Quantitative analysis method and device for stable isotope labeled recombinant human growth hormone

Abstract

The invention discloses a quantitative analysis method and device for stable isotope labeled recombinant human growth hormone, and belongs to the technical field of quantitative analysis of protein, the method comprises the following steps: S1, preliminarily measuring the purity of stable isotope labeled recombinant human growth hormone based on chromatography or electrophoresis; s2, on the basis of preliminarily determining the purity of the stable isotope labeled recombinant human growth hormone in the step S1, measuring the stable isotope labeled amino acid replacement efficiency through amino acid analysis-isotope dilution mass spectrometry; and S3, based on the stable isotope labeled amino acid replacement efficiency, measuring the content of the stable isotope labeled recombinant human growth hormone through peptide fragment quantitative analysis-isotope dilution mass spectrometry. According to the method, the isotope dilution mass spectrometry is adopted to eliminate system errors possibly introduced in the pretreatment step and the measurement process, and the accuracy and credibility of the measurement result can be remarkably improved. According to the method, traceable quantitative analysis of the content of the stable isotope labeled recombinant human growth hormone can be realized, and the measurement result can be traced to the international system of units.

Description

Technical Field

[0001] The invention belongs to the technical field of protein quantitative analysis, and in particular relates to a method and a device for quantitative analysis of recombinant human growth hormone labeled with stable isotope. Background Art

[0002] Isotope dilution mass spectrometry has the characteristics of high accuracy and good reproducibility, and is currently the preferred method for quantitative analysis of protein content. The CCQM Protein Working Group organized multiple rounds of measurement and comparison of human serum growth hormone content based on isotope dilution mass spectrometry. This method uses stable isotope-labeled full-length recombinant human growth hormone as an internal standard for sample pretreatment and quality control during quantitative analysis. It can effectively eliminate systematic errors in sample processing and measurement, and is the development trend of future medical measurements. This method requires the addition of stable isotope-labeled recombinant human growth hormone as an internal standard. Therefore, it is urgent to develop a method that can achieve high-accuracy quantitative analysis of stable isotope-labeled recombinant human growth hormone. Summary of the invention

[0003] In view of this, the present invention provides a method and device for quantitative analysis of recombinant human growth hormone labeled with stable isotope.

[0004] The present invention adopts the following technical solution:

[0005] A method for quantitative analysis of stable isotope labeled recombinant human growth hormone, the method comprising the following steps:

[0006] S1 Preliminary measurement of the purity of stable isotope labeled recombinant human growth hormone based on chromatography or electrophoresis;

[0007] S2, based on the preliminary determination of the purity of the stable isotope-labeled recombinant human growth hormone in step S1, measuring the substitution efficiency of the stable isotope-labeled amino acid by amino acid analysis-isotope dilution mass spectrometry;

[0008] S3 measures the content of stable isotope-labeled recombinant human growth hormone by peptide quantitative analysis-isotope dilution mass spectrometry based on the stable isotope-labeled amino acid replacement efficiency.

[0009] Furthermore, the amino acid sequence of the stable isotope-labeled recombinant human growth hormone is: LCLPW LQEGSAFPTIPLSRL FDNAM LRAHR LHQLA FDTYQ EFEEA YIPKE QKYSF LQNPQ TSLCFSESIP TPSNREETQQ KSNLE LLRIS LLLIQ SWLEP VQFLR SVFAN SLVYG ASDSN VYDLLKDLEE GIQTL MGRLEDGSPR TGQIF KQTYS KFDTN SHNDD ALLKN YGLLY CFRKD MDKVETFLRI VQCRS VEGSC GF, and the direction of the sequence is N-terminus-C-terminus; lysine in the amino acid sequence is replaced by lysine labeled with stable isotope, and lysine and arginine in the amino acid sequence are replaced by lysine and arginine labeled with stable isotope, respectively, and the C and N elements in the isotope-labeled lysine and arginine are respectively. 13 C and 15 N.

