KR20010064976A - Process for the purification of recombinant human erythropoietin - Google Patents

Abstract

PURPOSE: Provided is the purification method of recombinant erythropoietin (rEPO) from animal cell culture medium. Erythropoietin is hematopoietic hormone excreted from kidney. CONSTITUTION: The purification process comprises the steps of: concentrating the animal cell culture medium by diafiltration through the membrane with 5-20 K mol. wt. cut-off (MWCO) capacity, the cell strain used being CHO (KCLRF-BP-00018) and BHK (KCLRF-BP-00024); precipitating the impurity by adding 20-120mM of divalent metal salts and reacting 1/2-2 hours, preferably, using 80mM of ZnCl2 and reacting at 4°C for 1 hour and centrifuging; adding the supernatant solution to the resin column and carrying out the anion exchange chromatography with 100mM, 250mM and 500mM NaCl step gradient solutions. the product being extracted by 100mM concentration fraction; going through hydrophobic chromatography with 0-70% ethanol linear gradient extraction, the product being extracted preferably by 0-30% concentration ethanol and finally going through anion exchange chromatography once more to collect the highly in vivo active erythropoietin in the isomer band of pI 3.5-4.5.

Description

Process for the purification of recombinant human erythropoietin

The present invention relates to a method for purifying recombinant erythropoietin from the culture supernatant of animal cells producing recombinant erythropoietin (rEPO).

More specifically, the present invention is a divalent metal cation precipitation step; Anion exchange chromatography step; And a hydrophobic chromatography step, and the like, in addition to the step of concentrating the cell culture supernatant such as ultrafiltration and / or an additional secondary anion exchange chromatography step, to a simplified recombinant erythropoietin purification method. It is about.

Erythropoietin (EPO) is a hematopoietic hormone secreted by the kidney and is a circulating glycoprotein that acts specifically on erythroid cells and regulates the differentiation and proliferation of erythroid progenitor cells in bone marrow (Carnot et al., Et al. Compt.Rend. , 143: 384, 1906. Erythropoietin is mainly produced in the liver of the fetus or in the kidney of an adult, and its production is increased in the hypoxic state of the blood to regulate the production of red blood cells.

Natural human erythropoietin is a monomeric glycoprotein consisting of 165 amino acids with a total molecular weight of about 34,000 Daltons and a molecular weight of about 18,000 Daltons in protein (Wang et al., Endocrinology , 116: 2286, 1985). About 40% of the human erythropoietin molecule is composed of sugars, and the sugar chain portion plays an essential role in the in vivo activity of erythropoietin. Therefore, when the sugar content in the erythropoietin molecule is reduced, the half-life of the erythropoietin molecule in the blood is significantly reduced and the in vivo activity is lost (Goto et al., Biotechnology , 6:67, 1988). This phenomenon has been reported to be caused by the loss of sialic acid at the end of the erythropoietin sugar chain, and the absorption of galactose and the recognition of the receptor present in the liver to absorb and degrade erythropoietin. Based on these facts, the role of sialic acid present at the end of erythropoietin sugar chains and the effect of sialic acid degrading enzyme, an enzyme that degrades erythropoietin on the bioactivity of erythropoietin have been actively studied. In addition, studies on sugar structures other than sialic acid and studies on sugar structure and erythropoietin activity are being conducted in parallel.

Natural human erythropoietin was first reported in 1906 through the study of the possibility of hematopoietic regulators and was named erythropoietin in 1957 (Jacobson et al ., Nature , 179: 633, 1957). Subsequent studies have shown that human erythropoietin was the first to successfully mass-purify and purify from urine in patients with aplasticity (Miyake et al ., J. Biol. Chem ., 252: 5558, 1977) in 1977. N-terminal sequence of erythropoietin was reported by Yanagawa et al . (Yanagawa et al ., J. Biol. Chem ., 295: 2707, 1984). Two years later, Lai et al . The total amino acid sequence was determined and reported (Lai et al ., J. Biol. Chem . 261: 3116, 1986).

With the remarkable development of genetic engineering technology in the 1980s, mass production of useful physiologically active substances has been developed, enabling mass production of erythropoietin and stable supply as a clinical treatment.

