Method for identifying polypeptide sample mass spectrum detection adduct and application thereof

Abstract

The invention relates to the field of analysis, in particular to a method for identifying a polypeptide sample mass spectrum detection adduct and application of the method. The method comprises the following steps: performing chromatographic detection and mass spectrometric detection on a polypeptide sample; respectively performing in-source collision analysis under the first energy and the second energy, extracting an ion current, and calculating a difference value of a relative principal component ratio of the compound under the condition of the second energy and the first energy; wherein the second energy is greater than the first energy; if the difference value of the relative principal component ratio of the compound under the condition of the second energy and the first energy is greater than or equal to 0.05%, judging that the compound is a mass spectrometric detection adduct; if the difference value of the relative principal component ratio of the compound under the condition of the second energy and the first energy is lt; if the content is 0.05%, judging that the compound is an impurity. The method can accurately identify the polypeptide sample mass spectrometric detection adduct, and has the advantages of short analysis time, high analysis sensitivity, simplicity in operation and the like.

Description

Technical Field

[0001] The present invention relates to the field of analysis, and in particular to a method for identifying polypeptide sample adducts by mass spectrometry detection and its application. Background Art

[0002] Polypeptides are a class of compounds formed by multiple amino acids connected by peptide bonds, usually composed of 10 to 100 amino acid molecules, and their connection method is the same as that of proteins, with a relative molecular mass of less than 10,000. Polypeptides are commonly found in organisms. So far, tens of thousands of polypeptides have been found in organisms. They are widely involved in and regulate the functional activities of various systems, organs, tissues and cells in the body, and play an important role in life activities.

[0003] In recent years, peptide drugs synthesized using modern biotechnology have become one of the hot spots in drug research and development. Due to their wide indications, high safety and significant efficacy, they have been widely used in the prevention, diagnosis and treatment of diseases such as tumors, hepatitis, diabetes, and AIDS, and have broad development prospects. However, due to the side effects that are prone to occur during the synthesis of peptide drugs and the diversity of peptide structures, Separation and purification The process is also extremely challenging, resulting in the inability to fully guarantee the purity and quality of peptide drugs. The immunogenicity of peptide impurities is an important issue. Some peptide impurities can produce immunogenicity through a variety of mechanisms, such as epitope presentation, mimicking pathogens, and activating pattern recognition receptors of the innate immune system, which may lead to adverse side effects such as the production of drug-resistant antibodies and allergic reactions. Therefore, the impurity research and control of peptide drugs is one of the core contents of drug quality control.

[0004] Liquid chromatography-mass spectrometry (LC-MS) is used to study peptide impurities, which has the advantages of short analysis time, high analytical sensitivity, and simple operation. However, some adducts are easily produced during mass spectrometry detection, resulting in some false positive impurities. At present, there is no relevant literature report on the research on mass spectrometry detection of adducts of peptide samples.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In view of the defects in the existing analytical methods for determining adducts by mass spectrometry detection of polypeptide samples, the present invention aims to provide a method for identifying adducts by mass spectrometry detection of polypeptide samples and its application, which adopts liquid chromatography-mass spectrometry (LC-MS) to determine adducts by mass spectrometry detection of polypeptide samples, and has the advantages of short analysis time, high analysis sensitivity and simple operation.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] One aspect of the present invention relates to a method for identifying a polypeptide sample adduct by mass spectrometry detection, comprising the following steps:

[0009] (a) performing chromatography and mass spectrometry on the polypeptide sample; performing in-source collision analysis at a first energy and a second energy, respectively, extracting ion currents, and calculating the difference in relative principal component ratios of the compound under the second energy and the first energy conditions;

[0010] wherein the second energy is greater than the first energy;

[0011] (b) if the difference between the relative main component ratio of the compound under the second energy condition and the first energy condition is ≥0.05%, the compound is judged to be a mass spectrometry detection adduct; if the difference between the relative main component ratio of the compound under the second energy condition and the first energy condition is <0.05%, the compound is judged to be an impurity.

