KR100297927B1 - Method of Purifying Human Erythropoietin - Google Patents

Abstract

The present invention relates to a method for purifying human erythropoietin from a fluid, comprising sequentially performing first anion exchange chromatography, immunosorbent chromatography and second anion exchange chromatography on a fluid containing human erythropoietin. Not only can human EPO be purified quickly, simply and economically, but also with high purity and high titer to satisfy the efficacy, safety and stability as a therapeutic protein drug.

Description

Method for Purifying Human Erythropoietin

The present invention relates to a method for purifying human erythropoietin (hereinafter abbreviated as "EPO"), and more particularly, fluid containing human EPO by performing anion exchange chromatography before and after immunosorbent chromatography. The present invention relates to a method for purifying human EPO that can not only rapidly and economically purify human EPO, but also purify to high purity and high titer to satisfy efficacy, safety and stability as a therapeutic protein drug. .

EPO is a glycoprotein with a molecular weight of 34,000 to 39,000, first identified among colony stimulating factors (CSFs). In vivo, EPO is mainly produced in the kidneys and is known to be produced in small amounts in the liver, and promotes the growth and differentiation of erythroid progenitor cells such as colony forming unit erythroid (CFU-E) and burst forming unit erythroid (BFU-E). Because it plays a role in the production of mature red blood cells, it can be widely used in the treatment of anemia due to renal dysfunction, myeloid anemia, rheumatoid anemia, anemia related to cancer or anticancer drugs, and AIDS anemia.

On the other hand, as a host for producing a recombinant protein as a therapeutic protein drug, E. coli , which generally produces a target protein in the form of an inclusion body, is used. However, proteins such as EPO, which have many disulfide bonds or are essential for glycosylation to be active, cannot be produced using a microbial host, so mammalian cells, such as Chinese Hamster Ovary (CHO) Cells are used as hosts. In this case, the recombinant protein expressed in the mammalian host cell is secreted into the culture medium. The culture medium contains a large amount of serum proteins, and among them, protein components having properties similar to the desired recombinant protein are difficult to isolate. In addition, there are also proteins derived from host cells, including various protease, sugar degrading enzymes, and inhibitors, which require rapid separation of the target protein as much as possible. It also contains DNA, endotoxin, etc., and it is extremely difficult to completely eliminate all impurities in the purification process after the recovery of the culture solution, and at the same time to fill up the speed.

Currently, separation and purification of recombinant proteins after the recovery of the culture is mostly carried out by chromatography, for example, ion exchange chromatography, hydrophobic binding chromatography, reverse phase chromatography, hydroxyapatite chromatography, gel filtration chromatography, Chemical adsorption chromatography;

As a method for purifying an industrial production scale EPO, US Pat. No. 4,667,016 (Applicant; Por-Hsiung Lai et al., Filing date; June 20, 1985, Patent holder; Kirin-Amgen), Examples of the patent According to the method described in 2, the culture supernatant was concentrated, dialyzed and filtered, followed by ion exchange chromatography-reverse phase chromatography-ion exchange chromatography-gel filtration to obtain a purity of about 98% and a yield of about 30%. Was obtained. However, the method has a problem in that the entire purification process has a lot of steps and complicated to increase the time and cost required for purification, and there is a risk of unnecessary loss during each step.

In addition, US Pat. No. 4,465,624 (applicant; Chiba et al.) Described using immunosorbent chromatography as a method for purifying EPO, in which case the immunosorbent column can be reused about 10 times. However, the preparation of an immunosorbent column is typically performed on the resin from the production, isolation and purification of monoclonal antibodies from hybridomas prepared by cell fusion of spleen cells and myeloma cells of immunized animals. Since the process requires a lot of effort, time, and cost until the adsorption of, the method has a problem that it is practically impossible to apply industrially.

Also, in Ghanem et al., "Purification and biological activity of a recombinant human erythropoietin produced by lymphoblastoid cells", Preparative Biochemistry, 24 (2), 1994, 127-142, immunosorbent chromatography and ion exchange chromatography A method for purifying EPO according to a two step method has been disclosed, but satisfactory results in terms of efficiency and reusable number of immunosorbent columns have not been achieved.

