CN110618202B

CN110618202B - Method for detecting protein purity

Info

Publication number: CN110618202B
Application number: CN201810633150.4A
Authority: CN
Inventors: 柯潇; 岳菊; 郑强; 雷刚; 白灏
Original assignee: Chengdu Kanghong Biotechnologies Co Ltd
Current assignee: Chengdu Kanghong Biotechnologies Co Ltd
Priority date: 2018-06-20
Filing date: 2018-06-20
Publication date: 2022-07-08
Anticipated expiration: 2038-06-20
Also published as: CN110618202A

Abstract

The invention provides a method for detecting protein purity, which adopts size exclusion-high performance liquid chromatography to detect, and phosphate buffer solution as a mobile phase can be used for content determination of dimer impurities in protein biological products. The protein purity detection method can well separate dimeric protein from monomeric protein, has the advantages of rapidness, simplicity, low detection limit, high sensitivity and the like, and is suitable for industrial application.

Description

Method for detecting protein purity

Technical Field

The invention relates to the field of biological medicine, in particular to a method for detecting protein purity.

Background

With the rapid development of biotechnology and pharmaceutical industry, more and more protein and polypeptide drugs are developed and applied clinically. Antibodies and fusion proteins are common protein drugs, most of the drug proteins are expressed by CHO cells, the glycosylation degree is high, polymers are generated by aggregation in cell culture and purification production process flows, and in addition, the storage conditions and the self-stability of the protein drugs also have different degrees of influence on the generation of the polymers. Protein aggregation has great influence on the safety and effectiveness of protein drugs, dimeric protein may influence the membrane penetration property due to larger molecular weight, so that the bioavailability is reduced, the aggregation of antibody protein may also generate immunogenicity, and potential risks are brought to patients after administration, so the detection of protein purity becomes one of important indexes for quality control of protein drugs.

There are two common methods for detecting protein drug purity, one is non-reducing protein electrophoresis, and the other is Size Exclusion Chromatography (SEC). The non-reducing protein electrophoresis needs to be operated under a denaturing condition, which may affect the content of polymer components, and the SEC is a method for separating proteins mainly according to the difference of molecular size by using the unique characteristics of a porous gel stationary phase, and the detection conditions are mild and do not have great influence on the natural form of the proteins. Therefore, the SEC method is more suitable for the quality control of products in industry. However, when the currently disclosed size exclusion chromatography method is used for detecting dimer impurities in protein, the problems of poor separation degree or incapability of separating the dimer impurities often occur, and some methods have poor stability and low detection sensitivity and are not suitable for separating and detecting the low-concentration dimer impurities.

In order to further ensure the quality safety of protein drugs, a rapid, simple, sensitive and stable size exclusion chromatography detection method is needed to be established.

Disclosure of Invention

The invention aims to provide a rapid, simple and convenient detection method with high sensitivity and good stability, which is used for separating and detecting dimer protein impurities. The invention provides the following technical scheme:

a method for detecting protein purity adopts size exclusion-high performance liquid chromatography for detection, and a mobile phase in chromatographic conditions of the detection method is phosphate buffer solution; wherein the phosphate buffer is an aqueous solution comprising 150mM PB and 100mM NaCl; the protein is selected from an antibody or a fusion protein, preferably, the antibody comprises SEQ ID NO: 2 and the light chain sequence shown in SEQ ID NO: 3; the fusion protein comprises SEQ ID NO: 1.

In the chromatographic condition of the method for detecting the protein purity, the pH value of the mobile phase is 6.5-7.0, so that a good detection effect can be realized, and the pH value is preferably 7.0.

Furthermore, in the chromatographic conditions of the detection method, the detection wavelength is preferably 220nm, the chromatographic column is preferably a TSK-GEL G3000SWXL type GEL exclusion chromatographic column, the flow rate of the mobile phase is preferably 0.5mL/min, and the column temperature is 20-30 ℃, preferably 25 ℃.