[0010] Furthermore, step S1 comprises: performing purity analysis on the stable isotope-labeled recombinant human growth hormone by reverse phase liquid chromatography or size exclusion chromatography.

[0011] Furthermore, step S2 includes: taking a stable isotope-labeled recombinant human growth hormone solution and placing it in an ampoule, adding 500 microliters of concentrated hydrochloric acid thereto after drying, wherein the concentration of the concentrated hydrochloric acid is 6 mol / L; replacing the air in the ampoule with high-purity nitrogen, hot-melt sealing the ampoule, and then placing it in a high-temperature oven for reaction, wherein the oven temperature is 100°C to 150°C; after the reaction is completed, drying with nitrogen to remove the hydrochloric acid, and then re-dissolving with 1000 microliters of formic acid-water solution; and then performing liquid chromatography-mass spectrometry analysis.

[0012] Furthermore, step S3 includes: the quantitative peptide segments used for quantitative analysis include: LFDNAM LR (denoted as T2), SNLELLR (denoted as T8), LEDGSPR (denoted as T12) or TGQIFK (denoted as T13); at least two of the quantitative peptide segments are synthesized by chemical methods, and each of the quantitative peptide segments includes a non-isotope labeled quantitative peptide segment and a stable isotope labeled quantitative peptide segment; the labeling method of the stable isotope labeled quantitative peptide segment is: the lysine in the peptide segment is replaced by the stable isotope labeled lysine, and the arginine in the peptide segment is replaced by the stable isotope labeled arginine.

[0013] Furthermore, step S3 includes: taking a stable isotope-labeled recombinant human growth hormone solution and a quantitative peptide solution and placing them together in a low adsorption centrifuge tube; adding ammonium bicarbonate or Tris-HCl buffer solution to adjust the pH value of the solution to 8; adding DTT solution to react at 95 degrees Celsius for 10 minutes; after the reaction solution is cooled to room temperature, adding IAA solution, and reacting in a light-proof environment for 30 minutes; adding DTT solution again to react to remove excess IAA in the solution, and the reaction time is 30 minutes; adding 1 microgram of trypsin to the reaction solution, and reacting at 37°C for 16 hours; and then performing LC-MS / MS analysis.

[0014] Furthermore, the LC-MS / MS analysis includes: analyzing and detecting the quantitative peptide segment in a multiple reaction monitoring mode, and calculating the protein content according to the peak area ratio of the non-isotope labeled quantitative peptide segment and the stable isotope labeled quantitative peptide segment.

[0015] Furthermore, the LC-MS / MS analysis includes: preparing non-isotope labeled quantitative peptides and corresponding stable isotope labeled quantitative peptide solutions of different mass ratios, establishing a quantitative analysis standard working curve of the non-isotope labeled quantitative peptides and the stable isotope labeled quantitative peptides, calculating the content of the stable isotope labeled peptides obtained by trypsin hydrolysis in the sample, and calculating the content of the stable isotope labeled recombinant human growth hormone in the sample to be tested.

[0016] Furthermore, the LC-MS / MS analysis includes: the peptide content of the quantitative peptide solution is subjected to traceable quantitative analysis using amino acid hydrolysis-isotope dilution mass spectrometry.

[0017] A stable isotope labeled recombinant human growth hormone quantitative analysis device, the device adopts the above method to perform analysis and detection, the device comprises:

[0018] A module for preliminarily measuring protein purity, which preliminarily measures the purity of stable isotope-labeled recombinant human growth hormone based on chromatography or electrophoresis;

[0019] A module for measuring the substitution efficiency of stable isotope-labeled amino acids, wherein the module measures the substitution efficiency of stable isotope-labeled amino acids based on amino acid analysis-isotope dilution mass spectrometry;

[0020] A protein content measurement module, wherein the module measures the content of stable isotope labeled recombinant human growth hormone based on peptide quantitative analysis-isotope dilution mass spectrometry.