[0012] The method for identifying polypeptide sample mass spectrometry adducts can accurately identify polypeptide sample mass spectrometry adducts and has the advantages of short analysis time, high analysis sensitivity and simple operation.

[0013] Preferably, the first energy is 0 eV.

[0014] Preferably, the second energy is 20-40 eV.

[0015] Preferably, the mobile phase A of the chromatographic detection is ultrapure water containing 0.05 vol% to 0.1 vol% trifluoroacetic acid, and the mobile phase B is acetonitrile containing 0.05 vol% to 0.1 vol% trifluoroacetic acid.

[0016] Preferably, the elution gradient of the chromatographic detection is as follows:

[0017] 0min, 90%-95% mobile phase A, 5%-10% mobile phase B; 4min, 55%-60% mobile phase A, 40%-45% mobile phase B; 28min, 45%-50% mobile phase A, 50%-55% mobile phase B; 35min, 5%-10% mobile phase A, 90%-95% mobile phase B; 36min, 90%-95% mobile phase A, 5%-10% mobile phase B; 41min, 90%-95% mobile phase A: 5%-10% mobile phase B.

[0018] Preferably, the conditions for the chromatographic detection are as follows:

[0019] Detection wavelength 210-220 nm; flow rate 0.3-0.4 mL / min; injection volume 10-20 μL; column temperature 55-60°C; injection plate temperature 5-10°C.

[0020] Preferably, the chromatographic column for chromatographic detection comprises: an ACQUITY UPLC BEH C18 chromatographic column.

[0021] Preferably, the conditions for mass spectrometry detection are as follows:

[0022] The detection mode was positive mode; the scanning range was 200-2000 m / z.

[0023] Preferably, the concentration of the polypeptide sample is 1-5 mg / mL.

[0024] Another aspect of the present invention also relates to a method for analyzing impurities in a polypeptide sample, including the method for identifying adducts of a polypeptide sample by mass spectrometry detection.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The method for identifying a polypeptide sample mass spectrometry adduct provided by the present invention determines whether a compound is a mass spectrometry adduct by changing the energy of the collision in the source and comparing the change in the relative main component ratio of the compound under different energies. The method has a short analysis time. When the method described in the present invention is used for detection, it only takes a few hours from sample pretreatment to result analysis. The operation is simple, the analysis is fully automated, and the detection is completed by an instrument. No matter whether there is an experimental basis or not, the operation can be performed as long as the operation training is carried out. DETAILED DESCRIPTION

[0027] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be appreciated by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, all belong to the scope of protection of the present invention. If specific conditions are not indicated in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0028] One aspect of the present invention relates to a method for identifying a polypeptide sample adduct by mass spectrometry detection, comprising the following steps:

[0029] (a) performing chromatography and mass spectrometry on the polypeptide sample; performing in-source collision analysis at a first energy and a second energy, respectively, extracting ion currents, and calculating the difference in relative principal component ratios of the compound under the second energy and the first energy conditions;

[0030] wherein the second energy is greater than the first energy;

[0031] (b) if the difference between the relative main component ratio of the compound under the second energy condition and the first energy condition is ≥0.05%, the compound is judged to be a mass spectrometry detection adduct; if the difference between the relative main component ratio of the compound under the second energy condition and the first energy condition is <0.05%, the compound is judged to be an impurity.

[0032] The energy of in-source CID in mass spectrometry is usually used to remove neutral molecules bound to the analyte to be detected, thereby improving the signal-to-noise ratio of the analyte to be detected. However, when the energy of in-source collision reaches a certain value, it will cause in-source fragmentation of the analyte to be detected, such as molecular breakage or neutral loss.

[0033] The present invention determines whether a compound is a mass spectrometry detection adduct by changing the energy of the collision in the source and comparing the changes in the relative main component ratio of the compound under different energies.

