WO2014204023A1 - Novel purification method for darbepoetin alpha - Google Patents

Abstract

The present invention relates to a high-purity purification method for darbepoetin alpha having a high content of sugar chains and, particularly, to a purification method for darbepoetin alpha comprising the steps of: (a) eluting a fraction comprising darbepoetin alpha by applying a biological emulsion comprising darbepoetin alpha to blue resin chromatography; (b) eluting a fraction comprising darbepoetin alpha by applying an eluate produced from step (a) to hydroxyapatite resin chromatography; and (c) eluting a fraction comprising darbepoetin alpha by applying an eluate produced from step (b) to anion-exchange resin chromatography.

Description

Novel method of purifying darbepoetin alfa

The present invention relates to a high-purity purification method of dabepoetin alfa having a high sugar chain content. Specifically, (a) a biological fluid containing dabepoetin alfa is subjected to blue resin chromatography to obtain a fraction containing dabepoetin alfa. Eluting; (b) subjecting the eluate produced in step (a) to hydroxyapatite resin chromatography to elute the fraction comprising dabepoetin alfa; And (c) subjecting the eluate produced in step (b) to anion exchange resin chromatography to elute the fraction comprising dabepoetin alfa.

Darbepoetin Alfa (NESP) is a glycoprotein (see PCT patent application US94 / 02957) that replaces five amino acids in the molecule of erythropoietin (EPO) and adds two N-sugar chains. Biochemical properties such as molecular weight and isoelectric point are distinguished. Darbepoetin alfa is an erythrocyte production promoting protein such as EPO.

In addition to sugar chains, dabepoetin alfa has a plasma half-life of about 3 times longer than erythropoietin in mice, rats, dogs, and humans (Pedrazzoli P, Cinieri S, Lorusso V, Gamucci T, Secondino S, Silvestris N 2007 Nov-Dec, 27 (6C), 4419-24; Anticancer Res.), The maximum number of structural isoforms according to the sialic acid content of dabepoetin alfa (22), more than 14 of erythropoietin. Structural subtypes with high sugar chains and sialic acid content of dabepoetin alfa have a low isoelectric point and these structural subtypes are known to have high biological activity in vivo (US Pat. No. US09 / 48239).

Conventional purification of dabepoetin alfa proceeds with anion exchange resin alone or with anion exchange resin and C4 resin (PCT patent application US1994 / 09257), anion exchange resin and cation exchange resin, and then anion exchange resin. (See PCT patent application US09 / 48239). In particular, US Pat. No. 09/48239 discloses a method for purifying dabepoetin alfa with high sialic acid content having an isoelectric point of 4.5 or less so that dabepoetin alfa flows out through at least one cation exchange resin. However, the method of US Pat. No. 09/48239, which includes purification of several steps of cation exchange resin and concentration and dialysis to repeat the process of obtaining and concentrating the protein through flow, has a disadvantage in that the process takes a long time. As such, the conventional purification process of dabepoetin alfa has to go through a complicated process using chromatography using several stages of resin, and as a result, the cost and time are enormous, and thus, a necessity of a simple process is required. .

The present inventors have made diligent efforts to find a method for purifying dabepoetin alfa with high purity and high yield in a simple purification process. As a result, the dabepoetin alfa is prepared through a combination process of blue resin, hydroxyapatite resin and ion exchange resin. The present invention was completed by confirming that dabepoetin alfa having a high purity of a sugar chain content of more than 95% can be purified from the cell culture solution.

It is an object of the present invention to provide a method for purifying high purity dabepoetin alfa having structural subtypes with high sugar chains and sialic acid content.

The method of the present invention is a method for separating novel dabepoetin alpha, which is convenient and easy to apply to production, in a large amount of high purity dabepoetin alpha having a structural subtype having high sugar chain and sialic acid content from dabepoetin alpha cell culture medium. It can be purified and can be usefully used for the preparation of protein therapeutics.

FIG. 1 is a diagram showing the results obtained by measuring the purity of dabepoetin alfa separated using blue resin chromatography by C4 HPLC analysis chromatography.

FIG. 2 is a diagram showing the results of measurement of eluate purity of dabepoetin alfa separated using anion exchange resin chromatography by C4 HPLC analysis chromatography.

Figure 3 is a diagram showing the SDS-PAGE results showing the difference in the washing effect according to the inclusion of urea in the buffer solution.

