CN112430252A - Chromatography method for improving recovery rate of target protein - Google Patents
Chromatography method for improving recovery rate of target protein Download PDFInfo
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- CN112430252A CN112430252A CN202011265249.7A CN202011265249A CN112430252A CN 112430252 A CN112430252 A CN 112430252A CN 202011265249 A CN202011265249 A CN 202011265249A CN 112430252 A CN112430252 A CN 112430252A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21004—Trypsin (3.4.21.4)
Abstract
The invention relates to a chromatography method for improving the recovery rate of target protein, which comprises the following steps: taking a solution containing target protein as a raw material, adding salt, adjusting the pH value to obtain a treated sample solution, filling a chromatographic medium into a chromatographic column, controlling the sample loading temperature of the treated sample solution, and performing chromatographic operation. According to the invention, salts are added into the sample, and the pH value and the temperature of the sample solution are adjusted, so that the combination environment of the target protein and the chromatography medium is improved, the capture efficiency of the sample is improved, and the recovery rate is up to more than 95%.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, and relates to a chromatography method for improving the recovery rate of target protein.
Background
Protein drugs are one of the important development directions in the field of biopharmaceuticals, and the research or preparation of protein drugs requires a proper protein separation and purification technique to recover target proteins from a solution containing the target proteins. Protein separation and purification utilizes the inherent similarity and difference between different proteins to remove protein and non-protein impurities. Chromatographic techniques based on adsorption mechanisms are one of the most commonly used methods for protein isolation and purification.
Adsorption chromatography is a purification technique for separating a sample by adsorption and desorption between a stationary phase and a mobile phase. The forces of adsorption thereof typically include: electrostatic attraction between oppositely charged groups between the sample being separated and the chromatographic medium, hydrogen bonding, orientation between dipolar molecules, van der waals forces, and the like. Common chromatographic media include affinity filler, ion exchange filler, adsorption resin filler and the like. The adsorption performance between the chromatographic medium (or chromatographic packing) and the protein determines the separation and purification effect. The adsorption performance is influenced by the nature or state of the protein and the nature of the solution, in addition to the mechanism of action between the sample and the chromatographic medium.
The current research on protein separation and purification mainly includes two aspects: firstly, preparation and modification research of a chromatography medium: for example, CN101415692B, CN201310168576 and CN101827648A prepare different protein separation and purification chromatography media; luhuili (influence of chromatography medium aperture and ligand density on protein adsorption performance and antibody separation [ D ]]Zhejiang university, 2014), asterinamin (preparation and application of three adsorption separation materials based on grafted polymer chain [ D)]Northwest university, 2012) and the like. The methods need to develop special chromatographic packing and corresponding chromatographic processes, have poor universality, complex and long-time development process, and have large commercial risk of customized chromatographic packing. On the other hand, the improvement research of the chromatography process is mainly used for separating and purifying proteins by optimizing the conductivity or concentration or pH value of the buffer salt or improving the elution mode: such as CN1260249C and its respective cases, by changing the conductivity and/or pH of the buffer to isolate the polypeptide; CN106496302A by using different pH and/or concentrationRemoving different impurities in the protein by adopting a multi-step elution mode through a Tris buffer solution; CN102382168A adding proper amount of protein binding metal ion (Fe) into protein mixture and chromatography buffer solution3+、Ca2+) To improve the resolution of the target protein and the hetero-protein in ion exchange chromatography, the method can be applied only to proteins having a binding effect with metal ions, with the aim of maintaining the tertiary structure of the target protein. The method mainly aims to remove the foreign protein or improve the resolution, and the recovery rate of the target protein is not optimized, so that the recovery rate is low.
Disclosure of Invention
In order to solve the technical problems, the chromatographic method for improving the recovery rate of the target protein is provided, the sample environment is improved to improve the binding force between the protein and the chromatographic medium, so that the amino acid groups bound with the chromatographic medium are fully exposed, the effective contact part between the protein and the chromatographic medium is increased, and the temperature of the sample is controlled to improve the binding force between the protein and the chromatographic medium, so that the recovery rate is improved.
The technical scheme of the invention is as follows:
a chromatographic method for increasing recovery of a target protein, the method comprising the steps of:
s1: sample solution treatment: taking a solution containing target protein as a raw material, adding salt, and adjusting the pH value to obtain a treated sample solution;
s2: carrying out adsorption chromatography: filling a chromatographic column with a chromatographic medium;
s3: and controlling the sample loading temperature of the treated sample solution, and performing chromatography operation.