[0021] Beneficial effects:

[0022] The present invention adopts isotope dilution mass spectrometry to eliminate the systematic errors that may be introduced in the pre-treatment steps and the measurement process, and significantly improves the accuracy and credibility of the measurement results. The analysis method of the present invention can realize the traceable quantitative analysis of the content of stable isotope-labeled recombinant human growth hormone, and the measurement results can be traced back to the international system of units. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 A schematic diagram of the amino acid sequence of the stable isotope-labeled recombinant human growth hormone of the present invention;

[0025] Figure 2 This is a schematic diagram of the results of measuring the purity of hGH* using the SDS-PAGE method in the present invention;

[0026] Figure 3 This is a schematic diagram of the results of measuring the purity of hGH* using reverse phase chromatography in the present invention;

[0027] Figure 4 Schematic diagram of extracted ion chromatograms of lysine, arginine and heavy isotope-labeled amino acids thereof in the present invention;

[0028] Figure 5 Schematic diagram of LC-MS / MS spectra of peptide segments T2* / T2 and T13* / T13 after hGH* digestion in the present invention. DETAILED DESCRIPTION

[0029] In order to better understand the technical solution of the present invention, the embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0030] It should be clear that the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Embodiment 1:

[0032] A method for quantitative analysis of stable isotope labeled recombinant human growth hormone, the method comprising the following steps:

[0033] S1 Preliminary measurement of the purity of stable isotope labeled recombinant human growth hormone based on chromatography or electrophoresis;

[0034] S2, based on the preliminary determination of the purity of the stable isotope-labeled recombinant human growth hormone in step S1, measuring the substitution efficiency of the stable isotope-labeled amino acid by amino acid analysis-isotope dilution mass spectrometry;

[0035] S3 measures the content of stable isotope-labeled recombinant human growth hormone by peptide quantitative analysis-isotope dilution mass spectrometry based on the stable isotope-labeled amino acid replacement efficiency.

[0036] Furthermore, the amino acid sequence of the stable isotope-labeled recombinant human growth hormone is: LCLPW LQEGSAFPTIPLSRL FDNAM LRAHR LHQLA FDTYQ EFEEA YIPKE QKYSF LQNPQ TSLCFSESIP TPSNREETQQ KSNLE LLRIS LLLIQ SWLEP VQFLR SVFAN SLVYG ASDSN VYDLLKDLEE GIQTL MGRLEDGSPR TGQIF KQTYS KFDTN SHNDD ALLKN YGLLY CFRKD MDKVETFLRI VQCRS VEGSC GF, and the direction of the sequence is N-terminus-C-terminus; lysine in the amino acid sequence is replaced by lysine labeled with stable isotope, and lysine and arginine in the amino acid sequence are replaced by lysine and arginine labeled with stable isotope, respectively, and the C and N elements in the isotope-labeled lysine and arginine are respectively. 13 C and 15 N.

[0037] Furthermore, step S1 includes: analyzing the stable isotope-labeled recombinant human growth hormone solution to confirm that it is a single band in gel electrophoresis; and then using reversed-phase liquid chromatography or exclusion chromatography to perform purity analysis on the stable isotope-labeled recombinant human growth hormone; the purity analysis includes: setting the detection wavelength of the UV detector at 210-220 nm, and using the peak area normalization method to perform peak area statistics.

[0038] Further, step S2 includes: taking a stable isotope-labeled recombinant human growth hormone solution and placing it in an ampoule, wherein the protein content of the stable isotope-labeled recombinant human growth hormone solution is 0.1 micrograms to 10 micrograms; after drying, adding 500 microliters of concentrated hydrochloric acid thereto, wherein the concentration of the concentrated hydrochloric acid is 6 mol / L; after replacing the air in the ampoule with high-purity nitrogen, hot-melt sealing the ampoule, and then placing it in a high-temperature oven for reaction, wherein the oven temperature is 100°C to 150°C; after the reaction is completed, nitrogen blowing is performed to dry the hydrochloric acid, and then the solution is redissolved with 1000 microliters of formic acid-water solution; and then liquid chromatography-mass spectrometry analysis is performed, wherein the liquid chromatography-mass spectrometry analysis includes: taking the peak area ratio of isotope-labeled lysine to lysine and the peak area ratio of isotope-labeled arginine to arginine as the replacement efficiency of the isotope-labeled amino acids.

