KR20080082537A - A novel method for purifying recombinant erythropoietin - Google Patents

Abstract

A method for purifying a recombinant erythropoietin is provided to obtain the erythropoietin with high purity and high yield through a simple column purification process. A method for purifying a recombinant erythropoietin comprises the steps of: (a) applying a biological fluid solution including the recombinant erythropoietin to a hydrophobic chromatography; (b) subjecting an effluent generated from the step(a) to virus inactivation; and (c) subjecting the virus inactivated solution to anion chromatography, wherein the biological fluid solution is an animal cell culture solution, a bead size of a resin of the hydrophobic chromatography is less than 34 micrometers and the virus inactivation is performed by adding an organic solvent such as ethanol, acetonitrile and isopropanol thereto. Further, the functional group of the hydrophobic chromatography is selected from phenyl, octyl, isopropyl, butyl and ethyl.

Description

A novel method for purifying recombinant erythropoietin {A Novel method for purifying recombinant erythropoietin}

The present invention relates to a method for purifying recombinant erythropoietin from biological fluids producing recombinant erythropoietin (rEPO).

More specifically, the simplified recombination may include a high resolution hydrophobic chromatography step, a virus inactivation step, and an anion exchange chromatography step, and may further include a concentration step of a cell culture such as ultrafiltration. Erythropoietin purification method.

Erythropoietin (EPO) is a hormone that promotes red blood cell formation in higher organisms and is a glycoprotein composed of 166 amino acids and several types of sugars (Carnot, et al., Compt. Rend., 143, 384, 1906). In addition, erythropoietin is an indispensable factor for the formation of red blood cells in the blood and is used for diagnosis and treatment of blood-related diseases such as anemia.

Natural human erythropoietin is a monomeric acid glycoprotein with a total molecular weight of about 34 kDa and a protein molecular weight excluding sugar of about 18 kDa. In addition, since the sugar chain has 4 parts of the amino acid and this sugar chain plays an essential role in biological activity, when the sugar content of erythropoietin is reduced, its biological half-life is rapidly reduced and it is known to lose biological activity (Masato H. et al. , J. Biol. Chem., 267, 7703-7709, 1992).

In normal people, EPO is maintained at about 15-30 mU, or 0.01 mM, per ml of blood (Gaarcia, JF, Lab. Clin. Med., 99, 624-635, 1982), but diseases such as aplastic anemia In patients with erythropoietin, which is 1000 times higher than normal humans, erythropoietin is produced separately from their blood or urine (White et al., Rec. Progr. Horm. Res., 16, 219, 1960; Espada et al., Biochem.Med., 3, 475, 1970; Fisher, Pharmacol. Rev., 24, 459, 1972). However, this method has a limited supply of raw materials for mass production of erythropoietin, and low urinary concentration of erythropoietin in normal urine, as well as an active inhibitor, requires a high purification technology to remove it. (US Pat. Nos. 4,397,840, 4,303,650 and 3,865,810). In addition, a method of extracting erythropoietin from sheep plasma has been developed, but it is known that sheep EPO can act as an antigen to humans (Goldwasser, Control cell. Dif. Develop., Part A, 487-). 494, 1981). Sugimoto et al. Have reported a method for producing erythropoietin by propagating human lymphocytes, including Namalwa, BALL-1, NALL-1, TALL-1 and JBL in animals (US patent). 4,377,513), since this method uses human tumor cells, studies to secure safety are required.

In addition, methods for cloning human EPO genes and expressing them in animal cells (Korean Patent Publication Nos. 89-1011 and 93-5917) and yeast or insect cells (Korean Patent Publication No. 95-5988) have been developed. However, yeast or insect cells mainly produce high mannose type glycoproteins and have low glycosylation capacity, and thus cannot produce complex types of glycoproteins such as human erythropoietin. The sugar chain structure is not suitable as a host cell for expressing human erythropoietin since there is a risk of causing antigenicity in the human body. In addition, the production of human erythropoietin using animal cells can produce EPO that is most similar to the natural form, but the expression rate is very low, there is a disadvantage that the production cost is expensive.