[0034] The relative main component ratio of mass spectrometry adducts changed significantly at different energies, with a ratio change of ≥0.05%, while the ratio of other non-mass spectrometry adducts remained basically unchanged, with a ratio change of <0.05%. The reason for the above phenomenon is that the energy of the collision in the source has different effects on mass spectrometry adducts and non-mass spectrometry adducts, resulting in different ratio changes.

[0035] The method for identifying adducts of polypeptide samples by mass spectrometry detection has short analysis time and simple operation, and solves the problem of false positive impurities caused by adducts introduced into polypeptide samples during mass spectrometry detection.

[0036] Using the detection method of the present invention, the mass spectrometry detection adduct of the polypeptide sample is determined, and the detection time is less than 41 minutes.

[0037] Furthermore, the first energy is 0 eV.

[0038] Furthermore, the second energy is 20-40 eV, including but not limited to any point value of 20 eV, 24 eV, 28 eV, 32 eV, 36 eV or 40 eV or a range value between any two of them.

[0039] The present invention designs specific values ​​of the first energy and the second energy in combination with the effect of the energy of the collision within the source on the analyte to be measured. Within this energy range, mass spectrometry analysis is performed on the samples to be measured respectively, and the changes in the proportion of the main components produced are sufficient to distinguish between mass spectrometry detection adducts and impurities.

[0040] Furthermore, the concentration of the polypeptide sample is 1-5 mg / mL, including but not limited to any point value of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL or 5 mg / mL or a range value between any two of them. Limiting the concentration of the polypeptide sample to a reasonable range is conducive to accurate separation of the compounds during chromatographic detection.

[0041] Furthermore, the solvent of the polypeptide sample includes but is not limited to: 45 vol% to 55 vol% (for example, it can be but is not limited to any one of 45 vol%, 47 vol%, 49 vol%, 51 vol%, 53 vol% or 55 vol% or a range between any two) acetonitrile aqueous solution.

[0042] The present invention adopts liquid chromatography-mass spectrometry (LC-MS) for chromatographic detection and mass spectrometry detection, and the instrument used is Thermo Fisher Vanquish-QE Plus.

[0043] Furthermore, the chromatographic column for the chromatographic detection includes but is not limited to: ACQUITY UPLC BEH C18 chromatographic column.

[0044] Furthermore, the ACQUITY UPLC BEH C18 column has a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.7 μm.

[0045] ACQUITY UPLC BEH C18 column is a high-performance liquid chromatography column produced by Waters and widely used in ultra-high performance liquid chromatography (UPLC) separation of various analytes. It has the advantages of strong versatility, good peak symmetry and high chemical stability. It is widely used in pharmaceutical analysis, environmental analysis, food analysis and other fields, and is particularly suitable for separation and analysis tasks requiring high resolution and high sensitivity.

[0046] Further, the mobile phase A of the chromatographic detection is ultrapure water containing 0.05 vol% to 0.1 vol% (for example, it can be but not limited to any one of 0.05 vol%, 0.06 vol%, 0.07 vol%, 0.08 vol%, 0.09 vol% or 0.1 vol% or any range between two) trifluoroacetic acid, and the mobile phase B is acetonitrile containing 0.05 vol% to 0.1 vol% (for example, it can be but not limited to any one of 0.05 vol%, 0.06 vol%, 0.07 vol%, 0.08 vol%, 0.09 vol% or 0.1 vol% or any range between two) trifluoroacetic acid. Using the above mobile phase A and mobile phase B, different components in the sample can be effectively separated.

[0047] Furthermore, mobile phase A and mobile phase B are ultrasonically degassed for 10 to 15 minutes, including but not limited to any one of 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes or 15 minutes or a range between any two of them.

[0048] The acetonitrile used in the present invention is all mass spectrometry grade acetonitrile.