[0039] Furthermore, step S3 includes: the quantitative peptides released by trypsin for quantitative analysis include: LFDNAMLR (denoted as T2), SNLELLR (denoted as T8), LEDGSPR (denoted as T12) or TGQIFK (denoted as T13); at least two of the quantitative peptides are synthesized by chemical methods, and each of the quantitative peptides includes a non-isotope labeled quantitative peptide and a stable isotope labeled quantitative peptide; the labeling method of the stable isotope labeled quantitative peptide is as follows: the lysine in the peptide is replaced by the stable isotope labeled lysine, and the arginine in the peptide is replaced by the stable isotope labeled arginine.

[0040] Furthermore, step S3 includes: taking a stable isotope-labeled recombinant human growth hormone solution, wherein the protein content of the stable isotope-labeled recombinant human growth hormone solution is 0.1 micrograms to 50 micrograms, and placing it together with the quantitative peptide solution in a low adsorption centrifuge tube; adding ammonium bicarbonate or Tris-HCl buffer solution to adjust the pH value of the solution to 8; adding DTT solution to react at 95 degrees Celsius for 10 minutes; after the reaction solution is cooled to room temperature, adding IAA solution, and reacting in a light-proof environment for 30 minutes; adding DTT solution again to react to remove excess IAA in the solution, and the reaction time is 30 minutes; adding 1 microgram of trypsin to the reaction solution, and reacting it at 37°C for 16 hours; after the reaction is completed, desalting is performed, and after drying, it is re-dissolved with 0.1% formic acid-water solution, and LC-MS / MS analysis is performed.

[0041] Furthermore, the LC-MS / MS analysis includes: chromatographic separation using reversed-phase liquid chromatography, mass spectrometry data acquisition using a triple quadrupole mass spectrometer or a quadrupole-linear ion trap tandem mass spectrometer, analyzing and detecting the quantitative peptides in a multiple reaction monitoring mode, and calculating the protein content based on the peak area ratio of the non-isotope labeled quantitative peptides and the stable isotope labeled quantitative peptides.

[0042] Furthermore, the LC-MS / MS analysis includes: preparing non-isotope labeled quantitative peptides and corresponding stable isotope labeled quantitative peptide solutions with different mass ratios, establishing a quantitative analysis standard working curve of non-isotope labeled quantitative peptides and stable isotope labeled quantitative peptides with the mass ratio of the non-isotope labeled quantitative peptides and the corresponding stable isotope labeled quantitative peptides as the abscissa and the corresponding mass spectrum peak area ratio as the ordinate, calculating the content of stable isotope labeled peptides obtained by trypsin hydrolysis in the sample according to the regression equation of each quantitative peptide and the peak area ratio of each non-isotope labeled quantitative peptide and the corresponding stable isotope labeled quantitative peptide in the trypsin hydrolysis solution of the sample to be tested, and calculating the content of stable isotope labeled recombinant human growth hormone in the sample to be tested according to the molar mass ratio of the stable isotope labeled peptides to the stable isotope labeled recombinant human growth hormone.

[0043] Furthermore, the LC-MS / MS analysis includes: the peptide content of the quantitative peptide solution is subjected to traceable quantitative analysis using amino acid hydrolysis-isotope dilution mass spectrometry.

[0044] A stable isotope labeled recombinant human growth hormone quantitative analysis device, the device adopts the above method to perform analysis and detection, the device comprises:

[0045] A module for preliminarily measuring protein purity, which preliminarily measures the purity of stable isotope-labeled recombinant human growth hormone based on chromatography or electrophoresis;

[0046] A module for measuring the substitution efficiency of stable isotope-labeled amino acids, wherein the module measures the substitution efficiency of stable isotope-labeled amino acids based on amino acid analysis-isotope dilution mass spectrometry;

[0047] A protein content measurement module, wherein the module measures the content of stable isotope labeled recombinant human growth hormone based on peptide quantitative analysis-isotope dilution mass spectrometry.