The research on production of recombinant human erythropoietin began in 1986 by Amgen Inc. in the world for the first time in human cloning of human erythropoietin genome, and in 1984 by cloning of human genome cDNA by Genetic institute in USA. Amgen's Lin and Jacob of Genetic Institute have successfully expressed human erythropoietin in Chinese Hamster Ovary cells (CHO cells). Subsequently, Cilag, which received mass production, preclinical and clinical studies, and technology from Amgen in 1988, released the world's first licensed recombinant human erythropoietin manufactured in CHO cells in Switzerland, Belgium and France. It was released on the back. In the United States, it was first released by Amgen in 1989. Currently, recombinant human erythropoietin is widely used clinically in Japan and Europe. In line with this trend, researches on manufacturing processes for producing high quality erythropoietin are being actively conducted, and researches to improve product quality through improvement of the purification process, which is the final stage of the manufacturing process, are also actively conducted.

Purification methods known to date include a method using a monoclonal antibody against erythropoietin (Korean Patent 0153808), a combination of a Blue Sepharose column, a hydroxyapatite column and an ion exchange resin column (Korean Patent 0162517), a dye pro Chemical chromatography, cation exchange chromatography and gel filtration chromatography (Korean Patent 0177300).

In the case of purification using a single antibody against erythropoietin, there is an advantage in that erythropoietin can be purified with high purity, but it is difficult to manufacture a single antibody and requires a high cost, thereby degrading economic efficiency. In addition, the purification method disclosed in Korean Patent 0162517 and Korean Patent 0177300 has a drawback that the purification process is very difficult and there is no device for selecting high quality erythropoietin. In other words, the purification process by this method does not separate isomers of low biological activity from erythropoietin and simultaneously collects all isomers, so that a large amount of erythropoietin with low in vivo activity may be included to reduce its function. This is not so desirable.

Accordingly, the present inventors have conducted research to develop a simplified erythropoietin purification method, and as a result, the overall purification by separating erythropoietin of high purity through divalent metal cation precipitation method and simple chromatography of two to three steps. The present invention was completed by not only simplifying the process but also selecting high quality erythropoietin from the separated erythropoietin to produce a high quality erythropoietin formulation.

It is an object of the present invention to provide a method for purifying high quality erythropoietin in a minimal process that can replace the existing complex multi-step erythropoietin purification method.

Figure 1 shows a preferred embodiment of the erythropoietin purification method provided by the present invention,

2 shows a chromatogram of primary anion exchange chromatography, which is a first step purification step of the purification step of the present invention,

3 shows a chromatogram of hydrophobic chromatography in a two step purification process,

Figure 4 shows a chromatogram of the secondary anion exchange chromatography, a three-step purification process,

5a and 5b are high-speed reversed phase chromatographic analysis of erythropoietin purified separated through the purification process of the present invention,

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

MW: molecular weight label;

Lane 1: the culture supernatant;

Lane 2: the concentrate of the culture supernatant;

Lane 3: supernatant after zinc chloride precipitation step;

Lane 4: eluate of the primary anion exchange chromatograph;

Lane 5: eluent of hydrophobic chromatography;

Lane 6: eluent of secondary anion exchange chromatography,

7 is an isoelectric focusing test result for the erythropoietin elution fraction of the secondary anion exchange chromatography.

pI: isoelectric point marker;

Lane 1: elution fractions 12 to 14;

Lane 2: elution fractions 15 to 16;

Lane 3: elution fraction no. 17;

Lane 4: elution fraction no. 18;

Lane 5: elution fractions 19-24.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for separating and purifying recombinant erythropoietin from the culture supernatant of animal cells producing recombinant human erythropoietin.

The erythropoietin purification method is characterized in that it comprises a divalent cation precipitation step, anion exchange chromatography step and hydrophobic chromatography step.

Another feature of the erythropoietin purification method, in addition to the characteristic purification step may include the step of concentrating the cell culture by performing ultrafiltration or the like before the divalent metal cation precipitation step, and after the hydrophobic chromatography step That may include additional anion exchange chromatography steps.

Figure 1 shows a preferred embodiment of the erythropoietin purification method provided by the present invention. This purification process is roughly divided into divalent metal cation precipitation step such as zinc chloride and liquid chromatography step, of which pure water purification step using chromatography is two or three steps. Therefore, the purification process is greatly simplified compared with the conventional multi-stage purification process, but it is composed of a process that can obtain a pure purified product that meets all other clinical criteria including purity.

