WO2009066806A1 - Purification method of human erythropoietin - Google Patents

Purification method of human erythropoietin Download PDF

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Publication number
WO2009066806A1
WO2009066806A1 PCT/KR2007/005898 KR2007005898W WO2009066806A1 WO 2009066806 A1 WO2009066806 A1 WO 2009066806A1 KR 2007005898 W KR2007005898 W KR 2007005898W WO 2009066806 A1 WO2009066806 A1 WO 2009066806A1
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human erythropoietin
milk
chromatography
purifying
transgenic animal
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PCT/KR2007/005898
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French (fr)
Inventor
Bong Hyun Chung
Eun Gyo Lee
Joon Ki Jung
Kyung Mi Park
Seung Hui Lee
Jung Eun Baek
Won Kyong Chang
Jin Ki Park
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Korea Research Institute Of Bioscience & Biotechnology
Rural Development Administration
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Publication of WO2009066806A1 publication Critical patent/WO2009066806A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]

Definitions

  • the present invention relates to a method of purifying human erythropoietin, and more particularly, to a method of purifying human erythropoietin comprising the steps of (1) purifying a cell culture or pretreated milk of a transgenic animal by heparin chromatography, (2) purifying a fraction containing human erythropoietin obtained from the step (1) by reversed-phase chromatography, and (3) purifying the fraction containing human erythropoietin obtained from the step (2) by gel filtration chromatography.
  • the present invention is characterized in that, in a case where human erythropoietin is purified from milk of a transgenic animal, a pretreatment process for milk is performed before the step (1) and comprises the steps of (a) recovering a supernatant after centrifuging the milk of the transgenic animal, (b) adding a first precipitating agent to the supernatant for first precipitation of impurities, (c) adding a transition metal ion for second precipitation of impurities and (d) ultrafiltering a supernatant recovered from the step (c).
  • a pretreatment process for milk is performed before the step (1) and comprises the steps of (a) recovering a supernatant after centrifuging the milk of the transgenic animal, (b) adding a first precipitating agent to the supernatant for first precipitation of impurities, (c) adding a transition metal ion for second precipitation of impurities and (d) ultrafiltering a supernatant recovered from the step (c).
  • Natural human erythropoietin is a monomeric glycoprotein composed of 165 amino acids, which has a total molecular weight of about 34,000 daltons (-34 kDa) and a molecular weight of protein of about 18,000 daltons. It is known that erythropoietin has sugar chains occupying about 40% of the total molecular weight which are essential for in vivo activity, and contains N-linked sugar chains at Asns independently located at the 24 th , 38 th and 83 rd amino acids, and one mucin-type O- linked sugar chain at Ser located at the 126 th amino acid.
  • Natural human erythropoietin was first reported in 1906 as a result of research into the probability of the presence of hemopoietic growth factor, and was named in 1957 (Jacobson et al., Nature, 1957, vol. 179, p.633). Subsequent research led to the first successful large-scale isolation and purification of human erythropoietin from urine of a patient suffering from aplastic anemia by Miyake et al. in 1977 (Miyake et al., J.Biol. Chem. 1977, vol. 252, p.5558), an N-terminal sequence of erythropoietin was found by Yanagawa et al. (Yanagawa et al., J.
  • human erythropoietin is prepared by animal cell culture or a technique using a transgenic animal.
  • the preparation technique using a transgenic animal has advantages in many aspects compared to the technique using an animal cell.
  • the first advantage of the technique using a transgenic animal is that a high-quality recombinant protein may be obtained, which is almost the same as, but safer than, a natural type.
  • the protein may be mass-prepared on a scale of several grams per liter.
  • the foreign gene included therein is inherited by the offspring, thereby maintaining a constitutive production system.
  • human erythropoietin produced by animal cell culture is expressed in a protein-free media
  • human erythropoietin produced from the mammary gland of a transgenic animal is expressed in milk protein, whose which requires a more complicated purification method than the method for the animal cell culture.
  • Korean Patent Nos. 10-0153808 and 10-0297927 using immuno-adsorption chromatography Korean Patent Nos. 10-0162517 and 10- 0177300 using dye-affinity chromatography
  • Korean Patent No. 10-0065197 and U.S. Patent No. 4,677,195 using reversed-phase chromatography Since the human erythropoietin produced by animal cell culture generally has low pi, it is purified by ion exchange chromatography in conventional techniques.
  • An example of methods of purifying human erythropoietin produced by an insect cell is disclosed in Korean Patent No. 10-0321446 using positive ion exchange chromatography, lectin chromatography and gel filtration chromatography.
  • human erythropoietin can be purified from the milk of a transgenic animal with high purity and efficiency in a simple and fast process including: pretreating the milk of the transgenic animal by recovering a supernatant from the milk by centrifugation, adding a first precipitating agent and a transition metal ion to precipitate impurities, ultrafiltering the supernatant recovered from the previous the step, and then sequentially purifying the filtrate obtained from the previous the step using heparin, reversed-phase and gel filtration chromatographies.