[0048] Example 2

[0049] A method for quantitative analysis of stable isotope labeled recombinant human growth hormone.

[0050] (1) Obtaining stable isotope labeled recombinant human growth hormone protein:

[0051] Construct an expression system for lysine and arginine-deficient strains and add stable isotopes to the culture system 13 C. 15 N-labeled lysine ( 13 C 6 H 14 15 N 2 O 2 ), arginine ( 13C 6 H 14 15 N 4 O 2 ), after iterative cultivation, a high-purity stable isotope-labeled recombinant human growth hormone protein (hGH*) solution is obtained, and its protein sequence is as follows Figure 1 shown.

[0052] (2) Preliminary measurement of the purity of stable isotope-labeled recombinant human growth hormone based on chromatography or electrophoresis:

[0053] The purity of the protein was characterized by SDS-PAGE electrophoresis, and the electrophoresis test conditions were as follows: 22.4 μg of hGH* raw material and 22.4 μg of growth hormone standard material (hereinafter referred to as hGH CRM, WHO standard material, number NIBSC 98 / 574) were mixed with 1 / 4 volume of 5X electrophoresis loading buffer, boiled in a boiling water bath for 5 minutes and then loaded, separated by Mini-PROTEAN precast gel, electrophoresis voltage 120V, electrophoresis time 45 minutes. After the electrophoresis was completed, it was stained with Coomassie brilliant blue for 30 minutes, and after staining, it was decolorized with methanol-acetic acid solution and imaged. The results are shown in Error! Reference source not found., hGH* and hGH CRM show similar electrophoretic bands on gel electrophoresis, with a molecular weight of about 23 kDa, which indicates that the structure and charge properties of the proteins in the two samples are consistent. Since hGH* is replaced with stable isotope labeled lysine and arginine, its molecular weight is slightly larger and its band migration rate is slightly lower than that of hGH CRM. It can be seen from the band of hGH* that no obvious bands of other proteins are found in its lane, indicating that the purity of the solution is relatively high.

[0054] The measurement was performed using a Waters e2695 HPLC system (equipped with a PDA detector) and a ChromCore 300C4-T (5 μm, 4.6 × 250 mm) column; the mobile phase was H 2 O (containing 0.1% TFA, v / v), B phase is acetonitrile (containing 0.1% TFA, v / v); elution gradient: 0-5min, 2% B; 5-10min, 2%-30% B; 10-35min, 30%-65% B; 35-37.5min, 65% B; 37.5-40min, 65%-2% B; 40-50min, 2% B. UV detector @210nm; flow rate: 1mL / min; column temperature: 30℃; injection volume 30μL.

[0055] Impurities in hGH* solution are other proteins and protein aggregates. 210nm is selected as the detection wavelength to ensure sufficient signal response of organic matter. Figure 3 It can be seen that there are two compounds with high content in the solution, with retention times of 33.07min and 34.54min respectively. The signal at 33.07min is consistent with the retention time of hGH CRM 32.89min, which is the chromatographic response signal of hGH* protein. The signal at 34.54min is a protein impurity after UV spectroscopy analysis. The purity of hGH* protein in the sample was quantitatively analyzed by peak area normalization method, and the purity of hGH* was 94.06%.

