Peptide diagram detection and analysis method of thrombopoietin peptidomimetic

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a thrombopoietin peptidomimetic peptide diagram detection and analysis method which comprises the following steps: (1) performing buffer system replacement on thrombopoietin peptidomimetic through ultrafiltration centrifugation; (2) carrying out enzymolysis on the thrombopoietin peptidomimetic by adopting trypsin; and (3) detecting and analyzing the enzymolysis peptide fragment by using a reversed-phase high-performance liquid chromatography. The method disclosed by the invention has the beneficial effects that sample concentration and buffer solution replacement are performed through the ultrafiltration tube, and the sample subjected to enzymolysis is high in purity and can be directly detected by using reversed-phase high-performance liquid chromatography, so that the method for detecting and analyzing the peptide diagram of the thrombopoietin peptidomimetic is simple and convenient to operate, and the detection efficiency of thrombopoietin peptidomimetic drugs is improved; by controlling the chromatographic conditions, the accuracy and precision of high performance liquid chromatography detection are improved, and the quality analysis and control of thrombopoietin peptidomimetic drugs can be realized.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a peptide map detection and analysis method for thrombopoietin peptidomimetics. Background Art

[0002] Thrombopoietin peptidomimetic is a thrombopoietin (TPO) receptor agonist that specifically binds to the TPO receptor, activates cellular pathways, stimulates the proliferation, differentiation and maturation of megakaryocytes, promotes platelet production, and can significantly increase platelet levels and reduce the risk of bleeding.

[0003] Peptide mapping is based on the characteristics of the amino acid composition of proteins. It uses specific proteolytic enzymes to act on specific sites to break protein polypeptides into small fragments, and forms a characteristic fingerprint through certain separation and detection methods (such as high performance liquid chromatography). With the continuous development of the biopharmaceutical field, peptide mapping has become a key method in the characterization process of biological protein drugs, ensuring the high specificity of biological protein structure characterization. In the current traditional recombinant protein peptide mapping method, the buffer system is replaced by membrane dialysis, which has high time cost, complex operation, and introduces more impurities during the dialysis process. Therefore, how to design a peptide mapping method for thrombopoietin peptidomimetics that can simply and efficiently obtain protein hydrolysis samples with high purity while saving time and cost has become an urgent problem to be solved. Summary of the invention

[0004] In view of the above problems, the present invention provides a peptide map detection and analysis method for thrombopoietin peptidomimetics.

[0005] The present invention relates to a peptide map detection and analysis method for thrombopoietin peptidomimetics, comprising the following steps: (1) The thrombopoietin peptidomimetic is subjected to buffer replacement by ultrafiltration centrifugation; (2) Enzymatic hydrolysis of thrombopoietin peptidomimetic using trypsin; (3) Reverse-phase high performance liquid chromatography was used to detect and analyze the enzymatically hydrolyzed peptide fragments.

[0006] Preferably, in the step (1), the test sample and the reference substance are diluted to 1.0 mg / ml respectively with a 1% ammonium bicarbonate solution to obtain a test sample dilution solution and a reference substance dilution solution; an appropriate amount of blank buffer solution is taken and diluted with a 1% ammonium bicarbonate solution by the same multiple as the test sample to obtain a blank dilution solution; Take the test sample dilution, reference sample dilution and blank dilution, add them to 10kD ultrafiltration tubes respectively, and centrifuge for 30 minutes; discard the lower filtrate after centrifugation, add 1% ammonium bicarbonate solution with a mass percentage concentration to the 10kD ultrafiltration tube, centrifuge for 30 minutes, repeat the above centrifugation operation twice; rinse the filter membranes on both sides of the ultrafiltration tube with 1% ammonium bicarbonate solution with a mass percentage concentration, and collect the washing liquid in the ultrafiltration casing to obtain the test sample solution, reference sample solution and blank solution.

