CN114249793A - Recombinant protein purification method - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a recombinant protein purification method. The invention provides a recombinant protein purification method, which adopts mixed mode chromatography, takes cation composite filler as the filler of a chromatographic column, and the ligand of the cation composite filler can be directly injected under a high conductance value, namely, a sample can be directly loaded without changing liquid and keep higher loading capacity when the sample is in higher conductance, a pH linear elution mode adopted can obtain better resolution ratio on the composite mode filler, one-step chromatography can obtain 95% purity, and the purity can only reach 86% after the existing single-step purification by adopting salt concentration gradient elution.

Description

Recombinant protein purification method

Technical Field

The invention relates to the technical field of biology, in particular to a recombinant protein purification method.

Background

The capture process employed in downstream purification of recombinant proteins is not only the process most severely affected by the increased antibody titer produced by optimizing mammalian cell expression systems, but is also the most important purification step that determines overall yield, product quality and economic cost. Affinity chromatography is routinely used in the first capture step of protein purification processes due to its high selectivity, resulting in good overall yield and purity. However, recombinant proteins without affinity tags expressed by mammalian Cells (CHO) cannot be purified by affinity chromatography, and only the characteristics of the protein such as charge and hydrophobicity can be utilized to develop a purification process; when the recombinant protein is purified by using the ionic filler, the conductance of the cell culture supernatant is too high, and the conductance is reduced by liquid exchange or dilution, so that a lot of field, equipment and labor cost can be increased in large-scale purification.

Therefore, there is a need to develop a simple, efficient and cost-effective method for purifying recombinant proteins.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, an object of the present invention is to provide a method for purifying recombinant proteins, which uses mixed-mode chromatography, uses cation composite filler as the filler of a chromatography column, and the ligand thereof can be directly injected under a high conductance value, i.e. the sample can be directly loaded without changing the liquid and keep a higher loading capacity at a higher conductance, and the adopted pH linear elution mode can obtain a better resolution ratio on the composite-mode filler, and can obtain a purity (SEC-HPLC) of 95% by one-step chromatography, while the purity of the existing single-step purification method using salt concentration gradient elution can only reach 86%.

To this end, the present invention provides in a first aspect a method for purifying a recombinant protein. According to an embodiment of the invention, the method comprises the steps of:

loading a sample to be purified containing the recombinant protein to a chromatographic column, eluting the chromatographic column by using an elution buffer solution so as to obtain an eluent containing the recombinant protein, and collecting an elution peak;

the isoelectric point of the recombinant protein is 5-7;

the filler of the chromatographic column is suitable for mixed mode chromatography;

the elution mode of the elution treatment is pH linear elution and/or pH gradient elution.

In the present invention, the elution mode of the elution process is linear elution with pH, and it is also possible to switch to pH gradient elution depending on the result of the linear elution.

Because the recombinant protein expressed by CHO cells of mammalian cells has no affinity label, affinity chromatography cannot be used, the removal effect of other chromatography modes on Host Cell Protein (HCP) is not as good as that of the affinity chromatography, the components of the supernatant of a cell culture solution are complex, the conductance is high, the supernatant needs to be replaced by ion chromatography, and the process complexity and the cost are increased. By adopting the recombinant protein purification method provided by the invention, the sample can be directly loaded without changing the liquid at higher conductance and the higher loading capacity of 40mg/ml is kept. Furthermore, the recombinant protein purification method provided by the invention is also applicable to the purification of the recombinant protein with the affinity tag.

Ion chromatography and multi-mode chromatography containing ion mode generally rely on salt concentration elution, but the salt concentration elution resolution is not enough, and the separation effect of partial protein is not good. The method adopts pH linear elution instead of salt concentration gradient elution to obtain better separation effect, and the purity of the target (recombinant) protein obtained by adopting pH linear elution is far higher than that of the target (recombinant) protein obtained by adopting a salt concentration gradient elution mode.

