Mass spectrum identification method for human recombinant erythropoietin source and application

Abstract

The invention discloses a mass spectrum identification method for a human recombinant erythropoietin source, which comprises the following steps: S1, preparing a biotin-bound erythropoietin antibody A, and binding the erythropoietin antibody A with EPO (erythropoietin) in a sample to obtain a mixture 1; s2, performing co-incubation on magnetic beads coated with streptavidin and the mixture 1 to realize purification of erythropoietin; s3, carrying out reductive alkylation and trypsin digestion on the purified erythropoietin; and S4, carrying out qualitative detection on the peptide fragment of the erythropoietin subjected to enzyme digestion by adopting a mass spectrometer. According to the method, the characteristic glycopeptide containing the characteristic sialic acid in the CHO cell-derived rhEPO is detected by utilizing a mass spectrometry technology, and the method has good accuracy. Compared with a general immunological detection method, the method provided by the invention has higher detection flux, and provides a new thought and direction for rhEPO stimulant screening and rhEPO source identification in the biological medicine field.

Description

Technical Field

[0001] The invention relates to a mass spectrometry identification method for the source of human recombinant erythropoietin and its application, and relates to G01N, and specifically to a measurement and testing method. Background Art

[0002] Erythropoietin is an endogenous glycoprotein hormone in the human body. It is regulated by hypoxia-inducible factor and is mainly synthesized and secreted by the kidneys. It can stimulate the proliferation of red blood cells and promote hematopoiesis. Erythropoietin is widely used in clinical drugs, mainly for the treatment of anemic diseases such as chronic renal anemia and cancer-related anemia. rhEPO and its analogs can increase blood oxygen saturation, so they can also be used to produce stimulant drugs. The erythropoietin currently used is mostly erythropoietin cultured from mouse ovarian cells or mouse erythropoietin, but there is a certain risk of human rejection. In order to reduce rejection reactions, endogenous human erythropoietin is gradually used in clinical fields such as medicine. At present, the general detection method for erythropoietin stimulants in complex matrices such as serum / plasma is still based on gel electrophoresis combined with immunoblotting and chemiluminescence detection methods of sodium lauryl sarcosine. Since the bands of endogenous human EPO (erythropoietin) and rhEPO (recombinant human erythropoietin) are very close, the accuracy of the test results is not ideal, which can easily lead to inter-laboratory differences and affect the detection of stimulants, clinical diagnosis and medication.

[0003] Chinese invention patent CN202210601700.0 discloses a method for detecting the content of recombinant human erythropoietin, which uses high-performance liquid chromatography to detect the content of recombinant human erythropoietin. The detection method has high detection accuracy and simple operation, but it cannot distinguish between human and mouse erythropoietin. Chinese invention patent CN201610900013.3 discloses an erythropoietin stimulant kit and its detection method. The erythropoietin kit based on nucleic acid aptamer fluorescent probe has simple and fast detection operation and does not require complex sample processing and separation, but the distinction effect from mouse erythropoietin is not ideal and the detection accuracy is not high. Summary of the invention

[0004] In order to improve the accuracy of detecting the source of erythropoietin, the first aspect of the present invention provides a mass spectrometry identification method for the source of human recombinant erythropoietin, comprising the following steps:

[0005] S1 combines biotin with erythropoietin antibody to obtain erythropoietin antibody A, co-incubates the sample to be tested with erythropoietin antibody A, and the erythropoietin antibody A combines with the erythropoietin protein in the sample to obtain a mixture 1;

[0006] S2 uses streptavidin-coated magnetic beads to co-incubate with mixture 1 to purify erythropoietin;

[0007] S3 performs reductive alkylation on the purified erythropoietin and digests it with trypsin;

[0008] S4 uses a mass spectrometer to perform qualitative detection on the peptide fragments of erythropoietin after enzyme digestion.

[0009] As a preferred embodiment, the erythropoietin is selected from human endogenous erythropoietin (EPO) or recombinant human erythropoietin (rhEPO), and the recombinant human erythropoietin is selected from recombinant human erythropoietin cultured in mouse ovary cells (CHO) or recombinant human erythropoietin cultured in human embryonic kidney cells 293 (HEK293).

