KR100900033B1 - Purification Method of Human Erythropoietin - Google Patents

Abstract

The present invention comprises the steps of (1) purifying the milk of the cell culture or pretreated transgenic animals by heparin chromatography; (2) purifying the fraction containing human erythropoietin obtained from step (1) by reverse phase chromatography and (3) purifying the fraction containing human erythropoietin obtained from step (2) by gel filtration chromatography. A method for purifying human erythropoietin. In the present invention, when the human erythropoietin is purified from the milk of a transgenic animal, before the step (1), (a) recovering the supernatant by centrifuging the milk of the transgenic animal; (b) precipitating impurities by adding a primary precipitant to the supernatant; (c) precipitating impurities by adding transition metal ions; and (d) ultrafiltration of the supernatant recovered from the step.

Human erythropoietin, primary precipitant, transition metal ion, heparin chromatography, reverse phase chromatography, gel filtration chromatography

Description

Purification Method of Human Erythropoietin

The present invention relates to a method for purifying human erythropoietin, and more particularly, to (1) purifying milk of a cell culture medium or a pretreated transgenic animal by heparin chromatography; (2) purifying the fraction containing human erythropoietin obtained from step (1) by reverse phase chromatography and (3) purifying the fraction containing human erythropoietin obtained from step (2) by gel filtration chromatography. A method for purifying human erythropoietin. In the present invention, when the human erythropoietin is purified from the milk of a transgenic animal, before the step (1), (a) recovering the supernatant by centrifuging the milk of the transgenic animal; (b) precipitating impurities by adding a primary precipitant to the supernatant; (c) precipitating impurities by adding transition metal ions; and (d) ultrafiltration of the supernatant recovered from the step.

Natural human erythropoietin is a monomeric glycoprotein consisting of 165 amino acids with a total molecular weight of about 34,000 Daltons (~ 34 kDa) and a molecular weight of about 18,000 Daltons of protein. The sugar chain portion, which constitutes about 40% of the total molecular weight, plays an essential role in the in vivo activity of erythropoietin, and has an N-linked sugar chain at Asn at amino acids 24, 38 and 83 and one at Ser at 126th. It is known to have mucin type O-linked sugar chains.

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, 1957, vol. 179, p.633). Subsequent studies have shown that human erythropoietin was the first successful mass separation and purification from the urine of aplastic patients in 1977 (Miyake et al. J. Biol. Chem. 1977, vol. 252, p.5558). The study began to become full-scale and the N-terminal sequence of erythropoietin was reported by Yanagawa et al. (Yanagawa et al. J. Biol. Chem. 1984, vol. 295, p. 2707), and Lai et al. In human urine-derived erythropoietin samples. Erythropoietin total amino acid sequence was determined and reported (Lai et al. J. Biol. Chem. 1986, vol. 261, p. 3116).

Currently, human-derived erythropoietin is produced by animal cell culture and technology using a transgenic animal. Manufacturing with a transgenic animal has several advantages over manufacturing with an animal cell. Firstly, it is possible to obtain safer high quality recombinant protein which is almost the same as natural type. Second, the production of recombinant protein using transgenic animal is possible to mass production at the level of several grams per liter. When transgenic animals are produced that produce the recombinant proteins they need, the foreign genes they carry can be passed on to their offspring to maintain a permanent production system.

As discussed above, the purification method is different depending on two methods of producing human erythropoietin. In general, since erythropoietin produced in animal cell culture is expressed in non-protein-free media, and human erythropoietin produced in the mammary gland of transgenic animals is expressed in milk protein, the purification method is used for animal cell culture. It is more complicated than the method used.

Various purification methods have been disclosed in the case of culturing animal cells to produce human erythropoietin, and there are Korean Patent Registration No. 10-0153808 and Korean Patent Registration No. 10-0297927 using immunosorbent chromatography and dye affinity chromatography. There are Republic of Korea Patent No. 10-162517 and Republic of Korea Patent No. 10-0177300 characterized in that, there is Republic of Korea Patent No. 10-100065197 and US Patent 4,677,195 using reversed phase chromatography. In general, since human erythropoietin produced by culturing animal cells has a low pI, most conventional technologies are purified using ion exchange chromatography.

