Method for detecting and / or purifying protein polymer and application thereof

Abstract

The invention relates to a method for detecting and / or purifying a protein polymer and application thereof. The method comprises a step of detecting the protein polymer based on HPLC-SEC. The chromatographic conditions are as follows: a chromatographic column is a molecular size exclusion high performance liquid chromatographic column; and a PB buffer solution with the concentration of 50-200mM is used as a mobile phase. The method disclosed by the invention is good in separation degree, simple to operate, short in peak appearance time and short in detection time, and has good method reproducibility and stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, in particular to a method for detecting and / or purifying protein polymer and application thereof. BACKGROUND

[0002] Size Exclusion Chromatography (SEC) is based on the sieving effect of porous filler, and separates molecules according to the hydrodynamic volume, which is the "gold standard" for protein analysis. The macromolecules in the sample that exceed the exclusion limit cannot enter the gel pore and are excluded, and can only pass through the gap between the porous gel particles, and are eluted from the column first; the molecules smaller than the exclusion limit can enter the pores in the gel, and are eluted from the column in the order of size; the solvent molecules that dissolve the sample have the smallest molecular weight, and flow out of the column last, thereby realizing the separation of samples with different molecular sizes. SEC is a commonly used method for analyzing proteins, and is particularly suitable for detecting biological products such as monoclonal antibodies (mAb) and antibody drug conjugates (ADC).

[0003] The present inventors found in previous studies that mesenchymal stem cells cultured under certain pressure conditions can produce a protein polymer with nerve repair activity, but the previously disclosed method for analyzing the protein polymer has the disadvantages of low purity and poor separation degree.

[0004] Therefore, there is a need for a method for detecting and / or purifying protein polymer with high sensitivity, strong specificity, good repeatability, stable method and high analysis efficiency. SUMMARY

[0005] Problems to be solved by the invention

[0006] To overcome the shortcomings of the prior art, the present application provides a method for detecting and / or purifying protein polymer with high sensitivity, strong specificity, good repeatability and high separation degree.

[0007] Solution to the problem

[0008] The first object of the present application is to provide a method for detecting and / or purifying protein polymer, which comprises a detection step of protein polymer based on HPLC-SEC, and the HPLC-SEC chromatographic conditions are as follows:

[0009] Chromatographic column: molecular size exclusion high performance liquid chromatographic column;

[0010] Mobile phase: 50mM-200mM PB buffer solution.

[0011] Preferably, the molecular size exclusion high performance liquid chromatography column is selected from Agilent AdvanceBio SEC or Waters XBridge Protein BEH SEC column, preferably Agilent AdvanceBio SEC column.

[0012] Preferably, the pH of the mobile phase is 6.5-7.5.

[0013] Preferably, the flow rate of the mobile phase is 0.2 mL / min-1 mL / min.

[0014] Preferably, the mobile phase is isocratic elution.

[0015] Preferably, the column temperature of the chromatography column is 20℃-50℃.

[0016] Preferably, the detection wavelength is 250 nm-320 nm, preferably 280 nm.

[0017] Preferably, the injection amount is 50 μg-500 μg, preferably 50 μg-150 μg.

[0018] Preferably, the protein polymer is produced by stress-induced expression and / or secretion of in vitro cultured stem cells.

[0019] Preferably, the stress condition comprises at least one of the following: ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, low oxygen, high oxygen, high pH value, low pH value, ultrasound, terahertz electromagnetic wave, X-ray, microwave, alpha ray, beta ray, gamma ray, ion beam, high CO2, low CO2.

[0020] Preferably, the stress condition is ultraviolet irradiation.

[0021] Preferably, the ultraviolet irradiation intensity is controlled to be 10 μW / cm 2 -100 μW / cm 2 .

[0022] Preferably, the ultraviolet irradiation wavelength is controlled to be 290 nm-340 nm.

[0023] Preferably, the ultraviolet irradiation time is controlled to be 1 h-30 h.