[0056] (3) Measurement of stable isotope labeled amino acid substitution efficiency based on amino acid analysis-isotope dilution mass spectrometry:

[0057] Take 50μL of hGH* protein solution, desalt it and place it in a 2mL ampoule. After vacuum centrifugation and drying at 55℃, add 500μL of 6M hydrochloric acid solution to each tube, pass high-purity nitrogen to deoxygenate for 2min, seal the ampoule, and then place it in a 110℃ oven for hydrolysis for 48 hours. After the hydrolysis is complete, take it out and blow it dry with a nitrogen blower, then add 1mL of 0.1% formic acid aqueous solution to each tube for re-dissolution, filter the solution through a 0.22μm filter membrane, and place it in a 2mL liquid phase vial for detection. The measuring instrument is Shimadzu LC-20A UHPLC tandem AB 6500+ triple quadrupole mass spectrometer. Chromatographic method: Column: Waters ChromCoreAQ C18 column (1.8um, 2.1μm×100mm), mobile phase: 98% water / 2% acetonitrile (v / v, containing 0.8% formic acid), isocratic elution for 6min, flow rate of 250μL / min, column temperature set to 40℃, injection volume of 10μL, repeated injection three times, and the average value of the measurement was taken. Mass spectrometry data acquisition adopted MRM mode, in which the optimized MS 2 The mass spectrometry data acquisition method is shown in Table 1.

[0058] Table 1 MS of lysine, arginine and their stable isotope labeled amino acids 2 Mass spectrometry data acquisition method

[0059]

[0060] The extracted ion chromatograms of lysine, arginine and their isotope-labeled amino acids obtained based on the LC-MS / MS method are shown in Figure 4 According to the area normalization method, the labeling efficiency of stable isotope labeled lysine and arginine reached 93.19% (RSD=0.12%, n=3) and 93.59% (RSD=0.65%, n=3), respectively, as shown in Table 2.

[0061] Table 2 Labeling efficiency of hGH* stable isotope labeled lysine and arginine

[0062]

[0063] (4) Peptide quantitative analysis-isotope dilution mass spectrometry to measure the content of stable isotope-labeled recombinant human growth hormone:

[0064] The quantitative peptides of growth hormone LFDNAMLR (denoted as T2) and TGQIFK (denoted as T13) were chemically synthesized, and their corresponding isotope-labeled peptides were synthesized. The labeling method was as follows: lysine (K) and arginine (R) were labeled with stable isotope amino acids K*( 13 C 6 , 15 N 2 ) and R*( 13 C 6 , 15 N 4 ) was replaced. Three stable amino acids (leucine L, isoleucine I, and phenylalanine F) were used to establish an MRM mass spectrometry measurement method to quantify the synthesized isotope-labeled peptides. The chromatographic conditions were as follows: the chromatographic column was a ChromCore AQ C18 column (1.8 μm, 2.1 mm × 100 mm); the mobile phase was 98% water / 2% acetonitrile (v / v, containing 0.8% formic acid), isocratic elution for 6 min, and the flow rate was 250 μL / min. Ion source parameters and MS 2 The mass spectrometry data acquisition conditions are shown in Table 3.

[0065] Table 3 Mass spectrometry acquisition conditions and monitored ion pairs during HPLC-ID-MS analysis

[0066]

[0067] The standard curve was drawn with the mass ratio of amino acid standard substance to its labeled amino acid (0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1, 2:1) as the gradient. The concentrations of the synthetic isotope-labeled peptides T2* and T13* were measured to be 1.05 mg / g (RSD = 2.62%) and 0.86 mg / g (RSD = 1.56%), respectively.

[0068] Accurately weigh 20 micrograms of stable isotope labeled recombinant human growth hormone solution, and accurately weigh the solutions of the synthesized quantitative peptides T2 and T13 and place them in a low adsorption centrifuge tube, dilute to 100 μL with 50 mM ammonium bicarbonate solution, then add DTT solution and react at 95 degrees Celsius for 10 minutes, cool to room temperature, add IAA solution, and react for 30 minutes in a light-proof environment, add DTT solution again to remove excess IAA in the solution, add 1 microgram of trypsin to the solution after 30 minutes of reaction, and place it at a constant temperature of 37 degrees Celsius for 16 hours. After the reaction is completed, desalt, dry and re-dissolve with 0.1% formic acid-water solution, and perform LC-MS / MS analysis.