As a method for purifying human erythropoietin produced by insect cells, there is Korean Patent No. 10-0321446 using cation exchange chromatography, lectin chromatography, and gel filtration chromatography.

However, such purification methods are difficult to apply to the purification of human erythropoietin from the milk of transgenic animals. The reason for this is that there is a lot of glycoproteins similar to human erythropoietin in the milk of animals, and human erythropoietin produced by cultivation of animal and insect cells and human erythropoietin produced in the mammary gland of transgenic animals are different in the glycosylation and thus ion exchange chromatography or If lectin chromatography is used, the separation efficiency is lowered.

In order to purify human erythropoietin easily and quickly with high purity, the present invention recovers the supernatant by centrifuging the milk of a transgenic animal, adding a primary precipitant and a transition metal ion to precipitate impurities, and ultrafiltration of the supernatant recovered from the step. After the pretreatment process was performed, the filtrate recovered from the above step was purified sequentially by heparin chromatography, reverse phase chromatography, and gel filtration chromatography. As a result, human erythropoietin was easily and quickly obtained from the milk of the transgenic animal with high purity and high efficiency. It was confirmed that it could be purified and came to complete this invention.

The present invention comprises the steps of (1) purifying the milk of the cell culture or pretreated transgenic animals by heparin chromatography; (2) purifying the fraction containing human erythropoietin obtained from step (1) by reverse phase chromatography and (3) purifying the fraction containing human erythropoietin obtained from step (2) by gel filtration chromatography. A method for purifying human erythropoietin.

A cell culture, which is a raw material of the purification method according to the present invention, may be a culture of human cells that naturally express human erythropoietin or a culture of cells transformed to express human erythropoietin, preferably animal cells. . In the case of purifying human erythropoietin from the cell culture according to the present invention, the pretreatment may be omitted, but if necessary, the microfiltration may be carried out using a filtration membrane of 0.1 ~ 0.45 ㎛ size.

Another source, milk of transgenic animals, means milk obtained from an animal transformed to express human erythropoietin in the mammary gland. Milk from transgenic animals generally requires pretreatment because it consists of various components (impurities) such as proteins, sugars and fats. 'Pretreatment' means a process of removing impurities before performing the purification method in order to increase the purification efficiency of the purification method according to the present invention. In the case of purifying human erythropoietin from the milk of the transgenic animal according to the present invention, before the step (1), (a) recovering the supernatant by centrifuging the milk of the transgenic animal; (b) precipitating impurities by adding a primary precipitant to the supernatant; (c) precipitating impurities by adding transition metal ions; and (d) ultrafiltration of the supernatant recovered from the step. Hereinafter, each step will be described in detail.

In step (a), the supernatant is recovered by centrifuging the milk obtained from the transgenic animal. The milk of the transgenic animal in the present invention is centrifuged at 4,000 to 12,000 rpm, at 4 to 37 ° C. for 10 to 40 minutes. In general, centrifugation of animal milk separates it into three phases, forming fat in the top layer, colloidal phase in the middle layer, and insoluble complex in the bottom layer, respectively. The fat and insoluble complexes thus formed are removed and the colloidal phase is recovered and processed in the next step. Therefore, in the step (a) of the present invention, "supernatant" means a colloidal phase formed in the intermediate layer after removing the fat layer.

In step (b), the primary precipitate is added to the supernatant obtained according to step (a) to precipitate the impurities first. Here, ammonium sulfate, sodium sulfate, or potassium phosphate may be used as the primary precipitant, and their concentration is 10 to 60%, preferably 20 to 50%. After adding the primary precipitation agent of this concentration, the reaction is carried out at 4 to 37 ° C for 1 to 12 hours to precipitate impurities.