As such, it is difficult to produce clinically usable amounts of erythropoietin in nature and animals, and it is difficult to express large amounts by recombination of solubilized and pure erythropoietin without sugar chain or fusion protein form, which is known to contribute to biological activity in Escherichia coli. It is also difficult to obtain active erythropoietin with high purity.

After 1985 expression of natural human-like recombinant human erythropoietin in mammalian cells (Chinese Hamster Ovary Cells (CHO)), it was first released in 1989 in the United States and is now produced from recombinant human erythropoie cells from Chinese hamster ovary cells. Ethin is widely used clinically around the world. Therefore, until now, Chinese hamster ovary cells are mainly used for efficient production of erythropoietin. Therefore, in this condition, the most important task is to mass-produce low cost and high purity by simplifying the production process of erythropoietin and improving the purification process. to be.

Purification methods known to date include ion exchange chromatography, reverse phase chromatography, gel filtration (Korean Patent Publication No. 93-003665, US Pat. No. 4,667,016), blue sepharose column, hydroxyapatite column, ion exchange resin column Method (Korean patent 0162517), dye affinity chromatography and cation exchange chromatography, method using gel filtration chromatography (Korean patent 0177300), divalent metal cation precipitation, anion exchange chromatography, hydrophobic chromatography (phenyl ), A method using anion exchange chromatography (Korean Patent 10-0344059), a method using monoclonal antibody (Korean Patent 0153808), a method consisting of two steps of immunosorbent chromatography and ionic exchange chromatography on erythropoietin To purify erythropoietin accordingly [G hanem et al., "Purification and biological activity of a recombinant human erythropoietin produced by lymphoblastoid cells", Preparative Biochemistry, 24 (2), 1994, 127-142].

According to U.S. Patent No. 4,465,624, immunosorbent chromatography was used as a purification method of erythropoietin, and the column can be reused about 10 times, but in Korean Patent Registration No. 10-0297927, before the immunosorbent chromatography step, It is described that by applying ion exchange chromatography as an initial purification step to the immunosorbent column, the unit yield of about 80% can be maintained even after about 500 times of reusable number of columns. However, the purification of erythropoietin using immunosorbent chromatography is expensive for the preparation of the antibody, it is difficult to use in the actual process because the number of times of use is limited due to the decrease in yield. In addition, reverse phase chromatography used in Korean Patent Publication No. 93-003665 and US Patent No. 4,667,016 may affect the stability of erythropoietin due to excessive use of organic solvents during the purification process, and gel filtration. Has the disadvantage of not being a suitable method for producing protein on an industrial scale. In the purification method disclosed in Korean Patent No. 0162517 and Korean Patent No. 0177300, dye affinity chromatography is included in the purification process, but its purification is difficult because of its specific affinity, which is difficult to purify with high purity. This complex has a disadvantage in that it is inconvenient in the process, and has a disadvantage in that it cannot selectively separate high sialic acid erythropoietin with high in vivo activity.

As such, the conventional erythropoietin purification process requires a complicated process using three to four steps of chromatography, and the cost and time are enormous, and the present inventors have studied to solve the problems of the existing process. Repeated to complete the present invention.

Accordingly, it is an object of the present invention to provide a method for purifying and preparing phosphorus erythropoietin in high purity and high yield using only a simple column purification process.

In addition, another object of the present invention is to provide an erythropoietin purified by the method for purifying the recombinant erythropoietin.

In order to achieve the above object, in one embodiment the present invention comprises the steps of (a) applying a biological fluid containing recombinant erythropoietin to hydrophobic chromatography; (b) performing virus inactivation on the eluate produced in step (a); And (c) relates to a method for purifying recombinant erythropoietin comprising the step of anion exchange chromatography of the solution produced in step (b).

As used herein, "erythropoietin" refers to human erythropoietin, which includes natural human erythropoietin and human erythropoietin derivatives, and human erythropoietin derivatives are biologically similar or superior to natural human erythropoietin. Have activity. The human erythropoietin derivative includes a variant in which a natural human erythropoietin amino acid sequence is substituted, inserted or deleted, and includes a glycosylation pattern different from that of a natural human erythropoietin.