The invention further provides a method for detecting the purity of the protein, which comprises the following steps:

1) mobile phase configuration: weighing a proper amount of sodium dihydrogen phosphate, dissolving the sodium dihydrogen phosphate in ultrapure water to obtain a sodium dihydrogen phosphate solution, weighing a proper amount of disodium hydrogen phosphate, and dissolving the disodium hydrogen phosphate in the ultrapure water to obtain a disodium hydrogen phosphate solution; mixing appropriate amount of sodium dihydrogen phosphate solution and disodium hydrogen phosphate solution, adding NaCl, dissolving, adjusting pH to 6.5-7.0 with acid-base regulator, and filtering with microporous membrane; wherein the pH regulator is selected from sodium hydroxide and hydrochloric acid;

2) preparing a test solution: taking a proper amount of a protein sample to be detected, adding a buffer solution, and uniformly mixing to obtain a mixture;

3) and (3) chromatographic detection: injecting the sample prepared in the step 2) into a high performance liquid chromatograph for detection by adopting a high performance liquid chromatography and a gel size exclusion chromatographic column, wherein the mobile phase solution prepared in the step 1) is used, the column temperature is 25 ℃, the flow rate is 0.5mL/min, and the wavelength is 220 nm.

The inventor finds that the chromatographic detection conditions have a significant influence on the effect of size exclusion-high performance liquid chromatography (SEC-HPLC) on detecting the dimer in the protein in long-term experimental research, and the following is part of experimental contents.

1. Experimental Material

The experimental materials used in the present invention are as follows:

disodium hydrogen phosphate (dodecahydrate) purchased from Chengdu Kelong chemical reagent factory

Sodium dihydrogen phosphate (dihydrate) purchased from Chengdu Kelong chemical reagent factory

Anhydrous disodium hydrogen phosphate was purchased from Chengdu Kelong chemical reagent factory

Sodium chloride was purchased from Chengdu Dendron chemical industry reagent factory

Anhydrous sodium sulfate was purchased from Chengdu Kelong chemical reagent factory

Sodium hydroxide was purchased from Chengdu Dendrolon chemical reagent plant

Arg (arginine) from Shanghai-derived biosciences GmbH

Isopropanol (chromatographically pure) was obtained from Honeywell Burdick & Jackson, USA

Ethanol (chromatographically pure) was obtained from FISHER SCIENTIFIC, USA

Hydrochloric acid (chromatographically pure) purchased from Kyoto Colon Chemicals, Inc

20 PBS buffered saline was purchased from Biotech (Shanghai) Inc. (Sangon Biotech, used by diluting with distilled water to the desired fold)

Agilent 1260 high performance liquid chromatograph

TSK G3000 GEL column (TSK-GEL G3000SWXL, 7.8X 300mm, 5 μm)

2. Chromatographic conditions

Using an agilent high performance liquid chromatograph 1260, column: TSK G3000 GEL column (TSK-GEL G3000SWXL, 7.8X 300mm, 5 μm), mobile phase: 150mM (mM means mmol/L) PB +100mM NaCl, column temperature: 25 ℃, flow rate: 0.5mL/min, wavelength: 220nm, sample size: 10ul, elution time: and (3) 30 min.

3. Fluid phase system

In the present invention, PB refers to a phosphate buffer, i.e., a mixed solution prepared by dissolving sodium dihydrogen phosphate and disodium hydrogen phosphate in water, and the preparation method thereof is a conventional technique in the art. One exemplary configuration is as follows:

1) respectively preparing sodium dihydrogen phosphate solution and disodium hydrogen phosphate solution with corresponding concentrations in advance according to different concentration requirements

2) The dosage of the sodium dihydrogen phosphate solution and the dosage of the disodium hydrogen phosphate solution are selected according to different pH requirements, and if the pH is slightly different, the pH is adjusted to the required range by using an acid-base regulator, such as NaOH or HCl.

The part of the mobile phase examined by the inventor and the configuration method thereof are as follows:

mobile phase 1: 150mM PB +100mM NaCl

The configuration method comprises the following steps: 23.401g of sodium dihydrogen phosphate (dihydrate) was weighed out and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain solution A. 53.721g of disodium hydrogen phosphate (dodecahydrate) or 21.294g of anhydrous disodium hydrogen phosphate was weighed and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain a solution B. Weighing 585mL of the solution A and 915mL of the solution B, adding 8.775g of NaCl, dissolving, adjusting the pH value to 7.0 by using sodium hydroxide, and filtering by using a 0.22-micron filter membrane to obtain the product.

Mobile phase 2: 100mM disodium hydrogen phosphate (dodecahydrate) +100mM NaCl

The configuration method comprises the following steps: weighing 35.8g of disodium hydrogen phosphate (dodecahydrate) and 8.8g of sodium chloride in 800mL of ultrapure water, dissolving, adjusting the pH value to 6.7 by using hydrochloric acid, metering the volume to 1L, and filtering by using a 0.22-micron filter membrane to obtain the sodium hydrogen phosphate.