[0024] Preferably, the stem cells are selected from at least one of embryonic stem cells, mesenchymal stem cells, in vitro induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, MUSE cells, preferably one or more of umbilical cord-derived human mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, human adipose tissue-derived mesenchymal stem cells, human skin-derived mesenchymal stem cells.

[0025] A second object of the present application is to provide an application of the method according to any one of the above in protein polymer quality detection and / or separation and purification.

[0026] Effects of the application

[0027] The present application realizes effective separation of each component in the protein polymer by optimizing the HPLC-SEC chromatographic conditions, and obtains a target product with higher purity. The detection or purification method provided by the present application has the advantages of simple steps, rapid reaction, good repeatability, etc., and all common instruments and reagents are used, with low cost. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figures a and b are peak charts of two parallel experiments using chromatographic column 1 for the separation results of different chromatographic columns in Example 2;

[0029] Figure 2 Figures are comparative charts of sample injection amounts of 50 μg to 500 μg in Example 3;

[0030] Figure 3 Figures are linear charts of main peak and secondary peak concentrations and peak areas for sample injection amounts of 50 μg to 500 μg in Example 3;

[0031] Figure 4 Figures are separation result charts of mobile phase 1 in Example 4;

[0032] Figure 5 Figures are separation result charts of mobile phase 2 in Example 4;

[0033] Figure 6 Figures are comparative charts of analysis results of different flow rates on samples in Example 5;

[0034] Figure 7 Figures are Figure 6 magnified view of Figure 5;

[0035] Figure 8 Figures are comparative charts of analysis results of different column temperatures on samples in Example 6;

[0036] Figure 9UV 280nm detection result pattern in Example 7;

[0037] Figure 10 FLD(Ex:280nm / Em:350nm) detection result pattern in Example 7. DETAILED DESCRIPTION

[0038] In order to make the technical solutions and beneficial effects of the present application more obvious and easy to understand, the following will be described in detail by way of listing specific examples. The drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meaning as the technical and scientific terms in the technical field to which the present application belongs.

[0039] The present inventors have developed, for the first time, a method for detecting or purifying protein polymers. In the size exclusion-high performance liquid chromatography detection system and method of the present application, a specific mobile phase composition and optimized chromatographic conditions (such as selection of column type, column temperature, sample size, etc.) are combined to achieve effective separation of the components of protein polymers produced by MCs cells under stress. On this basis, the present application is completed.

[0040] The first object of the present application is to provide a method for detecting and / or purifying protein polymers, which comprises a detection step of protein polymers based on HPLC-SEC, and the HPLC-SEC chromatographic conditions are as follows:

[0041] Chromatographic column: molecular size exclusion high performance liquid chromatographic column;

[0042] Mobile phase: PB buffer solution with a concentration of 50mM-200mM.

[0043] In a certain example, the PB concentration in the buffer solution is 120mM-180mM, for example, 120mM, 125mM, 130mM, 140mM, 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM, 180mM, etc.

[0044] In a certain example, the molecular size exclusion high performance liquid chromatographic column is selected from Agilent AdvanceBio SEC or Waters XBridge Protein BEH SEC chromatographic column, preferably Agilent AdvanceBio SEC chromatographic column.

[0045] In a preferred example, the molecular size exclusion high performance liquid chromatographic column is selected from Agilent AdvanceBio SEC 2.7μm, 7.8 x 300 mm.

[0046] In certain examples, the pH of the mobile phase is 6.5-7.5, e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, etc.

[0047] In certain examples, the pH of the mobile phase is 6.8-7.2.

[0048] In certain examples, the flow rate of the mobile phase is 0.2 mL / min-1 mL / min, e.g., 0.2 mL / min, 0.25 mL / min, 0.3 mL / min, 0.35 mL / min, 0.4 mL / min, 0.45 mL / min, 0.5 mL / min, 0.55 mL / min, 0.6 mL / min, 0.65 mL / min, 0.7 mL / min, 0.75 mL / min, 0.8 mL / min, 0.85 mL / min, 0.9 mL / min, 0.95 mL / min, 1 mL / min, etc.