In one embodiment, the present invention comprises the steps of: (a) subjecting a biological fluid comprising dabepoetin alfa to blue resin chromatography to elute a fraction comprising dabepoetin alfa; (b) subjecting the eluate produced in step (a) to hydroxyapatite resin chromatography to elute the fraction comprising dabepoetin alfa; And (c) subjecting the eluate produced in step (b) to anion exchange resin chromatography to elute the fraction comprising darbepoetin alfa.

As used herein, the term “Darbepoetin alfa” is a recombinant form of the glycoprotein erythropoietin (EPO), in which two amino acids in the molecule of erythropoietin are substituted to add two N-sugar chains. As a glycoprotein, erythropoietin is distinguished from biochemical properties such as molecular weight and isoelectric point. Darbepoetin alfa induces red blood cell production and is used as a therapeutic agent for anemia associated with chemotherapy of renal failure or cancer. Dabepoetin alfa has a plasma half-life longer than erythropoietin due to the addition of sugar chains, and there may be various structural isodors depending on the sialic acid content. Structural subtypes with high sugar chains and sialic acid content of dabepoetin alfa have low isoelectric points, and these structural subtypes have high biological activity in vivo. Therefore, the selective purification of only structural subtypes having high sugar chains and sialic acid content to high purity is an important problem in protein therapeutics using the dabepoetin alfa, but the conventional purification process using a resin of several stages There was a problem of enormous amount of time. Therefore, the present inventors have developed a method for separating and purifying only the desired glycosylated dabepoetin alpha in a short time by a simple process.

Darbepoetin alfa separated and purified by the purification method of the present invention is characterized by high sugar chain content and sialic acid content, and preferably high sialic acid content having 14 to 22 moles per mole of dabepoetin alpha. Darbepoetin alfa having a sugar chain content and having an isoelectric point having an isoelectric point as low as pH 2.0 to 4.5 may be darbepoetin alfa, more preferably 3.5 or less.

In a specific embodiment of the present invention, darbepoetin alfa having an isoelectric point of 3.5 or less has a high sugar chain content. The higher the sugar chain content and the sialic acid content, the lower the isoelectric point of the subtypes of dabepoetin alfa.

As used herein, the term "biological fluid" refers to all cultures containing or derived from cells, cell components or cell products, including but not limited to cell cultures, cell culture supernatants, cell lysates, cell extracts, tissues Extracts, blood, plasma, serum, milk, urine, fractions thereof, and the like. In the purification method of the present invention, various types of biological fluids as described above may be used. Preferably, it may be a culture of yeast, plant cells or animal cells, more preferably animal cell cultures in which nucleotides encoding dabepoetin alfa have been transformed by genetic recombination, more preferably the animal The cell may be an animal cell culture in which the cell is CHO (Chinese hamster ovary).

In the case of using an animal cell culture solution or the like, as known in the art, it is more preferable to centrifuge the culture solution and use the supernatant thereof.

In a specific embodiment of the present invention, the inventors cultured CHO cells transformed with a vector containing dabepoetin alfa, using an ultrafiltration system using 50 mM sodium phosphate buffer in the supernatant of the culture. Diafiltration was used.

Each step of the present invention will be described in detail as follows.

Step (a) is a step of eluting the fraction containing darbepoetin alfa by applying biological fluid containing dabepoetin alfa to blue resin chromatography, preferably including dabepoetin alfa in the equilibrated blue resin. Biological fluids, or pH 6-9 buffer solution containing 0 to 100 mM NaCl, were added and adsorbed by diafiltration, and then washed with pH 6-9 buffer solution containing 0 to 0.1 M NaCl. Afterward, the step of eluting the fraction containing dabepoetin alfa with a buffer solution of pH 6 to 9 containing 0.1 to 1M NaCl.

As used herein, the term "blue resin chromatography" can be used interchangeably with "blue column" and is generally known to be capable of adsorbing about 10 mg of serum albumin per ml of resin as it is for removing serum albumin from a subject to be purified. It is a resin. In general, when the blue resin is used, the method suggested in the catalog is to maintain the pH of the buffer solution at 7, and the present inventors try to achieve a new effect by varying the target to be adsorbed by dabepoetin alfa instead of serum albumin. It was found that maintaining the pH of the buffer solution in the process of 6 to 9 can achieve the same effect as described above. In particular, it was confirmed that blue resin chromatography in the buffer solution can remove dyes and detergents in the culture solution containing dabepoetin alfa. That is, blue resin chromatography generally inhibits the adsorption of proteins as the pH is increased, but dabepoetin alfa can obtain the above effects by using adsorption. This configuration change is a great advantage in purifying dabepoetin alfa according to the present invention and is also a feature of the present invention.