[0049] Furthermore, the elution gradient of the chromatographic detection is as follows:

[0050] 0min, 90% to 95% mobile phase A, 5% to 10% mobile phase B; 4min, 55% to 60% mobile phase A, 40% to 45% mobile phase B; 28min, 45% to 50% mobile phase A, 50% to 55% mobile phase B; 35min, 5% to 10% mobile phase A, 90% to 95% mobile phase B; 36min, 90% to 95% mobile phase A, 5% to 10% mobile phase B; 41min, 90% to 95% mobile phase A: 5% to 10% mobile phase B. According to the specific mobile phase A and mobile phase B, the present invention designs the elution gradient of the chromatographic detection, improves the separation efficiency of each component, shortens the analysis time, and improves the detection sensitivity.

[0051] Furthermore, the conditions of the chromatographic detection are as follows:

[0052] Detection wavelength 210-220nm; flow rate 03-0.4mL / min; injection volume 10-20μL; column temperature 55-60℃; injection plate temperature 5-10℃. The chromatographic detection conditions will affect the separation effect, peak shape, peak height, and detection sensitivity. Setting the above chromatographic detection conditions is conducive to improving the separation effect, peak shape and peak height, and detection sensitivity.

[0053] Furthermore, the conditions for mass spectrometry detection are as follows:

[0054] The detection mode is positive mode; the scanning range is 200-2000m / z. By limiting the above mass spectrometry detection conditions, the sensitivity of mass spectrometry detection can be improved, a reasonable analysis range can be determined, the resolution can be improved, and the analysis time can be optimized.

[0055] The present invention adopts Thermo Fisher Xcalibur software to perform ion flow extraction.

[0056] The diluent and blank used in the present invention are both 50 vol% acetonitrile aqueous solution.

[0057] Another aspect of the present invention also relates to a method for analyzing impurities in a polypeptide sample, including the method for identifying adducts of a polypeptide sample by mass spectrometry detection.

[0058] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0059] Example 1

[0060] The method for identifying a polypeptide sample by mass spectrometry detection adduct provided in this embodiment comprises the following steps:

[0061] 1) Weigh an appropriate amount of polypeptide sample, add 50 vol% acetonitrile aqueous solution to make the final concentration 1 mg / mL to dissolve and obtain a polypeptide sample solution;

[0062] 2) Degas the prepared mobile phase A and mobile phase B by ultrasonication for 10 minutes respectively and set aside;

[0063] 3) Performing chromatographic detection and mass spectrometry detection on the polypeptide sample solution; performing in-source collision analysis at the first energy and the second energy, extracting ion current, and calculating the difference in the relative main component ratio of the compound under the second energy and the first energy conditions; the second energy is 40.0 eV and the first energy is 0.0 eV;

[0064] The mobile phase A for chromatographic detection was ultrapure water (v / v) containing 0.1% trifluoroacetic acid, and the mobile phase B was acetonitrile (v / v) containing 0.1% trifluoroacetic acid;

[0065] The elution gradient of the chromatographic detection is shown in Table 1:

[0066] Table 1 Elution gradient

[0067] Time (min) Mobile phase A% Mobile phase B% 0 90 10 4 60 40 28 50 50 35 5 95 36 90 10 41 90 10

[0068] The conditions for chromatographic detection are as follows:

[0069] Detection wavelength 214nm; flow rate 0.4mL / min; injection volume 10μL; column temperature 60℃; injection plate temperature 10℃;

[0070] The chromatographic columns used for chromatographic detection were: ACQUITY UPLC BEH C18 column;

[0071] The conditions for mass spectrometry detection were as follows:

[0072] The detection mode was positive mode; the scanning range was 200-2000 m / z; In-source CID: 0.0 eV and 40.0 eV;

[0073] 4) If the difference between the relative main component ratio of the compound under the second energy condition and the first energy condition is ≥0.05%, the compound is judged to be a mass spectrometry detection adduct; if the difference between the relative main component ratio of the compound under the second energy condition and the first energy condition is <0.05%, the compound is judged to be an impurity.

[0074] The identification results of the adducts detected by mass spectrometry of the polypeptide samples in this example are shown in Table 2.

