Quantitative detection method for all-round nuclease

Abstract

The invention discloses an all-round nuclease quantitative detection method, and belongs to the technical field of biology. The method comprises the following steps: firstly, carrying out enzymolysis and desalination pretreatment on an all-round nuclease sample; then, screening out a characteristic peptide fragment HAYTLNNNNSTTK with strong specificity from the enzymolysis peptide fragment by utilizing a high-resolution mass spectrum technology; and finally, based on the characteristic peptide fragment, establishing a quantitative standard curve between the characteristic peptide fragment and the content of the totipotent nuclease by using a liquid chromatography-tandem triple quadrupole mass spectrometry multi-reaction monitoring technology, thereby realizing accurate quantification of the trace totipotent nuclease residue in the biological product. The method is wide in linear range (19.8-1980 ng / mL), good in linear relation (R = 0.9998), high in sensitivity (the limit of quantitation is 198 pg) and excellent in accuracy and precision, is remarkably superior to a traditional ELISA method, and is particularly suitable for quality control and safety monitoring in the production process of complex biological products such as vaccines and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for quantitative determination of characteristic peptides and proteins, and more particularly to a method for quantitative detection of characteristic peptides and totipotent nucleases using liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Totipotent nucleases, also known as nonspecific nucleases (NUs), are derived from Serratia marcescens and are enzymes capable of degrading all forms of DNA and RNA. They are primarily used to remove residual nucleic acids from biological products such as vaccines. The presence of residual DNA in host cells poses potential risks of carcinogenicity and immunogenicity to drugs, affecting the quality and safety of antibody drugs. Therefore, it is necessary to control the residual DNA content. With the rapid development of antibody drug research and development in the biopharmaceutical industry, the application of totipotent nucleases in the production process of biological products is becoming increasingly widespread, playing an important role in improving product purity and safety. However, the detection technology for residual totipotent nucleases is relatively underdeveloped.

[0003] ELISA (Enzyme-Linked Immunosorbent Assay) is a commonly used traditional qualitative and quantitative detection method, characterized by high sensitivity and specificity. However, this method is relatively complex, and due to the complex matrix of biological products, false positives are prone to occur. The quality of the antibody greatly affects the accuracy and reproducibility of the method. In addition, its detection throughput is low, making it difficult to meet the current demand for high-throughput detection.

[0004] In recent years, targeted proteomics technology has been increasingly used in identification and quantification by enzymatically digesting proteins into peptides and then screening for representative characteristic peptides based on the amino acid sequence of the target species' protein. These characteristic peptides serve as unique "fingerprints" for specific proteins. Therefore, there is an urgent need to develop an accurate, sensitive, and high-throughput detection method to accurately quantify trace amounts of totipotent nuclease residues in biopharmaceuticals. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a quantitative detection method for totipotent nucleases based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). This method achieves accurate quantification of trace amounts of totipotent nuclease residues in biological products by screening characteristic peptides of totipotent nucleases and establishing a standard curve.

[0006] The technical solution of this invention: A quantitative detection method for a totipotent nuclease includes the following steps: 1. Perform pretreatment operations such as enzymatic digestion and desalting on the pluripotent nuclease sample; 2. Characteristic peptides were obtained by high-resolution mass spectrometry analysis and screening of enzymatically digested samples; 3. Using mass spectrometry multiple reaction monitoring (MRM) technology, a quantitative relationship between the characteristic peptides of totipotent nucleases and the content of totipotent nucleases was constructed, and then this quantitative relationship was used to quantitatively analyze the residues of totipotent nucleases in bioproducts.

[0007] Preferably, in step 1), the enzyme used for proteolytic digestion is trypsin, and the disulfide bond reducing and alkylating agents are DTT (dithiothreitol) and IAA (iodoacetamide), respectively.

[0008] Preferably, in step 2), the sample is analyzed using a Thermo Nanoscale Liquid Chromatography (LC) system EASY-nLC 1200 and a Thermo High Resolution Mass Spectrometer (Q-Exactive HF). LC conditions: The chromatographic column is a Thermo Scientific Acclaim PepMap. TM The 100A (75 µm × 2 cm, nanoViper 2Pk C18, 3 µm, 100A) and ThermoScientific Acclaim PepMap™ RSLC (75 µm × 25 cm, nanoViper 2Pk C18, 2 µm, 100A) were used. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 80% acetonitrile (0.1% formic acid) solution. Equilibration was performed with 92% of solution A, at a flow rate of 0.3 µL / min, with the following gradient elution program: 0–98 min, 8% → 28% mobile phase B; 98–113 min, 28% → 37% mobile phase B; 113–117 min, 37% → 100% mobile phase B; 117–120 min, 100% mobile phase B; injection volume 1 µL. Mass spectrometry conditions: analysis time 120 min; detection mode: positive ion; scan range 400-1800; primary resolution 60000, secondary resolution 15000; collision energy (CE) 28 eV; 20 fragment spectra acquired after each full scan (MS). 2 scan).

