator and slowly inverted 180°. A positive result (+) was recorded if the contents of the tube were a firm gel that did not deform and did not slide off the tube wall. A negative result (-) was recorded if no gel formed, or if a gel formed but could not maintain its integrity and slid off the tube wall. The test was valid only if the negative control tube was negative, and the positive control tube and the positive control tube of the test sample were positive; otherwise, it was considered invalid. The experimental results are shown in Table 5.

[0056] [Table 18]

[0057] Protein content analysis of protein aggregate test samples using the BCA method Main reagents and kits [Table 19]

[0058] Solution preparation Preparation of BCA working solution To prepare the required amount, 50 volumes of BCA reagent A were mixed with 1 volume of BCA reagent B (A:B=50:1), and the mixture was thoroughly and uniformly combined.

[0059] Preparation of BSA standards BSA standard systems were prepared according to the table below (microplate testing, linear range 20 μg / mL to 2000 μg / mL). [Table 20]

[0060] Sample testing and result processing Sample Measurement: 25 μL of standard and 25 μL of test sample were added to a microplate. 200 μL of BCA working solution was added to each well and shaken for 30 seconds to mix thoroughly and uniformly. The microplate was covered and incubated at 37°C for 30 minutes. After the microplate cooled to room temperature, the absorbance at a wavelength of 562 nm was measured using a microplate reader.

[0061] Results Processing: A standard curve was created based on the absorbance of the BSA standard (by subtracting the OD value of the blank well, i.e., the final reading, from the standard value). The X-axis represents protein concentration in μg / mL, and the Y-axis represents the final OD of 562 nm. The protein concentration of the sample was calculated from the standard curve and the sample dilution factor. Figure 4 shows the experimental results of protein concentration obtained by BCA testing.

[0062] Sterility testing and analysis of protein aggregate test samples Preparation of culture media: The prescribed amounts of thioglycolate fluid medium and triptycase soy broth liquid medium were prepared according to the instructions for use. After dissolution, the pH of the two media was adjusted to 7.1 and 7.3, respectively, using a pH meter, and then sterilized at 121°C for 15 minutes. After sterilization, the media were placed in a biological safety cabinet and left for 2 days to observe for contamination. Media confirmed to be sterile were used for sterility testing experiments.

[0063] Sample Culture: 100 μL of purified sample solution diluted with 5-fold cell medium was inoculated into 1 mL of thioglycolate fluid medium (FTM), or 100 μL of purified sample solution of the test specimen was inoculated into 1 mL of Tripticase soy broth liquid medium (TSB). The ratio of thioglycolate fluid medium to Tripticase soy broth liquid medium inoculations was 2:1, and a total of 6 tubes were used. The thioglycolate fluid medium was divided into two groups, with 2 tubes from each group, and cultured at 23°C and 33°C, respectively. The Tripticase soy broth liquid medium was cultured at 23°C. Cultures were maintained for at least 14 days. Negative Control: Simultaneously, 2 tubes from each treatment were co-cultured as negative controls at the corresponding temperature. Positive Control: Simultaneously, 2 tubes from each treatment were used as positive controls. Staphylococcus aureus (less than 100 cfu) was added to the tubes and cultured at the corresponding temperature. The positive controls should grow well within 72 hours.

[0064] Result interpretation: Negative controls should be free of turbidity, positive controls should be turbid, and test samples should be sterile-grown. In this test, due to the small volume, 5 mL of the corresponding culture medium was added to each tube at the time of imaging. The results of the sterility testing experiment are shown in Table 6 and Figure 5.

[0065] [Table 21] Notes: -: Sterile growth, +: Bacterial growth, N / A: Not applicable

[0066] During the culture period, all negative controls grew sterile, and all inoculated positive controls showed turbidity within 72 hours, indicating the validity of the experiment. None of the purified samples showed turbidity during the observation period.