Following step (b), in step (c) the transition metal ions are added to precipitate the impurities second. Here, the transition metal ion means a metal ion selected from the group consisting of Mn, Fe, Co, Ni, Cu and Zn, and their concentration is 5 to 500 mM, preferably 20 to 200 mM. After adding the transition metal ion of the said concentration, it reacts at 4-37 degreeC for 1 to 24 hours, and an impurity precipitates.

Meanwhile, in the present invention, steps (b) and (c) may be simultaneously performed. In this embodiment, the primary precipitant may be added only a part of the designated concentration and reacted at 4 to 37 ° C. for 1 to 6 hours to precipitate impurities. After adding a primary precipitant and adding transition metal ions of a predetermined concentration, impurities are precipitated by reacting at 4 to 37 ° C. for 1 to 24 hours.

The present invention is characterized by proceeding a total of two precipitation reactions as described above. In the first precipitation, hydrophobic proteins can be precipitated by increasing the salt concentration, and in the second precipitation, impurities can be effectively removed because they aggregate the fine particles formed by the first precipitation and induce binding with other impurities to promote the formation of precipitates. Can be.

In step (d), subsequent step (1) is to elute human erythropoietin by adjusting the salt concentration, and ultrafiltration is performed to remove various salts used in steps (b) and (c) and to reduce volume. In the present invention, it is preferable to further carry out the process of removing the fine precipitate by microfiltration of the supernatant obtained from step (3) before the ultrafiltration.

The milk of the transgenic animal pretreated in this manner is in a state where impurities are sufficiently removed to be suitable for efficiently performing the chromatography described below. In addition, the cell culture usually varies depending on the composition of the medium, but generally contains less impurities than milk, and thus is suitable for chromatography. Therefore, both the cell culture and the milk of the pretreated transgenic animal can be purified by the purification method according to the present invention. In particular, the method of purifying from the milk of the transformed animal according to the present invention is different from the method of purifying from animal cell culture utilizing sialic acid or other sugar chain moieties, and incomplete sugar chains (about 10 kDa in molecular weight are small). It can also be applied to the purification of erythropoietin.

In step (1) of the purification method according to the present invention, the milk of the cell culture or the pretreated transgenic animals is purified using heparin chromatography. Heparin chromatography is known to be particularly affinity for glycoproteins. In one embodiment of the present invention, a phosphate buffer solution containing a phosphate buffer solution as an equilibrium solution and sodium chloride as an elution solution may be used, and human erythropoietin is eluted at a concentration of 50 to 600 mM sodium chloride on a chromatogram. According to the present invention, by purifying human erythropoietin using heparin chromatography, the purification rate is faster and the adsorption capacity per unit gel is higher than that of other chromatography.

In step (2), fractions containing human erythropoietin obtained from the heparin chromatography are collected and purified by reverse phase chromatography. Step (1) and step (2) can proceed continuously because the fractions need not be treated before proceeding to step (2), simplifying the overall purification method. In one embodiment of the present invention, the equilibrium solution may be distilled water and the elution solution may be acetonitrile. Human erythropoietin is eluted in acetonitrile solution of about 30-70% on the chromatogram. Here, it is preferable to add trifluoroacetic acid to the equilibrium solution and the elution solution in order to reduce nonspecific adsorption of impure protein and silica resin. In addition, in reversed phase chromatography according to the present invention, it is preferable to use a gel having ligands of C1, C4, C6, C8, and C18. According to the present invention, the purification of human erythropoietin using reversed phase chromatography can accurately separate human erythropoietin and impurity proteins except for this.

In step (3), fractions containing human erythropoietin obtained from the reverse phase chromatography are collected and purified by gel filtration chromatography. In order to increase the purification efficiency by gel filtration chromatography, it is preferable to remove the acetonitrile solution from the fraction before proceeding to step (3). In one embodiment of the present invention, Tris buffer solution can be used as the equilibrium and elution solution. In the gel filtration chromatography according to the present invention, it is preferable to use a size exclusion gel that can be used to fractionate a protein corresponding to a molecular weight of 5 to 100 kDa. For example, a gel selected from the group consisting of Superrose 12, Superdex 75, Superdex 200, Sephadex 200, Sephadex 75, Sephacryl S-100, Sephacryl S-200 is used. Purification of human erythropoietin using gel filtration chromatography in accordance with the present invention allows removal of trace impurities contained in fractions and inactive human erythropoietin generated due to protein entanglement during the aforementioned purification steps.