The term "biological fluid" as used herein refers to any fluid containing or derived from a cell, cell component or cell product, including but not limited to cell culture, cell culture supernatant, cell lysate, cell extract, tissue 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, an animal cell culture medium in which nucleotides encoding erythropoietin is transformed by genetic recombination is used. More preferably, the animal cell culture medium 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 one specific embodiment of the present invention, T175 culture of X0GC / GEPO9647 (DXB11) CHO cell line transformed with recombinant human erythropoietin as described in the applicant's prior patent application (Korean Patent No. 10-2006-0100507). Subsequent passage in a container to amplify, amplify, incubate for about 48 hours in a thermostat and washed with water, and put 1 liter each of serum-free CHO-A-SFM containing sodium butyrate at 33 ℃, 5% CO ₂ By culturing in a thermostat and centrifuging, the supernatant of the cell culture solution in which human erythropoietin was expressed and secreted was used. The entire specification of Korean Patent Application No. 10-2006-0100507 is included as a reference of the present invention.

Another culture method was to culture human recombinant erythropoietin-transformed X0GC / GEPO9647 (DXB11) CHO cell line in a bioreactor for animal cells to produce human erythropoietin. Cell lines were expanded to ensure that they were inoculated into bioreactors. Incubate at 37 ° C. for 48 hours using serum-free medium (EX-CELL CHO medium, SAFC, Cat. # 14360C) in a bioreactor and increase the cell number, then lower the temperature to 33 ° C. for 8 or 9 days. Culture was performed to produce human erythropoietin.

Hydrophobic chromatography used in the present invention is any chromatography performed on a gel having a hydrophobic, suitably aromatic or aliphatic free charge ligand bound to various types of matrices on the market, which can be used as the gel. The resin preferably has a relatively small bead size to maintain high resolution. For example, a resin such as a source, a GE healthcare, a resource, or a GE healthcare may be used, but is not limited thereto. As the functional group of the resin that acts as a ligand, a phenyl, octyl, isopropyl, butyl or ethyl group is preferable. All of the normal phase hydrophobic chromatography used in the prior art uses a relatively high (50 μm or more) low resolution resin, whereas the bead size of the resin of the present invention is preferably 34 μm or less. Do.

The type of eluate that can be used in the hydrophobic chromatography of the present invention is not limited, but preferably tris or phosphate neutral (pH 6-8) buffer containing 2M ammonium sulfate may be used as the eluent. In addition, it is preferable that the erythropoietin culture solution is loaded onto the chromatography column using the buffer, and then the erythropoietin bound to the chromatographic resin is eluted with a linear concentration tool using the buffer solution. In addition, the volume of the buffer used in the hydrophobic chromatography of the present invention is preferably 10 times the column volume, that is, 10 column capacity.

In addition, the hydrophobic chromatography of the present invention may be added to the fraction to increase the conductivity of the fraction prior to the implementation.

In addition, the erythropoietin eluate collected in hydrophobic chromatography can then be desalted using a dialysis method.

In addition, anion exchange chromatography may be additionally performed as a protective column of the hydrophobic chromatography column, wherein the functional group of the anion exchange chromatography resin is preferably selected from the group consisting of Q, QAE and DEAE.

In a specific embodiment of the present invention, the addition of ammonium sulfate to the erythropoietin expression medium concentrated by ultrafiltration to a final concentration of 2M was loaded on a hydrophobic chromatography column filled with Source 15 iso resin. The loaded sample eluted erythropoietin using a linear gradient over 10 column volumes, ranging from 20 mM Tris (pH 7.5) with 2 M ammonium sulfate to 20 mM Tris (pH 7.5).

When high resolution hydrophobic chromatography is used as the first purification column according to the present invention, one column chromatography can purify EPO of at least 80% purity, preferably at least 85%, more preferably at least 90%. Can be.

