CN114878728A - Novel antibody peptide pattern detection reduction method - Google Patents
Novel antibody peptide pattern detection reduction method Download PDFInfo
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- CN114878728A CN114878728A CN202210486844.6A CN202210486844A CN114878728A CN 114878728 A CN114878728 A CN 114878728A CN 202210486844 A CN202210486844 A CN 202210486844A CN 114878728 A CN114878728 A CN 114878728A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
The invention relates to a novel antibody peptide pattern detection reduction method, which uses cysteine to reduce antibody protein. The detection method is suitable for the peptide graph analysis of the antibody, and can reduce the pollution to the environment and the harm to the human body in the analysis process of the peptide graph of the antibody.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a novel antibody peptide map detection reduction method, and particularly relates to application of a novel reducing agent in a sample preparation process.
Background
Peptide Mapping (Peptide Mapping) is to use proteolytic enzyme with strong specificity to act on a special Peptide chain site to crack a polypeptide into small fragments according to the molecular weight of protein and the polypeptide and the composition characteristics of amino acid, and then to form a characteristic fingerprint spectrum by a certain separation detection means (common high performance liquid chromatography). With the continuous development of the field of biopharmaceuticals, peptide mapping analysis has become a key step in the characterization process of biological therapeutic drugs, and the so-called "bottom-up" characterization of biological drugs can be realized, so that the complete sequence coverage of biological drug molecules is ensured.
2020, the inspection method of peptide map in pharmacopoeia <3405> of the people's republic of China mentions that when the test sample is a complex macromolecule, it is necessary to perform operations such as reduction of disulfide bond and the like as necessary to eliminate the blocking effect of its higher structure on the cracking agent. Currently, in peptide mapping of proteins containing disulfide bonds, disulfide bonds are generally completely reduced using Dithiothreitol (DTT) at a concentration of about 20 mM. However, since Dithiothreitol (DTT) with a final concentration of about 20mM is diluted from 1M DTT, experimenters are reminded to wear a lab coat, wear a gas mask and operate in a fume hood, and properly dispose of experimental wastes during the experiment considering that 1M DTT has a strong pungent smell, has a high risk of biotoxicity and environmental pollution, etc., but the harm of DDT and other reducing agents is still not negligible. We believe that there is a need to further optimize this reduction process to develop new methods with equivalent reduction effects while reducing environmental pollution and the impact on laboratory safety of laboratory personnel so that peptide mapping analysis can be performed more safely to meet the needs of the current protein biopharmaceutical analysis industry.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a novel method for detecting and reducing an antibody peptide diagram aiming at the condition of great harmfulness of the existing antibody peptide diagram analysis method, the harmfulness of a sample preparation process to experimenters and the environment can be reduced by using a novel reducing agent, and the accuracy of the analysis result of the antibody peptide diagram is ensured.
The invention discloses a novel antibody peptide pattern detection reduction method, which uses a novel reducing agent to reduce antibody protein, wherein the reducing agent is selected from monothiol compound cysteine; the antibody is selected from recombinant murine antibodies, human murine chimeric antibodies, humanized monoclonal antibodies, fully human monoclonal antibodies, antibody-conjugated drugs, antibody fusion proteins, antibody fragments and bispecific antibodies.
As a preferred scheme of the invention, the method comprises the following steps:
1) preparing an antibody peptide map detection denaturation buffer solution containing cysteine;
2) incubating the antibody sample with the prepared denaturation buffer;
3) incubating a sample by a denaturation buffer solution, and adding a cysteine aqueous solution to quench alkylation reaction;
4) and replacing the antibody sample incubated by the denaturation buffer solution with an enzyme digestion buffer solution, carrying out enzyme digestion on the monoclonal antibody sample, and carrying out high performance liquid chromatography analysis.
As a preferred embodiment of the present invention, the concentration of free thiol groups in the denaturation buffer is greater than 1mM, preferably 10-80mM, more preferably 20-40 mM.
In a preferred embodiment of the present invention, the denaturant of the denaturation buffer is selected from guanidine hydrochloride, and the buffer is selected from Tris-HCl buffer or phosphate buffer, wherein the buffer with a pH value ranging from 6.0 to 7.0 is selected from phosphate buffer; selecting Tris-HCl buffer solution as the buffer solution with the pH value ranging from 7.5 to 11.0; the Tris-HCl buffer solution is preferably a Tris-HCl buffer solution with the pH value ranging from 7.5 to 11.0, and is more preferably a Tris-HCl buffer solution with the pH value of 8.6 +/-0.1.
In a preferred embodiment of the present invention, the incubation temperature of the denaturation buffer is 37 ℃.
In a preferred embodiment of the present invention, the incubation time of the denaturation buffer is 1 hour.