An object of the present invention is to solve the above-mentioned problems of the prior art, it is possible to purify human EPO from a fluid containing human EPO quickly, simply and economically, as well as to purify to high purity and high titer, so that the therapeutic protein It is to provide a method for purifying human EPO that can satisfy the efficacy, safety and stability as a pharmaceutical.

BRIEF DESCRIPTION OF THE DRAWINGS The figure which shows the result of comparing the pi (# 2) of the EPO fraction obtained by this invention with the thing (# 1) of the standard product through isoelectric point fractionation method;

2 is a reverse phase HPLC chromatogram of EPO fractions obtained according to the present invention; And

3 is a comparison of the in vivo activity of EPO fractions 1 (-◆-) and 2 (-■-) obtained according to the present invention by biological efficacy test using multi-blood cell mice with that of the standard product (-○-) Graph showing one result.

The present invention provides a method for purifying human EPO from a fluid, characterized by sequentially performing a first anion exchange chromatography, an immunosorbent chromatography and a second anion exchange chromatography on a fluid containing human EPO. .

According to the method of the present invention, in the first anion exchange chromatography, a fluid containing human EPO is bound to the anion exchange resin at pH 8.0, eluting binding proteins other than human EPO at pH 4.0 to 4.5, and pH 7.0 to Eluting human EPO at 8.0, and in immunosorbent chromatography, the obtained human EPO fraction is adsorbed to an immunosorbent column to which human EPO-specific antibodies are attached at pH 8.0, eluting nonspecific adsorbed protein at pH 7.0, eluting human EPO at pH 2.0-2.5, in a second anion exchange chromatography, the obtained human EPO fraction is bound to an anion exchange resin at pH 8.0, eluting binding proteins other than human EPO at pH 4.0-4.5, Human EPO elutes at pH 7.0-8.0.

The anion exchange chromatography is carried out using a DEAE resin, in particular the first anion exchange chromatography is more preferably a batch process. In addition, it is preferable to add a denaturant selected from the group consisting of guanidine hydrochloric acid and urea at the time of eluting proteins other than human EPO, and more preferably the concentration of the denaturant is 2 to 6 M.

Immunosorbent chromatography is performed using human EPO-specific monoclonal antibodies, preferably further eluting at pH 8.8 before elution of human EPO, with 500-1,000 mM sodium chloride as eluent more preferred. Do. It is also preferred to exchange the eluent with the formulation buffer in the second anion exchange chromatography.

Hereinafter, the present invention will be described in more detail.

EPO has been shown to be effective as an agent for treating anemia in chronic renal failure. To date, the importance of EPO is increasing. Accordingly, the present inventors have completed the present invention as a result of continuous research to develop a method for purifying human EPO to be quick, simple, economical and high purity and high titer.

According to the present invention, immunosorption chromatography is introduced as a step in the purification method of human EPO, thereby allowing a high level of purification in a single step, reducing purification time and cost, and reducing the number of steps in the entire purification method. Reduction to minimize unnecessary losses, and the protein of interest is concentrated during purification to provide convenience for carrying out subsequent steps.

As mentioned above, the most important consideration when introducing immunosorbent chromatography at industrial scale is whether the column is reusable. Therefore, in the present invention, by performing ion exchange chromatography as an initial purification step before the immunosorbent chromatography step, as well as the purification of the concentrate, EPO is an acidic protein having a low pKa value. After the protein was removed, the number of reusable columns of the column was increased by applying it to an immunosorbent column. According to the present invention, the unit yield was about 80% even after about 500 times of use.

In addition, human EPO is a sialic acid protein in which about 40% of its molecular weight is combined with sialic acid, which is a carbohydrate. The presence or absence of sialic acid does not affect EPO activity in in vitro, but sialic acid is active in in vivo. As essential for sialic acid dropping, galactose residues are exposed because they are recognized by galactose receptors in hepatocytes and are captured and metabolized in hepatocytes. Thus, when performing isoelectric focusing, EPO with full activity is located below the pI value of 4, which is due to the low pKa value of sialic acid (pKa = 2.6) (Lukowsky et al. , Can. J. Biochem., 50, 1972, 909-917]. The present invention includes a process of selecting a high titer EPO having a high sialic acid content on an anion exchange resin by using a pKa value of the EPO depending on the sialic acid content.