Preferably, the target protein in step S1 is insulin or an insulin analogue or a protease or a polypeptide.
Preferably, the chromatographic medium in step S2 is affinity chromatography packing or macroporous adsorption chromatography or ion exchange chromatography packing or reverse phase chromatography packing.
Preferably, the salt in step S1 is sulfate, citrate, acetate or a combination thereof.
Further preferably, the sulfate is magnesium sulfate or sodium sulfate or ammonium sulfate, the citrate is sodium citrate, and the acetate is sodium acetate or ammonium acetate.
More preferably, the addition amount of the salt is 0.05-0.5 mol/L.
More preferably, the amount of the salt added is 0.1 to 0.3 mol/L.
Preferably, the pH is adjusted to be more than 1 unit above or below the isoelectric point of the target protein in step S1.
Further preferably, the acidic substance for adjusting the pH value is phosphoric acid or glacial acetic acid or citric acid or hydrochloric acid; the alkaline substance for adjusting the pH value is sodium hydroxide, ammonia water or potassium hydroxide.
Preferably, in step S3: the sample solution loading temperature is 25-30 ℃.
Preferably, in step S3: the chromatographic process is as follows:
balancing: washing the chromatographic column with an equilibration solution;
secondly, loading: loading the treated sample on a column;
③ flushing: flushing with a balancing liquid or a flushing liquid;
and fourthly, elution: eluting with an eluent, and collecting a main peak;
fifth, regeneration: and (4) carrying out regeneration treatment on the filler by using the regeneration liquid.
Wherein the flow rate of the sample loading, the washing and the elution is 60-210 cm/h.
The invention has the beneficial effects that:
(1) according to the invention, salts are added into the sample, the pH value of the sample solution is adjusted, so that amino acid groups combined with the chromatographic medium are fully exposed, the effective contact part between the protein and the chromatographic medium is increased, and the temperature of the sample is controlled to improve the binding force between the protein and the chromatographic medium. By the regulation and control, the capture efficiency of the sample is greatly improved, and the recovery rate is up to more than 95%.
(2) The invention improves the binding force between protein and chromatography medium, thus greatly improving the flow rate of the steps of loading, eluting and the like without reducing the recovery rate. The flow rate of sample loading, washing and elution is generally below 135cm/h, and can be increased to 150-210 cm/h after salts are added, so that the working hours are shortened by more than 40%, and the production efficiency is greatly improved.
(3) The invention is based on improving the binding force between the protein and the chromatography medium, so the invention can be applied to other chromatography media with the same chromatography mechanism and similar properties, and has strong adaptability. The method does not need to change the original chromatographic medium and chromatographic process, can improve the recovery rate by only carrying out simple sample treatment, has simple operation and low implementation cost, and is easy for commercialization and large-scale application.
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FIG. 1 is a schematic view of the process of the present invention.
Detailed Description
The invention will be further illustrated by the following examples. The examples are intended to illustrate the invention only and do not limit the scope of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and the preferred embodiments described herein are exemplary only.
In the method, a chromatography system is an AKTAavantan 25 system of American general electric company, a chromatography column is an XK16 glass column, and a detector detects the wavelength of 280 nm; the materials and the reagents are analytically pure. And (3) detecting the concentration of the target protein by using a Waterse2695 high performance liquid chromatograph.
The recovery rate calculation method comprises the following steps:
example 1
A chromatographic method for increasing recovery of a target protein, the method comprising the steps of:
s1: sample solution treatment: taking a solution containing target protein as a raw material, adding salt, and adjusting the pH value to obtain a treated sample solution;
s2: carrying out adsorption chromatography: filling a chromatographic column with a chromatographic medium, wherein the filling height is 28 cm;
s3: and controlling the sample loading temperature of the treated sample solution, and performing chromatography operation.
The target protein in step S1 is insulin glargine precursor.
Preferably, the chromatographic medium in step S2 is an affinity chromatography packing.
Preferably, the salts in step S1 are magnesium sulfate with different concentrations.
Preferably, in the step S1, the pH value is adjusted to 4.8, and the alkali is adjusted to be 4mol/L sodium hydroxide solution.
Preferably, in step S3: the sample solution loading temperature was 27 ℃.