In the method for purifying human erythropoietin according to the present invention, various impurities can be sufficiently removed from the milk of a transgenic animal by a pretreatment process, thereby reducing the amount of sample to be purified by subsequent chromatography and shortening the purification time. have. In addition, heparin, reversed phase and gel filtration chromatography are selected and sequentially processed from various chromatographic methods known in the art for the milk of cell cultures or pretreated transgenic animals, thereby easily and quickly high purity of human erythropoietin. And high efficiency purification.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Centrifugation

40 ml of milk obtained from pigs transformed with human erythropoietin was centrifuged at 12,000 rpm and 4 ° C for 30 minutes. The layers are separated into three phases, the fat of the top layer and the insoluble complex of the bottom layer are removed and the colloidal phase of the middle layer is recovered. At this time, the amount of erythropoietin lost was about 1 to 2%.

Example 2: Ammonium Sulfate and Transition Metal Precipitation

20% ammonium sulfate was added to the product recovered through Example 1, and reacted at a temperature of 4 ° C. for 1 hour to precipitate impurities. Subsequently, ammonium sulfate was added to 35%, followed by zinc chloride at a concentration of 60 mM to precipitate impurities at 4 ° C. for 2 hours. The product thus obtained was centrifuged at 6,000 rpm for 30 minutes at 4 ° C to obtain a supernatant.

Example 3: Microfiltration and Ultrafiltration

The supernatant obtained in Example 3 was subjected to microfiltration using a 0.45 μm filter. Subsequently, the microfiltrate was ultrafiltration (Millipore, MWCO 3K) to remove salts and sugars smaller than 3 kDa by passing through an ultrafiltration membrane. Here, difiltration and concentration were performed while diluting with a phosphate buffer solution in an amount of 10 times the initial volume.

Example 4: Heparin Chromatography

Heparin chromatography was performed using an AKTAFPLC system (GE, USA), and a HiTrap Heparin HP from GE with 5 ml of heparin resin was used as a column. The column was equilibrated with 10 mM sodium phosphate buffer (equilibration) at pH 7.0 at a flow rate of 3 ml / min. The protein adsorbed on the heparin gel was eluted using an equilibrium solution containing 2 M sodium chloride as the elution solution. For each fraction, enzyme immunoassay confirmed human erythropoietin activity.

100 µl of the dilution of the fraction for analysis was added to each well of the antibody adsorption plate of the enzyme immunoassay kit, and the mixture was mixed well and allowed to react at room temperature for 2 hours. The remaining reaction solution of each well was removed, washed four times with 400 μl of washing solution, and 200 μl of enzyme antibody conjugate solution was added thereto, followed by further reaction at room temperature for 2 hours. After the reaction filtrate was removed and washed in the above manner, 200 µl of the substrate-coloring solution included in the kit was added to the wells and developed for 25 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. Using the logarithmic coordinate system, the standard curve was prepared using the horizontal axis and the absorbance as the vertical axis, and the erythropoietin activity in the international standard solution and fractions was determined from the prepared standard curve, and the final activity of the fractions was determined by multiplying the dilution factor.

Heparin chromatography confirmed that the human erythropoietin eluted at a concentration of 50 ~ 600 mM sodium chloride (Fig. 2). The fraction confirmed the activity was recovered and subjected to reverse phase chromatography described below.

Example 5: Reversed Phase Chromatography

Reversed phase chromatography was performed using an HPLC system (Varian, USA), and C8 column of Vydac (USA) was used as a column. Distilled water was used as the equilibrium solution, and acetonitrile was used as the elution solution. In order to reduce nonspecific adsorption of impure protein and silica resin, 0.1% trifluoroactic acid was added to the equilibrium solution and the elution solution. Each fraction was subjected to enzyme immunoassay according to the same procedure as in Example 4 to confirm human erythropoietin activity.