[0007] Preferably, in step (2), a trypsin solution with a mass concentration of 0.5 mg / ml is added to the test solution, the reference solution and the blank solution in the ultrafiltration tube, respectively, and an enzymatic hydrolysis reaction is carried out at 37° C. After the enzymatic hydrolysis is completed, an acetic acid solution with a mass percentage concentration of 50% is added to terminate the enzymatic hydrolysis, and the supernatant is collected by centrifugation to obtain a test supernatant, a reference supernatant and a blank supernatant.

[0008] Preferably, the volume ratio of the test solution, the reference solution, the blank solution to the trypsin solution is 10:1, and the volume ratio of the test solution, the reference solution, the blank solution to the acetic acid solution is 10:1.

[0009] Preferably, the step (3) includes sample preparation, reversed-phase high performance liquid chromatography detection and experimental result analysis; in the reversed-phase high performance liquid chromatography detection, the chromatographic conditions are: Chromatographic column: Kromasil 300-5-C18 column, 4.6×250 mm, 5 μm; Guard column: Kromasil 300-5-C18 3.0-4.6mm guard starter kit; Mobile phase A: 0.1% trifluoroacetic acid-water solution; Mobile phase B: 0.1% trifluoroacetic acid-acetonitrile solution; Mobile phase C: 70% acetonitrile-water solution; Detection wavelength: 214nm; Injection volume: 50 μl; Flow rate: 1.0ml / min; Column temperature: 35°C; Sample pan temperature: 4°C; Elution mode: gradient elution.

[0010] Preferably, the procedure of the gradient elution is: .

[0011] Preferably, in the sample configuration, a blank supernatant is used as a blank enzyme digestion solution; a reference supernatant is used to prepare three parallel samples, recorded as enzyme digestion reference solution 1, enzyme digestion reference solution 2 and enzyme digestion reference solution 3; a test sample supernatant is used as a test sample solution; an equal volume of the reference sample supernatant and the test sample supernatant are mixed to obtain a 1:1 mixed solution of enzyme digestion reference sample-test sample.

[0012] Preferably, in the reversed-phase high performance liquid chromatography detection, the injection order is enzyme cleavage blank solution, enzyme cleavage reference solution 1, enzyme cleavage reference solution 2, enzyme cleavage reference solution 3, enzyme cleavage test solution and enzyme cleavage reference-test solution 1:1.

[0013] Preferably, in the reverse phase high performance liquid chromatography detection, the integration parameter is: Integration algorithm: Apextrack, apex detection; Integration interval: 4.5-40; Peak width: 10s; Threshold: 300; Peak integration: Peak start% 0.5%, Peak end% 0.5%; Minimum height: 0μV; Minimum area: 300000.