The recombinant protein purification method according to the embodiment of the present invention may further have at least one of the following additional technical features:

according to an embodiment of the invention, the sample to be purified is a mammalian cell culture fluid expressing the recombinant protein;

optionally, the mammalian cell is selected from any one of CHO cell and HEK293 cell.

According to an embodiment of the present invention, the method further comprises, prior to loading, subjecting the cell culture fluid to a pretreatment comprising acid precipitation;

optionally, the acid is acetic acid and/or citric acid.

The components of the cell culture solution are complex, the cell culture solution generally needs to be subjected to centrifugation and filtration or deep filtration to remove particulate matters such as cells and cell debris, and the operation is complex.

According to an embodiment of the present invention, the pH of the acid precipitate is in the range of pH4.2 to 4.8, preferably pH4.4 to 4.6, and more preferably pH 4.5.

The pH value of the acid precipitate is too high, so that the HCP removing effect is poor; if the pH of the acid precipitate is too low, the target protein may be unstable and precipitation or other structural changes may easily occur.

According to an embodiment of the invention, the filler suitable for mixed mode chromatography is a cationic composite filler;

optionally, the cationic composite filler is selected from at least one of ESHMUNO HCX, Capto MMC and Capto MMC Impres.

The trapping chromatographic packing can be selected from cationic composite packing of different companies, such as Capto MMC from GE.

According to the embodiment of the invention, the conductivity value of the elution buffer is 8-15 ms/cm, preferably 8-12 ms/cm, and the pH value is 4.5-8.5.

According to an embodiment of the present invention, the buffer in the elution buffer is selected from at least one of phosphate, acetate, citrate, carbonate, Tris, and further comprises a soluble sodium salt and a soluble potassium salt.

According to an embodiment of the invention, the buffer composition of the elution buffer may be NaPO₄NaAc, Tris and NaCl.

NaPO₄Means Na₂HPO₄And NaH₂PO₄The two reagents are mixed in a ratio such that the final pH is that of a particular pH (e.g., 4.5). The concentration of the buffer may be adjusted as appropriate according to the concentration conventionally used in the art.

According to an embodiment of the present invention, the method further comprises the step of column equilibration with an equilibration buffer prior to loading;

optionally, the conductivity value of the balance buffer solution is 8-12 ms/cm, the pH value is 4.2-4.8, and the preferred pH value is 4.4-4.6;

optionally, the buffer of the equilibration buffer is selected from at least one of phosphate, acetate, citrate, carbonate, Tris, and further comprises a soluble sodium salt, a soluble potassium salt.

The column equilibration is performed such that the chromatography column is maintained at a pH and conductivity value close to that of the sample.

According to an embodiment of the invention, the method further comprises a step of re-equilibration with a re-equilibration buffer while loading the sample to be purified onto the chromatography column;

optionally, the re-equilibration buffer is of the same composition as the equilibration buffer. The re-equilibration step may rinse the column of residual impurities that are not bound to the column.

According to the embodiment of the invention, the linear flow speed of the elution buffer is 100-200 cm/h.

In the present invention, the target protein/recombinant protein refers to a protein produced by selecting an appropriate expression system using gene recombination technology, and herein refers to a recombinant protein expressed by mammalian cells.

Capture/capture chromatography refers to the separation of the protein of interest from the cell culture broth, removing most of the impurities, relative to subsequent purification (further removal of trace impurities).

Gradient elution/linear elution means that elution is not performed by one solution, but two solutions are mixed in a certain ratio, and the ratio is gradually changed, so that the mixed solution gradually reaches a proper elution condition.

The target protein expressed by the mammalian cell expression system without an affinity tag cannot be subjected to affinity chromatography, other chromatography methods have poor effect on removing Host Cell Protein (HCP) compared with the affinity chromatography, and the control of the HCP becomes a difficult problem in the process. Affinity chromatography cannot be used, the components of cell culture solution supernatant are complex, the conductivity is high, and the use of ion chromatography requires solution exchange aiming at the supernatant, so that the process complexity and the cost are increased. Ion chromatography and multi-mode chromatography containing ion mode generally rely on salt concentration elution, but the salt concentration elution resolution is not enough, and the separation effect of partial protein is not good.