The purification method of the present invention includes a step of inactivating the potential virus after hydrophobic chromatography, for the virus safety of the eluate. Generally, in addition to the so-called S / D (solvent / cleaning) method, which inactivates the virus by damaging the lipid envelope protecting the virus, thermal inactivation methods such as pasteurization (heat treatment at 60 ° C. for 10 hours) It can be applied to increase viral stability and there is an example using an affinity chromatography process. Filtration through nanofilters can also be used. It is well known in the art that each of these is effective in itself and may combine two process steps based on different principles together in one manufacturing process for maximum virus safety.

Preferably, the inactivation treatment is performed using an organic solvent selected from the group consisting of ethanol, acetonitrile and isopropanol in order to inactivate the potential virus present in the eluate. More preferably ethanol, wherein the concentration of ethanol is 40 to 50% (v / v), the treatment time is 3 hours or more, the concentration of EPO is preferably 5 mg / ml or less. As a specific example, the present inventors mixed the biological fluid containing diafiltration and concentrated recombinant erythropoietin by ultrafiltration with ethanol such that the final ethanol concentration is 50% (v / v), and then room temperature The mixture was left to stir slowly for 3 hours.

The present invention includes performing anion exchange chromatography after the process.

As used herein, “anion exchange chromatography” refers to the separation of molecules according to their charge by binding a negatively charged (or acidic) molecule to a positively charged support. , Basic and neutral) can be easily separated by this technique.

Resin that may be used in the anion exchange chromatography of the present invention is Sephadex, Sepharose, Sauce, Mono, Mini (trade name, GE healthcare) and the like, but the functional group of the resin is not limited to Q (Quaternary amine), Resin which is DEAE (DiEthylAminoEthyl) or QAE (Quaternary Amino Ethyl) can be used. Preferably the functional group of the resin is Q or DEAE.

Anion exchange 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 recombinant erythropoietin from impurities.

In addition, the anion exchange column used in the anion exchange chromatography of the present invention may be equilibrated with an aqueous buffer prior to loading the eluate.

Preferably, the elution of the anion exchange chromatography of the present invention loads the purified sample by hydrophobic chromatography on a column equilibrated with 20mM Tris (pH7.5) buffer, and then 20mM Tris (pH7.5) containing 1M NaCl. The buffer may be used in a linear concentration gradient method of at least 10 column volumes.

In a specific embodiment of the present invention, the ultrafiltration and ethanol treated samples with 20 mM Tris (pH7.5) buffer in a Source Q (XK50 / 25, 500 ml) column equilibrated with 20 mM Tris (pH7.5) buffer are 10-fold. After diluting the mixture, the mixture was loaded at a flow rate of 12 ml / min, and then bound, and the protein was eluted using an 18-column elution solvent in a linear concentration gradient (sodium chloride concentration from 0 M to 1 M).

By performing anion exchange chromatography after the hydrophobic chromatography and the virus inactivation step, only active erythropoietin of high sialic acid can be easily selected from all erythropoietins.

In a preferred embodiment, in addition to the purification method according to the invention may comprise the step of concentrating a biological fluid containing recombinant erythropoietin. Specifically, the concentration step may be carried out before the purification step or after the hydrophobic chromatography step. The step of concentrating the biological fluid may use any concentration method known in the art without limitation, and preferably ultrafiltration.

Ultrafiltration is a method of fractionating a substance dissolved or dispersed in a liquid by particle size, and is usually capable of separation, concentration, and purification of molecules or colloidal particles having molecular weights of several thousand to hundreds of thousands.

The performance of the ultrafiltration membrane is expressed by the molecular weight of cut-off (MWCO), which means that substances above the fractional molecular weight of the membrane are excluded. MWCO is usually expressed as the molecular weight of a globular protein that can be excluded by 90% or more. The main functions of these ultrafiltration membranes are diafiltration, purification and concentration.

In the concentration step of the present invention, more preferably, ultrafiltration is performed using an ultrafiltration membrane having a resolution (MWCO) of 5 to 10K in size. The final ultrafiltration (dialysis) filtration dilution factor is preferably 150 to 200 times. In addition, the final concentration of the preferred concentrated erythropoietin is 1 to 2 mg / ml when performed before the step of purification, and 10 to 15 mg / ml when performed after the step of hydrophobic chromatography. Including such a concentration step can reduce the volume of the biological fluid containing recombinant erythropoietin, remove impurities below the resolution of the filtration membrane present in the biological fluid, and facilitate loading on the chromatography. . As a specific example, the present inventors wash the ultrafiltration membrane (Filtron Omega filter, Fall, MWCO 10K) before and after the hydrophobic chromatography step using 3L of 0.5N NaOH and 6L of sterile water to express erythropoietin The culture was injected and concentrated.