Another advantage of the present invention is the elimination of the exchange step into the formulation buffer, which is required for the purification of the majority of pharmaceutical proteins. In general, the exchange is accomplished by gel filtration. For this purpose, an operation such as condensation, which is inevitably performed, not only causes unnecessary loss but also causes the entire process to fail due to operational defects. In the present invention, the composition of the liquid used for eluting the EPO fragment is finally composed of the components required for the final product (excluding stabilizers), thereby eliminating the operation step, time, risk of loss, and the like.

In the present invention, the fluid containing human EPO can be of either natural or recombinant origin, blood or urine of natural origin, and genetically transformed mammals of recombinant origin. May be a culture of cells.

For example, recombinant human EPO contained in mammalian cell culture can be purified by the following method. The dialysis, filtered and concentrated cultures are batchwise bound to an anion exchange resin, such as DEAE, previously equilibrated with a buffer of pH 8.0 consisting of 10 mM Tris by a suitable method. The use of batch solutions with high protein concentrations and viscosities, such as culture concentrates, has the advantage of rapid and mass handling. As the anion exchange resin, any of DEAE Sepharose (Pharmacia), DEAE Toyo Pearl (Tosho), and DEAE Sephadex A50 (Pharmacia) can be used. Batch method can be carried out simply by adding the culture concentrate, stirring, standing, settling, and filtration, if there is a protein binding to the ion exchange resin more strongly than the target protein, more complicatedly the first ion When added to the exchange resin, the purification effect can be enhanced by continuous batch eluting before the target protein binds and adding it to the second anion exchange resin. The batch method prevents the column from filling up when it is performed columnar, but in order to remove proteins other than human EPO, the pH must be lowered to pH 4.0 to 4.5, which is near the pKa value of human EPO. At this time, in order to prevent protein entanglement and loss by isoelectric precipitation, a denaturant such as guanidine hydrochloric acid and urea is added. It is preferable that the denaturant concentration be 2 to 6 M because at this concentration, the desired steps can be smoothly performed by completely dissolving other proteins precipitated near the isoelectric point without damaging the activity of EPO. . After washing and eluting human EPO is eluted with 150-250 mM sodium chloride at pH 7.0-8.0. The human EPO containing fractions obtained as above are applied to an immunosorbent column to which human EPO-specific antibodies are attached at pH 8.0. The human EPO-specific antibody is preferably a monoclonal antibody that ensures a high degree of uniformity and affinity of the antibody. The nonspecific adsorbate is then eluted off by salt and high pH. That is, the nonspecific adsorbent protein is eluted off at pH 7.0, which is then preferably further eluted at pH 8.8. This is because proteins that are nonspecifically adsorbed by hydrophobic bonds are rarely removed by salts at acidic to neutral pH, and can be removed by increasing the electrostatic repulsion by inducing the adsorbed protein and the matrix to the same charge at high pH. At this time, it is appropriate to use 500 ~ 1,000mM sodium chloride as the eluent, because the process can be effectively carried out by using a high salt basic buffer solution, non-specific binding while preventing the elution loss of EPO at the salt concentration This is because the protein can be removed. Then, EPO specifically adsorbed to the monoclonal antibody is eluted at pH 2.0-2.5. Simultaneously with EPO elution, the pH is adjusted to 7.0-9.0, and the chromatography is re-run by addition to an anion exchange column acting on the same principle as the first step, at which time the eluate is exchanged into the formulation buffer.

EXAMPLE

Hereinafter, the configuration and effects of the present invention will be described in more detail based on the preferred embodiments, but this is only to aid the understanding of the present invention and is not intended to limit the scope of the present invention.

EXAMPLE

Extracellularly secreted recombinant human EPO secreted from CHO cells grown in a roller bottle containing serum medium was properly concentrated, dialyzed and filtered, followed by a series of chromatographic procedures.