In a preferred embodiment of the present invention, the concentration of the antibody is 1 mg/mL.
As a preferred embodiment of the present invention, the cysteine for quenching the alkylation reaction in step 3) is added in an amount of 25% of the molar amount of cysteine in the denaturation buffer.
As a preferred embodiment of the present invention, the denaturation buffer contains the following components:
(1) the reducing agent is selected from monothiol compound cysteine, and the concentration of total free sulfhydryl is 20-40 mM;
(2) the denaturant is selected from 6M guanidine hydrochloride;
(3) Tris-HCl buffer 360 mM;
the pH value is 8.6 +/-0.1.
Antibodies, antibody-conjugated drugs, antibody fusion proteins, antibody fragments, and diabodies.
The invention has the beneficial effects that:
the peptide map detection and reduction method containing the novel reducing agent can accurately represent the accuracy of the analysis of biopharmaceuticals such as antibody peptide maps, and simultaneously remarkably reduce the safety problem brought by the traditional reduction method and reduce the pollution to the environment and the harm to human bodies.
Drawings
FIG. 1 Structure of monothiol compound cysteine;
FIG. 2 peptide mapping analysis of mAb-A in example 1 with 20mM Dithiothreitol (DTT);
FIG. 3 peptide mapping analysis of mAb-A at various concentrations of cysteine in example 2.
FIG. 4 peptide mapping analysis of mAb-A in buffer at various pH with 40mM cysteine in example 3.
Detailed Description
The present invention will be further described with reference to the following specific examples. It must be noted that the following examples are intended to illustrate the invention, but not to limit it.
The invention considers the problems that Dithiothreitol (DTT) has extremely strong pungent smell, higher biotoxicity and environmental pollution risk, and the like, and therefore, the invention considers the search of an alternative reducing agent.
Cysteine is a common amino acid in organisms, and has a molecular formula C 3 H 7 NO 2 S, molecular weight 121.16, the structure of which is shown in FIG. 1. The cysteine side chain contains free sulfydryl, is not applied to detection and analysis of a peptide diagram of an antibody, and is a micromolecule thiol compound which is worthy of being developed.
Example results of peptide mapping analysis of recombinant anti-ricin humanized monoclonal antibody by 120 mM Dithiothreitol (DTT)
The recombinant anti-ricin humanized monoclonal antibody (10mg/mL) was diluted to a concentration of 1mg/mL with a denaturing buffer (6M guanidine hydrochloride, 360mM Tris, 2mM EDTA, pH 8.6. + -. 0.1) and the blank solution was diluted at the same ratio. Adding 20 μ L of 1M DTT solution, briefly vortexing, water-bathing at 37 deg.C for 1 hr, taking out the sample, and placing in a roomAnd (4) warming. Add 50. mu.L of 1M IAM solution and vortex briefly until well mixed. Incubate at room temperature in the dark for 15min, take out the sample and add 10. mu.L of 1M DTT quenching alkylation reaction. Subsequently replaced with digestion buffer (25mM Tris, 2mM CaCl) 2 pH 8.2), adding 100. mu.L of 0.125mg/mL trypsin solution to 800. mu.L of the solution after replacement, digesting the monoclonal antibody protein by enzyme digestion, and performing high performance liquid chromatography analysis, wherein the result is shown in FIG. 2.
Example 2 peptide mapping analysis of the recombinant anti-ricin humanized monoclonal antibody against cysteine at various concentrations
Denaturing buffers (6M guanidine hydrochloride, 360mM Tris, 2mM EDTA, pH 8.6. + -. 0.1) were prepared containing different concentrations of cysteine, 1mM, 5mM, 10mM, 20mM, 40mM and 80mM, respectively. The recombinant anti-ricin humanized monoclonal antibody (10mg/mL) was diluted to a concentration of 1mg/mL with the above denaturation buffer containing cysteine at different concentrations, and the blank solution was diluted at the same ratio. After a short vortex, the sample was taken out and left at room temperature after 1h of water bath at 37 ℃. Each group was added with 1.25 times the molar amount of the corresponding cysteine IAM (1M in IAM) and vortexed briefly until homogeneous. Incubating for 15min at room temperature in dark place, taking out a sample, and adding cysteine with the molar weight 0.2 times of that of IAM to quench alkylation reaction. Subsequently replaced with digestion buffer (25mM Tris, 2mM CaCl) 2 pH 8.2), adding 100. mu.L of 0.125mg/mL trypsin solution to 800. mu.L of the solution after replacement, digesting the monoclonal antibody protein by enzyme digestion, and performing high performance liquid chromatography analysis, wherein the result is shown in FIG. 3.