Further preferably, in step S3: the chromatographic process is as follows:
balancing: washing the column with equilibration solution for 3CV (CV, column volume);
secondly, loading: loading the treated sample on a column;
③ flushing: rinsing with 3.5CV of rinsing solution;
and fourthly, elution: eluting with an eluent, and collecting a main peak;
fifth, regeneration: regenerating the filler 3CV by using the regeneration liquid;
the flow rate of loading, washing and elution was 210 cm/h.
The results of the embodiment are shown in table 1, the combination between the protein and the chromatography medium is improved by adding magnesium sulfate with different concentrations into the sample, and when the dosage of the magnesium sulfate is 0.1-0.3 mol/L, the recovery rate of the target protein reaches more than 95.0%, and the highest recovery rate reaches 98.5%. However, when the concentration of magnesium sulfate is too high, the binding of protein to the chromatography medium is inhibited, resulting in a decrease in recovery.
Table 1 example 1 data
Example 2
A chromatographic method for increasing recovery of a target protein, the method comprising the steps of:
s1: sample solution treatment: taking a solution containing target protein as a raw material, adding salt, and adjusting the pH value to obtain a treated sample solution;
s2: carrying out adsorption chromatography: filling a chromatographic column with a chromatographic medium, wherein the filling height is 28 cm;
s3: and controlling the sample loading temperature of the treated sample solution, and performing chromatography operation.
The target protein in step S1 is insulin glargine precursor.
Preferably, the chromatographic medium in step S2 is an affinity chromatography packing.
Preferably, the salt in step S1 is magnesium sulfate, and the addition amount is 0.15 mol/L.
Preferably, in the step S1, the pH value is adjusted to 4.8, and the alkali is adjusted to be 4mol/L sodium hydroxide solution.
Preferably, in step S3: the sample solution controls different loading temperatures.
Further preferably, in step S3: the chromatographic process is as follows:
balancing: washing the chromatographic column 3CV by using an equilibrium solution;
secondly, loading: loading the treated sample on a column;
③ flushing: rinsing with 3.5CV of rinsing solution;
and fourthly, elution: eluting with an eluent, and collecting a main peak;
fifth, regeneration: regenerating the filler 3CV by using the regeneration liquid;
the flow rate of loading, washing and elution was 210 cm/h.
The results of this example are shown in Table 2, where different sample temperatures have different effects on the binding force between the protein and the chromatographic medium. With the higher layer of the sample temperature, the binding force is enhanced, and the recovery rate of the target protein is increased. When the temperature of the sample is above 27 ℃, the recovery rate of the target protein is up to more than 98%.
Table 2 example 2 data
Example 3
A chromatographic method for increasing recovery of a target protein, the method comprising the steps of:
s1: sample solution treatment: taking a solution containing target protein as a raw material, adding salt, and adjusting the pH value to obtain a treated sample solution;
s2: carrying out adsorption chromatography: filling a chromatographic column with a chromatographic medium, wherein the filling height is 28 cm;
s3: and controlling the sample loading temperature of the treated sample solution, and performing chromatography operation.
The target protein in step S1 is insulin glargine precursor.
Preferably, the chromatographic medium in step S2 is an affinity chromatography packing.
Preferably, the salt in step S1 is magnesium sulfate, and the addition amount is 0.15 mol/L.
Preferably, the step S1 adjusts the pH of the sample solution differently.
Preferably, in step S3: the sample solution loading temperature was 27 ℃.
Further preferably, in step S3: the chromatographic process is as follows:
balancing: washing the chromatographic column 3CV by using an equilibrium solution;
secondly, loading: loading the treated sample on a column;
③ flushing: rinsing with 3.5CV of rinsing solution;
and fourthly, elution: eluting with an eluent, and collecting a main peak;
fifth, regeneration: regenerating the filler 3CV by using the regeneration liquid;
the flow rate of loading, washing and elution was 210 cm/h.
As the sample is required to be dissolved in the mobile phase in the chromatographic operation process, the pH value needs to avoid the isoelectric point of the protein. In this example, the influence of different pH values on the adsorption characteristics was examined for insulin glargine precursor (PI is 6.9), and it can be seen from table 3 that the recovery rates in the chromatographic process are not much different and are all above 95% when the pH value is between 4.5 and 5.0.