[0049] In certain examples, the mobile phase is isocratic.

[0050] In certain examples, the column temperature of the chromatographic column is 20°C-50°C, e.g., 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, etc.

[0051] In certain examples, the column temperature of the chromatographic column is 20°C-40°C.

[0052] In certain examples, the detection wavelength is 250 nm-320 nm, e.g., 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, etc.

[0053] In certain examples, the detection wavelength is 280 nm.

[0054] In certain examples, the injection amount is 50 μg-500 μg, e.g., 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, etc.

[0055] In certain examples, the injection amount is 50 μg-150 μg.

[0056] In an example, the protein polymer is produced by stress-induced expression and / or secretion from in vitro cultured stem cells.

[0057] In an example, the stress condition comprises at least one of ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, low oxygen, high oxygen, high pH, low pH, ultrasound, terahertz electromagnetic wave, X-ray, microwave, alpha ray, beta ray, gamma ray, ion beam, high CO2, low CO2.

[0058] In an example, the stress condition is ultraviolet irradiation.

[0059] In an example, the ultraviolet irradiation intensity is controlled to be 10 μW / cm 2 ~ 100 μW / cm 2 , for example, 10 μW / cm 2 , 20 μW / cm 2 , 25 μW / cm 2 , 30 μW / cm 2 , 35 μW / cm 2 , 40 μW / cm 2 , 45 μW / cm 2 , 50 μW / cm 2 , 55 μW / cm 2 , 60 μW / cm 2 , 65 μW / cm 2 , 70 μW / cm 2 , 75 μW / cm 2 , 80 μW / cm 2 , 85 μW / cm 2 , 90 μW / cm 2 , 95 μW / cm 2 , 100 μW / cm 2 , etc.

[0060] In an example, the ultraviolet irradiation wavelength is controlled to be 290 nm ~ 340 nm, for example, 290 nm, 295 nm, 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, etc.

[0061] In an example, the ultraviolet irradiation is UVB irradiation.

[0062] In some examples, the time of the ultraviolet irradiation is controlled to be 1h-30h, such as 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h.

[0063] In some examples, the stem cell is selected from at least one of embryonic stem cell, mesenchymal stem cell, induced pluripotent stem cell, hematopoietic stem cell, neural stem cell, bone marrow stem cell, liver stem cell, muscle satellite cell, skin epidermal stem cell, intestinal epithelial stem cell, retinal stem cell, pancreatic stem cell, MUSE cell.

[0064] In some examples, the mesenchymal stem cell is selected from one or more of umbilical cord-derived human mesenchymal stem cell, human bone marrow-derived mesenchymal stem cell, human placenta-derived mesenchymal stem cell, human adipose tissue-derived mesenchymal stem cell, human skin-derived mesenchymal stem cell.

[0065] In some examples, the protein polymer is derived from culture supernatant, and the preparation method of the protein polymer comprises: filtering the supernatant after culture to obtain the protein polymer.

[0066] In some examples, the protein polymer is derived from intracellular, and the preparation method of the protein polymer comprises: filtering the culture medium after stress culture, collecting cells, washing with normal saline, lysing the cells with water, and then filtering to collect the protein polymer stock solution.

[0067] In preferred examples, the protein polymer comprises at least one of the following proteins:

[0068] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0069] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens;

[0070] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0071] sp|P62736|ACTA_HUMAN Actin,aortic smooth muscle OS=Homo sapiens;

[0072] sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens;

[0073] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0074] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0075] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0076] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens;

[0077] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens;

[0078] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens;

[0079] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0080] sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens;

[0081] sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens;

[0082] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens;

[0083] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0084] A second object of the present application is to provide the use of the method according to any of the above in protein polymer quality detection and / or separation and purification.

[0085] To better illustrate the embodiments of the present application, further examples are made below by examples.