[0010] As a preferred embodiment, the recombinant human erythropoietin is selected from recombinant human erythropoietin cultured in mouse ovary cells (CHO) or recombinant human erythropoietin cultured in human embryonic kidney cells 293 (HEK293).

[0011] As a preferred embodiment, the biotin is biotin N-hydroxysuccinimide ester (biotin-7-NHS), the concentration of the biotin is 3-7 mg / mL, the concentration of the erythropoietin antibody is 1-3 mg / mL, and the mass ratio of the biotin to the erythropoietin antibody is (3-9):1.

[0012] As a preferred embodiment, the biotin is biotin N-hydroxysuccinimide ester, the concentration of the biotin is 5 mg / mL, the concentration of the erythropoietin antibody is 1 mg / mL, and the mass ratio of the biotin to the erythropoietin antibody is 6.6:1.

[0013] As a preferred embodiment, the conditions for the binding of biotin and erythropoietin antibody are incubation at 300-900 rpm in the dark for 1-5 hours.

[0014] As a preferred embodiment, the conditions for the binding of biotin and erythropoietin antibody are incubation at 600 rpm and room temperature in the dark for 3 hours.

[0015] As a preferred embodiment, the erythropoietin antibody A is a biotin-modified antibody purified by Sephadex G-25 gel filtration column.

[0016] As a preferred embodiment, the sample to be tested is diluted with a PBS solution, the volume ratio of the sample to be tested after dilution with the PBS solution to erythropoietin antibody A is 1:(1-3), the concentration of the erythropoietin antibody A is 1-5 μg / mL, the co-incubation temperature of the sample to be tested and the erythropoietin antibody A is 1-5°C, and the co-incubation time is 2-12h.

[0017] As a preferred embodiment, the sample to be tested is diluted with a PBS solution, the volume ratio of the sample to be tested after dilution with the PBS solution to erythropoietin antibody A is 1:1, the concentration of the erythropoietin antibody A is 4 μg / mL, the co-incubation temperature of the sample to be tested and the erythropoietin antibody A is 4°C, and the co-incubation time is 2-12h.

[0018] As a preferred embodiment, the volume ratio of the sample to be tested to the PBS solution is 1:(1-2); preferably, the volume ratio of the sample to be tested to the PBS solution is 1:1.

[0019] As a preferred embodiment, the sample to be tested is selected from one of human serum, human plasma, and human urine, and the amount of the sample to be tested is 100-500 μL.

[0020] As a preferred embodiment, the sample to be tested is serum or plasma.

[0021] As a preferred embodiment, the serum or plasma is thawed and then centrifuged, and the centrifugation temperature is 2-8°C, selected from one of 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, and 8°C.

[0022] As a preferred embodiment, the centrifugation time is 10-30 min, selected from one of 10 min, 12 min, 14 min, 15 min, 17 min, 18 min, 20 min, 22 min, 25 min, 27 min, and 30 min.

[0023] As a preferred embodiment, the centrifugal force is 8000-22000g, selected from one of 8000g, 10000g, 12000g, 14000g, 16000g, 18000g, 20000g, and 22000g.

[0024] As a preferred embodiment, the specific steps of step S2 include: first washing the streptavidin-coated magnetic beads with TWEEN 20 prepared with PBS solution, then separating the magnetic beads by magnetic attraction using a magnetic stand, then washing the streptavidin-coated magnetic beads again with PBS solution, then incubating with mixture 1, then eluting erythropoietin with an eluent, washing the streptavidin-coated magnetic beads again with TWEEN 20 prepared with PBS solution, then separating the magnetic beads by magnetic attraction using a magnetic stand, and finally washing the streptavidin-coated magnetic beads again with PBS solution.

[0025] As a preferred embodiment, the concentration of TWEEN 20 in the PBS solution is 0.1-0.3 wt %.

[0026] As a preferred embodiment, the concentration of TWEEN 20 in the PBS solution is 0.1 wt %.

[0027] As a preferred embodiment, the volume ratio of the streptavidin-coated magnetic beads to the erythropoietin antibody A is 1:(8-12); the co-incubation conditions of step S2 are incubation at 35-40°C and 800-1200 rpm for 1-3h.