Animal cells that can be used in the erythropoietin purification method of the present invention include all animal cells capable of producing recombinant erythropoietin. Erythropoietin is described by the erythropoietin amino acid sequence determined by Lai et al . (Lai et al ., J. Biol. Chem . 261: 3116, 1986). However, in addition to the natural erythropoietin, animal cells capable of producing erythropoietin variants may also be used in the purification method of the present invention. In an embodiment of the present invention, CHO cell line (DCHE715 cell line, Korean Patent Application No. 98-32997, Accession No. KCLRF-BP-00018) and BHK cell line (DBHE318 cell line, Korean patent) that produces human erythropoietin recombined with animal cells Application No. 99-37463, Accession No. KCLRF-BP-00024).

Animal cells transformed by inserting recombinant human erythropoietin can be cultured by adding an inducer in a serum-free medium, and the culture supernatant can be collected to recover erythropoietin present in the supernatant.

The erythropoietin purification method of the present invention will be described in detail for each step as follows.

(1) Cell culture medium concentration step using ultrafiltration

In the method for purifying erythropoietin of the present invention, a cell culture medium obtained by culturing animal cells may be used directly for a purification step. Preferably, the cell culture solution may be filtered through a filtration membrane, and then a divalent metal cation precipitation step may be performed.

The filtration membrane is preferably a molecular weight cut-off (MWCO) 5 ~ 20 K size filtration membrane, the volume of the culture supernatant collected by ultrafiltration to reduce the capacity to adjust the capacity to facilitate later work do. Through this process, there is almost no loss of erythropoietin, and the reduction of the volume can greatly reduce the time required for sample injection when performing chromatography, and also remove impurities in the culture medium including impurities caused by cell disruption. Can be.

(2) divalent metal cation precipitation step

In the divalent metal cation precipitation step of the erythropoietin purification method of the present invention, the divalent metal salt is added to the culture solution of the animal cells, reacted, and centrifuged to precipitate impurities. Examples of divalent metal cations include calcium (Ca ²⁺ ), zinc (Zn ²⁺ ), copper (Cu ²⁺ ), nickel (Ni ²⁺ ), cobalt (Co ²⁺ ), and they are generally calcium chloride ( CaCl ₂ ), zinc chloride (ZnCl ₂ ), cobalt chloride (CoCl ₂ ), copper sulfate (CuSO ₄ ), nickel sulfate (NiSO ₄ ) and the like in the form of metal salts are added to the culture.

Through the divalent metal cation precipitation step, most of the low glycosylated protein and non-glycosylated protein except for the highly glycosylated protein are precipitated. According to US Pat. No. 5,276,141, glycoproteins such as the gp120 protein, HIV gp160 protein, mucin, and alpha-1-glycoprotein of human immunodeficiency virus (HIV) are the sugar chain portion of the total protein weight. It is classified as a high glycoprotein which accounts for about 20-40%, and since it does not form a complex when reacting with a divalent metal cation, it is known that it can be separated from a low glycoprotein and a nonglycoprotein. In the case of erythropoietin, the sugar chain portion is a high glycoprotein, which accounts for about 40% of the total protein weight, so that it can be separated from the impurity protein using a precipitation method using a divalent metal cation.

In the erythropoietin purification method of the present invention, the divalent metal cation is preferably added at a concentration of 20 to 120 mM, reacted for about 30 minutes to 2 hours, and then precipitated by centrifugation. Particularly, zinc chloride is about 80 mM. More preferably, it is added at a concentration and reacted at about 4 ° C. for about 1 hour, followed by centrifugation to precipitate impurities. In the present invention, by treating the metal cation up to 20mM ~ 120mM through a preliminary experiment, the reaction conditions are also treated for 1 hour, 2 hours, 4 hours at 4 ℃, room temperature, 37 ℃ respectively, optimum divalent metal cation precipitation conditions Was determined. As a result of the preliminary experiments, erythropoietin is preferable when the reaction of 40 to 120 mM zinc chloride (ZnCl ₂ ) at 4 ° C. for 1 hour has a great effect of removing impurities. Particularly, 80 mM zinc chloride is reacted at 4 ° C. for 1 hour. In this case, it was found that the removal effect of erythropoietin was the smallest while the impurities were removed the most.

Low glycoproteins and nonglycoproteins precipitated by the divalent cation precipitation method can be easily removed by centrifugation and erythropoietin in the supernatant can be recovered. In addition, since the purity improvement effect of 70% or more can be expected without any additional purification process in a short time can be improved the efficiency of the purification step to be performed later.