Reversed phase chromatography showed that human erythropoietin eluted with acetonitrile concentration between 30% and 70% (Fig. 3). The identified fraction was recovered and subjected to gel filtration chromatography described below. For this purpose, acetonitrile was completely removed using a vacuum evaporator.

Example 6: Gel Filtration Chromatography

Gel filtration chromatography was performed using AKTAexplorer system (GE, USA). At this time, proteins were fractionated by size using a Superdex 200 column (GE), and 50 mM Tris buffer solution at pH 7.0 was used. Through this process, human erythropoietin of high purity exhibiting a purity of 99% or more could be separated (FIG. 4).

Example 7: Purity Analysis Using Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis was performed on each step product (sample) to confirm whether the impurity protein is removed by the purification method according to the present invention and whether the desired human erythropoietin is purified. An electrophoretic gel (12%) was installed in the electrophoresis device and filled with transfer buffer. 5 μl of each sample was mixed with 25 μl of sample buffer, heated at 95 ° C. for 5 minutes, centrifuged at 12,000 rpm for 3 to 4 minutes, and 30 μl of each well was injected with a molecular weight standard (BIO-RAD). . After injection, electrophoresis was performed at a constant voltage of 150 V. When the development was completed, the gel was taken out and subjected to Coomassie staining.

The electrophoresis results are shown in Figure 5, it was confirmed that the impurities protein is removed and only human erythropoietin is purified. In FIG. 5, lane M is a protein size marker (Bio-Rad, Precision), lane 1 is a milky liquor, lane 2 is a supernatant after centrifugation, lane 3 is a supernatant after simultaneous precipitation of ammonium sulfate and zinc chloride, and lane 4 is microfiltration and The supernatant after ultrafiltration, lane 5 represents a heparin chromatography purified fraction, lane 6 represents a reverse phase chromatography purified fraction, and lane 7 represents a gel filtration chromatography purified fraction.

Figure 1 shows briefly a method for purifying human erythropoietin from the milk of a transgenic animal according to the present invention.

2 is a chromatogram obtained while purifying human erythropoietin using heparin chromatography in the purification method according to the present invention.

3 is a chromatogram obtained while purifying human erythropoietin using reverse phase chromatography in the purification method according to the present invention.

4 is a chromatogram obtained while purifying human erythropoietin using gel filtration chromatography in the purification method according to the present invention.

Figure 5 shows the results of electrophoresis of the fractions obtained by step of the purification method according to the present invention.

Claims (6)

(1) purifying the milk of the cell culture or pretreated transgenic animal by heparin chromatography; (2) purifying the fraction containing human erythropoietin obtained from step (1) by reverse phase chromatography and (3) purifying the fraction containing human erythropoietin obtained from step (2) by gel filtration chromatography. Method for Purifying Human Erythropoietin. The method of claim 1, The pretreatment comprises the steps of: (a) recovering the supernatant by centrifuging the milk of the transgenic animal; (b) precipitating impurities by adding a primary precipitant to the supernatant; (c) secondary precipitation of impurities by the addition of transition metal ions; and (d) ultrafiltration of the supernatant recovered from said step. The method of claim 2, The process of purifying human erythropoietin, characterized in that in step (b) the primary precipitant is selected from the group consisting of ammonium sulfate, sodium sulfate and potassium phosphate. The method of claim 2, The transition metal ion of step (c) is a method for purifying human erythropoietin, characterized in that the metal ion selected from the group consisting of Mn, Fe, Co, Ni, Cu and Zn. The method of claim 1, Reverse phase chromatography of step (2) is a method for purifying human erythropoietin, characterized in that the gel using a ligand as C1, C4, C6, C8, C18. The method of claim 1, Gel filtration chromatography of step (3) is a method for purifying human erythropoietin, characterized in that using a size exclusion gel that can be used to fractionate a protein corresponding to a molecular weight of 5 ~ 100 kDa.

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