Mixed-mode chromatography (MMC) is a novel biological separation method, ligand of the MMC has multiple interactions of hydrophobicity, static electricity, hydrogen bond and the like, and the MMC has the characteristics of high adsorption capacity, good selectivity, mild elution condition and the like and can be used for replacing a classical affinity chromatography method.

The invention aims to solve the problems and provides a recombinant protein purification method, which adopts mixed mode chromatography, wherein a step of pretreating a cell culture solution enables HCP (host cell protein) to be greatly reduced (by about 50%), a cation composite filler (Capto MMC composite filler) is used as a filler of a chromatographic column, a ligand of the cation composite filler can be directly injected under a high conductance value, namely, a sample can be directly loaded without changing liquid and keep higher loading capacity of 40mg/ml when the sample is at a higher conductance, and in contrast, the loading capacity of the cation filler SP HP under the same conductance condition is lower than 5 mg/ml; the pH linear elution mode adopted can obtain better resolution ratio on the composite mode packing, one-step chromatography can obtain 95% purity (SEC-HPLC), and salt concentration gradient elution can only achieve 86% purity after single-step purification.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows the HCP (host cell protein) concentration in the supernatant obtained by pretreatment of cell culture broth with acids at various pH values;

FIG. 2 shows the results of the salt concentration gradient elution of the Capto MMC capture chromatography in comparative example 1;

FIG. 3 shows the pH linear elution results of Capto MMC capture chromatography in example 2.

Detailed Description

According to some specific embodiments of the present invention, there is provided a method for purifying a recombinant protein comprising the steps of:

1) sample clarification: removing cells and debris particles by two-step centrifugation, centrifuging for 10min at 1000g, centrifuging the supernatant at 6000g for 30min, and collecting the supernatant;

2) sample pretreatment: dropwise adding 5M acetic acid into the supernatant to adjust the pH to 4.2-4.8, and incubating for 5-30 min;

3) and (3) capture chromatography:

filling: capto MMC (GE)

Column balancing: the buffer composition is as follows: 25mM NaPO₄(Na₂HPO₄And NaH₂PO₄A reagent in which the two reagents are mixed in a certain proportion so that the final pH is 4.5), 50mM NaAc, 25mM Tris, 60mM NaCl, pH4.5, and the conductance value of the equilibrium buffer solution is 8-12 ms/cm;

loading: loading the pretreated sample on a Capto MMC purification column, wherein the conductance value of the culture solution is not additionally adjusted and is 9-11 ms/cm;

rebalancing: the buffer composition is as follows: 25mM NaPO₄，50mM NaAc，25mM Tris，60mM NaCl，pH4.5；

Elution buffer composition: 25mM NaPO₄50mM NaAc, 25mM Tris, 60mM NaCl, pH 4.5-8.5, and the conductivity value of the elution buffer solution is 8-15 ms/cm;

linear flow velocity (flow rate): 100-200 cm/h;

and collecting an elution peak.

The sample pretreatment method may also use caprylic acid precipitation technique. The two-step centrifugation method of the sample clarification step can be replaced with a disposable depth filter (Merck 20 MS). Sample clarification step a disposable depth filter is often used in medium scale production, but in small scale sample preparation, often without a filter of the appropriate size, a two step centrifugation would be applied. The chromatographic packing adopts a composite mode packing which can tolerate high-salt sample loading, and the elution mode adopts pH linear elution instead of salt concentration gradient elution, so that a better separation effect can be obtained. The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 pH optimization of acid in cell culture broth pretreatment step

The cell culture broth was CHO cell culture broth expressing fibrise, 202 amino acids, PI 5.32.