The blue resin may be used without limitation as long as it can specifically adsorb and elute dabepoetin alfa, preferably Blue-sepharose 6 (GE company), Affi-gel blue (Affi-gel) blue, Bio-Rad) or Royopearl blue (Toyopearl blue, Tosho) can be used. In a specific embodiment of the present invention, Blue Sepharose 6 was used.

Preferably, the step of diluting the biological fluid with a pH 6-9 buffer containing 0-100 mM NaCl or concentrating and dialysis before step (a) may further increase the purification yield. That is, before the biological fluid is added to the blue resin and adsorbed, the biological fluid may be diluted with a buffer solution of pH 6 to 9 containing 0 to 100 mM NaCl, or concentrated and dialyzed to increase the purification yield. This can be obtained by performing diafiltration using ultrafiltration. Diafiltration using this ultrafiltration method removes less than 10,000 MWCO (Molecuar weight cut off) low-molecular substances (e.g., surfactants, dyes, small peptides, sugar components, etc.) in the culture medium. In addition, it is possible to improve the column adsorption efficiency by exchanging the buffer solution with the blue resin chromatography equilibration buffer solution.

In addition, the ultrafiltration method is capable of fractionating a substance dissolved or dispersed in a liquid by particle size, and is usually capable of separating, concentrating and purifying molecules or colloidal particles having a molecular weight of several thousand to several hundred thousand. The performance of the ultrafiltration membrane is expressed in fractional molecular weight (M.W.C.O., Molecular weight of cut-off), which means that substances above the fractional molecular weight of the membrane are excluded. M.W.C.O. is usually expressed as the molecular weight of a globular protein that can be excluded by at least 90%. The main functions of these ultrafiltration membranes are diafiltration, purification and concentration.

As the buffer used in each washing and elution step, sodium phosphate buffer, potassium phosphate buffer or Tris buffer may be preferably used.

In a specific embodiment of the present invention, the culture obtained by expressing dabepoetin alfa from CHO cells is diafiltered with an ultrafiltration system with 50 mM sodium phosphate buffer (pH 8.0), and then the sample is 50 mM sodium phosphate buffer. After binding to an XK-50 column filled with Blue Sepharose 6 resin equilibrated with pH 8.0 at a flow rate of 2.5 ml / min, 50 mM sodium phosphate buffer containing 0.1 M NaCl (pH 8.0) was added to 2.5. After washing to remove impurity proteins in 3 column volumes at a ml / min flow rate, 50 mM sodium phosphate buffer (pH 8.0) containing 0.7 M NaCl was used with a 3-column elution solvent at 2.5 ml / min flow rate. The solution containing ethyn alpha was eluted. As a result, the purity was about 88% (FIG. 1).

Step (b) is a step of eluting the fraction containing dabepoetin alfa by applying the eluate generated in step (a) to hydroxyapatite resin chromatography, preferably sequentially in the equilibrated hydroxyapatite resin The eluate recovered from the blue resin chromatography step or the eluate diafiltered with a buffer solution of pH 6 to 8 containing 0 to 100 mM NaCl was loaded, washed with a buffer solution of pH 6 to 8, and then loaded. It may be a step of obtaining a fraction containing darbepoetin alfa from the solution coming out without being attached to the resin in the wash.

In the present invention, the term "hydroxyapatite resin chromatography" may be used interchangeably with "hydroxyapatite column", and is filled with a hydroxyapatite resin, which is commonly used for removing nucleic acids such as DNA. will be. In particular, when the concentration of the phosphate buffer in which the resin is equilibrated is 5 to 10 mM or more, dabepoetin alpha protein is eluted without being adsorbed to the resin, and heterologous proteins and nucleic acids are adsorbed to the hydroxyapatite resin. This process also removes incomplete darbepoetin alfa. Therefore, the present invention almost completely removes incomplete darbepoetin alpha, endotoxin and nucleic acid material through the blue resin chromatography and the hydroxyapatite resin chromatography process and at the same time removes most of the heterologous proteins without any further treatment. By applying to the resin chromatography can be introduced to simplify the purification, automation, etc., there is an advantage that can shorten the process time. That is, the hydroxyapatite resin step of the present invention is characterized by a method of purifying using a method in which dabepoetin alpha does not adhere to the resin and impurities are attached to the resin.