The supernatant from which the precipitate is removed by centrifugation is desalted to perform primary anion exchange chromatography. Desalination is achieved by ultrafiltration, and the final salt concentration is preferably 5 mM or less by calculating the dilution factor.

(3) anion exchange chromatography step

In the anion exchange chromatography step of the erythropoietin purification method of the present invention, an anion exchange column chromatography is performed with an erythropoietin fraction separated in the divalent metal cation precipitation step, but stepwise of 100 mM, 250 mM and 500 mM sodium chloride Elution with step gradient solution, of which 100 mM sodium chloride fraction is separated into active fraction. In addition to this method, a protein may be eluted using a linear concentration gradient of sodium chloride to collect an erythropoietin active fraction, or a protein eluted method using calcium chloride as a salt other than sodium chloride may be used. In the former case, a linear concentration gradient was formed in 10 column volumes using 10 mM Tris buffer and 500 mM sodium chloride solution, and the protein eluted at a concentration of 75 mM to 120 mM was collected as an active fraction of erythropoietin. In the latter case, an active fraction of erythropoietin is obtained by forming a linear concentration gradient with 10 column volumes using 5 mM Tris buffer and 200 mM calcium chloride solution, or eluting the protein with stepwise concentration gradients using calcium chloride 5 mM, 20 mM, 100 mM, and 200 mM solutions. Collect it. At this time, erythropoietin is eluted in the 10 ~ 20mM fraction in the linear gradient, 20mM fraction in the step gradient. Among the above-mentioned methods, considering the process time and yield, erythropoietin purity, etc., the method using the step gradient of sodium chloride is most preferred.

In an embodiment of the present invention, the desalted supernatant after the divalent metal cation precipitation step is injected into a column filled with anion exchange column chromatography resin (e.g. DEAE Sepharose) and the stepwise concentrations of 100 mM, 250 mM and 500 mM sodium chloride. Eluate with gradient solution. Prior to sample injection, equilibrate the column with 10 mM Tris buffer (pH 8.0), etc., and at least 2 column volumes of solvent are injected at each step during elution. However, the type of resin, the size of the column, the type and amount of the equilibration buffer, the amount of the eluate, and the like can be modified according to conventional anion exchange chromatography techniques. Erythropoietin in the elution fraction was separated in the 100 mM sodium chloride concentration interval, most of the impure protein was eluted in the washing step of 250mM and 500mM (see Figure 2 ).

(4) hydrophobic chromatography step

In the hydrophobic chromatography step of the erythropoietin purification method of the present invention, the elution fraction containing erythropoietin in the anion exchange chromatography eluate was carried out to perform hydrophobic chromatography, but linear gradient of 0 to 70% ethanol Elution with a linear gradient solution, and the ethanol 10 ~ 35% concentration section is separated into active fractions.

In an embodiment of the present invention, the erythropoietin elution fraction of primary anion exchange chromatography is injected into a column filled with phenyl resin, and the erythropoietin is eluted by an ethanol linear concentration gradient. Prior to the injection of the sample, the column is equilibrated with 1M guanidine solution, and salt is added after the salt is added so that the final concentration of guanidine in the injection sample is 1M. However, the type of resin, the size of the column, the type of equilibration buffer, and the like can be modified according to conventional hydrophobic chromatography techniques.

Through the above purification process, the elution fraction of erythropoietin and the elution fraction of impurities are completely separated, and the endotoxin, host-derived DNA, and host-derived peptide are purified based on KFDA approval standard and test method. Erythropoietin of 99% purity removed below the test criteria can be purified (see FIG. 3 and Table 2 ).

The erythropoietin eluate collected in hydrophobic chromatography can be desalted using a dialysis method. At this time, it is preferable to calculate the dilution factor of the salt to the solvent so that the concentration of the final salt is 5 mM or less.

(5) additional anion exchange chromatography steps

Erythropoietin, a glycoprotein, is regulated by sialic acid (sialic acid or N-acetylneuraminic acid) at the end of sugar chains, and the higher the sialic acid content, the higher the in vivo activity. This is because the sialic acid at the end of the sugar chain performs a function of extending the half-life of erythropoietin, which requires a process of selecting high-quality erythropoietin. To this end, an additional anion exchange chromatography step is performed to fractionate the erythropoietin eluate.