The fibrise amino acid sequence is shown as SEQ ID NO: 2, as shown in the figure:

ERFAPRYIELVIVADHSVATKYNDNVTAILSWVHQLVNNIILFYRDLNVHFTLSAVEVWSNGDLINVQPEATVTLNLFGEWRERDLLNRRMHDNAQLLNNVALDDNTIGLAYDEGMCDPKYSVGIVKDHSAINRMVAATMAHEIGHNLGMNHDGSQCNCGGNGCVMSAVLMQQHSYQFSDCSKDEYQRYLTNHNPQCILNQP

the nucleic acid sequence encoding fibrise is set forth in SEQ ID NO: 1, and the following components:

gaacgctttgcgccgcgctatattgaactggtgattgtggcggatcatagcgtggcgaccaaatataacgataacgtgaccgcgattctgagctgggtgcatcagctggtgaacaacattattctgttttatcgcgatctgaacgtgcattttaccctgagcgcggtggaagtgtggagcaacggcgatctgattaacgtgcagccggaagcgaccgtgaccctgaacctgtttggcgaatggcgcgaacgcgatctgctgaaccgccgcatgcatgataacgcgcagctgctgaacaacgtggcgctggatgataacaccattggcctggcgtatgatgaaggcatgtgcgatccgaaatatagcgtgggcattgtgaaagatcatagcgcgattaaccgcatggtggcggcgaccatggcgcatgaaattggccataacctgggcatgaaccatgatggcagccagtgcaactgcggcggcaacggctgcgtgatgagcgcggtgctgatgcagcagcatagctatcagtttagcgattgcagcaaagatgaatatcagcgctatctgaccaaccataacccgcagtgcattctgaaccagccg

CHO cell culture fluid: the synthesized fibrolase gene sequence is inserted into an expression vector pCMV to construct an expression plasmid, and the PEI reagent transfects the plasmid into a CHO cell to obtain a cell culture solution through amplification culture.

The protein capture method comprises the following steps:

1) sample clarification: removing cells and debris particles by two-step centrifugation, centrifuging for 10min at 1000g, centrifuging the supernatant at 6000g for 30min, and collecting the supernatant;

2) sample pretreatment: when the cells are harvested, 5M acetic acid is dripped into the culture solution under the stirring state to adjust the pH value, and the cells are incubated for 30 min.

In the cell culture solution pretreatment step, the cell culture solution pH was adjusted to 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 with acetic acid, respectively. Host cell proteins were detected by HCP ELISA detection kit (purchased from Cygnus) and control samples were set up without acid precipitation.

The results in FIG. 1 show that the HCP (host cell protein) in the supernatant after pretreatment of the cell culture broth by the acid precipitation technique can be greatly reduced, and the HCP gradually decreases with decreasing pH, but no significant decrease of HCP is observed after the pH decreases to pH4.6, so that the pH of the acid precipitation is in the range of pH 4.2-4.8. In view of maintaining the stability of the target protein, pH4.5 was selected as the final pH of the acid precipitation.

Example 2pH Linear elution

The cell culture solution is CHO cell culture solution for expressing fibrise, and the protein capture method comprises the following steps:

1) sample clarification: removing cells and debris particles by two-step centrifugation, centrifuging for 10min at 1000g, centrifuging for 30min at 6000g, and collecting supernatant for 30 min;

2) sample pretreatment: dropwise adding 5M acetic acid into the supernatant to adjust the pH value to 4.5, and incubating for 30 min;

3) and (3) capture chromatography:

filling: capto MMC (GE);

column balancing: and (3) an equilibrium buffer: 25mM NaPO₄50mM NaAc, 25mM Tris, 60mM NaCl, pH4.5, and the conductivity value of the balance buffer solution is 10 ms/cm;

loading: loading the pretreated sample on a Capto MMC purification column, wherein the conductance value of the culture solution is not additionally adjusted and is 9-11 ms/cm;

rebalancing: and (3) an equilibrium buffer: 25mM NaPO₄，50mM NaAc，25mM Tris，60mM NaCl，pH4.5；

And (3) elution:

elution buffer a: 25mM NaPO₄，50mM NaAc，25mM Tris，60mM NaCl，pH4.5；

Elution buffer B: 25mM NaPO₄，50mM NaAc，25mM Tris，60mM NaCl，pH8.5；

In the elution process, the ratio of the elution buffer solution A to the elution buffer solution B is adjusted, the ratio of the elution buffer solution B is changed between 0% and 100%, the pH of the elution buffer solution is linearly changed between 4.5 and 8.5, pH linear elution is carried out, and the conductivity value of the elution buffer solution is 10 ms/cm;

linear flow velocity (flow rate): 200 cm/h;

and collecting an elution peak.