[0014] Preferably, in the experimental result analysis, the identification peaks include identification peak 1, identification peak 2, identification peak 3 and identification peak 4, and the relative retention times of identification peak 1, identification peak 2, identification peak 3 and identification peak 4 are 18.750min±0.5min, 22.897±0.5min, 25.069±0.5min, and 27.100±0.5min, respectively; Experimental establishment criteria: 1) No significant new peaks appear in the peptide map results. The criteria for determining significant new peaks are: the new peak area in the peptide map is ≥ 0.5% of the total peak area, and it can also be detected in the peptide map of the test sample: reference sample = 1:1 mixed solution, and the peak area is 50±20% of the peptide map of the test sample solution; 2) The peak shapes of the marked peaks of the enzyme digestion reference solution 1, enzyme digestion reference solution 2, and enzyme digestion reference solution 3 are consistent; Test result judgment criteria: 1) The peak shapes of the marker peaks of enzyme cleavage reference solution 1, enzyme cleavage reference solution 2, enzyme cleavage reference solution 3, enzyme cleavage test solution and enzyme cleavage reference-test solution 1:1 are consistent; 2) With marker peak 4 as the internal standard peak, the relative peak area ratio of each marker peak needs to meet the following requirements: the relative peak area ratio of the marker peak of the test sample to the relative peak area ratio of the marker peak of the reference sample should be between 100±15%; The calculation formula of relative peak area is: Among them, Area n is the peak area of ​​the nth peptide peak in the sample, n=1,2,3... Area 内标 is the peak area of ​​the internal standard peak.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are: (1) In the present invention, sample concentration and buffer exchange are performed by ultrafiltration tube, and the sample after enzymatic hydrolysis has high purity and can be directly detected by reversed-phase high performance liquid chromatography, so that the peptide map detection and analysis method of thrombopoietin peptidomimetics is easy to operate and improves the detection efficiency of thrombopoietin peptidomimetics; (2) The present invention improves the accuracy and precision of HPLC detection by controlling chromatographic conditions, thereby enabling quality analysis and control of thrombopoietin peptidomimetic drugs; (3) In the present invention, three enzyme cleavage reference solutions are set up to ensure the validity of the detection experiment through system suitability test, thereby ensuring the accurate identification of the thrombopoietin peptidomimetic. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment. Figure 1 The peptide map of the blank solution of enzyme digestion provided in Example 1; Figure 2 Overlapping peptide maps of enzyme digestion reference solutions 1, 2, and 3 provided in Example 1; Figure 3 The peptide map of the enzyme-digested test solution provided in Example 1; Figure 4 The peptide map of the 1:1 mixed solution of the enzyme digestion reference substance and the test substance provided in Example 1; Figure 5 Overlapping peptide maps of the enzyme cleavage reference substance 1, enzyme cleavage reference substance solution 2, enzyme cleavage reference substance solution 3, enzyme cleavage test substance solution and the enzyme cleavage reference substance-test substance 1:1 mixed solution provided in Example 1. DETAILED DESCRIPTION

[0017] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below in conjunction with embodiments.

[0018] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0019] Example 1 A peptide map detection and analysis method for thrombopoietin peptidomimetics, characterized in that it comprises the following steps: (1) Solution preparation: 1% ammonium bicarbonate solution: Weigh 1g of ammonium bicarbonate, dissolve it in ultrapure water and make up to 100ml.

[0020] 0.5 mg / ml trypsin solution: Take 20 μg of trypsin and add 40 μl of trypsin buffer to dilute to 0.5 mg / ml.

[0021] Preparation of blank buffer solution: weigh 1g polysorbate 20, add purified water to dissolve and dilute to 100ml to obtain polysorbate 20 solution; weigh 8g mannitol, 4g sucrose, and 0.32g histidine, adjust the pH to 5.0 with dilute hydrochloric acid, add water to dissolve, add 0.8ml prepared polysorbate 20 solution, mix well, dilute to 200ml, filter with a 0.22um filter membrane to obtain blank buffer solution.

[0022] (2) Sample solution dilution: Dilute the test sample and reference substance to 1.0 mg / ml respectively with 1% ammonium bicarbonate solution to obtain test sample dilution solution and reference substance dilution solution; take an appropriate amount of blank buffer solution and dilute it with 1% ammonium bicarbonate solution by the same multiple as the test sample to obtain blank dilution solution.

[0023] (3) Ultrafiltration fluid replacement: Take 400 μl of the test dilution, reference dilution and blank dilution, add them to 10kD ultrafiltration tubes respectively, and centrifuge at 4°C, 9600g for 30 min; discard the lower filtrate after centrifugation, add 200 μl of 1% ammonium bicarbonate solution to the ultrafiltration tube, centrifuge at 4°C, 9600g for 30 min, repeat the above centrifugation operation twice; take 200 μl of 1% ammonium bicarbonate solution in the ultrafiltration tube, and rinse the ultrafiltration tube membrane 10 times (5 times on each side of the membrane) with a pipette, then invert the ultrafiltration tube in another centrifuge sleeve, centrifuge at 4°C, 1000g for 3 min; take 1% ammonium bicarbonate solution into the ultrafiltration tube in the previous step twice, take 100 μl each time, rinse the ultrafiltration tube membrane 10 times (5 times on each side of the membrane), collect the washing liquid in the ultrafiltration sleeve, and obtain the test solution, reference solution and blank solution.