The hydroxyapatite resin chromatography was loaded with an eluate containing dabepoetin alfa obtained from the blue resin of step (a) and washed with a buffer solution of pH 6 to 8 containing 0 to 100 mM NaCl. It contains dabepoetin alfa, which has a lot of sugar chains in the solution that does not adhere to the resin during washing and washing. After washing, 0.1-0.7 M potassium phosphate buffer at pH 6-8 was flowed into the resin to elute the fraction containing dabepoetin alfa with low sugar chain content to remove it from the resin.

Preferably, the step of (b) further eluting the eluate with a pH 6-8 buffer containing 0-100 mM NaCl before applying the hydroxyapatite resin chromatography may increase the purification yield. .

As the buffer used in each washing and elution step, sodium phosphate buffer, potassium phosphate buffer or Tris buffer may be preferably used.

 In a specific embodiment of the present invention, the blue resin eluate was diafiltered with a buffer solution of pH 6 to 8, and then, in an XK-50 column filled with hydroxyapatite resin equilibrated with 10 mM sodium phosphate buffer solution (pH 7.0). After application, 10 mM sodium phosphate buffer (pH 7.0) was flowed in about 4 column volumes to elute dabepoetin alfa with high sugar chains.

Step (c) is a step of eluting the fraction containing dabepoetin alfa by applying the eluate generated in step (b) to anion exchange resin chromatography, preferably the hydroxyapatite resin in an anion exchange resin. The resulting eluate recovered from the chromatography step was adsorbed, washed first with a buffer of pH 4.0 or less containing 0 to 6 M urea, and washed twice with a buffer of pH 6 to 8, followed by 0 to Elution of the fraction containing dabepoetin alfa with a buffer containing 0.5 M NaCl.

By performing the step (c) it can be obtained high purity dabepoetin alfa of 98% or more of the isoelectric point 3.5 or less.

As used herein, the term "anion exchange resin chromatography" means that molecules can be separated according to their charge by binding a negatively charged (or acidic) molecule to a positively charged support. Acidic, basic and neutral) can be easily separated by this technique. As the resin that can be used in the anion exchange chromatography of the present invention, strong anion exchange resin and weak anion exchange resin can be used without limitation, for example, Sephadex, Sepharose, Sauce, Mono, Mini (trade name, GE healthcare), etc. Although not limited thereto, a resin having a functional group of the resin, such as Q (Quaternary amine), DEAE (DiEthylAminoEthyl), or QAE (Quaternary Amino Ethyl), may be used. Preferably, the functional group of the resin may be Q or DEAE, and most preferably Q-sepharose, which is a strong anion exchange resin, may be used.

Anion exchange resin chromatography can be performed by column chromatography, or in batch mode. For commercial manufacturing, it may be desirable to use a batch mode. The anion exchange resin is washed and eluted with a stepwise salt gradient or continuous salt gradient. Suitable stepwise or continuous salt gradients may be any that allow for the separation of the sustained human growth hormone from impurities.

In addition, the anion exchange resin used in the anion exchange resin chromatography of the present invention may be equilibrated with an aqueous buffer before adsorbing the culture.

Preferably, in the step (c), the first wash may be performed with a buffer solution containing urea to separate dabepoetin alfa, which is a structural isoform having the desired isoelectric point. This can be eliminated by separating and eluting undesired structural subtypes with different isotropic points in the anion exchange resin chromatography step according to the primary wash buffer. However, washing of urea-containing buffers may be more effective for removing structural isoforms with undesired isoelectric points than primary wash buffers without urea (FIG. 3).

The buffer used in each washing and elution step is preferably sodium acetate (sodium phosphate) buffer, citrate buffer, glycine-HCl (glycin-HCl) buffer or citric acid-sodium phosphate ( citric acid-sodium phosphate) buffer solution may be used.