[0028] As a preferred embodiment, the volume ratio of the streptavidin-coated magnetic beads to the erythropoietin antibody A is 1:10; the co-incubation condition of step S2 is incubation at 37° C. and 900 rpm for 2 h.

[0029] As a preferred embodiment, after co-incubation in step S2, erythropoietin is eluted with an eluent, and the eluent includes an aqueous citric acid solution and acetonitrile. The concentration of the aqueous citric acid solution is 0.01-0.03 mol / L, and the volume ratio of the aqueous citric acid solution to acetonitrile is (6-12):1. The elution conditions are vortexing at 500-1000 rpm for 8-12 min at 40-50°C.

[0030] As a preferred embodiment, after co-incubation in step S2, erythropoietin is eluted with an eluent, and the eluent includes an aqueous citric acid solution and acetonitrile. The concentration of the aqueous citric acid solution is 0.01 mol / L, the volume ratio of the aqueous citric acid solution to acetonitrile is 9:1, and the elution conditions are vortexing at 900 rpm for 10 min at 45°C.

[0031] As a preferred embodiment, in step S3, a reducing agent and an alkylating agent are used to carry out a reductive alkylation reaction, the concentration of the reducing agent is 5-15 mM, the concentration of the alkylating agent is 15-30 mM, the temperature of the reductive alkylation reaction is 50-60° C., and the reaction time is 40-60 min.

[0032] As a preferred embodiment, in step S3, a reducing agent and an alkylating agent are used to carry out a reducing alkylation reaction, the concentration of the reducing agent is 10 mM, the concentration of the alkylating agent is 20 mM, the temperature of the reducing alkylation reaction is 56° C., and the reaction time is 50 min.

[0033] As a preferred embodiment, the alkylating agent includes but is not limited to iodoacetamide (IAA) and chloroacetamide (CAA), and the reducing agent includes but is not limited to dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP).

[0034] As a preferred embodiment, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP); and the alkylating agent is chloroacetamide (CAA).

[0035] As a preferred embodiment, before the trypsin digestion in step S3, 20-40 μL of a 20-30 mM aqueous solution of ammonium bicarbonate is added to adjust the pH of the system; the concentration of the trypsin is 0.3-0.7 μg / μL, the amount of trypsin used is 2-4 μL, and the digestion conditions of the trypsin are 500-1000 rpm at 35-40°C for 2-12h, and then the reaction is terminated with a formic acid aqueous solution.

[0036] As a preferred embodiment, before the trypsin digestion in step S3, 30 μL of 25 mM ammonium bicarbonate solution is added to adjust the system pH; the concentration of the trypsin is 0.5 μg / μL, the amount of trypsin used is 4 μL, and the digestion conditions of the trypsin are 900 rpm at 37°C for 2-12h, and then the reaction is terminated with a formic acid aqueous solution.

[0037] As a preferred embodiment, the concentration of the formic acid aqueous solution is 10 wt %.

[0038] As a preferred embodiment, the mass spectrometer is a nano-liquid chromatography high-resolution electrostatic field orbital trap mass spectrometer.

[0039] As a preferred embodiment, the peptides are separated by nano-liquid chromatography, and the peptides are detected by high-resolution electrostatic field orbital trap mass spectrometer.

[0040] As a preferred embodiment, the conditions of the nano-liquid chromatography are: mobile phase A: 0.1wt% formic acid water; mobile phase B: 0.1wt% formic acid-80wt% acetonitrile aqueous solution; flow rate: 300nL / min; chromatographic column: self-packed C18 column (inner diameter 75μm, column length 200mm, particle size 1.9μm); column temperature: 60°C.

[0041] As a preferred embodiment, the mass spectrometry conditions are: nanoESI ion source (NSI) parameters: positive ion mode, ion source voltage 2.2 kV, ion transfer tube temperature 320°C, FAIMS gas flow 4.6 L / min, FAIMS voltage -60 V; mass spectrometry scanning mode and parameters: one-stage full scan (Full Scan) combined with parallel reaction monitoring (PRM) mode, the resolution of the first-stage full scan is 60000 (m / z 200), the PRM resolution is 15000 (m / z 200), the quadrupole isolation window is 2m / z, and the NCE is 15, 20, 35.