According to the purification method according to the present invention through this step, the medium in which human erythropoietin is expressed is concentrated by ultrafiltration, and most of the impurity proteins in the culture are removed by high resolution hydrophobic chromatography to remove 90% or more pure human erythropoietin. By effectively removing the remaining erythropoietin protein through additional anion exchange chromatography, only a more active human erythropoietin fraction can be obtained with high purity and high yield through a minimum process compared to the conventional method.

As another aspect, the present invention provides a recombinant erythropoietin purified by the purification method and a pharmaceutical composition comprising the same.

Matters concerning the preparation of the pharmaceutical composition and its effects are well known to those skilled in the art.

Hereinafter, the present invention will be described in detail by the following examples, but the present invention is not limited thereto.

Example

Example 1 Ultrafiltration of EPO Producing Animal Medium

Human erythropoietin cultures were cultured in T175 culture vessels using recombinant human erythropoietin X0GC / GEPO9647 (DXB11) cell line as described in detail in Korean Patent Application No. 10-2006-0100507, the applicant's prior patent application. Amplify up to 120 T175 culture vessels in steps and inoculate about 2.5x10 ⁸ cell lines per cell factory (Nunc, Cat # 170009), inoculate a total of 8 cell factories, and then incubate 37 degrees, 5 Incubated for about 48 hours in a% CO2 incubator. Serum-free CHO-A, a medium-production medium for erythropoietin with 0.3 mM sodium butyrate (Sigma, Cat # B-5887), washed twice with 1 liter per cell factory in phosphate buffer -SFM (Gibco Fomula # 05-5072FF) was added to each 1 liter and incubated in a 33 ℃, 5% CO ₂ incubator and was prepared by recovering the human erythropoietin expression supernatant for a total of nine times every two days.

In another culture method, recombinant human erythropoietin X0GC / GEPO9647 (DXB11) cell line as described above is cultured in a bioreactor for animal cells with a total volume of 7.5 L and an actual production volume of 5 L using serum free medium. Human erythropoietin was produced and the production method is briefly described as follows. In order to secure the number of cells needed to start cultivation in the bioreactor, the frozen cell line was incubated for 48 hours from the T175 culture vessel to the total volume of 250 ml spinner culture vessel for 1 hour to the total volume 1 L culture vessel, Thereafter, the bioreactor was inoculated to a concentration of 5 X 10 ₅ cells / ml. Incubate at 37 ° C. for 48 hours using serum-free medium (EX-CELL CHO medium, SAFC, Cat. # 14360C) in a bioreactor, increase the cell number, lower the temperature to 33 ° C. and add 0.3 mM sodium butyrate (Sodium butyrate, Sigma, Cat # B-5887) was added and cultured for 8 or 9 days to produce human erythropoietin.

The ultrafiltration membrane (Filtron Omega filter, Fall, MWCO 10K) was washed with 3L 0.5N NaOH and 6L sterile water, and then erythropoietin expression culture solution was injected and concentrated 40 times. There was little loss of EPO and the final concentration of concentrated erythropoietin was maintained at 2 mg / ml.

Example 2 Hydrophobic Chromatography Step

Hydrophobic chromatography was loaded by adding 3M ammonium sulfate solution to a final concentration of 2M in erythropoietin expression medium concentrated by ultrafiltration. In the present invention, a column filled with Source 15 iso (GE healthcare) resin was used for hydrophobic chromatography, and the loaded sample was solvent B (20 mM in Solvent A (2M ammonium sulfate + 20 mM Tris (pH 7.5)). Proteins were eluted using a linear gradient of 10 column volumes up to Tris (pH 7.5)) (see FIG. 2).