Mobile phase 3: 1% isopropanol +200mM PB

15.6g of sodium dihydrogen phosphate (dihydrate) is weighed and dissolved in 180mL of ultrapure water, and the volume is adjusted to 200mL to obtain solution A. 75.2g of disodium hydrogen phosphate (dodecahydrate) was weighed out and dissolved in 380mL of ultrapure water, and the volume was adjusted to 420mL to obtain solution B. Mixing the solution A and the solution B, adding 15.5mL of isopropanol into 914.5mL of ultrapure water, adjusting the pH value to 7.0, and filtering with a 0.22-micron filter membrane to obtain the compound.

Mobile phase 4: 20mM PB +100mM NaCl

3.12g of sodium dihydrogen phosphate (dihydrate) is weighed and dissolved in 800mL of ultrapure water, and the volume is adjusted to 1L to obtain solution A. 7.163g of disodium hydrogen phosphate (dodecahydrate) or 2.839g of anhydrous disodium hydrogen phosphate was weighed and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain solution B. Weighing 585mL of the solution A and 915mL of the solution B, adding 8.775g of NaCl, dissolving, adjusting the pH value to 7.0 by using sodium hydroxide, and filtering by using a 0.22-micron filter membrane to obtain the product.

Mobile phase 5: 20mM disodium hydrogen phosphate (dodecahydrate) +150mM NaCL +200mM Arg

Weighing 7.16g of disodium hydrogen phosphate (dodecahydrate), 8.8g of sodium chloride and 42.2g of Arg in 800mL of ultrapure water, dissolving, adjusting the pH value to 7.2 by using hydrochloric acid, metering the volume to 1L, and filtering by using a 0.22 mu m filter membrane to obtain the sodium phosphate.

Mobile phase 6: 150mM PB +300mM NaCl

The configuration method comprises the following steps: 23.401g of sodium dihydrogen phosphate (dihydrate) was weighed out and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain solution A. 53.721g of disodium hydrogen phosphate (dodecahydrate) or 21.294g of anhydrous disodium hydrogen phosphate was weighed and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain a solution B. Weighing 585mL of the solution A and 915mL of the solution B, adding 26.325g of NaCl, dissolving, adjusting the pH value to 7.0 by using sodium hydroxide, and filtering by using a 0.22-micron filter membrane to obtain the product.

Mobile phase 7: 100mM PB +300mM NaCl

The configuration method comprises the following steps: 15.601g of sodium dihydrogen phosphate (dihydrate) was weighed out and dissolved in 800mL of ultrapure water, and the volume was made to 1L to obtain solution A. 35.814g of disodium hydrogen phosphate (dodecahydrate) or 14.196g of anhydrous disodium hydrogen phosphate was weighed and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain a solution B. Weighing 585mL of the solution A and 915mL of the solution B, adding 26.325g of NaCl, dissolving, adjusting the pH value to 7.0 by using sodium hydroxide, and filtering by using a 0.22-micron filter membrane to obtain the product.

Mobile phase 8: 100mM PB +100mM NaCl

The configuration method comprises the following steps: 15.601g of sodium dihydrogen phosphate (dihydrate) was weighed out and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain solution A. 35.814g of disodium hydrogen phosphate (dodecahydrate) or 14.196g of anhydrous disodium hydrogen phosphate was weighed and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain a solution B. Weighing 585mL of the solution A and 915mL of the solution B, adding 8.775g of NaCl, dissolving, adjusting the pH value to 7.0 by using sodium hydroxide, and filtering by using a 0.22-micron filter membrane to obtain the product.

Mobile phase 9: 50mM PB +100mM NaCl

The configuration method comprises the following steps: 7.800g of sodium dihydrogen phosphate (dihydrate) is weighed and dissolved in 800mL of ultrapure water, and the volume is adjusted to 1L to obtain solution A. 17.907g of disodium hydrogen phosphate (dodecahydrate) or 7.098g of anhydrous disodium hydrogen phosphate was weighed and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain a solution B. Weighing 585mL of the solution A and 915mL of the solution B, adding 8.775g of NaCl, dissolving, adjusting the pH value to 7.0 by using sodium hydroxide, and filtering by using a 0.22-micron filter membrane to obtain the product.