[0086] In the following examples, the reagents and instruments used are shown in Tables 1-3.

[0087] Table 1 Main instruments and equipment

[0088]

[0089]

[0090] Table 2 Main reagents and consumables

[0091] Name Manufacturer Catalogue No. NaH2PO4.2H2O Shanghai test 20040718 Na2HPO4-12H2O Shanghai test 10020318 DPBS BasaIMedia B210KJ 1.5 mL microcentrifuge tube Axygen MCT-150-C

[0092] Table 3 Test sample information

[0093] Name Source Batch No. Concentration Protein polymer stock solution Example 1 20240408 5.4 mg / mL C160 control Homemade by Yuan C160-240904-WN-15 2.10 mg / mL C160 protein stock solution Homemade by Yuan C160DS-20241001 2.48 mg / mL

[0094] Example 1: Preparation of protein polymer

[0095] Recover a small crystal P8 generation HUC-MSC into a T25 culture flask, add 2.5 mL of Huagang mesenchymal stem cell serum-free culture medium to each flask, and inoculate the cells at a density of 1 x 10 5

[0096] Take the cells and irradiate them under UVB ultraviolet conditions at a wavelength of 300-316 nm for 18 h.

[0097] The ultraviolet irradiation conditions are as follows:

[0098] Ultraviolet irradiation characteristics UVB (μW / cm 2 )]]> 40.05 Peak wavelength (nm) 306.0

[0099] After irradiation, the cells are harvested to obtain the protein polymer. Specifically, the medium supernatant is taken after 18 h of irradiation and the following operations are performed: the supernatant is filtered through a 0.22 μL filter and stored at 4°C.

[0100] Then the remaining cells are washed twice with 2 mL of normal saline, 1 mL of pure water is added and blown repeatedly, the cells are blown off from the bottom of the bottle, and the cells are lysed by repeated blowing for about 6 min, then filtered through a 0.22 μm filter and stored at 4°C for standby.

[0101] The intracellular proteins and / or protein polymers in the supernatant are collected for further detection or purification analysis.

[0102] Example 2: Column screening ​

[0103] Test sample treatment: The protein polymer stock solution (5.4 mg / mL) obtained according to the method of Example 1 was diluted 20 times with PBS to 270 μg / mL, 50 μL was injected, and Agilent AdvanceBio SEC 2.7 μm, 7.8 x 300 mm (column 1), Agilent AdvanceBio SEC 2.7 μm, 4.6 x 300 mm (column 2), Waters XBridge Protein BEH SEC 7.8 x 300 mm (column 3), to investigate the separation effect of different columns.

[0104] Mobile phase preparation: Mobile phase 1 (50 mmol / L PB, 150 mmol / L NaCl): Weigh 2.5 g of sodium dihydrogen phosphate dihydrate, 30.08 g of disodium hydrogen phosphate dodecahydrate, and 17.52 g of sodium chloride, add water to 2000 mL, mix well. Filter with a 0.20 μm filter membrane to obtain the mobile phase, pH 7.0.

[0105] The column screening conditions are shown in Table 4:

[0106] Table 4

[0107]

[0108] The results are shown in Figure 1 , and Figure 1 It can be seen that the three peak elution curves corresponding to the three columns each have a main peak (peak area ratio about 93.0%) and a secondary peak (peak area ratio about 5.0%), and the Agilent AdvanceBio SEC 2.7 μm, 7.8 x 300 mm has better separation effect, and the main peak and the secondary peak detected have no overlap, and has excellent separation degree. Therefore, this column (column 1) is selected for subsequent experiments.

[0109] At the same time, two parallel experiments were performed using column 1 in this example, and the results of the two parallel experiments are shown in Figure 1 , and Figure 1 It can be seen that the peak graphs of the two experiments are basically the same, indicating that the method for detecting and / or purifying protein polymers provided by the application has good repeatability.