The result shows that the liquid phase spectrogram under the action of cysteine with different concentrations is almost the same as that when DTT is used as a reducing agent, the reduction effect of cysteine is better, and the peptide map detection can be realized by replacing DTT. Among them, the concentration of cysteine is preferably 20 to 80mM, more preferably 20 to 40 mM.
Example 3 peptide mapping analysis of recombinant anti-ricin humanized monoclonal antibody against buffer solutions of different pH containing 40mM cysteine
Preparing denaturing buffers with different pH values containing 40mM cysteine, 6M guanidine hydrochloride and 2mM EDTA, wherein the buffer pairs are selected from Tris-HCl and phosphate, the buffer with the pH value ranging from 6.0 to 7.0 is selected from phosphate buffer pairs, and the buffer with the pH value ranging from 7.5 to 11.0 is selected from Tris-HCl buffer pairs, thereby preparing the following buffers with different pH values: 6.0, 7.0, 7.5, 8.6, 10.0, 11.0, etc.
The recombinant anti-ricin humanized monoclonal antibody (10mg/mL) was diluted to a concentration of 1mg/mL with the above denaturation buffer containing 40mM cysteine at various pH's, and the blank solution was diluted in the same ratio. After a short vortex, the sample was taken out and left at room temperature after 1h of water bath at 37 ℃. Add 50. mu.L of 1M IAM solution at the ratio corresponding to cysteine and vortex briefly until well mixed. Incubate at room temperature in the dark for 15min, take out the sample and add 125. mu.L 80mM cysteine to quench the alkylation reaction. Subsequently replaced with digestion buffer (25mM Tris, 2mM CaCl) 2 pH 8.2), adding 100. mu.L of 0.125mg/mL trypsin solution to 800. mu.L of the solution after replacement, digesting the monoclonal antibody protein by enzyme digestion, and performing high performance liquid chromatography analysis, wherein the result is shown in FIG. 4.
As a result, the liquid phase spectrum of the denatured buffer containing 40mM cysteine at various pH values was almost the same as that of DTT as a reducing agent, and it was revealed that the reducing effect of cysteine was more consistent among the above buffers at various pH values, among which Tris-HCl buffer having a pH value in the range of 7.5 to 11.0 is preferable, and Tris-HCl buffer having a pH value of 8.6. + -. 0.1 is more preferable.
Claims (8)
1. A novel antibody peptide pattern detection reduction method is characterized in that a novel reducing agent is used in the reduction process of an antibody sample; the reducing agent is selected from cysteine; the antibody is selected from recombinant murine antibody, human murine chimeric antibody, humanized monoclonal antibody, fully human monoclonal antibody, antibody-conjugated drug, antibody fusion protein, antibody fragment or bispecific antibody.
2. The peptide graph test reduction method of claim 1, comprising the steps of:
1) preparing an antibody peptide map detection denaturation buffer solution containing cysteine;
2) incubating the antibody sample with the prepared denaturation buffer;
3) incubating a sample by a denaturation buffer solution, and adding a cysteine aqueous solution to quench alkylation reaction;
4) and replacing the antibody sample incubated by the denaturation buffer solution with an enzyme digestion buffer solution, carrying out enzyme digestion on the monoclonal antibody sample, and carrying out high performance liquid chromatography analysis.
3. The peptide graph detection reduction method according to claim 2, wherein the concentration of free thiol in the denaturation buffer is greater than 1mM, preferably 10-80mM, more preferably 20-40 mM.
4. The peptide graph test reduction method according to claim 2, wherein the denaturant of the denaturation buffer is selected from guanidine hydrochloride, and the buffer is selected from Tris-HCl buffer or phosphate buffer, wherein the buffer with the pH value ranging from 6.0 to 7.0 is selected from phosphate buffer; selecting Tris-HCl buffer solution as the buffer solution with the pH value ranging from 7.5 to 11.0; the Tris-HCl buffer solution is preferably a Tris-HCl buffer solution with the pH value ranging from 7.5 to 11.0, and is more preferably a Tris-HCl buffer solution with the pH value of 8.6 +/-0.1.
5. The peptide graph detection reduction method according to claim 2, wherein the incubation temperature of the denaturation buffer is 37 ℃.
6. The peptide graph test reduction method according to claim 2, wherein the incubation time of the denaturation buffer is 1 hour.
7. The peptide graph assay reduction method of claims 2-6, wherein the concentration of the antibody is 1 mg/mL.
8. The peptide graph test reduction method according to any one of claims 2 to 6, wherein the denaturation buffer contains the following components:
(1) the reducing agent is selected from monothiol compound cysteine, and the concentration of total free sulfhydryl is 20-40 mM;
(2) the denaturant is selected from 6M guanidine hydrochloride;
(3) Tris-HCl buffer 360 mM;
the pH value is 8.6 +/-0.1.
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