  • the present invention is directed to a method of preparing human erythropoietin, comprising the steps of (1) purifying a cell culture or pretreated milk of a transgenic animal by heparin chromatography, (2) purifying a fraction containing human erythropoietin obtained in the step (1) by reversed-phase chromatography, and (3) purifying a fraction containing human erythropoietin obtained in the step (2) by gel filtration chromatography.
  • the term 'a cell culture' is used in the purification method according to the present invention and may be a culture of human cells naturally expressing human erythropoietin or a culture of cells, preferably animal cells, transformed to express human erythropoietin.
  • a pretreatment the step may be omitted and it may be purified by microfiltration using a 0.1 to 0.45 ⁇ m filtering membrane if necessary.
  • milk of a transgenic animal' refers to milk obtained from a mammary gland of an animal transformed to express human erythropoietin.
  • the milk of the transgenic animal generally requires pretreatment since it is composed of various elements (impurities) such as proteins, sugars, lipids and the like.
  • 'pretreatment' refers to a process of removing impurities before performing the purification method in order to increase purification efficiency of the purification method according to the present invention.
  • the pretreatment process comprises the steps of (a) recovering a supernatant from milk of a transgenic animal by centrifugation, (b) adding a first precipitating agent to the supernatant for first precipitation of impurities, (c) adding a transition metal ion for second precipitation of impurities and (d) ultrafiltering a supernatant recovered from the previous the step, before the step (1) in the purification method.
  • step (a) milk obtained from a transgenic animal is centrifuged to recover a supernatant.
  • the milk of the transgenic animal in the present invention is centrifuged at 4,000 to 12,000 rpm and 4 to 37 ° C for 10 to 40 minutes.
  • the milk of the animal is centrifuged into 3 layers, in which an uppermost layer is a lipid, a middle layer is a colloid, and a lowermost layer is an insoluble complex.
  • the lipid and insoluble complex are removed, and the colloid is recovered to be treated in the next the step.
  • the 'supernatant' in the step (a) of the present invention refers to the colloid formed in the middle layer obtained after the removal of the lipid layer.
  • a first precipitating agent is added to the supernatant obtained according to the step (a) for first precipitation of impurities.
  • the first precipitating agent may be ammonium sulfate, sodium sulfate or potassium phosphate, which may be used at a concentration of 10 to 60%, and preferably 20 to 50%. After adding the first precipitating agent at the above-mentioned concentration, the mixture is reacted for 1 to 12 hours at 4 to 37 ° C to precipitate impurities.
  • the transition metal ion may be a metal ion selected from the group consisting of Mn, Fe, Co, Ni, Cu and Zn, which may be used at a concentration of 5 to 50OmM, and preferably 20 to 20OmM.
  • the mixture is reacted for 1 to 24 hours at 4 to 37 ° C to precipitate impurities.
  • the steps (b) and (c) may be simultaneously performed, in which a first precipitating agent is added at a partial predetermined concentration, and the mixture is reacted for 1 to 6 hours at 4 to 37 " C to precipitate impurities. Then, the remaining first precipitating agent and the predetermined concentration of the transition metal ion are added, and the mixture is reacted for 1 to 24 hours at 4 to 37 ° C to precipitate impurities.
  • the present invention is characterized by performing twice a precipitation reaction as described above.
  • the first precipitation reaction increases salt concentration to precipitate a hydrophobic protein.
  • the second precipitation reaction agglomerates minute particles formed in the first precipitation, induces binding to other impurities in order to stimulate the formation of a precipitate, and thus may effectively remove the impurities.
  • step (d) ultrafiltration is performed to remove all kinds of salts used in the steps (b) and (c) and reduce the volume, since the subsequent the step (1) regulates the salt concentration and extracts human erythropoietin.
  • fine precipitates may be removed by additional microfiltration with respect to the supernatant obtained from the step (c).
  • both the cell culture and the pretreated milk of a transgenic animal can be purified by the method according to the present invention.
  • the method of purifying the milk from a transgenic animal according to the present invention is different from the purification method using an animal cell culture employing sialic acid or another sugar chain, and may be applied to the purification of erythropoietin having an imperfect sugar chain (having a smaller molecular weight of about 10 kDa).
  • a cell culture or pretreated milk of a transgenic animal is purified by heparin chromatography. It is known that the heparin chromatography has excellent affinity to glycoprotein in particular.
  • a phosphate buffer may be used as an equilibrium solution, and a phosphate buffer containing sodium chloride (NaCl) may be used as an elution solution. Human erythropoietin is eluted at a NaCl concentration of 50 to 60OmM on the chromatogram.