Comparative example 1 salt Linear elution

The cell culture solution is CHO cell culture solution for expressing fibrise, and the protein capture method comprises the following steps:

1) sample clarification: removing cells and debris particles by two-step centrifugation, centrifuging for 10min at 1000g, centrifuging for 30min at 6000g, and collecting supernatant for 30 min;

2) sample pretreatment: dropwise adding 5M acetic acid into the supernatant to adjust the pH value to 4.5, and incubating for 30 min;

3) and (3) capture chromatography:

filling: capto MMC (GE);

column balancing: and (3) an equilibrium buffer: 50mM NaAc, 60mM NaCl, pH 4.5;

loading: loading the pretreated sample on a Capto MMC purification column, wherein the conductance value of the culture solution is not additionally adjusted and is 9-11 ms/cm;

rebalancing: and (3) an equilibrium buffer: 50mM NaAc, 60mM NaCl, pH 4.5;

and (3) elution:

elution buffer a: 50mM NaAc, 60mM NaCl, pH 4.5;

elution buffer B: 50mM NaAc, 1M NaCl, pH 4.5;

in the elution process, the salt concentration of the elution buffer solution is linearly changed by adjusting the proportion of the elution buffer solution A to the elution buffer solution B and changing the proportion of the elution buffer solution B between 0 and 100 percent, and salt linear elution is carried out;

linear flow velocity (flow rate): 100-200 cm/h;

and collecting an elution peak.

Comparative example 1 the results of salt linear elution are shown in figure 2 and example 2 the results of pH linear elution are shown in figure 3. In addition, the filler in example 2 above was replaced with SP HP (normal cationic filler) for protein purification.

The purity and recovery of the recombinant protein obtained by using different fillers and elution conditions are shown in the following table 1:

TABLE 1 comparison of different fillers and elution conditions

The results in table 1 show that the composite mode filler has a higher loading than the conventional ionic filler and is suitable for use in such protein purification. The elution mode of the composite mode packing comprises salt linear elution and pH linear elution, and although the recovery rate of the recombinant protein is equivalent in the two elution modes, the pH elution has better resolution and the purity of the obtained protein is higher.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Sequence listing

<110> Fushan Hanteng Biotech Co., Ltd

CANTONBIO Co.,Ltd.

<120> method for purifying recombinant protein

<130> PIDC3203309

<160> 2

<170> PatentIn version 3.5

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<211> 606

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gaacgctttg cgccgcgcta tattgaactg gtgattgtgg cggatcatag cgtggcgacc 60

aaatataacg ataacgtgac cgcgattctg agctgggtgc atcagctggt gaacaacatt 120

attctgtttt atcgcgatct gaacgtgcat tttaccctga gcgcggtgga agtgtggagc 180

aacggcgatc tgattaacgt gcagccggaa gcgaccgtga ccctgaacct gtttggcgaa 240

tggcgcgaac gcgatctgct gaaccgccgc atgcatgata acgcgcagct gctgaacaac 300

gtggcgctgg atgataacac cattggcctg gcgtatgatg aaggcatgtg cgatccgaaa 360

tatagcgtgg gcattgtgaa agatcatagc gcgattaacc gcatggtggc ggcgaccatg 420

gcgcatgaaa ttggccataa cctgggcatg aaccatgatg gcagccagtg caactgcggc 480

ggcaacggct gcgtgatgag cgcggtgctg atgcagcagc atagctatca gtttagcgat 540

tgcagcaaag atgaatatca gcgctatctg accaaccata acccgcagtg cattctgaac 600

cagccg 606

<210> 2

<211> 202

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Glu Arg Phe Ala Pro Arg Tyr Ile Glu Leu Val Ile Val Ala Asp His