[0042] The applicant uses antibody enrichment-mass spectrometry to identify rhEPO protein in a complex body fluid matrix, and the source of recombinant human erythropoietin in human serum is identified with high accuracy, achieving accurate identification of the source of erythropoietin. It is speculated that the possible reason is that erythropoietin from different sources is identified by immunological method, the diffusion effect is obvious, and the difficulty of judgment is relatively high. Mass spectrometry detection is performed on cells containing rhEPO protein, and characteristic glycopeptide PRM qualitative fragment analysis is performed, the corresponding specificity is higher, and it is easier to detect the peak. When using immunological method for identification, cloned antibody purification columns are used for enrichment, and then gel electrophoresis separation is performed. The bands of endogenous human EPO and rhEPO are very close, and accurate separation cannot be achieved. Therefore, the effect is more diffuse, and it is not easy to achieve accurate identification.

[0043] The second aspect of the present invention provides an application of a mass spectrometry identification method for the source of human recombinant erythropoietin, which is applied to the identification of the source of erythropoietin.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The mass spectrometry identification method for the source of human recombinant erythropoietin described in the present invention is based on the method of antibody enrichment-mass spectrometry identification of the source of rhEPO protein in a complex body fluid matrix, and uses mass spectrometry technology to detect characteristic glycopeptides containing characteristic sialic acid in rhEPO derived from CHO cells, with good accuracy.

[0046] (2) Compared with the commonly used immunological detection methods, the mass spectrometry identification method for the source of human recombinant erythropoietin of the present invention has a higher detection throughput, and provides a new idea and direction for rhEPO doping screening and rhEPO source identification in the biomedical field.

[0047] (3) The mass spectrometry identification method for the source of human recombinant erythropoietin described in the present invention has high mass spectrometry detection accuracy and strong specificity, and can distinguish the sources of endogenous human erythropoietin, recombinant human erythropoietin cultured from mouse ovarian cells, and recombinant human erythropoietin cultured from human embryonic kidney cells.

[0048] (4) The mass spectrometry identification method for the source of human recombinant erythropoietin described in the present invention has simple and fast detection operation, rapid separation and accurate detection.

[0049] (5) The mass spectrometry identification method for the source of human recombinant erythropoietin described in the present invention can realize the rapid screening of rhEPO stimulants, detect the biopharmaceutical source of rhEPO, and reduce the risk of human rejection. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a flow chart for mass spectrometry identification of recombinant human erythropoietin derived from human serum;

[0051] Figure 2 The characteristic glycopeptide structure and secondary mass spectrum of recombinant human erythropoietin derived from mouse ovarian cells;

[0052] Figure 3 This is the PRM extracted ion map of the characteristic glycopeptide of recombinant human erythropoietin derived from mouse ovarian cells;

[0053] Figure 4 This is a comparison chart of the mass spectrometry results of characteristic glycopeptide detection of recombinant human erythropoietin cultured in mouse ovarian cells, recombinant human erythropoietin cultured in human embryonic kidney cells, and endogenous human erythropoietin;

[0054] Figure 5 This is a comparison chart of the immunological identification results of recombinant human erythropoietin cultured from mouse ovarian cells, recombinant human erythropoietin cultured from human embryonic kidney cells, and endogenous human erythropoietin. DETAILED DESCRIPTION

[0055] Example 1

[0056] A mass spectrometry identification method for the source of human recombinant erythropoietin comprises the following steps:

[0057] S1 combines biotin with erythropoietin antibody to obtain erythropoietin antibody A, co-incubates the sample to be tested with erythropoietin antibody A, and the erythropoietin antibody A combines with the erythropoietin protein in the sample to obtain a mixture 1;

[0058] S2 uses streptavidin-coated magnetic beads to co-incubate with mixture 1 to purify erythropoietin;

[0059] S3 performs reductive alkylation on the purified erythropoietin and digests it with trypsin;

[0060] S4 uses a mass spectrometer to perform qualitative detection on the peptide fragments of erythropoietin after enzyme digestion.

[0061] The erythropoietin includes three groups, including human endogenous erythropoietin, recombinant human erythropoietin cultured in mouse ovarian cells (R&D Systems, 11264-TC), and recombinant human erythropoietin cultured in human embryonic kidney cells 293 (HEK293) (Acro Biosystems, H4214).