  • the heparin chromatography purifies the human erythropoietin faster and with higher adsorption efficiency per unit gel than other types of chromatography, and thus may highly concentrate the human erythropoietin.
  • a fraction containing the human erythropoietin obtained by the heparin chromatography is harvested and then purified by reversed-phase chromatography.
  • no treatment of the fraction is required, so that the steps (1) and (2) may be continuously performed, and thus the overall purification method is simple.
  • distilled water may be used as an equilibrium solution, and acetonitrile may be used as an elution solution.
  • the human erythropoietin is eluted by about 30 to 70% of an acetonitrile solution in the chromatogram.
  • trifluoroacetic acid may be added to both the equilibrium solution and the elution solution.
  • a gel having Cl, C4, C6, C8 and Cl 8 as ligands may be employed for the reversed-phase chromatography according to the present invention. Accordingly, the human erythropoietin may be completely separated from the impurity protein by the purification method using the reversed-phase chromatography according to the present invention.
  • a fraction containing the human erythropoietin obtained by the reversed-phase chromatography is harvested and then purified by gel filtration chromatography.
  • the acetonitrile solution may be removed from the fraction before the step (3).
  • tris buffer may be used as both equilibrium and elution solutions.
  • a size-exclusion gel may be used to separate proteins by size, each having a molecular weight of 5 to 10OkDa.
  • the gel is one selected from the group consisting of Superose 12, Superdex 75, Superdex 200, Sephadex 200, Sephadex 75, Sephadex S-100, Sephacryl S-100 and Sephacryl S-200. Accordingly, even very small amounts of impurities contained in the fraction and inactive human erythropoietin produced by protein coagulation in the above- described purification the step may be removed by purifying the human erythropoietin using the gel filtration chromatography according to the present invention.
  • human erythropoietin According to the purification method of human erythropoietin according to the present invention, several impurities may be sufficiently removed from the milk of a transgenic animal through pretreatment, which may reduce the amount of a sample to be purified by subsequent chromatography and reduce the overall purification time.
  • human erythropoietin may be efficiently and thoroughly purified in a simple and fast process by sequentially performing heparin, reversed-phase and gel filtration chromatographies, from among the well-known chromatography techniques in the art, of a cell culture or pretreated milk of the transgenic animal.
  • FIG. 1 is a schematic diagram of a method of purifying human erythropoietin from milk of a transgenic animal according to the present invention.
  • FIG. 2 shows a chromatogram obtained from purification of human erythropoietin using heparin chromatography during the purification method according to the present invention.
  • FIG. 3 shows a chromatogram obtained from purification of human erythropoietin using reversed-phase chromatography during the purification method according to the present invention.
  • FIG. 4 shows a chromatogram obtained from purification of human erythropoietin using gel filtration chromatography during the purification method according to the present invention.
  • FIG. 5 shows electrophoresis results for fractions obtained in the steps of the purification method according to the present invention.
  • Example 2 Precipitation of ammonium sulfate and transition metal 20% ammonium sulfate was added to the recovered result of Example 1, and the mixture was reacted at 4°C for one hour to precipitate impurities. Thereafter, ammonium sulfate was further added up to a total of 35%, and 6OmM zinc chloride was added at 4°C for 2 hours to precipitate impurities. The resulting product was centrifuged at 6,000 rpm and 4 ° C for 30 minutes, thereby obtaining a supernatant.
  • Example 2 The supernatant obtained in Example 2 underwent microfiltration using a 1.45 ⁇ m filter. Then, salts and sugars smaller than 3 kDa were removed from the microfiltrate by ultrafiltration through an ultrafiltration membrane (Millipore, MWCO 3K). Here, the resulting filtrate was diluted with a phosphate buffer to a volume 10 times larger than the initial volume for diafiltration and concentration.
  • Example 4 Heparin chromatography Heparin chromatography was performed using an AKTAFPLC system (GE,
  • HiTrap Heparin HP (GE) and HiTrap Heparin HP (GE), in which 5ml heparin resin was filled and used as a column.
  • the column was equilibrated with 1OmM sodium phosphate buffer (an equilibrium solution; pH 7.0) at a flow rate of 3ml/min.
  • the equilibrated solution containing 2M sodium chloride was used as an elution solution to elute protein adsorbed to a heparin gel.
  • Each fraction was monitored by enzyme-linked immunoabsorbent assay (ELISA) to confirm the activity of human erythropoietin.
  • ELISA enzyme-linked immunoabsorbent assay
  • a lOO ⁇ l dilute of the fraction was added to each well in an antibody-adsorbed plate of an ELISA kit and well mixed, and was reacted at room temperature for 2 hours. The remaining reactant in each well was removed and each well was washed with a 400 ⁇ l washing solution four times. Afterward, a 200 ⁇ l enzyme-antibody conjugates solution was added and reacted at room temperature for 2 hours. The remaining reactant was removed and washed in the same manner as described above, and a 200 ⁇ l substrate-dye solution was added to the well and developed at room temperature for 25 minutes. After the development was completed, a lOO ⁇ l reaction blocking solution was added to block the reaction and its absorbance was measured at 450nm.