Mobile phase 10: 50mM PB +200mM NaCl

The configuration method comprises the following steps: 7.800g of sodium dihydrogen phosphate (dihydrate) is weighed and dissolved in 800mL of ultrapure water, and the volume is adjusted to 1L to obtain solution A. 17.907g of disodium hydrogen phosphate (dodecahydrate) or 7.098g of anhydrous disodium hydrogen phosphate was weighed and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain a solution B. Weighing 585mL of the solution A and 915mL of the solution B, adding 17.550g of NaCl, dissolving, adjusting the pH value to 7.0 by using sodium hydroxide, and filtering by using a 0.22-micron filter membrane to obtain the product.

Mobile phase 11: 250mM PB +50mM NaCl

The configuration method comprises the following steps: 39.000g of sodium dihydrogen phosphate (dihydrate) was weighed out and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain solution A. 89.535g of disodium hydrogen phosphate (dodecahydrate) or 35.490g of anhydrous disodium hydrogen phosphate was weighed and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L to obtain a solution B. Weighing 585mL of the solution A and 915mL of the solution B, adding 4.387g of NaCl, dissolving, adjusting the pH value to 7.0 by using sodium hydroxide, and filtering by using a 0.22-micron filter membrane to obtain the product.

4. Preparation of test article

And taking a proper amount of protein A or protein B, adding 1 × PBS buffer salt solution, and preparing 0.1mg/mL test solution.

Wherein, the protein A is a polypeptide containing SEQ ID NO: 2 and the light chain sequence shown in SEQ ID NO: 3, an antibody having a heavy chain sequence set forth in seq id no; protein B is a polypeptide comprising SEQ ID NO: 1 in sequence shown in the specification.

5. Results of the experiment

Under the condition that other experimental conditions are the same and only the mobile phase composition is different, protein samples of the same batch are detected, the content of dimer impurities in the samples is calculated by adopting an area normalization method, and the coefficient of variation (CV/%) of the content of dimer in the samples of the same batch is calculated by adopting the following calculation formula.

CV ═ 100% (standard deviation SD/Mean)%

Meanwhile, according to the general rule of section 4 in pharmacopoeia of the people's republic of China (2015 th edition)0514 "size exclusion chromatography", when using size exclusion chromatography to detect biomacromolecules, the calculation formula of the separation degree (R) of the dimer is:

h represents a dimer peak height, H represents a valley height between the dimer and the monomer, and R should not be less than 2.0.

The effect of separating the dimers under different mobile phase conditions is shown in table 1 below:

TABLE 1 Effect of protein dimer separation under different mobile phase conditions

According to the results in the table, the problems of substandard separation degree, tailing of monomer sample peak, large base line fluctuation, unstable result of dimer content (CV%), high detection limit and the like can occur when different mobile phases are adopted for separation detection, and when the mobile phase 1 is adopted for protein separation detection, the dimer protein and the monomer protein can be well separated, the base line is stable, the dimer content is stable, the detection limit of the method is low, and the sensitivity is good.

Drawings

FIG. 1 is a chromatogram detection map of protein B in comparative example I

FIG. 2 is a chromatogram detection map of protein A in comparative example II

FIG. 3 is a chromatogram detection map of protein B in comparative example II

FIG. 4 is a chromatogram detection map of protein B in comparative example III

FIG. 5 is the chromatogram detection map of protein A in example I

FIG. 6 is the chromatogram detection map of protein B in example two

Wherein, 1 is a dimeric protein chromatographic peak, 2 is a monomeric protein sample chromatographic peak, and 3 is a protein fragment chromatographic peak.

Detailed Description

Comparative example 1

1. Fluid phase system

Solution A: 50mM NaH₂PO₄: 7.9g of sodium dihydrogen phosphate (dihydrate) was weighed out and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L.

Solution B: 50mM Na₂HPO₄: disodium hydrogen phosphate (dodecahydrate) 17.9g was weighed out and dissolved in 800mL of ultrapure water, and the volume was made to 1L.

Weighing 510mL of the solution A and 490mL of the solution B, adding 17.55g of NaCl into a beaker, adjusting the pH value to 6.8 by using sodium hydroxide after dissolution, and filtering by using a 0.22-micron filter membrane to obtain the product.

2. Preparation of test article

And respectively taking a proper amount of protein A and a proper amount of protein B, adding 1 × PBS buffer salt solution, and preparing to the required concentration.

3. Chromatographic conditions

An Agilent high performance liquid chromatograph 1260, a TSKgel G3000SWXL type (300mm multiplied by 7.8mm) gel exclusion chromatographic column, with 0.05mol/L phosphate buffer solution (pH 6.8) to 0.3mol/L sodium chloride solution as a mobile phase, a flow rate of 1mL/min, a DAD detector, and a detection wavelength of 220nm are used.