Example 3 Diafiltration and Concentration of EPO Using Ultrafiltration (Diafiltration + Concentration)

The ultrafiltration membrane (Filtron Omega filter, Fall, MWCO 10K) was washed with 3L NaOH and 6L sterilized water, and then hydrophobic chromatographic eluate was diluted 5 times and injected into diafiltration and concentrated. The final diafiltration dilution multiple was 170-fold, the final concentration of concentrated erythropoietin was 10 mg / ml, and the ultrafiltration membrane resolution (MWCO) was 10K size.

Example 4 Ethanol Treatment Step

Diafiltration and concentrated erythropoietin are mixed with ethanol solution and incubated at room temperature. At this time, the concentration of erythropoietin was 2 mg / ml, the concentration of ethanol was 50%, it was incubated with stirring slowly for 3 hours at room temperature.

Example 5 Anion Exchange Chromatography Step

20mM Tris (pH7.5) buffer diafiltration and ethanol treated samples were diluted 10-fold in a Source Q (XK50 / 25, 500ml) column equilibrated with 20mM Tris (pH7.5) buffer and loaded at 12ml / min. Then, the protein was eluted using a linear concentration gradient of 18 column volumes by a linear concentration gradient (sodium chloride concentration 0 M → 1 M). (See Figure 3)

[ Example 6 Refined EPO Analysis of

The erythropoietin purified through two-step column chromatography was subjected to identification and purity test according to the test method of erythropoietin listed in EP. Purity of erythropoietin was analyzed by size exclusion chromatography (SEC) polyacrylamide gel electrophoresis (SDS PAGE), and EPO material was identified by immunoblotting, peptide mapping, and N-terminal sequencing. In the test, the content of isomers was determined by capillary electroporesis (CE), host-derived DNA and protein were quantified, and the content analysis test of sialic acid was performed.

[ Experimental Example 1  Purity Analysis Using Size Exclusion Chromatography]

Dimers and multimers in erythropoietin were analyzed using a size exclusion column (silica-base, 7.5x300 mm, TSKG3000).

-Exclusion HPLC analysis conditions are as follows.

   device ; Agilent's HP1100

   Precolumn; TOSHO TSK-Guard SW (7.5x75mm)

   Bone column; TOSHO TSK -G3000 SW (7.5x300mm)

  Mobile phase; 1.5 mM potassium dihydrogen phosphate, 8.1 mM disodium hydrogen phosphate, 0.4 M sodium chloride, pH7.4

   Flow rate; 0.5 ml / min

   Detection wavelength; 214nm

   Injection amount; 50 μl

The analytical results are shown in FIG. 4 and showed more than 99% purity.

[ Experimental Example 2 Polyacrylic Amide  Gel Electrophoresis (SDS-PAGE)  Purity Analysis Using]

40 μl of erythropoietin with a reducing buffer was injected into the 12.5% SDS-PAGE gel, followed by electrophoresis. The gel was dyed by shaking for 5 minutes while soaking Coomasie brilliant blue R-250 for use as a protein dye in a dyeing solution made to 0.1% in 30% methanol and 10% acetic acid solution. Soaking in a destaining solution consisting of% methanol and 10% acetic acid solution to remove non-specifically bound dye was confirmed the distribution and concentration of the band. The results are shown in FIG. 5, and as can be seen from the results, erythropoietin appears as a single disulfide bond, and the position and intensity of the band are consistent with the international standard EPO BRP. Therefore, the erythropoietin purified by the purification method of the present invention had the same molecular weight as EPO BRP without other impurities having a molecular weight, and the final purified product showed purity of 99% or more.

[ Experimental Example 3 Weston Blot  Used EPO  Confirmation analysis]

Western blots are experiments that use specific binding reactions of antigen-antibodies, so that qualitative analysis of those proteins can be performed using antibodies of specific proteins as probes. Therefore, SDS-PAGE gels were western blotted to confirm purified erythropoietin and to demonstrate that the international standard was the same as EPO BRP. After the sample solution was mixed 1: 1 with non-reducing buffer, a total of 20 μl was loaded onto a 12.5% Bio-Rad gel and electrophoresed. The gel was transferred to PVDF membrane and incubated with 1% Skim milk blocking solution. The gel was incubated with anti-human EPO antibody added according to the manufacturer's protocol while shaking at room temperature for 1 hour. Rinse the membrane with TBST buffer for 30 minutes, add peroxidase labeled anti-rabbit antibody, shake incubate at room temperature for 1 hour, and then wash with TBST for 1 hour. It was developed by exposing to a film. As a result, a single band was identified at the same position as that of the international standard EPO BRP, and since no other band was detected, it was confirmed that there were no variants and cleavage fragments showing different mobile phases on the gel. The results are shown in FIG.