Anion exchange chromatography, which may be additionally performed in the erythropoietin purification method of the present invention, is performed after the hydrophobic chromatography step, preferably eluted with a linear gradient solution of 0 to 500 mM sodium chloride, of which 45 to 175 mM concentration. The interval can be separated into active fractions. Protein elution method using calcium chloride can be used as a salt other than sodium chloride, and the concentration gradient condition uses a linear concentration gradient. Using 5 mM Tris buffer (pH 8.0) and 200 mM calcium chloride solution, a linear concentration gradient was formed in 10 column volumes, and the protein was eluted to collect the active fraction of erythropoietin. Erythropoietin is eluted in 10 ~ 20mM fraction. Among the above methods, considering the process time and yield, erythropoietin purity, fractional resolution, and the like, a method using a linear concentration gradient of sodium chloride is most preferred.

In the embodiment of the present invention, the sample after dialysis is injected into a column filled with Q resin, and further secondary anion exchange chromatography is performed by a linear gradient of 0 to 500 mM sodium chloride. Secondary anion exchange chromatography was performed by injecting a dialysis sample into a column equilibrated with 10 mM Tris buffer (pH 8.0), forming a sodium chloride concentration gradient, and then fractionating the eluted protein to a constant fraction size. (See FIG. 4 ). However, the type of resin, the size of the column, the type of equilibration buffer, and the like can be modified according to conventional anion exchange chromatography techniques.

Erythropoietin is mainly eluted in the 45-175mM section through anion exchange chromatography, and erythropoietin between pI 3.5 and 4.5 is included through isoelectric focusing analysis by fractionating the erythropoietin eluted from this section. Only fractions were collected. Each of the eluted fractions is identified by isoelectric focusing (IEF), and then only the fractions having a pI range of 3.5 to 4.5 are collected (see FIG. 7 ). Through this process, high-purity erythropoietin with a purity of 99% or more can be separated, and only high quality erythropoietin with high bioactivity can be selected without a separate purification process.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Collection of Cell Culture Supernatant

DCHE715 cell line (stabilized recombinant erythropoietin producing CHO cell line (Korean Patent Application No. 98-32997, Accession No. KCLRF-BP-00018)) and DBHE318 cell line, which is a stabilized recombinant erythropoietin producing baby hamster kidney (BHK) cell line (Korea) Patent application No. 99-37463, Accession No. KCLRF-BP-00024), respectively, was serum-free and cultured to collect 100 L of the culture supernatant, which was used in Example 2 and below. At this time, the erythropoietin production CHO and BHK cells were cultured at 37 ° C. for 6 days and 8 days, respectively, and the concentrations of the cells were 7.5% and 8.1% of the initial inoculation concentrations, respectively. The DCHE715 cell line was selected from methotrexate (MTX) selection medium in a CHO cell line transformed with the vector pDEP173 having the gene sequence of human erythropoietin, and the DBHE318 cell line was transformed with the vector pDEP521 containing the human erythropoietin gene. Among the converted BHK cell lines were selected in MTX selection medium.

Example 2 Concentration of Cell Culture Supernatant

A filtration membrane (Pellicon 2 Cassette Filter, Millipore, MWCO 10K) was fitted with a pump (MasterFlex, Millipore, Inc.) and the inside of the filtration system was washed with 60 L of tertiary sterile water. After the washing of the filter was completed, the supernatant of the culture supernatant of CHO cells was injected into the filter through a pump, and the filtrate was recovered and reinjected. After the ultrafiltration was completed, the volume of the culture supernatant concentrate was 3-4 L.

Example 3 Zinc Chloride Pretreatment

A 1 M zinc chloride (ZnCl ₂ ) solution was prepared using tertiary sterile water and added to the concentrate of Example 2 such that the final concentration was 80 mM. After the precipitation with 80 mM zinc chloride added was left for 1 hour at 4 ℃ to perform the precipitation reaction, the concentrate was dispensed into a centrifuge container and centrifuged at 4 ℃ for 20 minutes at a speed of 3000rpm. After the centrifugation, the supernatant was recovered so that the precipitates were not mixed. Purity analysis and protein concentration measurement using reverse phase high performance liquid chromatography (RP-HPLC) were performed (see Experimental Example 1).

As a result, the purity of the concentrate pretreated with zinc chloride was 75-80% and about 40% of impurities were removed.