[0110] Example 3: Screening of injection amount

[0111] Test sample treatment: The protein polymer prepared and diluted according to the method of Example 1 (5.0 μg / μL) was respectively injected 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL to investigate whether the main peak and the secondary peak peak area and concentration were linearly related. Except for the different injection amounts, other chromatographic conditions were the same as in Example 2.

[0112] The separation results of different injection amounts are shown in Figure 2 The main peak and the secondary peak concentration and peak area linear graph are shown in Figure 3 The main peak and the secondary peak area and peak area ratio are shown in Table 5:

[0113] Table 5

[0114]

[0115]

[0116] From the above results, when the injection amount is 100 μg and above, with the increase of the injection amount, the peak number is stable (excluding less than 0.1% peak, which may be baseline fluctuation peak). In the range of 50 μg-500 μg injection amount, the peak area of the main peak and the secondary peak is linearly related to the injection amount, and the percentage content is stable, and the RSD is less than 2.0%, and the result is not affected by the injection amount. Considering the column load and the sample waste caused by large volume injection, the injection amount is preferably 100 μg.

[0117] Example 4: Mobile phase screening

[0118] The above protein polymer test sample was taken to screen the mobile phase, in addition to the mobile phase 1 in Example 1, the separation effect of the mobile phase 2 was also investigated in this example.

[0119] Mobile phase preparation: Mobile phase 2 (150 mmol / L PB): 16.85 g of sodium dihydrogen phosphate dihydrate and 68.76 g of disodium hydrogen phosphate dodecahydrate were weighed into a 2 L beaker, 1900 mL of ultrapure water was added, completely mixed, and then diluted to 2000 mL. After filtration with a 0.20 μm filter membrane, ultrasonic treatment was performed to obtain the mobile phase, pH 7.0.

[0120] The chromatographic conditions are shown in Table 6:

[0121] Table 6

[0122]

[0123] The separation spectrum of the mobile phase 1 is shown in Figure 4 The separation spectrum of the mobile phase 2 is shown in Figure 5 Figure 4 and Figure 5 ​It can be seen that the 150 mM PB (pH 7.0) mobile phase has better data in terms of separation, theoretical plate number, and tailing between the main peak and the secondary peak, so mobile phase 2 was selected as the mobile phase used in subsequent experiments.

[0124] Example 5: Flow rate screening

[0125] Sample Preparation: A 20 μL injection of the protein polymer (5.0 μg / μL) prepared according to the method of Example 1 was used to investigate the analytical effects of different flow rates (0.20 mL / min, 0.35 mL / min, 0.5 mL / min, 0.7 mL / min, or 1.0 mL / min). The mobile phase was Mobile Phase 2 (150 mM PB, pH 7.0). Other chromatographic conditions were the same as in Example 4.

[0126] The separation spectrum is shown in Figure 6, and the enlarged diagram is shown in Figure 6. Figure 7 The main peak analysis results of the samples at different flow rates are shown in Table 7.

[0127] Table 7

[0128]

[0129] As can be seen from the table above, when the flow rate is 0.35 mL / min, the number of detected peaks is the largest, and the peak height and separation of the main peak are the best. This flow rate is the recommended flow rate for the chromatographic column, and 0.35 mL / min is determined as the optimal flow rate for this method.

[0130] Example 6: Column temperature selection

[0131] Sample treatment: 20 μL of the diluted protein polymer (5.0 μg / μL) prepared by the method of Example 1 was injected. In addition to the column temperature in Example 4, this example also investigated the analytical effects of different column temperatures (20°C, 40°C, 50°C, 60°C, or 70°C) on the sample.

[0132] Separation pattern Figure 8 As shown in Table 8:

[0133] Table 8

[0134]

[0135] From Table 8 and Figure 8 As can be seen, as the column temperature increases, the main peak resolution gradually decreases. The main peak height initially increases and then decreases, reaching its highest point at 50°C. The resolution continues to decrease, from 2.63 at 20°C to 1.78 at 70°C. The peak area remains relatively stable. When the column temperature is above 60°C, the number of peaks decreases to 5. Considering the main peak height, main peak resolution, main peak area, and peak number, 30°C is the column temperature selected for the analysis of protein aggregate components.