[0062] The sample to be tested is serum, and the amount used is 100 μL; the serum is thawed and centrifuged, the centrifugation time is 10 min, the centrifugal force is 12000 g, the centrifugation temperature is 4°C, and after removing the bottom precipitate, the supernatant is transferred to a new tube for standby use.

[0063] The biotin is biotin N-hydroxysuccinimide ester, the concentration of the biotin is 5 mg / mL, the concentration of the erythropoietin antibody is 1 mg / mL, and the mass ratio of the biotin to the erythropoietin antibody is 6.6:1.

[0064] The conditions for the biotin to bind to the erythropoietin antibody are incubation at 600 rpm and room temperature in the dark for 3 hours.

[0065] The erythropoietin antibody A is a biotin-modified antibody purified by Sephadex G-25 gel filtration column.

[0066] The sample to be tested is diluted with a PBS solution, the volume ratio of the sample to be tested after dilution with the PBS solution to the erythropoietin antibody A is 1:1, the concentration of the erythropoietin antibody A is 4 μg / mL, the co-incubation temperature of the sample to be tested and the erythropoietin antibody A is 4°C, and the co-incubation time is 10 hours. The volume ratio of the sample to be tested to the PBS solution is 1:1.

[0067] The specific steps of step S2 include: first washing the streptavidin-coated magnetic beads with TWEEN 20 prepared with PBS solution, then separating the magnetic beads by magnetic attraction using a magnetic stand, then washing the streptavidin-coated magnetic beads again with PBS solution, then incubating with mixture 1, then eluting erythropoietin with an eluent, washing the streptavidin-coated magnetic beads again with TWEEN 20 prepared with PBS solution, then separating the magnetic beads by magnetic attraction using a magnetic stand, and finally washing the streptavidin-coated magnetic beads again with PBS solution.

[0068] The concentration of TWEEN 20 prepared in the PBS solution is 0.1 wt %.

[0069] The volume ratio of the streptavidin-coated magnetic beads to the erythropoietin antibody A is 1:10; the co-incubation condition of step S2 is incubation at 37° C. and 900 rpm for 2 hours.

[0070] After the co-incubation in step S2, erythropoietin is eluted with an eluent, wherein the eluent includes a citric acid aqueous solution and acetonitrile, the concentration of the citric acid aqueous solution is 0.01 mol / L, the volume ratio of the citric acid aqueous solution to acetonitrile is 9:1, and the elution condition is vortexing at 900 rpm for 10 min at 45°C.

[0071] In step S3, a reducing agent and an alkylating agent are used to carry out a reducing alkylation reaction. The concentration of the reducing agent is 10 mM, the concentration of the alkylating agent is 20 mM, the temperature of the reducing alkylation reaction is 56° C., and the reaction time is 50 min.

[0072] The reducing agent is tris(2-carboxyethyl)phosphine (TCEP); and the alkylating agent is chloroacetamide (CAA).

[0073] Before the trypsin digestion in step S3, 30 μL of 25 mM ammonium bicarbonate solution was added to adjust the system pH to 8; the concentration of the trypsin was 0.5 μg / μL, the amount of trypsin used was 4 μL, and the digestion conditions of the trypsin were 900 rpm at 37° C. for 2-12 h, and then the reaction was terminated with a formic acid aqueous solution.

[0074] The concentration of the formic acid aqueous solution is 10 wt %.

[0075] The injection column is processed as follows:

[0076] (1) Cut two layers of C18 membrane and place them at the lower end of a 200 μL pipette tip. Add 200 μL of acetonitrile to the pipette tip to activate the column, centrifuge at 1000 g for 30 s at room temperature, and discard the effluent. Add 200 μL of 50 wt % acetonitrile aqueous solution to the pipette tip to activate the column, centrifuge at 1000 g for 30 s at room temperature, and discard the effluent. Add 200 μL of 0.1 wt % trifluoroacetic acid aqueous solution to the pipette tip to equilibrate the column, centrifuge at 1000 g for 30 s at room temperature, and discard the effluent.