  • a standard curve was plotted with the activity of the standard solution on the x axis and with the absorption on the y axis using a logarithmic coordinate system. From the standard curve, the activities of erythropoietin in the world standard solution and the fraction were measured and multiplied by a dilution multiple, thereby measuring the final activity of the fraction. It was confirmed by the heparin chromatography that human erythropoietin was eluted at a sodium chloride concentration of 50 to 60OmM (refer to FIG. 2). The fraction whose activity was confirmed was recovered and underwent reversed-phase chromatography, which will be described below.
  • the reversed-phase chromatography was performed using an HPLC system (Virian, USA) and a C8 column (Vydac, USA). Distilled water was used as an equilibrium solution and acetonitrile was used as an elution solution. In order to reduce non-specific adsorption between protein impurities and silica resin, 0.1% trifluoroacetic acid was added to each equilibrium solution and elution solution. ELISA was performed on each fraction according to the same procedure as described in Example 4 to confirm the activity of human erythropoietin.
  • each product obtained in each the step (sample) was analyzed by polyacryl amide gel electrophoresis.
  • a gel (12%) for electrophoresis was put into an electrophoresis device, and the device was filled with mobile buffer.
  • 5 ⁇ l of each sample was mixed with 25 ⁇ l of sample buffer, and the mixture was boiled at 95 ° C for 5 minutes and then centrifuged at 12,000 rpm for 3 to 4 minutes.
  • 30 ⁇ l of the sample mixture was injected into each well together with a prestained standard (Bio- Rad). Afterward, electrophoresis of the samples was performed at a constant voltage of 150V, and then the gel was stained with Kumasi after development.
  • FIG. 5 The results of the electrophoresis are shown in FIG. 5, in which it can be confirmed that protein impurities were removed and only human erythropoietin remained.
  • lane M shows a protein size marker (Bio-Rad, Precision)
  • lane 1 shows undiluted milk
  • lane 2 shows a supernatant obtained after centrifugation
  • lane 3 shows a supernatant obtained after simultaneous precipitation of ammonium sulfate and zinc chloride
  • lane 4 shows a supernatant obtained after microfiltration and ultrafiltration
  • lane 5 shows a fraction purified by heparin chromatography
  • lane 6 shows a fraction purified by reversed-phase chromatography
  • lane 7 shows a fraction purified by gel filtration chromatography.

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Abstract

The present invention discloses the a method of purifying human erythropoietin, the method of the present invention comprises the steps of (1) purifying a cell culture or pretreated milk of a transgenic animal by heparin chromatography, (2) purifying a fraction containing human erythropoietin obtained from the step (1) by re versed-phase chromatography, and (3) purifying a fraction containing human erythropoietin obtained from the step (2) by gel filtration chromatography. When human erythropoietin is purified from milk of a transgenic animal, the milk of the transgenic animal is pretreated before performing the step (1), and this pretreatment process comprises the steps of (a) recovering a supernatant from the milk of the transgenic animal by centrifugation, (b) adding a first precipitating agent to the supernatant for first precipitation of impurities, (c) adding a transition metal ion for second precipitation of impurities, and (d) ultrafiltering the supernatant recovered from the step (c).

Description

[DESCRIPTION] [Invention Title]
PURIFICATION METHOD OF HUMAN ERYTHROPOIETIN
[Technical Field] The present invention relates to a method of purifying human erythropoietin, and more particularly, to a method of purifying human erythropoietin comprising the steps of (1) purifying a cell culture or pretreated milk of a transgenic animal by heparin chromatography, (2) purifying a fraction containing human erythropoietin obtained from the step (1) by reversed-phase chromatography, and (3) purifying the fraction containing human erythropoietin obtained from the step (2) by gel filtration chromatography. The present invention is characterized in that, in a case where human erythropoietin is purified from milk of a transgenic animal, a pretreatment process for milk is performed before the step (1) and comprises the steps of (a) recovering a supernatant after centrifuging the milk of the transgenic animal, (b) adding a first precipitating agent to the supernatant for first precipitation of impurities, (c) adding a transition metal ion for second precipitation of impurities and (d) ultrafiltering a supernatant recovered from the step (c). [Background Art]
Natural human erythropoietin is a monomeric glycoprotein composed of 165 amino acids, which has a total molecular weight of about 34,000 daltons (-34 kDa) and a molecular weight of protein of about 18,000 daltons. It is known that erythropoietin has sugar chains occupying about 40% of the total molecular weight which are essential for in vivo activity, and contains N-linked sugar chains at Asns independently located at the 24th, 38th and 83rd amino acids, and one mucin-type O- linked sugar chain at Ser located at the 126th amino acid.