[ Experimental Example 4 Peptide Mapping  Used EPO  Confirmation analysis]

5 μl of Trypsin (1 mg / ml) was added to 0.25 ml of erythropoietin at a concentration of 1.0 mg / ml of final purified erythropoietin at a concentration of 1.0 mg / ml. After reacting for 18 hours, the reaction was terminated by freezing. The results are shown in FIG. 7 and confirmed that they are consistent with the international standard EPO BRP.

HPLC conditions for peptide analysis are as follows.

Column: Butylsilyl silica gel R (0.25m * 4.8mm)

Mobile phase A: 0.06% TFA (trifluoroacetic acid) in DW (distilled water)

Mobile phase B: 0.06% TFA. / 90% ACN (acetonitrile) in D.W

In the case of purifying recombinant erythropoietin using the purification method according to the present invention, the purification process can be minimized by two steps of hydrophobic and anion exchange chromatography, and the conventional complex chromatographic purification process is generally a final processing step. High resolution hydrophobic high pressure purification chromatography used in the first process can be subjected to one column chromatography to purify erythropoietin of at least 80% purity, preferably at least 85% and more preferably at least 90%. In addition, anion exchange chromatography can be performed to select only active erythropoietin of high sialic acid out of the total erythropoietin.

1 shows a preferred embodiment of the purification process provided by the present invention,

Figure 2 shows the chromatogram of the hydrophobic chromatography step of the purification process provided by the present invention,

3 shows a chromatogram of an anion exchange chromatography step,

4 is a size exclusion chromatography analysis of erythropoietin purified through the purification process of the present invention,

5 is a polyacrylamide gel electrophoresis result of erythropoietin purified through the purification process of the present invention,

MW: molecular weight labeling;

Lane 1: international standard BRP EPO;

Lane 2: erythropoietin purified through the purification process of the present invention,

6 is a result of Western blot of erythropoietin purified through the purification process of the present invention,

MW: molecular weight labeling;

Lane 1: international standard BRP EPO;

Lane 2: erythropoietin purified through the purification process of the present invention,

Figure 7 shows the peptide mapping analysis of erythropoietin purified through the purification process of the present invention.

Claims (11)

(a) subjecting the biological fluid containing recombinant erythropoietin to hydrophobic chromatography; (b) performing virus inactivation on the eluate produced in step (a); And (c) anion exchange chromatography of the virus-inactivated solution according to step (b). The method of claim 1, wherein the biological fluid is animal cell culture. The method of claim 2, wherein the animal cell used for the production of the animal cell culture solution is a Chinese hamster ovary cell (CHO) strain. The purification method according to claim 1, wherein a bead size of the resin of the hydrophobic chromatography is 34 μm or less. The method of claim 1, wherein the functional group of the resin of the hydrophobic chromatography is one selected from the group consisting of phenyl, octyl, isopropyl, butyl, and ethyl. The method according to claim 1, comprising performing anion exchange chromatography as a protective column of the hydrophobic chromatography column of step (a). The purification method according to claim 6, wherein the functional group of the anion exchange chromatography resin is one selected from the group consisting of Q, QAE and DEAE. The method of claim 1, wherein the virus inactivation step comprises adding an organic solvent selected from the group consisting of ethanol, acetonitrile, and isopropanol. The method of claim 8, wherein the concentration of the organic solvent is 10 to 70% (v / v). The purification method according to claim 1, wherein the functional group of the anion exchange chromatography resin is one selected from the group consisting of Q, QAE and DEAE. Recombinant erythropoietin purified by the method of claim 1.

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