[0077] (2) adding the supernatant obtained after step S3 to the pipette tip, centrifuging at 1000 g for 30 s at room temperature, collecting the effluent, and repeating the loading three times; adding 200 μL of 0.1 wt % trifluoroacetic acid aqueous solution to the pipette tip to rinse the column to remove nonspecific adsorption, centrifuging at 1000 g for 30 s at room temperature, and discarding the effluent; adding 200 μL of an eluent prepared from 50 wt % acetonitrile aqueous solution and 0.1 wt % trifluoroacetic acid aqueous solution to the pipette tip, centrifuging at 1000 g for 30 s at room temperature, collecting the effluent, repeating the operation, and combining the effluent;

[0078] (3) The effluent was concentrated to dryness by centrifugation, and 20 μL of 0.1 wt % formaldehyde aqueous solution was added to re-dissolve the sample. 500 ng of the sample was taken for testing, and data was collected using nano-liquid chromatography tandem high-resolution mass spectrometry (Ultimate 3000-Orbitrap Exploris 480).

[0079] The mass spectrometer is a nano-liquid chromatography high-resolution electrostatic field orbital trap mass spectrometer; nano-liquid chromatography is used to separate peptide segments, and a high-resolution electrostatic field orbital trap mass spectrometer is used to detect peptide segments.

[0080] The conditions of the nano-liquid chromatography are: mobile phase A: 0.1wt% formic acid water; mobile phase B: 0.1wt% formic acid-80wt% acetonitrile aqueous solution; flow rate: 300nL / min; chromatographic column: self-filled C18 column (inner diameter 75μm, column length 200mm, particle size 1.9μm); column temperature: 60°C.

[0081] The elution gradient was as follows: 0-4 min, 5 vol% B; 4-5 min, 5-10 vol% B; 5-40 min, 10-37 vol% B; 40-60 min, 37-70 vol% B; 60-61 min, 98 vol% B, 61-67 min, 98 vol% B.

[0082] The mass spectrometry conditions are as follows: nanoESI ion source (NSI) parameters: positive ion mode, ion source voltage 2.2 kV, ion transfer tube temperature 320°C, FAIMS gas flow 4.6 L / min, FAIMS voltage -60 V; mass spectrometry scanning mode and parameters: one-stage full scan (Full Scan) combined with parallel reaction monitoring (PRM) mode, the resolution of the first-stage full scan is 60000 (m / z 200), the PRM resolution is 15000 (m / z 200), the quadrupole isolation window is 2 m / z, and the NCE is 15, 20, 35.

[0083] The mass spectrometry detection parameters containing peptides are shown in Table 1:

[0084] Table 1

[0085]

[0086] The mass spectrometry identification flow chart is shown in Figure 1 ; The characteristic glycopeptide structure and secondary mass spectrum of recombinant human erythropoietin derived from mouse ovarian cells are shown in Figure 2 ; The PRM extracted ion map of the characteristic glycopeptide of recombinant human erythropoietin derived from mouse ovarian cells is shown in Figure 3 .

[0087] Example 2

[0088] A method for identifying human recombinant erythropoietin, using an immune identification method as follows:

[0089] (1) Serum samples containing recombinant human erythropoietin were first enriched by MAIIA monoclonal antibody purification column, separated by sarcosyl sodium gel electrophoresis (SAR-PAGE), and then subjected to immunoblotting and chemiluminescence detection. The detection equipment was a CCD camera (LAS, 4000, Fujifilm, Tokyo, Japan), and the EPO marker was Dynepo (Shire, Hampshire, UK).

[0090] The results of mass spectrometry and immunoassay for the identification of the source of recombinant human erythropoietin are shown in Figure 4 and Figure 5 . Figure 4 Middle: A-2, human serum sample supplemented with rhEPO expressed by CHO cells; B-2, human serum sample supplemented with rhEPO expressed by HEK293 cells; C-2, blank human serum sample containing endogenous human EPO. Figure 5 Middle: A-1, human serum sample supplemented with rhEPO expressed by CHO cells; B-1, human serum sample supplemented with rhEPO expressed by HEK293 cells; C-1, blank human serum sample containing endogenous human EPO.