[0069] Chromatographic separation was performed by reversed phase liquid chromatography, the chromatographic column was Aeris Peptide XB-C18 column (3.6 μm, 2.1×250 mm, Phenomenex), elution conditions: phase A was water (containing 0.1% FA), phase B was acetonitrile (containing 0.1% FA), elution gradient: 0-6 min, 98% A, 6-15 min, 98%-91% A, 15-70 min, 91%-74.5% A, flow rate was 0.3 mL / min, column temperature was set to 45°C. Mass spectrometry data were collected using an AB 6500+ triple quadrupole mass spectrometer, and the selected quantitative peptides and their isotope-labeled peptides were detected in multiple reaction monitoring mode. MS 2 The mass spectrometry data acquisition conditions are shown in Table 4. The extracted ion chromatograms of peptides T2, T13 and their isotope-labeled peptides based on the LC-MS / MS method are shown in Figure 5 shown.

[0070] Table 4 MS analysis of peptides that can be used for traceable quantitative analysis of hGH* protein 2 Mass spectrometry data acquisition conditions

[0071]

[0072] A series of mixed solutions of T2* / T2 and T13* / T13 with different mass ratios were prepared and analyzed by liquid chromatography-mass spectrometry. The mass ratio of the injected high-purity quantitative peptide and its corresponding isotope-labeled peptide was used as the horizontal coordinate, and the mass spectrum peak area ratio was used as the vertical coordinate to establish a quantitative analysis standard working curve of the quantitative peptide / isotope-labeled peptide. According to the regression equation of each quantitative peptide and the peak area ratio of each quantitative peptide in the enzymatic hydrolyzate to the corresponding isotope-labeled peptide, the content of the isotope-labeled peptide obtained by enzymatic hydrolysis in the measured sample was calculated. Then, according to the molar mass ratio of the isotope-labeled peptide to the stable isotope-labeled recombinant human growth hormone, the content of the stable isotope-labeled recombinant human growth hormone in the measured sample was calculated.

[0073] After calculation, the contents of stable isotope-labeled recombinant human growth hormone in the samples measured by the two quantitative peptides T2* and T13* were 0.251 mg / g (RSD=2.12%, n=3) and 0.237 mg / g (RSD=1.88%, n=3), respectively. The average of the two was taken as the final measured concentration, which was 0.244 mg / g. The quantitative peptides T2* and T13* used in this measurement have been assigned values ​​by amino acid standard substances by the AAA-IDMS method, respectively. Therefore, the content of stable isotope-labeled recombinant human growth hormone in the measurement results can also be traced back to the international system of units through an uninterrupted traceability chain.

[0074] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for quantitative analysis of stable isotope labeled recombinant human growth hormone, characterized in that: The method comprises the following steps: S1 Preliminary measurement of the purity of stable isotope labeled recombinant human growth hormone based on chromatography or electrophoresis; S2, based on the preliminary determination of the purity of the stable isotope-labeled recombinant human growth hormone in step S1, measuring the substitution efficiency of the stable isotope-labeled amino acid by amino acid analysis-isotope dilution mass spectrometry; S3 measures the content of stable isotope-labeled recombinant human growth hormone by peptide quantitative analysis-isotope dilution mass spectrometry based on the stable isotope-labeled amino acid replacement efficiency.

2. The method according to claim 1, characterized in that: The amino acid sequence of the stable isotope-labeled recombinant human growth hormone is: LCLPW LQEGS AFPTIPLSRL FDNAM LRAHR LHQLA FDTYQ EFEEA YIPKE QKYSFLQNPQ TSLCF SESIP TPSNR EETQQ KSNLE LLRIS LLLIQ SWLEP VQFLR SVFAN SLVYG ASDSNVYDLL KDLEE GIQTL MGRLE DGSPR TGQIF KQTYS KFDTN SHNDD ALLKN YGLLY CFRKD MDKVETFLRI VQCRS VEGSC GF, and the direction of the sequence is N-terminus-C-terminus; lysine and arginine in the amino acid sequence are replaced by stable isotope-labeled lysine and arginine, respectively, and the C and N elements in the isotope-labeled lysine and arginine are respectively. 13 C and 15 N.