[0024] (4) Enzymatic hydrolysis of thrombopoietin peptidomimetics by trypsin: Take 400 μl of the test solution, reference solution and blank solution in the ultrafiltration tube, add 40 μl of trypsin solution with a mass concentration of 0.5 mg / ml respectively, and place in a water bath at 37°C for 18 h; after the enzyme digestion is completed, add 40 μl of acetic acid solution with a mass percentage concentration of 50% to terminate the enzyme digestion, centrifuge at 4°C, 9600g for 10 min, collect the supernatant, and obtain the test supernatant, reference supernatant and blank supernatant.

[0025] (5) Sample configuration: Take 200 μl of blank supernatant as blank enzyme digestion solution; take 110 μl of each reference supernatant to prepare 3 parallel samples, recorded as reference solution 1, reference solution 2 and reference solution 3; take 200 μl of test sample supernatant as test sample solution; take 100 μl of reference supernatant and test sample supernatant respectively, mix well and use as 1:1 mixed solution of reference-test sample.

[0026] (6) Mobile phase configuration for HPLC: Mobile phase A: Pipette 2.0 ml of TFA into 2000 ml of ultrapure water, mix well, and degas by ultrasonication for 20 min.

[0027] Mobile phase B: Pipette 1.0 ml of TFA into 1000 ml of acetonitrile (chromatographic grade), mix well, and degas by ultrasonication for 20 min.

[0028] Mobile phase C: Measure 350 ml of acetonitrile (chromatographic grade) and 150 ml of ultrapure water, mix well, and degas by ultrasonication for 20 minutes.

[0029] (7) Equilibration and flushing before HPLC injection: After flushing the pipelines and the injection nozzle with mobile phases A, B, and C respectively, install the chromatographic column at a low flow rate according to the flow direction marked on the chromatographic column. First, flush 10 column volumes with 100% mobile phase B at a flow rate of 1.0 ml / min, then flush 10 column volumes with 100% mobile phase A at a flow rate of 1.0 m / min, and finally balance the chromatographic column with 98% mobile phase A and 2% mobile phase B at a flow rate of 1.0 ml / min for 20 column volumes.

[0030] (8) The chromatographic conditions of high performance liquid chromatography are: The model of high performance liquid chromatography is: Waters e2695; Chromatographic column: Kromasil 300-5-C18 column, 4.6×250 mm, 5 μm; Guard column: Kromasil 300-5-C18 3.0-4.6mm guard starter kit; Mobile phase A: 0.1% trifluoroacetic acid-water solution; Mobile phase B: 0.1% trifluoroacetic acid-acetonitrile solution; Mobile phase C: 70% acetonitrile-water solution; Detection wavelength: 214nm; Injection volume: 50 μl; Flow rate: 1.0ml / min; Column temperature: 35°C; Sample pan temperature: 4°C; Elution mode: gradient elution.

[0031] The gradient elution program was: .

[0032] (9) High performance liquid chromatography batch analysis table editing: Enter the sample bottle number, sample name, injection volume, and injection order; the injection order is: one injection of mobile phase (mobile phase A: mobile phase B = 98:2), one injection of enzyme digestion blank solution, one injection of enzyme digestion reference solution 1, one injection of enzyme digestion reference solution 2, one injection of enzyme digestion reference solution 3, one injection of enzyme digestion test solution, and one injection of a 1:1 mixed solution of enzyme digestion reference and test solution.

[0033] (10) Post-injection flushing of HPLC: The column was flushed with 100% mobile phase C at a flow rate of 1.0 ml / min for 30 min.

[0034] (11) Integration parameters of HPLC: Integration algorithm: Apextrack, apex detection; Integration interval: 4.5-40; Peak width: 10; Threshold: 300; Peak integration: Peak start % is 0.5%, Peak end % is 0.5%; Minimum height: 0μV; Minimum area: 300000.