In a specific embodiment of the present invention, an XK-50 column filled with Q Sepharose FF resin equilibrated with 10 mM sodium phosphate buffer solution (pH 7.0) in the eluate containing darbepoetin alfa obtained in step (b) ( GE Healthcare) was flowed into the column at a flow rate of 2.5 ml / min, and then washed again by flowing 10 mM sodium phosphate buffer (pH 7.0) to about 3 column volumes. After washing sequentially with a buffer solution of pH 3.5 or less containing 6M urea and pH 6 ~ 8 buffer containing no urea, Fractions containing dabepoetin alfa were eluted with a pH 6-8 buffer solution containing 0-0.5 M NaCl. At this time, in the process of washing with a buffer solution of pH 3.5 or less containing 6M urea and a buffer solution of pH 6 to 8 was removed the dabepoetin alfa having a low sugar chain content. The eluate containing only dabepoetin alfa with high sugar chains and low isoelectric point was diafiltered by ultrafiltration using PBS (pH 7.45) buffer containing 150 mM NaCl. As a result of confirming the purity of the recovered eluate, it was confirmed that the purity of darbepoetin alfa with high sugar chains and low isoelectric point was about 98%.

Eluates containing low isoelectric dabepoetin alfa obtained from anion exchange resin may be subjected to additional buffer exchange processes. The buffer exchange process can be carried out by gel filtration or concentration and diafiltration.

In another aspect, the present invention provides darbepoetin alfa purified by the above purification method.

As another aspect, the present invention provides a method for producing darbepoetin by the above purification method.

As another aspect, the present invention provides dabepoetin produced by the above purification method.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Dabepoetin Alpha Purification by Blue Sepharose Column

About 2 L of the culture medium obtained by expressing dabepoetin alpha from CHO cells transformed with the vector containing dabepoetin alpha was transferred to 50 mM sodium phosphate buffer (pH 8.0) using an ultrafiltration system (molecular weight cut off 10,000). Diafiltration was performed.

About 20 ml of Blue-sepharose 6 (GE Healthcare, Inc.) resin was charged into an XK-50 column (GE Healthcare Inc.) and the column was equilibrated with sufficient flow of 50 mM sodium phosphate buffer (pH 8.0). About 0.1-0.2 L of the diafiltration solution was flowed on the prepared Blue Sepharose 6 column at a flow rate of 2.5 mL / min, and then the column was washed with 50 mM sodium phosphate buffer solution (pH 8.0) by about 3 CV (column volume).

50 mM sodium phosphate buffer solution containing 0.1 M NaCl (pH 8.0) was flowed at a rate of 2.5 ml / min at about 3 CV to remove impurities, and 2.5 ml of 50 mM sodium phosphate buffer solution (pH 8.0) containing 0.7 M NaCl was removed. The solution containing dabepoetin alfa was recovered by flowing about 3 CV at a flow rate of / min.

The purity of the recovered eluate was measured by C4 HPLC analysis chromatography.

Example 2: Purification of dabepoetin alfa by hydroxyapatite column

Before adsorbing the Blue Sepharose column eluate obtained in Example 1 to the hydroxyapatite resin, diafiltration was performed using an ultrafiltration system (molecular weight cutoff 10,000) with a 10 mM buffer of pH 6-8. .

About 20 ml of hydroxyapatite (GE Healthcare) resin was filled in an XK-50 column (GE Healthcare) and the column was equilibrated with sufficient flow of 10 mM sodium phosphate buffer (pH 7.0). About 0.1 L of the diafiltration solution was flowed to the prepared hydroxyapatite column at a flow rate of 2.5 mL / min, and then 10 mM sodium phosphate buffer solution (pH 7.0) was flowed about 4 CV (column volume).

In this case, it contains dabepoetin alfa, which has a lot of sugar chains in the solution that does not adhere to the resin in loading and washing, and collects these solutions to proceed to the next process. After washing, a buffer containing 0.1-0.7 M potassium phosphate pH 6-8 was flowed into the resin to elute the fraction containing dabepoetin alfa with less sugar chains to remove it from the resin. In the hydroxyapatite resin step of the present invention, dabepoetin alfa does not adhere to the resin, and impurities are purified using a method of adhering to the resin. Thus, the solution applied to the anion exchange column of Example 3 below is a solution eluted from loading and washing.

Example 3: Darbepoetin Alpha Purification by Anion Exchange Column

Approximately 20 mL of Q Sepharose FF (GE Healthcare) resin was charged to an XK-50 column (GE Healthcare) to equilibrate the column with sufficient flow of 10 mM sodium phosphate buffer (pH 7.0).