[0075] Table 2 Identification results of peptide sample adducts detected by mass spectrometry

[0076]

[0077] It can be seen from Table 2 that the compounds named as unknown impurities in Table 2 have no significant change in the relative main component ratio under different In-source CID (0.0 / 40.0eV) parameters (ratio change <0.05%), and are real impurities in the polypeptide sample; on the contrary, the compounds named as mass spectrometry detected adducts in Table 2 have significant changes in the relative main component ratio under different In-source CID (0.0 / 40.0eV) parameters (ratio change ≥0.05%).

[0078] Based on the above analysis, it can be quickly determined that these compounds with a relative change of ≥0.05% in the ratio of the main component are mass spectrometry detection adducts, rather than real impurities in the polypeptide sample. As can be seen from the above, the present invention has a good effect, quickly and accurately determines mass spectrometry detection adducts, and eliminates false positive impurities caused by detection.

[0079] Example 2

[0080] The difference between this embodiment and Embodiment 1 is that the first energy is 0.0 eV and the second energy is 30.0 eV.

[0081] The identification results of the adducts detected by mass spectrometry of the polypeptide samples in this example are shown in Table 3.

[0082] Table 3 Identification results of peptide sample adducts detected by mass spectrometry

[0083]

[0084]

[0085] It can be seen from Table 3 that the compounds named as unknown impurities in Table 3 have no significant change in the relative main component ratio under different In-source CID (0.0 / 30.0eV) parameters (ratio change <0.05%), and are real impurities in the polypeptide sample; on the contrary, the compounds named as mass spectrometry detected adducts in Table 3 have significant changes in the relative main component ratio under different In-source CID (0.0 / 30.0eV) parameters (ratio change ≥0.05%).

[0086] Based on the above analysis, it can be quickly determined that these compounds with a relative change of ≥0.05% in the ratio of the main component are mass spectrometry detection adducts, rather than real impurities in the polypeptide sample. As can be seen from the above, the present invention has a good effect, quickly and accurately determines mass spectrometry detection adducts, and eliminates false positive impurities caused by detection.

[0087] Example 3

[0088] The difference between this embodiment and Embodiment 1 is that the first energy is 0.0 eV and the second energy is 20.0 eV.

[0089] The identification results of the adducts detected by mass spectrometry of the polypeptide samples in this example are shown in Table 4.

[0090] Table 4 Identification results of peptide sample mass spectrometry adducts

[0091]

[0092] It can be seen from Table 4 that the compounds named as unknown impurities in Table 4 have no significant change in the relative main component ratio under different In-source CID (0.0 / 20.0 eV) parameters (ratio change <0.05%), and are real impurities in the polypeptide sample; on the contrary, the compounds named as mass spectrometry detected adducts in Table 4 have significant changes in the relative main component ratio under different In-source CID (0.0 / 20.0 eV) parameters (ratio change ≥0.05%).

[0093] Based on the above analysis, it can be quickly determined that these compounds with a relative change of ≥0.05% in the ratio of the main component are mass spectrometry detection adducts, rather than real impurities in the polypeptide sample. As can be seen from the above, the present invention has a good effect, quickly and accurately determines mass spectrometry detection adducts, and eliminates false positive impurities caused by detection.

[0094] Comparative Example 1

[0095] The difference between this comparative example and Example 1 is that the second energy is 10 eV.

[0096] The identification results of the adducts detected by mass spectrometry of the polypeptide samples of this comparative example are shown in Table 5.

