Mixed polypeptide analysis method

Abstract

The invention discloses a mixed polypeptide analysis method, which uses a volume exclusion chromatographic column in a mixed mode, carries out separation by combining the hydrophobicity difference of samples on the basis of a common volume exclusion chromatographic column, and can effectively solve the problem that the common volume exclusion chromatographic column is difficult to effectively separate samples with small molecular weight difference. Especially, different polypeptide components with similar molecular weights in the mixed polypeptide can be effectively separated, the steps are simple and easy to operate, and rapid and efficient analysis of the components of the mixed polypeptide can be realized without connecting other analysis methods in series.

Description

Technical Field

[0001] The invention relates to a liquid chromatography analysis method, in particular to a mixed polypeptide analysis method. Background Art

[0002] Peptides are biologically active molecules composed of amino acids connected by peptide bonds. They are widely present in organisms and have important biological functions and pharmaceutical values. In recent years, with the rapid development of biotechnology and the pharmaceutical industry, peptides have been increasingly used in new drug development, diagnostic reagents, functional foods and other fields. However, the complexity of peptide molecules and the mixed peptides often generated during the preparation process have brought great challenges to their separation and purification.

[0003] At present, the main methods for peptide separation include the following:

[0004] 1. Electrophoresis techniques, such as capillary electrophoresis and two-dimensional electrophoresis, are commonly used to analyze and separate peptide samples. This method has the characteristics of high resolution, but the equipment cost is high and it is difficult to apply it to industrial production on a large scale. In addition, the separation conditions during the electrophoresis process may cause some peptides to degrade.

[0005] 2. Ultrafiltration technology uses the pore size selectivity of the membrane to separate polypeptides of different molecular weights. This method is easy to operate and suitable for large-scale separation. However, for polypeptides with similar molecular weights, its separation accuracy is low, and long-term use may cause membrane pollution and clogging problems;

[0006] 3. Reversed-phase high performance liquid chromatography is a separation method based on the hydrophobicity difference of peptides. By changing the concentration of organic solvent in the mobile phase, gradient elution separation of peptides can be achieved. However, for complex peptide mixtures, this method may require multiple fractionation separations, which is complicated to operate and time-consuming. At the same time, high concentrations of organic solvents may affect the activity of peptides;

[0007] 4. Size exclusion chromatography is a classic separation technology based on differences in molecular size and shape. When the sample passes through a stationary phase containing a gel with a specific pore size, large molecules flow out preferentially, while small molecules are retained due to entering the gel pores. However, the resolution of this method is limited, especially for polypeptides with similar molecular weights, the separation effect is poor.

[0008] In summary, existing peptide separation technologies have their own limitations in terms of resolution, operating cost and scope of application. In particular, when a mixed peptide sample contains multiple components of similar molecular weight, existing technologies often cannot achieve efficient separation. Therefore, in order to meet the needs of scientific research and industrial production for high-purity peptides, it is of great theoretical significance and practical application value to develop a new peptide separation technology based on molecular weight differences that is highly selective, efficient and low-cost. Summary of the invention

[0009] Purpose of the invention: The purpose of the present invention is to provide an analytical method for separating different types of polypeptides in a mixed polypeptide.

[0010] Technical solution: The mixed polypeptide analysis method of the present invention comprises the following steps:

[0011] (1) preparing a mixed polypeptide sample to be tested;

[0012] (2) Setting the parameters of chromatographic conditions on the high performance liquid chromatograph;

[0013] (3) Injecting the sample solution to be tested into a high performance liquid chromatograph, analyzing and recording the chromatogram.

[0014] Preferably, in the mixed polypeptide analysis method of the present invention, the chromatographic condition parameters set on the high performance liquid chromatograph in step 2 include:

[0015] (21) Chromatographic column, mixed mode size exclusion chromatography column;

[0016] (22) an eluent comprising a mobile phase A and a mobile phase B, wherein the mobile phase A is a trifluoroacetic acid-acetonitrile solution, wherein the concentration of trifluoroacetic acid in the trifluoroacetic acid-acetonitrile solution is 0.05-0.2%, and the mobile phase B is a trifluoroacetic acid-water solution, wherein the concentration of trifluoroacetic acid in the trifluoroacetic acid-water solution is 0.05-0.2%;

[0017] (23) Elution mode: isocratic elution, wherein the content of mobile phase A is 10-50%: the content of mobile phase B is 50-90%, and the flow rate is 0.3-0.7 mL / min;

[0018] (24) Elution temperature: column temperature is 23-27°C;

[0019] (25) Detection wavelength, the wavelength is 272-280nm.

[0020] Preferably, in the mixed polypeptide analysis method of the present invention, the mixed mode size exclusion chromatography column in step 21 is a Sepax BioMix SEC chromatography column with a specification of 7.8×300 mm, 3 μm, In the eluent described in step 22, the concentration of trifluoroacetic acid in mobile phase A is 0.08-0.12%, and the concentration of trifluoroacetic acid in mobile phase B is 0.08-0.12%; in step 23, the content of mobile phase A is 25-35%, the content of mobile phase B is 65-75%, and the flow rate is 0.4-0.6 mL / min; in step 24, the column temperature is 24-26° C.; in step 25, the detection wavelength is 275-277 nm.