Example 4 Solvent Exchange of Pretreatment Liquid

A filtration membrane (Pellicon 2 Cassette Filter, Millipore Co., MWCO 10K) was fitted with a pump (MasterFlex, Millipore Co.) and the inside of the filtration apparatus was washed with 60 L of tertiary sterile water. After washing the filter, the sterilized water of 10 times volume is added to the zinc chloride pretreatment solution to dilute the pretreatment solution 10 times, and the diluent is injected into the filter device through the pump, and the filtrate is recovered and reinjected. Ultrafiltration was performed. The concentrate concentrated by ultrafiltration was re-diluted with 10 times the volume of tertiary sterile water to repeat the ultrafiltration twice more so that the concentration of salt in the concentrate was 5 mM or less.

Example 5 Primary Anion Exchange Chromatography (IEC-I)

Anion exchange resin (DEAE-Sepharose Fast Flow, Amersham-pharmacia biotech) was charged in 200 × 500 columns and equilibrated by 2 column volumes with 10 mM Tris buffer (pH 8.0). The concentrate of Example 4 was injected into the equilibrated column and the column was washed with 10 mM Tris buffer (pH 8.0). 100 mM sodium chloride / 10 mM Tris buffer (pH 8.0), 200 mM sodium chloride / 10 mM Tris buffer (pH 8.0), and 500 mM sodium chloride / 10 mM Tris buffer (pH 8.0) were each injected in two column volumes to elute the protein and each eluate was RP. Analyzed using HPLC (see Experimental Example 1).

As a result of primary anion exchange chromatography, proteins were separated in the same manner as in FIG. 2. As a result of analyzing each protein eluate, erythropoietin was mostly detected in 100 mM sodium chloride eluate and the purity reached 95-98%.

Example 6 Hydrophobic Chromatography (HIC)

Hydrophobic chromatography resin (Phenyl-Sepharose Fast Flow, Amersham-pharmacia biotech) was packed in a column (FineLINE Pilot 35, Amersham-pharmacia biotech) and equilibrated with 1M guanidine hydrochloride solution. Guanidine was added to the erythropoietin eluate of primary anion exchange chromatography so as to have a final concentration of 1 M, and then dissolved in a column. As shown in FIG. 3 , a linear concentration gradient was formed at 10 column volumes from solvent A (1M guanidine) to solvent B (70% ethanol / 1M guanidine) to elute the protein. The protein eluate was fractionated and only fractions containing erythropoietin were collected after purity analysis using reverse phase high performance liquid chromatography (RP-HPLC) (see Experimental Example 1). Erythropoietin was eluted at a ethanol 10-35% concentration gradient with a high purity of 99%.

Example 7 Dialysis of Erythropoietin Eluate

Hydrophobic chromatography eluate was added to an activated dialysis membrane ( Spectra / Por ^?? Spectrum, MWCO 12-14K), and then sealed, and dialyzed three times at 6 hour intervals for 10-fold tertiary sterile water. Was set to 5 mM or less.

Example 8 Secondary Anion Exchange Chromatography

The dialysate was injected into an anion exchange column (Q-Sepharose Fast Flow, Amersham-pharmacia biotech) equilibrated with 10 mM Tris buffer (pH 8.0) and the remaining protein in the column was washed with 10 mM Tris buffer (pH 8.0). After washing was complete, a linear concentration gradient was formed in 10 column volumes from solvent A (10 mM Tris buffer, pH 8.0) to solvent B (500 mM sodium chloride / 10 mM Tris buffer, pH 8.0) to elute the protein (see FIG. 4 ). .

Erythropoietin was eluted extensively in concentration gradient zones ranging from 45 mM to 175 mM sodium chloride. The protein eluate was fractionated to a fraction size of 20 mL and isoelectric focusing (IEF) was performed on each fraction of erythropoietin eluted (see Experimental Example 3). After analysis of the isoelectric focusing test, only the fraction containing erythropoietin having a pI range of 3.5 to 4.5 was collected and formulated. At this time, almost no impurities of endotoxin, host-derived DNA, and host-derived peptide were detected in erythropoietin distributed at pI 3.5-4.5.

Experimental Example 1 Purity Analysis Using Reversed Phase High Performance Liquid Chromatography

The purity of the cell culture supernatant, its concentrate, zinc chloride pretreatment and protein eluate collected at each purification step was measured using reverse phase high performance liquid chromatography (RP-HPLC). Purity analysis using RP-HPLC was performed by taking 100 μl of each sample and injecting it into an HPLC column to separate proteins in the sample by acetonitrile linear concentration gradient.

RP-HPLC apparatus and conditions were as follows.