[0009] Preferably, in step 2), the data from the instrument is analyzed using the Proteome Discoverer 2.5 mass spectrometry database to screen for characteristic peptides.

[0010] Preferably, in step 3), the method is established using a Thermo Vanquish ultra-high performance liquid chromatograph in tandem with a Thermo TSQ Quantiva triple quadrupole mass spectrometer. Liquid chromatography conditions: The column is a Waters ACQUITY UPLC® HSS T3 column (2.1 × 100 mm, 1.8 µm). Mobile phase A is 0.1% formic acid solution, and mobile phase B is acetonitrile. Equilibrate with 5% B solution, flow rate 0.2 mL / min, gradient elution program: 0–0.5 min, 5% mobile phase B; 0.5–2 min, 5% → 40% mobile phase B; 2–2.5 min, 40% → 60% mobile phase B; 2.5–4 min, 90% mobile phase B; 4.1–5 min, 90% → 5% mobile phase B; injection volume 2 µL. Mass spectrometry conditions: ion source: ESI+; acquisition mode: MRM; scan time: 5 min; capillary voltage: 3.5 kV (positive ion); ion transmission tube temperature: 350 ℃; nebulization temperature: 350 ℃; sheath gas: 40 Arb; auxiliary gas: 10 Arb; detection ion pairs: m / z 587.7→578.512; m / z 587.7→752.375; m / z 587.7→966.655.

[0011] Preferably, the sequence of the characteristic peptide is HAYTLNNNSTTK.

[0012] Preferably, the linear correlation coefficient R² of this method can reach 0.9998, demonstrating high accuracy and sensitivity.

[0013] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention is the first to apply targeted proteomics technology to the quantitative detection of pluripotent nucleases. By screening, the highly specific characteristic peptide HAYTLNNNSTTK is obtained, which effectively avoids interference from complex biological sample matrices.

[0014] 2. The liquid chromatography-tandem mass spectrometry (LC-MS / MS) quantitative method established in this invention has a wide linear range (1-100 ng / mL, equivalent to 19.8-1980 ng / mL for totipotent nucleases), good linearity (R²=0.9998), high sensitivity (limit of quantification for characteristic peptides is 5 ng / mL, and limit of quantification for totipotent nucleases is 198 pg), high accuracy (recovery rate 85.87%~111.37%), and good precision (RSD 0.77%).

[0015] 3. This method is relatively simple to operate, highly automated, and has high throughput, making up for the shortcomings of traditional methods such as ELISA. It is particularly suitable for high-throughput and accurate quantitative analysis of trace totipotent nuclease residues in complex biological products such as vaccines, providing reliable technical support for the quality control and safety evaluation of biological products. Attached Figure Description

[0016] Figure 1 It is the standard curve for the quantitative detection method of omnipotent nuclease by mass spectrometry; Figure 2 This is a graph showing the results of a specificity experiment. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0018] Example 1 1. Main Instruments and Materials Main instruments: Nanoscale liquid chromatograph EASY-nLC 1200 (Thermo Fisher Scientific), high resolution mass spectrometer Q-Exactive HF (Thermo Fisher Scientific), ultra-high performance liquid chromatograph Vanquish (Thermo Fisher Scientific), triple quadrupole mass spectrometer TSQ Quantiva (Thermo Fisher Scientific), analytical balance (FA-1004, Shanghai Sunny Hengping Scientific Instruments Co., Ltd.), centrifuge (TGL-16M, Xiangyi Centrifuge Instrument Co., Ltd.), heat-collecting constant heating magnetic stirrer (DF-101S, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.), nitrogen blowing concentration device (MTN-5800, Tianjin Autoshine Instruments Co., Ltd.).