4. Results of the experiment

Table 2 results of protein dimer detection according to comparative example method

As can be seen from the data in the table, when the method of the first comparative example is used for detecting the dimer in the protein A or the dimer in the protein B, the separation degree of the dimer in the protein B does not reach the standard, and accurate qualitative and quantitative determination cannot be carried out, so that the method is not suitable for the industrial quality control of products.

Comparative example No. two

1. Fluid phase system

0.02mol/L sodium dihydrogen phosphate: 3.1g of sodium dihydrogen phosphate (dihydrate) is weighed and dissolved in 800mL of ultrapure water, and the volume is adjusted to 1L.

0.15 mol/LNaCl: 8.8g of sodium chloride is weighed and dissolved in 800mL of ultrapure water, and the volume is adjusted to 1L.

95% ethanol solution: measuring 190mL of ethanol, adding 10mL of ultrapure water, and uniformly mixing.

Weighing 900mL of 0.02mol/L sodium dihydrogen phosphate, 900mL of 0.15mol/L NaCl and 200mL of 95% ethanol solution, uniformly mixing, and filtering with a 0.22 mu m filter membrane to obtain the product.

2. Preparation of test article

Wherein, the protein A is a polypeptide containing SEQ ID NO: 2 and the light chain sequence shown in SEQ ID NO: 3; protein B is a polypeptide comprising SEQ ID NO: 1 in sequence shown in the specification.

3. Chromatographic conditions

An Agilent high performance liquid chromatograph 1260 is used, a TSK G3000 GEL chromatographic column (TSK-GEL G3000SWXL, 7.8 x 300mm, 5 mu m), 0.02mol/L sodium dihydrogen phosphate solution, 0.15mol/L sodium chloride solution and 95% ethanol (45: 10) are used as a mobile phase, the flow rate is 0.6mL/min, the detection wavelength is 222nm, and the sample feeding amount is as follows: 20 ul.

4. Results of the experiment

TABLE 3 results of protein dimer detection versus the comparative example method

The two kinds of protein A and protein B with different concentrations in the same batch are respectively detected, and the result does not effectively separate out dimer impurities in the protein sample (see attached figures 2 and 3), which indicates that the method is not suitable for the quality control of products.

Comparative example No. three

1. Fluid phase system

Solution A: 100mM NaH2PO 4: 15.6g of sodium dihydrogen phosphate (dihydrate) was weighed out and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L.

Solution B: 100mM Na2HPO 4: disodium hydrogen phosphate (dodecahydrate) 35.8g was weighed out and dissolved in 800mL of ultrapure water, and the volume was made to 1L.

Weighing 390mL of solution A, 610mL of solution B, putting the solution B in a beaker, adding Na2SO414.2g, adjusting the pH value to 7.0 by using sodium hydroxide after dissolution, and filtering by using a 0.22-micron filter membrane to obtain the nano-particles.

2. Preparation of test article

3. Chromatographic conditions

Using agilent high performance liquid chromatograph 1260, TSK G3000 GEL chromatography column (TSK-GEL G3000SWXL, 7.8 × 300mm, 5 μm), column temperature: 25 ℃, flow rate: 0.6mL/min, wavelength: 214nm, sample size: 20 ul.

4. Results of the experiment

TABLE 4 results of protein dimer detection by the third method of comparative example

According to the results in the table, when the method is used for detecting the dimer in the protein A, the stability of the method is poor relative to the invention, the detection limit is improved by one order of magnitude, and the detection of the low-concentration dimer impurity is not facilitated, when the method is used for detecting the dimer in the protein B, the dimer impurity in the sample cannot be effectively separated (see figure 4), and the method is not suitable for the quality control of the product.

Example one

1. Fluid phase system

Solution A: 23.4g of sodium dihydrogen phosphate (dihydrate) was weighed out and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L.

Solution B: 53.7g of disodium hydrogen phosphate (dodecahydrate) was weighed out and dissolved in 800mL of ultrapure water, and the volume was adjusted to 1L.

Weighing 585mL of the solution A and 915mL of the solution B in a beaker, adding 8.8g of NaCl, adjusting the pH value to 7.0 by using sodium hydroxide after dissolution, and filtering by using a 0.22-micron filter membrane to obtain the product.

2. Preparation of test article

Taking appropriate amount of protein A, taking 1 × PBS buffer solution as solvent, and preparing by stepwise dilution method to obtain protein A with concentration of 0.1mg/mL

A test solution.