3. The method according to claim 2, characterized in that: Step S1 includes: performing purity analysis on the stable isotope-labeled recombinant human growth hormone by reverse phase liquid chromatography or size exclusion chromatography.

4. The method according to claim 3, characterized in that: Step S2 includes: taking a stable isotope-labeled recombinant human growth hormone solution and placing it in an ampoule, adding 500 microliters of concentrated hydrochloric acid after drying, wherein the concentration of the concentrated hydrochloric acid is 6 mol / L; replacing the air in the ampoule with high-purity nitrogen, hot-melt sealing the ampoule, and then placing it in a high-temperature oven for reaction, wherein the oven temperature is 100°C to 150°C; after the reaction is completed, drying with nitrogen to remove the hydrochloric acid, and then re-dissolving with 1000 microliters of formic acid-water solution; and then performing liquid chromatography-mass spectrometry analysis.

5. The method according to claim 4, characterized in that: Step S3 includes: the quantitative peptides used for quantitative analysis include: LFDNAM LR, SNLELLR, LEDGSPR or TGQIFK; at least two of the quantitative peptides are synthesized by chemical methods, each of the quantitative peptides includes a non-isotope labeled quantitative peptide and a stable isotope labeled quantitative peptide; the labeling method of the stable isotope labeled quantitative peptide is: lysine in the peptide is replaced by lysine labeled by the stable isotope, and arginine in the peptide is replaced by arginine labeled by the stable isotope.

6. The method according to claim 5, characterized in that: Step S3 includes: taking a stable isotope labeled recombinant human growth hormone solution and a quantitative peptide solution and placing them together in a low adsorption centrifuge tube; adding ammonium bicarbonate or Tris-HCl buffer solution to adjust the solution pH to 8; adding DTT solution to react at 95 degrees Celsius for 10 minutes; after the reaction solution is cooled to room temperature, adding IAA solution, and reacting in a light-proof environment for 30 minutes; adding DTT solution again to react and remove excess IAA in the solution, the reaction time is 30 minutes; adding 1 microgram of trypsin to the reaction solution, and reacting at 37°C for 16 hours; and then performing LC-MS / MS analysis.

7. The method according to claim 6, characterized in that: The LC-MS / MS analysis includes: analyzing and detecting the quantitative peptide segment in a multiple reaction monitoring mode, and calculating the protein content according to the peak area ratio of the non-isotope labeled quantitative peptide segment and the stable isotope labeled quantitative peptide segment.

8. The method according to claim 7, characterized in that: The LC-MS / MS analysis comprises: preparing non-isotope labeled quantitative peptides and corresponding stable isotope labeled quantitative peptide solutions of different mass ratios, establishing a quantitative analysis standard working curve of the non-isotope labeled quantitative peptides and the stable isotope labeled quantitative peptides, calculating the content of the stable isotope labeled peptides obtained by trypsin hydrolysis in the sample, and calculating the content of the stable isotope labeled recombinant human growth hormone in the sample to be tested.

9. The method according to claim 8, characterized in that: The LC-MS / MS analysis includes: the peptide content of the quantitative peptide solution is subjected to traceable quantitative analysis using amino acid hydrolysis-isotope dilution mass spectrometry.

10. A stable isotope labeled recombinant human growth hormone quantitative analysis device, the device adopts the method according to any one of claims 1 to 9 for analysis and detection, the device comprising: A module for preliminarily measuring protein purity, which preliminarily measures the purity of stable isotope-labeled recombinant human growth hormone based on chromatography or electrophoresis; A module for measuring the substitution efficiency of stable isotope-labeled amino acids, wherein the module measures the substitution efficiency of stable isotope-labeled amino acids based on amino acid analysis-isotope dilution mass spectrometry; A protein content measurement module, wherein the module measures the content of stable isotope labeled recombinant human growth hormone based on peptide quantitative analysis-isotope dilution mass spectrometry.

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