- Device :

HPLC pumps (LC 22, Bruker)

Detector (LAMDA 1000, BISCHOFF)

Hi-Pore Reversed phase column (RP-304, BIO-RAD)

Chromatography Conditions

Solvent A; 20% acetonitrile / 0.1% trifluoroacetic acid

Solvent B; 80% Acetonitrile / 0.1% Trifluoroacetic Acid

Flow rate; 0.5 mL / min

Detection wavelength; 280 nm

Development time; 45 minutes

Concentration gradient; See Table 1

Deployment time (minutes) % Solvent A % Solvent B 0 70 30 10 70 30 35 20 80

37 20 80 38 70 30 45 70 30

5 shows the results of analyzing the purified products according to the above conditions.

Experimental Example 2 Purity Analysis Using Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Purity of the cell culture supernatant, its concentrate, zinc chloride pretreatment and protein eluate collected at each purification step was measured using polyacrylamide gel electrophoresis.

SDS-PAGE gel (Pre-cast gel, gradient 4-20%, NOVEX) was placed on an electrophoresis device (Xcell II Mini-Cell, NOVEX) and filled with mobile buffer. 50 μl of each sample was mixed with 50 μl of sample buffer, heated at 95 ° C. for 5 minutes, centrifuged at 10,000 rpm for 3 to 4 minutes, and 25 μl were injected into each well with a molecular weight standard. After injection, electrophoresis was performed at a voltage of 130V, and when the development was completed, the gel was taken out and subjected to Coomassie staining. The SDS-PAGE results are shown in FIG. 6 and the final erythropoietin purified showed more than 99% purity.

Experimental Example 3 Isoelectric Focusing Test

Each fraction eluted in Example 8 was concentrated to a concentration of about 600,000 IU / mL, and then the concentrate and the sample buffer were mixed 1: 1. An IEF gel (IEF PAGE pH 3-7, NOVEX) was placed on an electrophoresis device (Xcell II Mini-Cell, NOVEX), and 20µl of each sample and 5µl of a low pI kit (pH 2.5 ~ 6.5, Pharmacia) Was injected into each well and electrophoresed for 2 hours at 2W. After the electrophoresis was completed, the gel was fixed by stirring slowly for 30 minutes in a fixed solution and Coomassie stained. The band distribution of each sample was read after decolorizing so that the band was clearly distinguished by a decolorizing solution. As shown in FIG. 7 , the erythropoietin fraction eluted in the secondary anion exchange chromatography was evenly distributed over a wide range of pi, and only erythropoietin, which appeared in the range of pi 3.5-4.5, was collected.

Experimental Example 4 In vitro Activity Assay by Enzyme Immunoassay

In vitro activity of the cell culture supernatant, its concentrate, zinc chloride pretreatment, and the protein eluate collected at each purification step was measured by enzyme-immunoassay, and the yield of each purification step was calculated. 100 μl of analytical dilution was added to each well of the antibody adsorption plate in the Quantikine ™ IVD ™ ELISA (R & D System), the standard solution (86 IU / ample, NIBSC) diluted appropriately with the sample dilution, and 100 μl of the sample solution was added to the mixture and allowed to react at room temperature for 1 hour. The remaining reaction solution of each well was removed, washed three times with 250 μl of washing solution, and 200 μl of enzyme antibody conjugate solution was added thereto, followed by further reaction at room temperature for 1 hour.

After the reaction filtrate was removed and washed in the above manner, 200 µl of the substrate-chromic solution prepared in the elution was added to the wells and developed for 20 minutes at room temperature. After the completion of color development, the reaction stop solution was added to 100μl to stop the reaction and the absorbance was measured at 450nm. Prepare a standard curve using the horizontal axis as the activity of the standard solution (log mIU / mL) and the absorbance (A _{450 nm} ) as the standard axis. Calculated.

Table 2 shows an example of the purification step of the purified products obtained by performing the above purification process from the serum-free culture of the DCHE715 cell line, a CHO cell line.