Natural human erythropoietin was first reported in 1906 as a result of research into the probability of the presence of hemopoietic growth factor, and was named in 1957 (Jacobson et al., Nature, 1957, vol. 179, p.633). Subsequent research led to the first successful large-scale isolation and purification of human erythropoietin from urine of a patient suffering from aplastic anemia by Miyake et al. in 1977 (Miyake et al., J.Biol. Chem. 1977, vol. 252, p.5558), an N-terminal sequence of erythropoietin was found by Yanagawa et al. (Yanagawa et al., J. Biol. Chem. 1984, vol. 295, p.2707), and the total amino acid sequence of erythropoietin was determined from a human urine-derived erythropoietin sample by Lai et al. (Lai et al., J. Biol. Chem. 1986, vol. 261, p. 3116).
Now, human erythropoietin is prepared by animal cell culture or a technique using a transgenic animal. The preparation technique using a transgenic animal has advantages in many aspects compared to the technique using an animal cell. The first advantage of the technique using a transgenic animal is that a high-quality recombinant protein may be obtained, which is almost the same as, but safer than, a natural type. Secondly, the protein may be mass-prepared on a scale of several grams per liter. Thirdly, once the animal for preparing the recombinant protein of interest is transformed, the foreign gene included therein is inherited by the offspring, thereby maintaining a constitutive production system.
As described above, various purification methods have been developed for the two different methods of producing human erythropoietin. Generally, human erythropoietin produced by animal cell culture is expressed in a protein-free media, and human erythropoietin produced from the mammary gland of a transgenic animal is expressed in milk protein, whose which requires a more complicated purification method than the method for the animal cell culture.
Various purification methods for human erythropoietin produced by animal cell culture are disclosed in Korean Patent Nos. 10-0153808 and 10-0297927 using immuno-adsorption chromatography, Korean Patent Nos. 10-0162517 and 10- 0177300 using dye-affinity chromatography, and Korean Patent No. 10-0065197 and U.S. Patent No. 4,677,195 using reversed-phase chromatography. Since the human erythropoietin produced by animal cell culture generally has low pi, it is purified by ion exchange chromatography in conventional techniques. An example of methods of purifying human erythropoietin produced by an insect cell is disclosed in Korean Patent No. 10-0321446 using positive ion exchange chromatography, lectin chromatography and gel filtration chromatography.
However, the above-described purification methods are difficult to apply to the purification of human erythropoietin in the milk of a transgenic animal. There are many glycoproteins similar to human erythropoietin in the milk of the animal, and the human erythropoietin produced in the mammary gland of the transgenic animal and the human erythropoietin produced by the animal and insect cell culture exhibit different glycosylation, so that they are isolated with low efficiency using ion exchange chromatography or lectin chromatography. [Disclosure]
[Technical Problem]
The present inventors found that human erythropoietin can be purified from the milk of a transgenic animal with high purity and efficiency in a simple and fast process including: pretreating the milk of the transgenic animal by recovering a supernatant from the milk by centrifugation, adding a first precipitating agent and a transition metal ion to precipitate impurities, ultrafiltering the supernatant recovered from the previous the step, and then sequentially purifying the filtrate obtained from the previous the step using heparin, reversed-phase and gel filtration chromatographies.
[Technical Solution]
The present invention is directed to a method of preparing human erythropoietin, comprising the steps of (1) purifying a cell culture or pretreated milk of a transgenic animal by heparin chromatography, (2) purifying a fraction containing human erythropoietin obtained in the step (1) by reversed-phase chromatography, and (3) purifying a fraction containing human erythropoietin obtained in the step (2) by gel filtration chromatography.
The term 'a cell culture' is used in the purification method according to the present invention and may be a culture of human cells naturally expressing human erythropoietin or a culture of cells, preferably animal cells, transformed to express human erythropoietin. When human erythropoietin is purified from a cell culture according to the present invention, a pretreatment the step may be omitted and it may be purified by microfiltration using a 0.1 to 0.45μm filtering membrane if necessary.
The term 'milk of a transgenic animal' refers to milk obtained from a mammary gland of an animal transformed to express human erythropoietin. The milk of the transgenic animal generally requires pretreatment since it is composed of various elements (impurities) such as proteins, sugars, lipids and the like. The term 'pretreatment' refers to a process of removing impurities before performing the purification method in order to increase purification efficiency of the purification method according to the present invention. When human erythropoietin is purified from milk of a transgenic animal according to the present invention, the pretreatment process comprises the steps of (a) recovering a supernatant from milk of a transgenic animal by centrifugation, (b) adding a first precipitating agent to the supernatant for first precipitation of impurities, (c) adding a transition metal ion for second precipitation of impurities and (d) ultrafiltering a supernatant recovered from the previous the step, before the step (1) in the purification method. Each of the steps will be explained in detail below.