1. Batch DEAE Anion Exchange Chromatography

The culture concentrate was added to DEAE Sephadex A50 resin (manufactured by Pharmacia) pre-equilibrated with 10 mM Tris at pH 8.0, then stirred for several minutes and allowed to settle for 30 minutes to settle and filter. After binding, the resin was first washed with 3 volumes of 10 mM acetate chloride / 2M guanidine hydrochloric acid at pH 4.0 and secondly with 6 volumes of 20 mM sodium phosphate / 15 mM sodium chloride at pH 6.5. The EPO-containing fractions were eluted with 20 mM sodium phosphate / 250 mM sodium chloride at pH 8.0 after removing the EPO having a low sialic acid protein and sialic acid content.

2. Immunosorbent Chromatography

Tween 20 (0.02%) was added to the obtained product from 1, and the cells cloned after fusion of mouse myeloma-derived SP2 / 0 cells and B lymphocytes isolated from the mouse spleen immunized with EPO were cloned into a plastic microcapillary tube. Columns filled with triazine activated agarose resin (ACL) with EPO-specific monoclonal antibodies attached as a result of incubation on the surface of the microcapillary bundles and recovered from the recovered cultures And then adsorbed with 100 mM Tris / 0.02% Tween 20 at pH 8.0. Thereafter, non-specifically adsorbed proteins were removed by sequentially washing with 10 mM sodium phosphate / 1 M sodium chloride / 0.02% Tween 20 at pH 7.0 and 100 mM Tris / 0.5 M sodium chloride at pH 8.8. Thereafter, EPO was eluted with 100 mM citric acid at pH 2.0 while simultaneously raising the pH to 8.0 by dropwise addition of 1M Tris.

3. Columnar DEAE Anion Exchange Chromatography

The obtained product from 2 was exchanged in the same manner as the composition of the equilibration buffer by repeatedly concentrating and diluting using an ultrafiltration apparatus having a 10,000 MW blocker. Then, the obtained product was added to a DEAE Toyopearl (Tosho Co.) column equilibrated with 10 mM Tris / 20 mM sodium chloride at pH 8.0, and the column was washed by the same method as in the above 1, followed by sialic acid bound to the column. The high EPO containing fractions were eluted with 20 mM sodium phosphate / 100 mM sodium chloride at pH 7.2.

4. Result Analysis

Through the isoelectric point fractionation method, the isoelectric point range of the EPO fraction obtained from the above was compared with an International Reference Standard, and the results are shown in FIG. 1. 1, it can be seen that the pI of the EPO fraction of the present invention is distributed in 3.5 to 4.2, so that the EPO fraction of the present invention contains a high titer EPO having a higher sialic acid content than the standard product.

The EPO fraction obtained from above was also analyzed by reverse phase HPLC (20-70% acetonitrile (0.1% TFA), absorbance; 220 nm), and the results are shown in FIG. 2. As shown in Figure 2, the EPO fraction according to the present invention exhibits at least about 99% purity.

In addition, since impure proteins induce an immune response and exhibit detrimental activity when applied to humans, the method described in Table 1 shows whether the EPO fraction according to the present invention meets the requirements for parenteral administration of pharmaceutical products. As a result, it was confirmed that the EPO fraction according to the present invention satisfies the requirements for parenteral administration of pharmaceutical products.

Analysis method and result of EPO fraction Analysis method Measure result ELISA analysis Host Cell Protein ≤100ppm LAL Analysis * Pyrogen ≤1.0EU / 10,000Unit Dot blot hybridization analysis ** DNA contaminants ≤10pg DNA / 10,000Unit

Limlus Amebocyte Lysate assay (USP 22 <85 'Bacterial Endotoxin')

** Dot Blot Hybridization assay (Leonard G. Davis et al., Methods in molecular biology, 1986, 84-87 and 147-149, Elsevier Science Publishing Co., New York)

Next, in order to investigate the biological activity of the EPO composition according to the present invention, the biological efficacy test using polycythemic mice (see polycythemic bioassay; P. Mary Cotes et al., Nature, 191, 1961, 1065-1067) Was carried out. That is, when ⁵⁹ Fe is injected into an animal, it is inserted only into newly formed red blood cells. Therefore, allow enough time for complete metabolism of labeled ⁵⁹ Fe in the plasma after injection, and measure the amount of erythrocyte production by measuring the amount of label in peripheral blood. By doing so, the biological activity of the EPO can be confirmed. The results of comparing the in vivo activity of the final EPO fractions 1 and 2 obtained by performing the purification method according to the present invention through the above method with the standard product are shown in FIG. 3, and as shown in FIG. 3. The EPO fraction showed biological activity comparable to the standard.