1 5 10 15

Ser Val Ala Thr Lys Tyr Asn Asp Asn Val Thr Ala Ile Leu Ser Trp

20 25 30

Val His Gln Leu Val Asn Asn Ile Ile Leu Phe Tyr Arg Asp Leu Asn

35 40 45

Val His Phe Thr Leu Ser Ala Val Glu Val Trp Ser Asn Gly Asp Leu

50 55 60

Ile Asn Val Gln Pro Glu Ala Thr Val Thr Leu Asn Leu Phe Gly Glu

65 70 75 80

Trp Arg Glu Arg Asp Leu Leu Asn Arg Arg Met His Asp Asn Ala Gln

85 90 95

Leu Leu Asn Asn Val Ala Leu Asp Asp Asn Thr Ile Gly Leu Ala Tyr

100 105 110

Asp Glu Gly Met Cys Asp Pro Lys Tyr Ser Val Gly Ile Val Lys Asp

115 120 125

His Ser Ala Ile Asn Arg Met Val Ala Ala Thr Met Ala His Glu Ile

130 135 140

Gly His Asn Leu Gly Met Asn His Asp Gly Ser Gln Cys Asn Cys Gly

145 150 155 160

Gly Asn Gly Cys Val Met Ser Ala Val Leu Met Gln Gln His Ser Tyr

165 170 175

Gln Phe Ser Asp Cys Ser Lys Asp Glu Tyr Gln Arg Tyr Leu Thr Asn

180 185 190

His Asn Pro Gln Cys Ile Leu Asn Gln Pro

195 200

Claims (10)

1. A method of purifying a recombinant protein, comprising the steps of:

loading a sample to be purified containing the recombinant protein to a chromatographic column, eluting the chromatographic column by using an elution buffer solution so as to obtain an eluent containing the recombinant protein, and collecting an elution peak;

the isoelectric point of the recombinant protein is 5-7;

the filler of the chromatographic column is suitable for mixed mode chromatography;

the elution mode of the elution treatment is pH linear elution and/or pH gradient elution.

2. The method according to claim 1, wherein the sample to be purified is a mammalian cell culture fluid expressing the recombinant protein;

optionally, the mammalian cell is any one selected from a CHO cell and a HEK293 cell.

3. The method of claim 2, further comprising, prior to loading, subjecting the cell culture fluid to a pretreatment comprising acid precipitation;

optionally, the acid is acetic acid and/or citric acid.

4. The method according to claim 3, wherein the pH of the acid precipitation is in the range of pH 4.2-4.8, preferably pH 4.4-4.6.

5. The method of claim 1, wherein the filler suitable for mixed mode chromatography is a cationic composite filler;

optionally, the cationic composite filler is selected from at least one of ESHMUNO HCX, Capto MMC and Capto MMC Impres.

6. The method according to claim 1, wherein the conductivity of the elution buffer is 8 to 15ms/cm and the pH is 4.5 to 8.5.

7. The method of claim 1, wherein the buffer in the elution buffer is selected from at least one of phosphate, acetate, citrate, carbonate, Tris, and further comprises a soluble sodium salt and a soluble potassium salt.

8. The method of claim 1, further comprising the step of column equilibration with an equilibration buffer prior to loading;

optionally, the conductivity value of the balance buffer solution is 8-12 ms/cm, the pH value is 4.2-4.8, and the preferred pH value is 4.4-4.6;

optionally, the buffer of the equilibration buffer is selected from at least one of phosphate, acetate, citrate, carbonate, Tris, and further comprises a soluble sodium salt, a soluble potassium salt.

9. The method according to claim 1 or 8, further comprising a step of re-equilibration with a re-equilibration buffer while loading the sample to be purified onto the chromatography column;

optionally, the re-equilibration buffer is of the same composition as the equilibration buffer.

10. The method according to claim 1, wherein the linear flow rate of the elution buffer is 100 to 200 cm/h.

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