[0097] Table 5 Identification results of peptide sample adducts detected by mass spectrometry

[0098]

[0099]

[0100] It can be seen from Table 5 that the compounds named as unknown impurities in Table 5 have no significant change in the relative main component ratio under different In-source CID (0.0 / 10.0 eV) parameters (ratio change <0.05%), which are real impurities in the polypeptide sample; however, most of the compounds named as mass spectrometry detection adducts in Table 5 have significant changes in the relative main component ratio under different In-source CID (0.0 / 10.0 eV) parameters (ratio change ≥ 0.05%), but the names of mass spectrometry detection adduct 1, spectrum detection adduct 2, spectrum detection adduct 3 and spectrum detection adduct 4 in Table 5 should actually be mass spectrometry detection adducts (based on Examples 1 to 3), but in the case of this comparative example, that is, the relative main component ratio of these mass spectrometry detection conjugates under the In-source CID (0.0 / 10.0 eV) parameters has not changed significantly (ratio change <0.05%), because the second energy is too low, resulting in no significant change in the relative main component ratio of the mass spectrometry detection adduct. This comparative example illustrates the effects of the first energy and the second energy defined in the present invention. The judgment rule proposed by the present invention can be obtained only within a reasonable range. If the energy requirements are not met, misjudgment will occur and the method of the present invention cannot be used.

[0101] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A method for identifying polypeptide sample adducts by mass spectrometry detection, characterized in that: The following steps are involved: (a) performing chromatography and mass spectrometry on the polypeptide sample; Performing in-source collision analysis at a first energy and a second energy respectively, extracting ion currents, and calculating the difference in relative principal component ratios of the compound under the second energy and the first energy conditions; wherein the second energy is greater than the first energy; (b) if the difference between the relative main component ratio of the compound under the second energy condition and the first energy condition is ≥0.05%, the compound is judged to be a mass spectrometry detection adduct; if the difference between the relative main component ratio of the compound under the second energy condition and the first energy condition is <0.05%, the compound is judged to be an impurity.

2. The method for identifying polypeptide sample adducts by mass spectrometry detection according to claim 1, characterized in that: The first energy is 0 eV.

3. The method for identifying polypeptide sample adducts by mass spectrometry detection according to claim 1, characterized in that: The second energy is 20-40 eV.

4. The method for identifying polypeptide sample adducts by mass spectrometry detection according to claim 1, characterized in that: The mobile phase A of the chromatographic detection is ultrapure water containing 0.05 vol% to 0.1 vol% trifluoroacetic acid, and the mobile phase B is acetonitrile containing 0.05 vol% to 0.1 vol% trifluoroacetic acid.

5. The method for identifying polypeptide sample adducts by mass spectrometry detection according to claim 1, characterized in that: The elution gradient of the chromatographic detection is as follows: 0min, 90%-95% mobile phase A, 5%-10% mobile phase B; 4min, 55%-60% mobile phase A, 40%-45% mobile phase B; 28min, 45%-50% mobile phase A, 50%-55% mobile phase B; 35min, 5%-10% mobile phase A, 90%-95% mobile phase B; 36min, 90%-95% mobile phase A, 5%-10% mobile phase B; 41min, 90%-95% mobile phase A: 5%-10% mobile phase B.

6. The method for identifying polypeptide sample adducts by mass spectrometry detection according to claim 1, characterized in that: The conditions of the chromatographic detection are as follows: Detection wavelength 210-220 nm; flow rate 0.3-0.4 mL / min; injection volume 10-20 μL; column temperature 55-60°C; injection plate temperature 5-10°C.

7. The method for identifying polypeptide sample adducts by mass spectrometry detection according to claim 1, characterized in that: The chromatographic column for the chromatographic detection includes: an ACQUITY UPLC BEH C18 chromatographic column.

8. The method for identifying polypeptide sample adducts by mass spectrometry detection according to claim 1, characterized in that: The conditions for mass spectrometry detection are as follows: The detection mode was positive mode; the scanning range was 200-2000 m / z.

9. The method for identifying polypeptide sample adducts by mass spectrometry detection according to claim 1, characterized in that: The concentration of the polypeptide sample is 1-5 mg / mL.

10. A method for analyzing impurities in a polypeptide sample, characterized in that: The invention comprises the method for identifying adducts of polypeptide samples by mass spectrometry detection as described in any one of claims 1 to 9.

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