Purification steps Capacity (mL) EPO titer (IU / mL) Protein concentration (mg / mL) Inactive (IU / mg) Endotoxin (EU / mL) water(%) Culture supernatant 104,000 7,565 0.237 31,941 ND 46 concentrate 3,400 229,086 7.018 32,644 ND 48.9 Zinc Chloride Pretreatment 3,400 219,830 3.219 70,256 ND 75.5 Tier 1 (IEC-I) 3,000 133,294 1.453 91,756 ND 96 Tier 2 (HIC) 3,000 129,885 1.283 101,267 ND 99 Tier 3 (IEC-II) 400 110,145 0.866 127,243 ND > 99 EPO stock ^a 350 125,877 0.985 127,845 0.1 > 99 * a: EPO stock solution formulated after the three steps of purification ND: not determined

Experimental Example 5 Impurity Detection of Endotoxin, Host-Derived DNA, and Host-Derived Peptide

(5-1) endotoxin confirmation test

After diluting the sample solution and the standard solution at an appropriate dilution ratio, 50 μl was aliquoted into a 96 well plate, and 50 μl of LAL (Limulus Amebocyte Lysate) solution was added thereto, followed by reaction at 37 ° C. for 10 minutes. 100 μl of the substrate color developing solution was added to each well, followed by color development at 37 ° C. for 10 minutes, and 100 μl of the reaction stop solution was added. The endotoxin of the test solution was calculated by converting the endotoxin of the test solution from the standard solution and multiplying it by the dilution factor. As a result, endotoxin was detected at 0.1 EU / mL or less in EPO stock solution purified purely by the three-step purification method of the present invention.

(5-2) Host derived DNA test

Proteins in the sample were removed by phenol / chloroform extraction, desalted and concentrated to a volume of 100 μl. Host-derived DNA standards were prepared and adsorbed onto the nitrocellulose membrane with the sample solution and hybridized using a probe labeled with Digoxygenin-dUTP. The color development was performed by adding the substrate color development solution, and the degree of color development was compared with the standard solution to calculate the host-derived DNA content in the sample solution. As a result, the host-derived DNA was detected to be less than 10 pg / 60,000 IU in the EPO stock purified by the three-step purification method of the present invention.

(5-3) Host derived peptide test

Primary antibodies to host-derived peptides were adsorbed on 96-well plates at 37 ° C. for 1 hour and the filtrate was washed. A sample solution and host-derived peptide standard solution were added to each well to react at 37 ° C, the filtrate was washed, and then enzyme-antibody conjugate was added to react at 37 ° C. Substrate color development solution is added to color at room temperature, absorbance at 490nm wavelength is measured and compared with the standard solution to calculate the host-derived peptide concentration of the sample solution. The final host-derived peptide concentration was converted by multiplying by the dilution factor of the sample solution. As a result, the host-derived peptide was detected to 78ng / 60,000 IU or less in EPO stock purified purely by the 3-step purification method of the present invention.

The erythropoietin purification method provided by the present invention comprises a culture supernatant concentration process, a simple divalent metal ion pretreatment, a chromatography step consisting of only two or three steps, and thus, the conventional purification method is complicated using the purification method. Compared to this, the purification time and cost can be reduced.

Claims (8)

In the method for purifying recombinant erythropoietin from the culture supernatant of animal cells producing recombinant human erythropoietin, An erythropoietin purification method comprising a divalent metal cation precipitation step, an anion exchange chromatography step and a hydrophobic chromatography step. The method for purifying erythropoietin according to claim 1, wherein the animal cell is a CHO or BHK cell line. The method for purifying erythropoietin according to claim 1, wherein the divalent metal cation precipitation step adds a divalent metal salt at a concentration of 20 to 120 mM, reacts for about 30 minutes to 2 hours, and then precipitates impurities by centrifugation. . The method for purifying erythropoietin according to claim 3, wherein the divalent metal cation precipitation step adds zinc chloride at a concentration of about 80 mM and reacts for about 1 hour at about 4 ° C, followed by centrifugation to precipitate impurities. The method for purifying erythropoietin according to claim 1, wherein the anion exchange chromatography step is eluted with a stepwise gradient solution of 100 mM, 250 mM and 500 mM sodium chloride, and the 100 mM sodium chloride fraction is separated into the active fraction. . The method for purifying erythropoietin according to claim 1, wherein the hydrophobic chromatography step is eluted with a 0-70% ethanol linear concentration gradient solution, and the ethanol 10-35% concentration section is separated into active fractions. The method for purifying erythropoietin according to claim 1, wherein the ultrafiltration through a filtration membrane (MWCO 5-20 K) is performed before the divalent metal cation precipitation step. The method of claim 1, further comprising the step of collecting only the fraction containing only an isomer band between the additional anion exchange pI 3.5 to 4.5 after the hydrophobic chromatography step. Method of Purification of Ethine.