[0136] Example 7: Detection wavelength screening

[0137] Sample treatment: 20 μL of protein polymer prepared by the method of Example 1 (5.0 μg / μL) was injected, except for the detection wavelength in Example 4, this example also investigated the effect of other detection conditions (FLD detection results map of Ex: 280 nm and Em: 350 nm).

[0138] The detection conditions were UV 280 nm and FLD Ex: 280 nm / Em: 350 nm, and the detection results map is shown in Figure 9 and Figure 10 .

[0139] The results show that the peak types of fluorescence detection and ultraviolet detection are similar, and there is no obvious difference in the percentage of main peaks and secondary peaks. Fluorescence can detect more peaks, but the content is low and almost none (peaks less than 0.1% may be baseline fluctuation peaks); in addition, there is no obvious difference between the ultraviolet and fluorescence detection results, so the protein characteristic absorption wavelength 280 nm (UV) is selected as the detection wavelength.

[0140] It should be understood that the above examples are exemplary and are not intended to include all possible embodiments encompassed by the claims. Various modifications and changes can also be made on the basis of the above examples without departing from the scope of the present disclosure. Similarly, any combination of the technical features of the above examples can also be made to form additional embodiments of the present application that can not have been explicitly described. Therefore, the above examples only express several embodiments of the present application, and do not limit the protection scope of the present application.

Claims

1. A method for detecting and / or purifying protein polymers, characterized in that: The method comprises a step of detecting protein polymers based on HPLC-SEC, wherein the HPLC-SEC chromatographic conditions are: Chromatographic column: size exclusion high performance liquid chromatography column; Mobile phase: PB buffer with a concentration of 50mM to 200mM.

2. The method according to claim 1, characterized in that The size exclusion high performance liquid chromatography column is selected from Agilent AdvanceBio SEC or Waters XBridge Protein BEH SEC chromatography column, preferably Agilent AdvanceBio SEC chromatography column.

3. The method according to claim 1, characterized in that The pH of the mobile phase is 6.5 to 7.5; Preferably, the flow rate of the mobile phase is 0.2 mL / min to 1 mL / min; Preferably, the mobile phase is isocratic elution.

4. The method according to claim 1, wherein The column temperature of the chromatographic column is 20°C to 50°C.

5. The method according to claim 1, wherein The detection wavelength is 250 nm to 320 nm, preferably 280 nm.

6. The method according to claim 1, characterized in that The injection volume is 50 μg to 500 μg, preferably 50 μg to 150 μg.

7. The method according to claim 1, characterized in that The protein polymer is produced by in vitro cultured stem cells through stress-induced expression and / or secretion; Preferably, the stress conditions include at least one of the following: ultraviolet irradiation, infrared irradiation, electromagnetic field, high temperature, low temperature, low oxygen, high oxygen, high pH, ​​low pH, ultrasound, terahertz electromagnetic waves, X-rays, microwaves, α rays, β rays, γ rays, ion beams, high CO2, and low CO2.

8. The method according to claim 7, characterized in that The stress condition is ultraviolet irradiation, Preferably, the UV irradiation intensity is controlled to be 10 μW / cm 2 ~100μW / cm 2 ; Preferably, the wavelength of ultraviolet irradiation is controlled to be 290nm to 340nm; Preferably, the ultraviolet irradiation time is controlled to be 1 hour to 30 hours.

9. The method according to claim 7, characterized in that The stem cells are selected from at least one of embryonic stem cells, mesenchymal stem cells, in vitro induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, liver stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, and MUSE cells, and are preferably one or more of umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and skin-derived mesenchymal stem cells.

10. Use of the method according to any one of claims 1 to 9 in the quality detection and / or separation and purification of protein polymers.

Images