[0067] Mass spectrometry was performed on the samples, and based on the mass spectrometry data, known proteins were matched to confirm that the protein aggregates contained the following two types of proteins. sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens, and sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens. Furthermore, the protein aggregate of the present application contains 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, and sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0068] Here, the content of Serum albumin protein is at least 38% of the total content of the protein aggregates, and the content of Serotransferrin protein is at least 2% of the total content of the protein aggregates.

[0069] Although the present invention has been described in more detail above, this should not be considered a limitation on the specific implementation of the present invention. Any simple derivation or substitution that does not depart from the concept of the present invention is within the scope of protection for a general expert in the art to which the present invention pertains.

Claims

1. A method for inspecting the quality of protein aggregates, characterized by comprising reverse-phase HPLC testing and at least one of the following methods: SDS-PAGE analysis, Mass spectrometry, Particle size inspection, Exosome surface marker testing, Sterility testing, and Endotoxin test; The conditions for the reversed-phase HPLC test are as follows: Mobile phase A is a 0.1% TFA aqueous solution, mobile phase B is a 0.1% TFA acetonitrile solution, and the gradient elution conditions are as follows: Table 1 It is said that, Alternatively, the conditions for reverse-phase HPLC testing are: Mobile phase A is an aqueous solution of 0.1% TFA, and mobile phase B is a 71.4% acetonitrile solution of 0.075% TFA. The chromatography conditions are as follows: Table 2 It is said that, Alternatively, the chromatography conditions are: Table 3 A quality inspection method that is considered to be the standard.

2. The chromatography column used for reverse-phase HPLC testing is a NanoMicro 300 C4-T 4.6 x 150, 5 μm column, preferably with a column temperature of 30°C, a flow rate of 0.8 mL / min, a test wavelength of 280 nm, and an injection volume of 25 μL, or The quality inspection method according to claim 1, characterized in that the chromatography column used for reverse-phase HPLC inspection is a chromatography column XBridge Protein BEH C4, 300 Å, 3.5 μm, 4.6 mm * 150 mm, the inspection wavelength is 220 nm, and the flow rate is 1.0 mL / min.

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

4. The quality inspection method according to claim 1, characterized in that, during SDS-PAGE analysis, a sample separation test is performed using a 4% to 20% precast gel, and the sample bands are mainly distributed between 11KD and 100KD, with the first band located between 75KD and 100KD and the second band located between 63KD and 75KD in order of decreasing molecular weight.

5. The quality inspection method according to claim 1, characterized in that, during particle size inspection, the particle size of the particles in the protein aggregate sample should be between 30 nm and 150 nm, preferably between 50 nm and 80 nm.

6. Exosome surface markers are identified by examining the expression status of the exosome-positive marker TSG101 / CD9 / HSP70 and the negative marker Calnexin, and it is expected that there will be no expression of exosome markers. Preferably, the method used for testing exosome surface markers is Western blotting. Furthermore, the procedure for testing exosome surface markers is as follows: The quality inspection method according to claim 1, characterized by including the following steps: specific binding of an antibody to a protein aggregate sample subjected to gel electrophoresis; cascade amplification of the test signal by a labeled secondary antibody; and detection of the absence of exosome marker expression by emission of a substrate chemiluminescent reagent.

7. The quality inspection method according to claim 1, characterized in that a sterility test is performed using a direct inoculation method.

8. The quality inspection method according to claim 1, characterized in that an endotoxin test is performed using the Limulus reagent method or the gel method, and the endotoxin content should be less than 10 EU / mL, preferably less than 2 EU / mL.

9. Mass spectrometry analysis revealed that the protein aggregates contained the following two types of proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens, and sp | P02787 | TRFE_HUMAN Serotransferrin OS=Homo sapiens; Preferably, further, 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, and The quality inspection method according to claim 1, characterized in that it contains at least one of the proteins of sp|P01024|C3 HUMAN Complete C3 OS = Homo sapiens.

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