In the step (a), milk obtained from a transgenic animal is centrifuged to recover a supernatant. The milk of the transgenic animal in the present invention is centrifuged at 4,000 to 12,000 rpm and 4 to 37 °C for 10 to 40 minutes. Generally, the milk of the animal is centrifuged into 3 layers, in which an uppermost layer is a lipid, a middle layer is a colloid, and a lowermost layer is an insoluble complex. The lipid and insoluble complex are removed, and the colloid is recovered to be treated in the next the step. Accordingly, the 'supernatant' in the step (a) of the present invention refers to the colloid formed in the middle layer obtained after the removal of the lipid layer.
In the step (b), a first precipitating agent is added to the supernatant obtained according to the step (a) for first precipitation of impurities. Here, the first precipitating agent may be ammonium sulfate, sodium sulfate or potassium phosphate, which may be used at a concentration of 10 to 60%, and preferably 20 to 50%. After adding the first precipitating agent at the above-mentioned concentration, the mixture is reacted for 1 to 12 hours at 4 to 37 °C to precipitate impurities.
In the step (c), following the step (b), a transition metal ion is added for second precipitation of impurities. Here, the transition metal ion may be a metal ion selected from the group consisting of Mn, Fe, Co, Ni, Cu and Zn, which may be used at a concentration of 5 to 50OmM, and preferably 20 to 20OmM. After adding the transition metal ion at the above-mentioned concentration, the mixture is reacted for 1 to 24 hours at 4 to 37 °C to precipitate impurities. In the present invention, the steps (b) and (c) may be simultaneously performed, in which a first precipitating agent is added at a partial predetermined concentration, and the mixture is reacted for 1 to 6 hours at 4 to 37 "C to precipitate impurities. Then, the remaining first precipitating agent and the predetermined concentration of the transition metal ion are added, and the mixture is reacted for 1 to 24 hours at 4 to 37 °C to precipitate impurities.
The present invention is characterized by performing twice a precipitation reaction as described above. The first precipitation reaction increases salt concentration to precipitate a hydrophobic protein. The second precipitation reaction agglomerates minute particles formed in the first precipitation, induces binding to other impurities in order to stimulate the formation of a precipitate, and thus may effectively remove the impurities.
In the step (d), ultrafiltration is performed to remove all kinds of salts used in the steps (b) and (c) and reduce the volume, since the subsequent the step (1) regulates the salt concentration and extracts human erythropoietin. In the present invention, before the ultrafiltration, fine precipitates may be removed by additional microfiltration with respect to the supernatant obtained from the step (c).
The milk of the transgenic animal pretreated as described above becomes suitable for effective performance of the subsequent chromatography due to sufficient removal of the impurities. Also, a common cell culture, dependant on the ingredients of the media, generally has a lower impurity level than the milk, so that the cell culture is suitable for chromatography. Thus, both the cell culture and the pretreated milk of a transgenic animal can be purified by the method according to the present invention. Particularly, the method of purifying the milk from a transgenic animal according to the present invention is different from the purification method using an animal cell culture employing sialic acid or another sugar chain, and may be applied to the purification of erythropoietin having an imperfect sugar chain (having a smaller molecular weight of about 10 kDa).
In the step (1) of the purification method according to the present invention, a cell culture or pretreated milk of a transgenic animal is purified by heparin chromatography. It is known that the heparin chromatography has excellent affinity to glycoprotein in particular. In one aspect of the present invention, a phosphate buffer may be used as an equilibrium solution, and a phosphate buffer containing sodium chloride (NaCl) may be used as an elution solution. Human erythropoietin is eluted at a NaCl concentration of 50 to 60OmM on the chromatogram. According to the present invention, the heparin chromatography purifies the human erythropoietin faster and with higher adsorption efficiency per unit gel than other types of chromatography, and thus may highly concentrate the human erythropoietin.
In the step (2), a fraction containing the human erythropoietin obtained by the heparin chromatography is harvested and then purified by reversed-phase chromatography. Before the step (2), no treatment of the fraction is required, so that the steps (1) and (2) may be continuously performed, and thus the overall purification method is simple. In one embodiment of the present invention, distilled water may be used as an equilibrium solution, and acetonitrile may be used as an elution solution. The human erythropoietin is eluted by about 30 to 70% of an acetonitrile solution in the chromatogram. Here, in order to reduce non-specific adsorption between an impurity protein and silica resin, trifluoroacetic acid may be added to both the equilibrium solution and the elution solution. Also, for the reversed-phase chromatography according to the present invention, a gel having Cl, C4, C6, C8 and Cl 8 as ligands may be employed. Accordingly, the human erythropoietin may be completely separated from the impurity protein by the purification method using the reversed-phase chromatography according to the present invention.