[0021] Preferably, the mixed polypeptide analysis method of the present invention is characterized in that, in the mixed polypeptide sample to be tested in step 1, the relative molecular weight difference of each polypeptide component is no more than 1000Da.

[0022] Preferably, in the mixed polypeptide analysis method of the present invention, in step 3, the volume of the sample injected into the high performance liquid chromatograph is 15-25 μL, and during the detection process, the separation degree of the mixed polypeptide is not less than 0.6.

[0023] Preferably, in the mixed polypeptide analysis method of the present invention, the high performance liquid chromatograph comprises an Agilent 1260 high performance liquid chromatograph equipped with an ultraviolet detector.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. It can effectively separate different polypeptide components with similar molecular weights in a mixed polypeptide; 2. It can effectively analyze the components of the mixed polypeptide without the need to use other methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the chromatogram of peptide A analysis based on a mixed-mode size exclusion chromatography column;

[0026] Figure 2 This is the chromatogram of peptide B analysis based on a mixed-mode size exclusion chromatography column;

[0027] Figure 3 This is the chromatogram of the analysis of mixed peptides A+B based on a mixed-mode size exclusion chromatography column;

[0028] Figure 4 This is the chromatogram of peptide A analysis based on a common size exclusion chromatography column;

[0029] Figure 5 This is the chromatogram of peptide B analysis based on a common size exclusion chromatography column;

[0030] Figure 6 This is the chromatogram of the analysis of mixed peptides A+B based on a common size exclusion chromatography column. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below.

[0032] sample:

[0033] Peptide A: semaglutide (molecular weight approximately 4113Da)

[0034] Polypeptide B: Liraglutide (molecular weight approximately 3751Da)

[0035] Example 1: Purity analysis of mixed polypeptides based on mixed-mode size exclusion chromatography columns 1. Prepare samples to be tested, including polypeptide A, polypeptide B, and a 1:1 mixture of polypeptides A+B, wherein the concentration of the polypeptides is 7 mg / mL; 2. Set the parameters of the chromatographic conditions on an Agilent 1260 high performance liquid chromatograph equipped with a UV detector:

[0036] (1) Chromatographic column, Sepax BioMix SEC chromatographic column, specification: 7.8×300 mm, 3 μm, Item No. 214300-7830;

[0037] (2) an eluent, comprising a mobile phase A and a mobile phase B, wherein the mobile phase A is a 0.1% trifluoroacetic acid-100% acetonitrile solution, and the mobile phase B is a 0.1% trifluoroacetic acid-water solution;

[0038] (3) Elution mode: isocratic elution, where the content of mobile phase A is 30%: the content of mobile phase B is 70%, and the flow rate is 0.5 mL / min;

[0039] (4) Column temperature is 25°C;

[0040] (5) Detection wavelength: 276 nm;

[0041] (3) Analysis and detection: Samples of polypeptide A, polypeptide B, and mixed polypeptide A+B were extracted and injected into a high performance liquid chromatograph with a sample volume of 20 μL for analysis and recording of the chromatograms.

[0042] Peptide A, peptide B, and mixed peptide A+B were analyzed and tested respectively. The results are as follows: Figure 1-3 As shown in the figure, from the perspective of retention time: the retention time of polypeptide A is 33.808min, the retention time of polypeptide B is 39.318min, and the retention time of mixed polypeptide A+B is 36.166min (polypeptide A) and 38.851min (polypeptide B), respectively. The retention time of polypeptide A in mixed polypeptide A+B is slightly later. The analysis reason may be that due to the differences in chemical structure and properties between different polypeptides, chemical properties may change due to some interaction force after mixing; from the perspective of separation effect: polypeptide A and polypeptide B in mixed polypeptide A+B show slight asymmetry due to the strong hydrophobic interaction with the chromatographic column, but these deviations do not affect the accuracy and repeatability of integration, which is usually acceptable. In addition, the separation degree of polypeptide A and polypeptide B in mixed polypeptide A+B reaches 0.63, with obvious peak-to-valley ratio, and effective separation is achieved. The peak area of ​​polypeptide A accounts for 50.21%, and the peak area of ​​polypeptide B accounts for 24.32%. The results are in line with expectations, indicating that this method can be effectively used for the analysis of various components of mixed polypeptides.