[0091] It can be seen that the mass spectrometry-based method can detect glycopeptides containing characteristic sialic acid in recombinant human erythropoietin derived from mouse ovarian cells, while there is no relevant signal in recombinant human erythropoietin derived from human embryonic kidney cells and human endogenous EPO. Based on the immune method, recombinant human erythropoietin from both sources showed a more obvious diffusion effect than endogenous human EPO, which is more difficult to judge.

Claims

1. A mass spectrometry method for identifying the source of human recombinant erythropoietin, characterized in that: The following steps are involved: S1 combines biotin with erythropoietin antibody to obtain erythropoietin antibody A, co-incubates the sample to be tested with erythropoietin antibody A, and the erythropoietin antibody A combines with the erythropoietin protein in the sample to obtain a mixture 1; S2 uses streptavidin-coated magnetic beads to co-incubate with mixture 1 to purify erythropoietin; S3 performs reductive alkylation on the purified erythropoietin and digests it with trypsin; S4 uses a mass spectrometer to perform qualitative detection on the peptide fragments of erythropoietin after enzyme digestion.

2. The mass spectrometry identification method for the source of human recombinant erythropoietin according to claim 1, characterized in that: The erythropoietin is selected from human endogenous erythropoietin or recombinant human erythropoietin, and the recombinant human erythropoietin is selected from recombinant human erythropoietin cultured from mouse ovarian cells or recombinant human erythropoietin cultured from human embryonic kidney cells.

3. The mass spectrometry identification method for the source of human recombinant erythropoietin according to claim 1, characterized in that: The biotin is biotin N-hydroxysuccinimide ester, the concentration of the biotin is 3-7 mg / mL, the concentration of the erythropoietin antibody is 1-3 mg / mL, and the mass ratio of the biotin to the erythropoietin antibody is (3-9):

1.

4. The mass spectrometry identification method for the source of human recombinant erythropoietin according to claim 1, characterized in that: The conditions for the biotin to bind to the erythropoietin antibody are incubation at 300-900 rpm at room temperature in the dark for 1-5 hours.

5. The mass spectrometry identification method for the source of human recombinant erythropoietin according to claim 1, characterized in that: The sample to be tested is diluted with a PBS solution, the volume ratio of the sample to be tested after dilution with the PBS solution to the erythropoietin antibody A is 1:(1-3), the concentration of the erythropoietin antibody A is 1-5 μg / mL, the co-incubation temperature of the sample to be tested and the erythropoietin antibody A is 1-5°C, and the co-incubation time is 2-12h.

6. The mass spectrometry identification method for the source of human recombinant erythropoietin according to claim 1, characterized in that: The sample to be tested is selected from one of human serum, human plasma and human urine, and the usage amount of the sample to be tested is 100-500 μL.

7. The mass spectrometry identification method for the source of human recombinant erythropoietin according to claim 1, characterized in that: After the co-incubation in step S2, the erythropoietin is eluted with an eluent, wherein the eluent includes an aqueous citric acid solution and acetonitrile, the concentration of the aqueous citric acid solution is 0.01-0.03 mol / L, the volume ratio of the aqueous citric acid solution to acetonitrile is (6-12):1, and the elution condition is vortexing at 500-1000 rpm at 40-50°C for 8-12 min.

8. The mass spectrometry identification method for the source of human recombinant erythropoietin according to claim 1, characterized in that: In step S3, a reducing agent and an alkylating agent are used to carry out a reducing alkylation reaction. The concentration of the reducing agent is 5-15 mM, the concentration of the alkylating agent is 15-30 mM, the temperature of the reducing alkylation reaction is 50-60° C., and the reaction time is 40-60 min.

9. The mass spectrometry identification method for the source of human recombinant erythropoietin according to claim 1, characterized in that: Before the trypsin digestion in step S3, 20-40 μL of a 20-30 mM aqueous solution of ammonium bicarbonate is added to adjust the pH of the system; the concentration of the trypsin is 0.3-0.7 μg / μL, the amount of trypsin used is 2-4 μL, and the digestion conditions of the trypsin are 500-1000 rpm at 35-40° C. for 2-12 hours, and then the reaction is terminated with a formic acid aqueous solution.

10. An application of the mass spectrometry identification method for the source of human recombinant erythropoietin according to any one of claims 1 to 9, characterized in that: Used in the identification of the source of erythropoietin.

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