[Comparative Example]

Immunosorbent chromatography and ion exchange, as described by Ghanem et al., "Purification and biological activity of a recombinant human erythropoietin produced by lymphoblastoid cells", Preparative Biochemistry, 24 (2), 1994, 127-142. EPO was purified according to the method consisting of two steps of chromatography. Table 2 compares the results of the examples and the comparative examples.

Analysis of EPO Fractions Obtained from Examples and Comparative Examples Example Comparative example Potency (IU / A ₂₈₀ ) 180,000-190,000 144,000-176,000 water(%) ≥99 ≥98 Reusable frequency of immunosorbent column (times) ≥500 ≥20 PI range by isoelectric point fractionation 3.5 to 4.2 4.0 to 5.0 yield(%) 30 50

From Table 2, it can be seen that the Example shows better results in the pH range according to the titer, purity, reusability of the immunosorbent column, isoelectric point fractionation method than the comparative example. However, although the method of Comparative Example 1 is shown to have a higher yield than Example 1 due to the fewer processes, it is a result of radioimmunoassay, and isoforms of low biological efficacy such as EPO. If it is preferable to exclude the case does not have a great meaning. In other words, if the results according to the in vivo titer and isoelectric point fractionation method is not the same can not be compared significantly. In particular, the reusable number of the immunosorbent column is an important measure of industrial applicability. When the purification method according to the embodiment is used, the number of reuse of the immunosorbent column (500 times) is compared with that of the comparative example (20 times). It can be improved by about 25 times. In addition, the human EPO fraction according to the present invention has already met the requirements as a parenteral protein drug.

According to the purification method of the present invention, not only can the human EPO be purified quickly and simply and economically from the fluid containing human EPO, but also can be purified to high purity and high titer, thereby improving the efficacy, safety and stability as a therapeutic protein medicine. Can be satisfied.

Claims (9)

First anion exchange chromatography for binding a fluid containing human erythropoietin to an anion exchange resin at pH 8.0, eluting binding proteins other than human erythropoietin at pH 4.0 to 4.5, and eluting human erythropoietin at pH 7.0 to 8.0; The human erythropoietin fraction obtained through the first anion exchange chromatography is adsorbed on an immunosorbent column to which human erythropoietin-specific antibody is attached at pH 8,0, eluting the nonspecific adsorption protein at pH 7.0, pH 2.0∼ Immunosorbent chromatography eluting human erythropoietin at 2.5; And binding the human erythropoietin fraction obtained through the immunosorbent chromatography to an anion exchange resin at pH 8,0, eluting binding proteins other than human erythropoietin at pH 4.0 to 4.5, and eluting human erythropoietin at pH 7.0 to 8.0. A second anion exchange chromatography is carried out sequentially to purify human erythropoietin from the fluid. The method of claim 1 wherein the first and second anion exchange chromatography is performed using a DEAE resin. The method of claim 1 wherein the first anion exchange chromatography is batchwise. 2. The method of claim 1, wherein in the first and second anion exchange chromatography, a denaturing agent selected from the group consisting of guanidine hydrochloric acid and urea is added upon elution of a binding protein other than human erythropoietin. The method according to claim 4, wherein the denaturant has a concentration of 2 to 6 M. The method of claim 1, wherein said immunosorbent chromatography is performed using human erythropoietin-specific monoclonal antibodies. The method of claim 1, wherein in the immunosorbent chromatography, further eluting at pH 8.8 before eluting human erythropoietin. 8. The method according to claim 7, further comprising eluting with 500 to 1,000 mM sodium chloride as eluent. The method according to claim 1, wherein in the second anion exchange chromatography, the eluent is exchanged with the preparation buffer in the elution of human erythropoietin.