About 0.2 L of the solution containing darbepoetin alfa obtained in Example 2 was flowed on the column at a flow rate of 2.5 ml / min, and then 10 mM sodium phosphate buffer solution (pH 7.0) was flowed about 3 CV (column volume) to open the column. Washed. After sequentially washing with a buffer solution of pH 3.5 or less containing 6M urea, and a buffer solution of pH 6 ~ 8 containing no urea, Fractions containing dabepoetin alfa were eluted with a pH 6-8 buffer solution containing 0-0.5 M NaCl. At this time, dabepoetin alfa with low sugar chain content was removed in the process of washing with a buffer solution of pH 3.5 or less containing 6M urea and a buffer solution of pH 6-8.

The eluate containing only dabepoetin alfa with high sugar chains and low isoelectric point was diafiltered with an ultrafiltration system (molecular weight cut off 10,000) using PBS (pH 7.45) buffer containing 150 mM NaCl.

As a result of measuring the purity of the recovered eluate through C4 HPLC analysis chromatography, as shown in FIG. 2, it was confirmed that the purity of dabepoetin alfa with high sugar chains and low isoelectric point was about 98%.

In addition, in the step of washing with a buffer of pH 3.5 or less containing the 6M urea, washing with a buffer of pH 4.0 containing 0M urea (that is, a buffer containing no urea) instead of the buffer solution As a result of SDS-PAGE analysis of dabepoetin alfa eluted from the group washed with a buffer solution of pH 4.0 containing 6M urea by SDS-PAGE, it was washed with a buffer containing urea rather than a buffer without urea. One group was found to be more effective in removing structural subtypes with undesired isoelectric points (FIG. 3).

Claims (11)

(a) subjecting the biological latex comprising dabepoetin alfa to blue resin chromatography to elute the fraction comprising dabepoetin alfa; (b) subjecting the eluate produced in step (a) to hydroxyapatite resin chromatography to elute the fraction comprising dabepoetin alfa; And (c) subjecting the eluate produced in step (b) to anion exchange resin chromatography to elute the fraction comprising darbepoetin alpha. The method of claim 1, wherein the darbepoetin alfa has a high sugar chain content of 14 to 22 moles per 1 mole of dabepoetin alfa, and has an isoelectric point having a low isoelectric point of pH 2.0 to 4.0. The method of claim 1, further comprising diluting or concentrating and dialysis the biological fluid with a pH 6-9 buffer containing 0-100 mM NaCl prior to step (a). The method of claim 1, further comprising diafiltration the eluate of step (b) with a buffer of pH 6 to 8 containing 0 to 100 mM NaCl before subjecting to hydroxyapatite resin chromatography. Way. According to claim 1, wherein the step (a) is a biological fluid containing diabepoetin alpha in the equilibrated blue resin, or biological fluids diafiltered with a buffer solution of pH 7 to 9 containing 0 to 100 mM NaCl After adsorbing, washing with a buffer of pH 7-9 containing 0-100 mM NaCl, eluting the fraction containing dabepoetin alfa with a buffer of pH 7-9 containing 0.4-1 M NaCl How to do. According to claim 1, wherein the step (b) is diafiltration with the eluate recovered from the blue resin chromatography step or the buffer solution of pH 6 to 8 containing 0 to 100 mM NaCl sequentially in the equilibrated hydroxyapatite resin Loading the eluate, and washing with a buffer solution of pH 6 to 8, and then obtaining a fraction containing dabepoetin alfa from the solution which does not adhere to the resin in loading and washing. The method of claim 1, wherein step (c) is performed by adding an eluate recovered from the hydroxyapatite resin chromatography step to an anion exchange resin and adsorbing the same, and then using a buffer solution having a pH of 4.0 or less containing 0-6 M urea. Washing first, washing second with a buffer of pH 6 to 8, and then eluting the fraction containing dabepoetin alfa with a buffer containing 0 to 0.5 M NaCl. The method of claim 7, wherein the first washing is performed with a buffer solution containing urea to separate dabepoetin alfa, which is a structural isoform having a desired isoelectric point. The method according to any one of claims 1 to 8, wherein the buffer used in the washing and eluting steps (a) and (b) is sodium phosphate buffer, potassium phosphate buffer. Solution or Tris buffer, the buffer used in the washing and elution step (c) is sodium acetate buffer, citrate buffer, glycine-HCl (glycin- HCl) buffer or citric acid-sodium phophate buffer. The method of claim 1, wherein the biological fluid is yeast, plant cell or animal cell culture. The method of claim 1, wherein the functional group of the anion exchange resin is any one selected from the group consisting of Q (Quaternary amine), DEAE (DiEthylAminoEthyl), and QAE (Quaternary Amino Ethyl).

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