[0035] (12) Analysis of experimental results: The peptide map of the blank solution is shown in Figure 1 ; Overlapping peptide maps of enzyme digestion reference solutions 1, 2, and 3 are shown in Figure 2 , the chromatographic peak information is shown in Table 1; the peptide map of the enzyme digestion test solution is shown in Figure 3 ; The peptide map of the 1:1 mixed solution of enzyme digestion reference substance and test substance is shown in Figure 4 ; Overlapping peptide maps of enzyme digestion reference substance 1, enzyme digestion reference substance solution 2, enzyme digestion reference substance solution 3, enzyme digestion test substance solution and enzyme digestion reference substance-test substance 1:1 mixed solution are shown in Figure 5 The chromatographic peak information is shown in Table 2; the relative peak area ratios of the marked peaks of the enzyme-cut test solution are shown in Table 3.

[0036] Table 1. Chromatographic peak results of overlapping peptide maps of enzyme digestion reference solutions 1, 2, and 3 Table 2. Chromatographic peak results of overlapping peptide maps of enzyme digestion control, test sample and 1:1 mixed solution of control and test sample Table 3. Results of relative peak area ratios of each marked peak in the enzyme digestion test solution Depend on Figure 1-5 As shown in Tables 1-3, the peak shapes of the identified peaks in the peptide map are consistent, and no significant new peaks appear. The relative peak area ratios of the identified peaks are all between 100±15%, indicating that this batch of test products meets the detection standards.

[0037] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes for application in other fields. However, any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A peptide map detection and analysis method for thrombopoietin peptidomimetics, characterized in that: The following steps are involved: (1) The thrombopoietin peptidomimetic is subjected to buffer replacement by ultrafiltration centrifugation; (2) Enzymatic hydrolysis of thrombopoietin peptidomimetic using trypsin; (3) Reverse-phase high performance liquid chromatography was used to detect and analyze the enzymatically hydrolyzed peptide fragments.

2. The peptide map detection and analysis method of thrombopoietin peptidomimetics according to claim 1, characterized in that: In the step (1), the test sample and the reference substance are diluted to 1.0 mg / ml respectively with a 1% ammonium bicarbonate solution to obtain a test sample dilution solution and a reference substance dilution solution; an appropriate amount of blank buffer is taken and diluted with a 1% ammonium bicarbonate solution by the same multiple as the test sample to obtain a blank dilution solution; Take the test sample dilution, reference sample dilution and blank dilution, add them to 10kD ultrafiltration tubes respectively, and centrifuge for 30 minutes; discard the lower filtrate after centrifugation, add 1% ammonium bicarbonate solution with a mass percentage concentration to the 10kD ultrafiltration tube, centrifuge for 30 minutes, repeat the above centrifugation operation twice; rinse the filter membranes on both sides of the ultrafiltration tube with 1% ammonium bicarbonate solution with a mass percentage concentration, and collect the washing liquid in the ultrafiltration casing to obtain the test sample solution, reference sample solution and blank solution.

3. The peptide map detection and analysis method of thrombopoietin peptidomimetic according to claim 1, characterized in that: In the step (2), the test solution, the reference solution and the blank solution in the ultrafiltration tube are taken, and a trypsin solution with a mass concentration of 0.5 mg / ml is added respectively, and an enzymatic hydrolysis reaction is carried out at 37° C. After the enzymatic hydrolysis is completed, an acetic acid solution with a mass percentage concentration of 50% is added to terminate the enzymatic hydrolysis, and the supernatant is collected by centrifugation to obtain the test supernatant, the reference supernatant and the blank supernatant.

4. The peptide map detection and analysis method of thrombopoietin peptidomimetics according to claim 3, characterized in that: The volume ratio of the test solution, the reference solution, the blank solution to the trypsin solution was 10:1, and the volume ratio of the test solution, the reference solution, the blank solution to the acetic acid solution was 10:

1.