[0019] Main materials: Trypsin (sequencing grade, Promega), acetonitrile (chromatographic grade, Thermo), methanol (chromatographic grade, Thermo), formic acid (HPLC grade, Shanghai Maclean Biotech Co., Ltd.), iodoacetamide (reagent grade, Vetec), dithiothreitol (reagent grade, Vetec), totipotent nuclease (Biogradetech), characteristic peptide of totipotent nuclease (synthesized by Gil Biochemical (Shanghai) Co., Ltd.), tris(hydroxymethyl)aminomethane (≥99.8%, Shanghai Aladdin Biotech Co., Ltd.), magnesium chloride hexahydrate (analytical grade, Xilong Scientific Co., Ltd.), sodium chloride (analytical grade, Xilong Scientific Co., Ltd.), Pierce™ Water (mass spectrometry grade, Thermo), SEP-PAK C18 solid-phase extraction column (Waters), Eclipse Plus C18 column (2.1×100 mm, 1.8µm, Agilent), ACQUITY UPLC ® HSS T3 column (2.1×100 mm, 1.8µm, Waters), Acclaim PepMapTM100 (75 µm×2 cm, nanoViper 2PkC18, 3 µm, 100A, Thermo Scientific), Acclaim PepMapTM RSLC (75 µm×25 cm, nanoViper2Pk C18, 2 µm, 100A, Thermo Scientific).

[0020] 2. Pretreatment of totipotent nuclease samples before enzymatic desalting (1) Sample digestion: Take 150 µg of totipotent nuclease sample, add 1 mol / L DTT solution to a final concentration of approximately 10 mmol / L, and react in a 37 ℃ water bath for 1 h; then add an appropriate amount of 1 mol / L IAA solution to a final concentration of 50 mmol / L, and incubate at room temperature in the dark for 45 min. Add trypsin at a ratio of m(enzyme):m(protein) = 1:30-1:50 and digest overnight in a 37 ℃ water bath.

[0021] (2) Sample desalting: The Waters SEP-PAKC18 solid-phase extraction column was activated sequentially with methanol, 70% acetonitrile (containing 0.1% formic acid), and 0.1% formic acid. The enzymatically digested sample was then added to the activated solid-phase extraction column, equilibrated with 0.1% formic acid, and then eluted with 70% acetonitrile (containing 0.1% formic acid). The eluent was then concentrated. After concentration, it was dissolved in 100 µL of 0.1% formic acid for detection.

[0022] 3. Instrumental analysis of totipotent nuclease samples and screening of characteristic peptides. (1) The processed sample is injected into a nano-level liquid chromatograph for separation and then detected by a Q-Exactive high-resolution mass spectrometer. The liquid chromatography and mass spectrometry conditions are the same as those described in step 2) of the [Summary of the Invention].

[0023] (2) The data from the instrument were analyzed using the Proteome Discoverer 2.5 mass spectrometry database. The specific species was Serratia marcescens. Parameter settings: Enzyme was No-Enzyme (Unspecific), Fixed modifications were Carbamidomethyl (C), Variable modifications were: Oxidation (M), Acetyl / +42.011 Da (N-Terminus), Met-loss / -131.040 Da (M) (N-Terminus), Met-loss+Acetyl / -89.030 Da (M) (N-Terminus), Peptide Mass Tolerance was 10 ppm, Fragment Mass Tolerance was 0.02 Da, and FDR (Findable Protein Detection Criterion) was ≤0.01.

[0024] (3) Screening is carried out in accordance with the principle of characteristic peptide screening. The principle includes: 1) deleting peptides containing modification sites (such as C, M); 2) deleting peptides with glutamine at the N end; 3) no NG or QG combination; 4) no DP or DG combination; 5) no continuous P and S; 6) peptide length of 7-21 amino acids; 7) no continuous R and K residues at the enzyme cleavage site.

[0025] (4) The characteristic peptide sequence obtained by screening is: HAYTLNNNSTTK, with a parent ion m / z of 587.7 and daughter ions m / z of 578.512, 752.375 and 966.655.

[0026] 4. Development of quantitative methods for totipotent nucleases (1) The characteristic peptides obtained from the above screening were synthesized, and a peptide quantification method was established using a Thermo Vanquish ultra-high performance liquid chromatograph in series with a Thermo TSQ Quantiva triple quadrupole mass spectrometer. The liquid chromatography and mass spectrometry conditions were the same as those described in step 3) of the [Summary of the Invention].

[0027] (2) Establishment of standard curve: The synthesized characteristic peptide was used to prepare standard solutions with concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. These solutions were then injected into an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) instrument for content determination. A standard curve was established with the standard concentration X (ng / mL) as the abscissa and the peak area Y (counts*min) as the ordinate: Y = 9.658X - 3.053. The linear range for quantitative detection of totipotent nuclease using this characteristic peptide was 1–100 ng / mL. Based on the conversion between the mass of totipotent nuclease protein and the characteristic peptide (conversion factor 19.8), the linear range for detecting totipotent nuclease content in the sample was 19.8–1980 ng / mL. The correlation coefficient of the regression equation was R² = 0.9998, indicating a good linear relationship (see [link to relevant documentation]). Figure 1 ).