[0043] Comparative Example 1: Purity analysis of mixed peptides based on ordinary size exclusion chromatography columns

[0044] 1. Prepare the samples to be tested, including polypeptide A, polypeptide B and a 1:1 mixture of polypeptide A+B, where the polypeptide concentration is 7 mg / mL;

[0045] 2. Set the parameters of the chromatographic conditions on an Agilent 1260 HPLC equipped with a UV detector:

[0046] (1) Chromatographic column: Zenix-C SEC-300 column, specification: 7.8×300 mm, 3 μm, Item No. 233300-7830;

[0047] (2) an eluent, comprising a mobile phase A and a mobile phase B, wherein the mobile phase A is a 0.1% trifluoroacetic acid-100% acetonitrile solution, and the mobile phase B is a 0.1% trifluoroacetic acid-water solution;

[0048] (3) Elution mode: isocratic elution, where the content of mobile phase A is 30%: the content of mobile phase B is 70%, and the flow rate is 0.5 mL / min;

[0049] (4) Column temperature is 25°C;

[0050] (5) Detection wavelength: 276 nm;

[0051] (3) Analysis and detection: Samples of polypeptide A, polypeptide B, and mixed polypeptide A+B were extracted and injected into a high performance liquid chromatograph with a sample volume of 20 μL for analysis and recording of the chromatograms.

[0052] Peptide A, peptide B, and mixed peptide A+B were analyzed and tested respectively. The results are as follows: Figure 4-6 As shown in the figure, from the perspective of retention time: the retention time of polypeptide A is 36.015min, the retention time of polypeptide B is 26.492min, and the retention time of polypeptide A+B is 25.733min. The retention times of polypeptide A and polypeptide B do not conform to the conventional size exclusion principle. The reason for the analysis may be that there is a strong interaction between the sample and the chromatographic column (such as adsorption, charge interaction, etc.), which slows down the diffusion rate of the molecules in the gel. In this case, the retention time will no longer be determined solely by the size of the molecule, but will be affected by the interaction force.

[0053] From the perspective of separation effect: polypeptide A and polypeptide B in mixed polypeptide A+B are not effectively separated, and the peaks overlap. The peak area of ​​mixed polypeptide A+B is 32207, the peak area of ​​polypeptide A is 26583, and the peak area of ​​polypeptide B is 6484. By comparing the peak areas, it can be seen that mixed polypeptide A+B contains polypeptide A and polypeptide B, with no obvious signs of separation, and component analysis cannot be performed.

[0054] In summary, through comparative analysis of Example 1 and Comparative Example 1, it is found that the analysis method of the present invention, by utilizing the hydrophobic difference between polypeptide A and polypeptide B, can effectively separate the mixed polypeptide A+B and perform purity analysis.

Claims

1. A mixed peptide analysis method, characterized in that: The following steps are involved: (1) preparing a mixed polypeptide sample to be tested; (2) Setting the parameters of chromatographic conditions on the high performance liquid chromatograph; (3) Injecting the sample solution to be tested into a high performance liquid chromatograph, analyzing and recording the chromatogram.

2. The mixed polypeptide analysis method according to claim 1, characterized in that: The chromatographic condition parameters set on the high performance liquid chromatograph in step 2 include: (21) Chromatographic column, mixed mode size exclusion chromatography column; (22) an eluent comprising a mobile phase A and a mobile phase B, wherein the mobile phase A is a trifluoroacetic acid-acetonitrile solution, wherein the concentration of trifluoroacetic acid in the trifluoroacetic acid-acetonitrile solution is 0.05-0.2%, and the mobile phase B is a trifluoroacetic acid-water solution, wherein the concentration of trifluoroacetic acid in the trifluoroacetic acid-water solution is 0.05-0.2%; (23) Elution mode: isocratic elution, wherein the content of mobile phase A is 10-50%: the content of mobile phase B is 50-90%, and the flow rate is 0.3-0.7 mL / min; (24) Elution temperature: column temperature is 23-27°C; (25) Detection wavelength, the wavelength is 272-280nm.

3. The mixed polypeptide analysis method according to claim 2, characterized in that: The mixed-mode size exclusion chromatography column in step 21 is a Sepax BioMix SEC chromatography column with specifications of 7.8×300 mm, 3 μm, 4. The mixed polypeptide analysis method according to claim 2, characterized in that: In the eluent of step 22, the concentration of trifluoroacetic acid in mobile phase A is 0.08-0.12%, and the concentration of trifluoroacetic acid in mobile phase B is 0.08-0.12%.

5. The mixed polypeptide analysis method according to claim 2, characterized in that: In step 23, the content of mobile phase A is 25-35%, the content of mobile phase B is 65-75%, and the flow rate is 0.4-0.6 mL / min.

6. The mixed polypeptide analysis method according to claim 2, characterized in that: The column temperature in step 24 is 24-26°C.

7. The mixed polypeptide analysis method according to claim 2, characterized in that: The detection wavelength in step 25 is 275-277 nm.

8. The mixed polypeptide analysis method according to claim 1, characterized in that: In the mixed polypeptide sample to be tested in step 1, the relative molecular weight difference of each polypeptide component is no more than 1000 Da.

9. The mixed polypeptide analysis method according to claim 1, characterized in that: In step 3, the volume of the sample injected into the high performance liquid chromatograph is 15-25 μL, and during the detection process, the separation degree of the mixed polypeptides is not less than 0.

6.

10. The mixed polypeptide analysis method according to claim 1, characterized in that: The high performance liquid chromatograph includes an Agilent 1260 high performance liquid chromatograph equipped with an ultraviolet detector.

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