5. The peptide map detection and analysis method of thrombopoietin peptidomimetics according to claim 3, characterized in that: The step (3) includes sample preparation, reversed-phase high performance liquid chromatography detection and experimental result analysis; in the reversed-phase high performance liquid chromatography detection, the chromatographic conditions are: Chromatographic column: Kromasil 300-5-C18 column, 4.6×250mm, 5μm; Guard column: Kromasil 300-5-C18 3.0-4.6mm guard starter kit; Mobile phase A: 0.1% trifluoroacetic acid-water solution; Mobile phase B: 0.1% trifluoroacetic acid-acetonitrile solution; Mobile phase C: 70% acetonitrile-water solution; Detection wavelength: 214nm; Injection volume: 50 μl; Flow rate: 1.0ml / min; Column temperature: 35°C; Sample pan temperature: 4°C; Elution mode: gradient elution.

6. The peptide map detection and analysis method of thrombopoietin peptidomimetics according to claim 5, characterized in that: The procedure of the gradient elution is: 。 7. The peptide map detection and analysis method of thrombopoietin peptidomimetics according to claim 5, characterized in that: In the sample configuration, a blank supernatant was taken as a blank enzyme digestion solution; a reference supernatant was taken to prepare three parallel samples, recorded as reference digestion solution 1, reference digestion solution 2, and reference digestion solution 3; a test sample supernatant was taken as a test sample digestion solution; an equal volume of reference sample supernatant and test sample supernatant were taken and mixed to obtain a 1:1 mixed solution of reference digestion-test sample.

8. The peptide map detection and analysis method of thrombopoietin peptidomimetics according to claim 5, characterized in that: In the reversed-phase high-performance liquid chromatography detection, the injection order is enzymatic blank solution, enzymatic reference solution 1, enzymatic reference solution 2, enzymatic reference solution 3, enzymatic test solution and enzymatic reference-test solution in a ratio of 1:

1.

9. The peptide map detection and analysis method of thrombopoietin peptidomimetics according to claim 5, characterized in that: In the reversed-phase high performance liquid chromatography detection, the integration parameters are: Integration algorithm: Apextrack, apex detection; Integration interval: 4.5-40; Peak width: 10s; Threshold: 300; Peak integration: Peak start% 0.5%, Peak end% 0.5%; Minimum height: 0μV; Minimum area: 300000.

10. The peptide map detection and analysis method of thrombopoietin peptidomimetics according to claim 5, characterized in that: In the experimental result analysis, the identification peaks include identification peak 1, identification peak 2, identification peak 3 and identification peak 4, and the relative retention times of identification peak 1, identification peak 2, identification peak 3 and identification peak 4 are 18.750min±0.5min, 22.897±0.5min, 25.069±0.5min, and 27.100±0.5min, respectively; Experimental establishment criteria: 1) No significant new peaks appear in the peptide map results. The criteria for determining significant new peaks are: the new peak area in the peptide map is ≥ 0.5% of the total peak area, and it can also be detected in the peptide map of the test sample: control sample = 1:1 mixed solution, and the peak area is 50±20% of the peptide map of the test sample solution; 2) The peak shapes of the marker peaks of enzyme digestion reference solution 1, enzyme digestion reference solution 2 and enzyme digestion reference solution 3 are consistent; Test result judgment criteria: 1) The peak shapes of the marker peaks of enzyme cleavage reference solution 1, enzyme cleavage reference solution 2, enzyme cleavage reference solution 3, enzyme cleavage test solution and enzyme cleavage reference-test solution 1:1 are consistent; 2) With marker peak 4 as the internal standard peak, the relative peak area ratio of each marker peak needs to meet the following requirements: the relative peak area ratio of the marker peak of the test sample to the relative peak area ratio of the marker peak of the reference sample should be between 100±15%; The calculation formula of relative peak area is: Among them, Area n is the peak area of ​​the nth peptide peak in the sample, n=1,2,3... Area 内标 is the peak area of ​​the internal standard peak.

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