[0028] 5. Methodological validation of the totipotent nuclease quantification method (1) Specificity verification: The buffer solution, reference solution (50 ng / mL), and sample solution were injected into the ultra-high performance liquid chromatography-mass spectrometry instrument for content determination. The chromatograms are shown below. Figure 2 As shown, this indicates that the method has good specificity.

[0029] (2) Limit of detection and limit of quantitation: The limit of detection for the characteristic peptide of the totipotent nuclease is 1 ng / mL (signal-to-noise ratio S / N=3), and the limit of quantitation is 5 ng / mL (signal-to-noise ratio S / N=10). Converted to totipotent nuclease in the sample, the corresponding limit of detection is 39.6 pg and the limit of quantitation is 198 pg.

[0030] (3) System suitability: The characteristic peptide stock solution was diluted to 50 ng / mL and injected 6 times consecutively. The peak area RSD was 2.07%, indicating that the system has good suitability.

[0031] (4) Accuracy: Standard solutions with concentrations of 5 ng / mL, 10 ng / mL, and 50 ng / mL prepared using negative buffer were analyzed by solid-phase extraction. The RSDs for the low, medium, and high concentration levels were 1.67%–8.71%, and the recoveries were 85.87%–111.37%, indicating that the method is accurate and reliable.

[0032] (5) Stability: 50 ng / mL of characteristic peptide solution was injected and measured at 0 h, 2 h, 4 h, 8 h, 16 h, 18 h and 24 h respectively. The RSD of the peak area was 8.26%, indicating that the sample had good stability within 24 h.

[0033] (6) Precision: Five parallel totipotent nuclease samples were pretreated and measured. The peak area RSD was 0.77%, indicating that the method has good precision.

[0034] The above verification results demonstrate that the method exhibits good linearity, high accuracy and sensitivity, strong specificity, and good stability, and can be used for the detection of trace amounts of totipotent nucleases in complex biological products.

[0035] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for quantitative detection of a totipotent nuclease, characterized in that, Includes the following steps: 1) Pretreatment of the totipotent nuclease sample by enzymatic digestion and desalting; 2) The enzymatically digested samples after step 1) were analyzed by high-resolution mass spectrometry to screen and obtain the characteristic peptides of the totipotent nuclease; 3) Based on the characteristic peptides screened in step 2), a quantitative relationship between the characteristic peptides and the content of totipotent nucleases is established using mass spectrometry multiple reaction monitoring technology, thereby enabling quantitative analysis of totipotent nuclease residues in biological products.

2. The method according to claim 1, characterized in that, In step 1), the enzyme used for proteolytic hydrolysis is trypsin, the disulfide bond reducing agent is dithiothreitol, and the alkylating agent is iodoacetamide.

3. The method according to claim 1, characterized in that, In step 2), analysis is performed using nano-level liquid chromatography-tandem high-resolution mass spectrometry. The liquid chromatography column is a C18 reversed-phase column, the mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is an 80% acetonitrile solution containing 0.1% formic acid, with gradient elution. Mass spectrometry detection is performed in positive ion mode, and fragment ion spectra are acquired after a full scan.

4. The method according to claim 1 or 3, characterized in that, In step 2), the mass spectrometry data is analyzed using ProteomeDiscoverer 2.5 software to screen for characteristic peptides.

5. The method according to claim 1, characterized in that, In step 3), analysis was performed using ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-MS / MS). The HPLC column was a Waters ACQUITY UPLC® HSS T3 column. Mobile phase A was 0.1% formic acid solution, and mobile phase B was acetonitrile, with gradient elution. Mass spectrometry detection was performed using electrospray ionization positive ion mode and multiple reaction monitoring mode. The monitored ion pairs included m / z 587.7→578.512, m / z 587.7→752.375, and m / z 587.7→966.

655.

6. The method according to claim 1 or 5, characterized in that, The amino acid sequence of the characteristic peptide is HAYTLNNNSTTK.

7. The method according to claim 1, characterized in that, The quantitative relationship is established by preparing a series of characteristic peptide standard solutions at different concentrations, and plotting a standard curve with concentration on the x-axis and mass spectrometry response peak area on the y-axis. The linear correlation coefficient R² of the standard curve is not less than 0.

999.

8. The method according to claim 1, characterized in that, The method is used to quantitatively detect trace amounts of totipotent nuclease residues in complex biological products such as vaccines.

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