Table 3 example 3 data
Example 4
A chromatographic method for increasing recovery of a target protein, the method comprising the steps of:
s1: sample solution treatment: taking a solution containing target protein as a raw material, adding salt, and adjusting the pH value to obtain a treated sample solution;
s2: carrying out adsorption chromatography: filling a chromatographic column with a chromatographic medium, wherein the filling height is 28 cm;
s3: and controlling the sample loading temperature of the treated sample solution, and performing chromatography operation.
The target protein in step S1 is insulin glargine precursor.
Preferably, the chromatographic medium in step S2 is an affinity chromatography packing.
Preferably, the salts in step S1 are different salts.
Preferably, the pH is adjusted to 4.8 in step S1.
Preferably, in step S3: the sample solution loading temperature was 27 ℃.
Further preferably, in step S3: the chromatographic process is as follows:
balancing: washing the chromatographic column 3CV by using an equilibrium solution;
secondly, loading: loading the treated sample on a column;
③ flushing: rinsing with 3.5CV of rinsing solution;
and fourthly, elution: eluting with an eluent, and collecting a main peak;
fifth, regeneration: regenerating the filler 3CV by using the regeneration liquid;
the flow rate of the sample loading, the washing and the elution is 90-210 cm/h.
The results of this example are shown in table 4, and when different salts such as sulfate, citrate, acetate, etc. or their complex is added to the sample, the recovery rate of the target protein is improved, and the recovery rate is about 95%. The method is mainly characterized in that after salts are added, amino acid groups combined with a chromatography medium in the protein are exposed, effective contact positions of the protein and the chromatography medium are increased, the combination capacity between the protein and the chromatography medium is improved, and the recovery rate is improved. However, when salts such as phosphate are added, these amino acid groups are folded inside, and thus the protein is not effectively bound to the chromatography medium during loading, and the recovery rate is greatly reduced.
When no salt is added, the binding force between the target protein and the chromatography medium is weak, the recovery rate is low, and even if measures such as flow rate reduction are taken, the improvement of the recovery rate is limited.
Therefore, the addition of sulfate, citrate, acetate or the like to the sample can improve the recovery rate, and at the same time, the flow rate can be increased to shorten the time and improve the production efficiency while ensuring the recovery rate.
Table 4 example 4 data
Example 5
A chromatographic method for increasing recovery of a target protein, the method comprising the steps of:
s1: sample solution treatment: taking a solution containing target protein as a raw material, adding salt, and adjusting the pH value to obtain a treated sample solution;
s2: carrying out adsorption chromatography: filling a chromatographic column with a chromatographic medium, wherein the filling height is 28 cm;
s3: and controlling the sample loading temperature of the treated sample solution, and performing chromatography operation.
The target protein in step S1 is insulin aspart precursor.
Preferably, the chromatographic medium in step S2 is an affinity chromatography packing.
Preferably, different salts are added in the step S1, and the addition amount is 0.1-0.3 mol/L.
Preferably, the pH is adjusted to 5.5 in step S1.
Preferably, in step S3: the sample solution loading temperature is 25-30 ℃.
Further preferably, in step S3: the chromatographic process is as follows:
balancing: washing the chromatographic column 4CV with an equilibration solution;
secondly, loading: loading the treated sample on a column;
③ flushing: washing 5CV with the balance liquid;
and fourthly, elution: eluting with an eluent, and collecting a main peak;
fifth, regeneration: regenerating the filler 3CV by using the regeneration liquid;
the flow rate of loading, washing and elution was 210 cm/h.
In this example, the insulin aspart precursor was used as the target protein, and chromatography was carried out using affinity chromatography packing, and the results of the examples are shown in Table 5. When salts such as magnesium sulfate are added into the sample, the recovery rate is up to more than 95%.
Table 5 example 5 data
Example 6
A chromatographic method for increasing recovery of a target protein, the method comprising the steps of:
s1: sample solution treatment: taking a solution containing target protein as a raw material, adding salt, and adjusting the pH value to obtain a treated sample solution;
s2: carrying out adsorption chromatography: filling a chromatographic column with a chromatographic medium, wherein the filling height is 18 cm;
s3: and controlling the sample loading temperature of the treated sample solution, and performing chromatography operation.
The target protein in the step S1 is recombinant trypsin.
Preferably, the chromatographic medium in step S2 is cation exchange chromatographic packing.
Preferably, different salts are added in the step S1, and the addition amount is 0.1-0.3 mol/L.
Preferably, the pH is adjusted to 5.3 in step S1.
Preferably, in step S3: the sample solution loading temperature is 25-30 ℃.