3. Chromatographic conditions

Using an agilent high performance liquid chromatograph 1260, column: TSK G3000 GEL column (TSK-GEL G3000SWXL, 7.8X 300mm, 5 μm), mobile phase: 150mM PB +100mM NaCl, column temperature: 25 ℃, flow rate: 0.5mL/min, wavelength: 220nm, sample size: 10ul, elution time: 30min

4. Results of the experiment

The content of dimer impurities in the samples was calculated by an area normalization method, and the coefficient of variation (CV/%) of the dimer content in the same batch of samples was calculated by the following calculation formula.

CV ═ 100% (standard deviation SD/Mean)%

Meanwhile, according to the relevant regulations in the general rules 0514 ' size exclusion chromatography ' of pharmacopoeia of the people's republic of China (2015 edition), when biomacromolecules are detected by adopting a size exclusion chromatography, the calculation formula of the separation degree (R) of the dimers is as follows:

TABLE 5 results of protein dimer detection by the method of example

Protein	Protein concentration/mg.mL^-1	Dimer content CV (n-3)/%)	Degree of dimer separation R	Dimer detection limit/mg
					A	0.1	0	5.2	0.0004mg

The method is used for detecting the dimer impurities in the protein A, has good sample separation degree, high precision and good accuracy, and is suitable for industrially detecting the low-concentration dimer impurities (see the attached figure 5).

Example two

1. Fluid phase system

In the same manner as in the first embodiment

2. Preparation of test article

Taking a proper amount of protein B, taking PBS buffer solution as a solvent, and preparing the protein B with the concentration of 0.1mg/mL by adopting a stepwise dilution method

And (4) a test solution.

3. Chromatographic conditions

4. Results of the experiment

CV is (standard deviation SD/Mean) 100%

h represents a dimer peak height, H represents a valley height between the dimer and the monomer, and R must not be less than 2.0.

TABLE 6 results of protein dimer detection by the second method of example

Protein	Protein concentration/mg.mL^-1	Dimer content CV (n-3)/%)	Degree of dimer separation R	Dimer detection limit/mg
					B	0.1	0	2.2	0.0003mg

The method is used for detecting the dimer impurities in the protein B, has good sample separation degree, high precision and good accuracy, and is suitable for industrially detecting the low-concentration dimer impurities (see the attached figure 6).

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Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys

210 215 220

Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

245 250 255

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

305 310 315 320

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

325 330 335

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

340 345 350

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

355 360 365

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

405 410 415

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

420 425 430

Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

Claims

1. A method for detecting protein and its dimer, adopt size exclusion-high performance liquid chromatography to detect, mobile phase is phosphate buffer solution in the chromatographic condition of the detection method; wherein the phosphate buffer is an aqueous solution comprising 150mM PB and 100mM NaCl, said protein being selected from an antibody or fusion protein, said antibody comprising the amino acid sequence of SEQ ID NO: 2 and the light chain sequence shown in SEQ ID NO: 3; the fusion protein comprises SEQ ID NO: 1; the pH of the mobile phase is 6.5-7.0; the chromatographic column is a TSK-GEL G3000SWXL type GEL exclusion chromatographic column.

2. The method for detecting proteins and dimers thereof according to claim 1, wherein the pH of the mobile phase is 7.0 in said chromatographic conditions.

3. The method for detecting proteins and dimers thereof according to claim 1, wherein the detection wavelength is 220nm in said chromatographic conditions.

4. The method for detecting proteins and dimers thereof according to claim 1, wherein the flow rate of the mobile phase in the chromatographic conditions is 0.5 mL/min.

5. The method for detecting proteins and dimers thereof according to claim 1, wherein the chromatographic conditions are a column temperature of 20-30 ℃.

6. The method for detecting proteins and dimers thereof according to claim 5, wherein the chromatographic conditions are at a column temperature of 25 ℃.

7. A method for detecting proteins and dimers thereof, comprising the steps of:

3) and (3) chromatographic detection: injecting the sample prepared in the step 2) into a high performance liquid chromatograph for detection by adopting a high performance liquid chromatography and a gel size exclusion chromatographic column, wherein the mobile phase solution prepared in the step 1) is used, the column temperature is 25 ℃, the flow rate is 0.5ml/min, and the wavelength is 220 nm; the chromatographic column is a TSK-GEL G3000SWXL type GEL exclusion chromatographic column; the protein is selected from an antibody or a fusion protein, wherein the antibody comprises SEQ ID NO: 2 and the light chain sequence shown in SEQ ID NO: 3; the fusion protein comprises SEQ ID NO: 1.