In the step (3), a fraction containing the human erythropoietin obtained by the reversed-phase chromatography is harvested and then purified by gel filtration chromatography. In order to increase the purification efficiency of the gel filtration chromatography, the acetonitrile solution may be removed from the fraction before the step (3). In one embodiment of the present invention, tris buffer may be used as both equilibrium and elution solutions. For the gel filtration chromatography according to the present invention, a size-exclusion gel may be used to separate proteins by size, each having a molecular weight of 5 to 10OkDa. For example, the gel is one selected from the group consisting of Superose 12, Superdex 75, Superdex 200, Sephadex 200, Sephadex 75, Sephadex S-100, Sephacryl S-100 and Sephacryl S-200. Accordingly, even very small amounts of impurities contained in the fraction and inactive human erythropoietin produced by protein coagulation in the above- described purification the step may be removed by purifying the human erythropoietin using the gel filtration chromatography according to the present invention. [Advantageous Effects] According to the purification method of human erythropoietin according to the present invention, several impurities may be sufficiently removed from the milk of a transgenic animal through pretreatment, which may reduce the amount of a sample to be purified by subsequent chromatography and reduce the overall purification time. In addition, human erythropoietin may be efficiently and thoroughly purified in a simple and fast process by sequentially performing heparin, reversed-phase and gel filtration chromatographies, from among the well-known chromatography techniques in the art, of a cell culture or pretreated milk of the transgenic animal. [ Description of Drawings ]
FIG. 1 is a schematic diagram of a method of purifying human erythropoietin from milk of a transgenic animal according to the present invention.
FIG. 2 shows a chromatogram obtained from purification of human erythropoietin using heparin chromatography during the purification method according to the present invention.
FIG. 3 shows a chromatogram obtained from purification of human erythropoietin using reversed-phase chromatography during the purification method according to the present invention.
FIG. 4 shows a chromatogram obtained from purification of human erythropoietin using gel filtration chromatography during the purification method according to the present invention.
FIG. 5 shows electrophoresis results for fractions obtained in the steps of the purification method according to the present invention. [Modes of the Invention] Hereinafter, examples of the present invention will be described in detail. However, the present invention is not limited to the examples disclosed below, but can be implemented in various modified forms. The present examples are provided to fully enable those of ordinary skill in the art to embody and practice the invention.
Example 1 : Centrifugation
40ml of milk obtained from a pig transformed with a human erythropoietin gene was centrifuged at 12,000 rpm and 4 "C for 30 minutes. The milk was separated into 3 layers, of which an uppermost layer of lipid and a lowermost layer of an insoluble complex were removed, and a middle layer of colloid was recovered. Here, the loss of erythropoietin was about 1 to 2%.
Example 2: Precipitation of ammonium sulfate and transition metal 20% ammonium sulfate was added to the recovered result of Example 1, and the mixture was reacted at 4°C for one hour to precipitate impurities. Thereafter, ammonium sulfate was further added up to a total of 35%, and 6OmM zinc chloride was added at 4°C for 2 hours to precipitate impurities. The resulting product was centrifuged at 6,000 rpm and 4°C for 30 minutes, thereby obtaining a supernatant.
Example 3 : Microfϊltration and Ultrafiltration
The supernatant obtained in Example 2 underwent microfiltration using a 1.45μm filter. Then, salts and sugars smaller than 3 kDa were removed from the microfiltrate by ultrafiltration through an ultrafiltration membrane (Millipore, MWCO 3K). Here, the resulting filtrate was diluted with a phosphate buffer to a volume 10 times larger than the initial volume for diafiltration and concentration.
Example 4: Heparin chromatography Heparin chromatography was performed using an AKTAFPLC system (GE,
USA) and HiTrap Heparin HP (GE), in which 5ml heparin resin was filled and used as a column. The column was equilibrated with 1OmM sodium phosphate buffer (an equilibrium solution; pH 7.0) at a flow rate of 3ml/min. The equilibrated solution containing 2M sodium chloride was used as an elution solution to elute protein adsorbed to a heparin gel. Each fraction was monitored by enzyme-linked immunoabsorbent assay (ELISA) to confirm the activity of human erythropoietin.
A lOOμl dilute of the fraction was added to each well in an antibody-adsorbed plate of an ELISA kit and well mixed, and was reacted at room temperature for 2 hours. The remaining reactant in each well was removed and each well was washed with a 400μl washing solution four times. Afterward, a 200μl enzyme-antibody conjugates solution was added and reacted at room temperature for 2 hours. The remaining reactant was removed and washed in the same manner as described above, and a 200μl substrate-dye solution was added to the well and developed at room temperature for 25 minutes. After the development was completed, a lOOμl reaction blocking solution was added to block the reaction and its absorbance was measured at 450nm. A standard curve was plotted with the activity of the standard solution on the x axis and with the absorption on the y axis using a logarithmic coordinate system. From the standard curve, the activities of erythropoietin in the world standard solution and the fraction were measured and multiplied by a dilution multiple, thereby measuring the final activity of the fraction. It was confirmed by the heparin chromatography that human erythropoietin was eluted at a sodium chloride concentration of 50 to 60OmM (refer to FIG. 2). The fraction whose activity was confirmed was recovered and underwent reversed-phase chromatography, which will be described below.