Further preferably, in step S3: the chromatographic process is as follows:
balancing: washing the chromatographic column 3CV by using an equilibrium solution;
secondly, loading: loading the treated sample on a column;
③ flushing: washing 2CV by using an equilibrium solution;
and fourthly, elution: eluting with an eluent, and collecting a main peak;
fifth, regeneration: 3.5CV of filler is regenerated by using regeneration liquid;
the flow rate of loading, washing and elution was 210 cm/h.
This example uses recombinant trypsin as the target protein and chromatography is carried out using cation exchange chromatography packing, and the results of the examples are shown in Table 6. It can be seen that the recovery of the target protein is also as high as 95%.
Table 6 example 6 data
Example 7
A chromatographic method for increasing recovery of a target protein, the method comprising the steps of:
s1: sample solution treatment: taking a solution containing target protein as a raw material, adding salt, and adjusting the pH value to obtain a treated sample solution;
s2: carrying out adsorption chromatography: filling a chromatographic column with a chromatographic medium, wherein the filling height is 100 cm;
s3: and controlling the sample loading temperature of the treated sample solution, and performing chromatography operation.
In the step S1, the target protein is a recombinant human insulin precursor.
Preferably, the chromatographic medium in step S2 is a macroporous adsorbent resin filler.
Preferably, different salts are added in the step S1, and the addition amount is 0.1-0.3 mol/L.
Preferably, the pH is adjusted to 4.5 in step S1.
Preferably, in step S3: the sample solution loading temperature is 25-30 ℃.
Further preferably, in step S3: the chromatographic process is as follows:
balancing: washing the chromatographic column 3CV by using an equilibrium solution;
secondly, loading: loading the treated sample on a column;
③ flushing: washing 3CV by using an equilibrium solution;
and fourthly, elution: eluting with an eluent, and collecting a main peak;
fifth, regeneration: 3.5CV of filler is regenerated by using regeneration liquid;
the flow rate of loading, washing and elution was 210 cm/h.
In this example, recombinant human insulin precursor was used as the target protein, and chromatography was performed using macroporous adsorbent resin packing, and the results of the examples are shown in Table 7. It can be seen that the recovery of the target protein is also as high as 95%.
Table 7 example 7 data
The above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A chromatographic method for increasing the recovery of a target protein, comprising the steps of:
s1: sample solution treatment: taking a solution containing target protein as a raw material, adding salt, and adjusting the pH value to obtain a treated sample solution;
s2: carrying out adsorption chromatography: filling a chromatographic column with a chromatographic medium;
s3: and controlling the sample loading temperature of the treated sample solution, and performing chromatography operation.
2. The method of claim 1, wherein the target protein in step S1 is insulin or an insulin analog or a protease or a polypeptide.
3. The method as claimed in claim 1, wherein the chromatographic medium in step S2 is affinity chromatography filler or macroporous adsorption chromatography filler or ion exchange chromatography filler or reverse phase chromatography filler.
4. The method of claim 1, wherein the salt in step S1 is sulfate, citrate, acetate or a combination thereof.
5. The method of claim 4, wherein the sulfate salt is magnesium sulfate or sodium or ammonium sulfate, the citrate salt is sodium citrate, and the acetate salt is sodium or ammonium acetate.
6. The method of claim 4, wherein the salt is added in an amount of 0.05 to 0.5 mol/L.
7. The method according to claim 1 or 2, wherein the pH is adjusted to a value higher or lower than the isoelectric point of the target protein by 1 unit or more in step S1.
8. The method according to claim 7, wherein the acidic substance for adjusting the pH value is phosphoric acid or glacial acetic acid or citric acid or hydrochloric acid; the alkaline substance for adjusting the pH value is sodium hydroxide, ammonia water or potassium hydroxide.
9. The method according to claim 1, wherein in step S3: the sample solution loading temperature is 25-30 ℃.
10. The method according to claim 1, wherein in step S3: the chromatographic process is as follows:
balancing: washing the chromatographic column with an equilibration solution;
secondly, loading: loading the treated sample on a column;
③ flushing: flushing with a balancing liquid or a flushing liquid;
and fourthly, elution: eluting with an eluent, and collecting a main peak;
fifth, regeneration: carrying out regeneration treatment on the filler by using a regeneration liquid;
wherein the flow rate of the sample loading, the washing and the elution is 60-210 cm/h.
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