Example 5 : Reversed-phase chromatography
The reversed-phase chromatography was performed using an HPLC system (Virian, USA) and a C8 column (Vydac, USA). Distilled water was used as an equilibrium solution and acetonitrile was used as an elution solution. In order to reduce non-specific adsorption between protein impurities and silica resin, 0.1% trifluoroacetic acid was added to each equilibrium solution and elution solution. ELISA was performed on each fraction according to the same procedure as described in Example 4 to confirm the activity of human erythropoietin.
It was confirmed by reversed-phase chromatography that human erythropoietin was eluted at an acetonitrile concentration of about 30 to 70% (refer to FIG. 3). The fraction from which the activity of human erythropoietin was confirmed was recovered and underwent gel filtration chromatography, which will be described below. Here, acetonitrile was completely removed using a vacuum evaporator.
Example 6: Gel filtration chromatography
Gel filtration chromatography was performed using an AKTAexplorer system (GE, USA). In this case, proteins were separated by size using a Superdex 200 column (GE), and 5OmM tris buffer (pH 7.0) was used. Through this process, human erythropoietin having a purity of at least 99% may be separated (refer to FIG. 4). Example 7: Purity analysis using polyacryl amide gel electrophoresis
In order to confirm whether the protein impurities were removed and target human erythropoietin was purified by the purification method according to the present invention, each product obtained in each the step (sample) was analyzed by polyacryl amide gel electrophoresis. A gel (12%) for electrophoresis was put into an electrophoresis device, and the device was filled with mobile buffer. 5μl of each sample was mixed with 25 μl of sample buffer, and the mixture was boiled at 95 °C for 5 minutes and then centrifuged at 12,000 rpm for 3 to 4 minutes. 30μl of the sample mixture was injected into each well together with a prestained standard (Bio- Rad). Afterward, electrophoresis of the samples was performed at a constant voltage of 150V, and then the gel was stained with Kumasi after development.
The results of the electrophoresis are shown in FIG. 5, in which it can be confirmed that protein impurities were removed and only human erythropoietin remained. In FIG. 5, lane M shows a protein size marker (Bio-Rad, Precision), lane 1 shows undiluted milk, lane 2 shows a supernatant obtained after centrifugation, lane 3 shows a supernatant obtained after simultaneous precipitation of ammonium sulfate and zinc chloride, lane 4 shows a supernatant obtained after microfiltration and ultrafiltration, lane 5 shows a fraction purified by heparin chromatography, lane 6 shows a fraction purified by reversed-phase chromatography, and lane 7 shows a fraction purified by gel filtration chromatography.
While the invention has been shown and described with reference to certain examples thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

[CLAIMS] [Claim 1 ] A method of purifying human erythropoietin, comprising the steps of:
(1) purifying a cell culture or pretreated milk of a transgenic animal by heparin chromatography;
(2) purifying a fraction containing human erythropoietin obtained from the step (1) by reversed-phase chromatography; and
(3) purifying a fraction containing human erythropoietin obtained from the step (2) by gel filtration chromatography. [Claim 2]
The method according to claim 1, wherein the milk is pretreated by the process comprising the steps of:
(a) recovering a supernatant from the milk of the transgenic animal by centrifugation; (b) adding a first precipitating agent to the supernatant for first precipitation of impurities;
(c) adding a transition metal ion for second precipitation of impurities; and
(d) ultrafiltering the supernatant recovered from the above the step. [Claim 3] The method according to claim 2, wherein the first precipitating agent in the step (b) is selected from the group consisting of ammonium sulfate, sodium sulfate and potassium phosphate. [Claim 4] The method according to claim 2, wherein the transition metal ion of the step (c) is one selected from the group consisting of Mn, Fe, Co, Ni, Cu and Zn. [Claim 5]
The method according to claim 1, wherein the reversed-phase chromatography in the step (2) is performed using a gel having Cl, C4, C6, C8 and Cl 8 as ligands. [Claim 6]
The method according to claim 1, wherein the gel filtration chromatography in the step (3) is performed using a size-exclusion gel able to separate proteins having a molecular weight of 5 to 10OkDa.
PCT/KR2007/005898 2007-11-21 2007-11-22 Purification method of human erythropoietin WO2009066806A1 (en)

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KR0177300B1 (en) * 1996-01-24 1999-04-01 성재갑 Method for Purifying Recombinant Erythropoietin
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