KR100321446B1 - Purification method of recombinant human erythropoietin(epo) expressed from recombinant insect cells - Google Patents

Abstract

PURPOSE: A purification method of recombinant human erythropoietin(EPO) expressed from recombinant insect cells is provided, thereby rapidly and effectively purifying the human EPO, maintaining its activity. CONSTITUTION: A purification method of recombinant human erythropoietin(EPO) expressed from recombinant insect cells comprises the steps of: (1) culturing insect cells transformed with a microorganism containing an EPO gene; (2) subjecting the supernatant of the cultured medium to filtration, dialysis and centrifugation; (3) subjecting the concentrated medium to cation exchange chromatography to recover an EPO containing fraction; (4) subjecting the EPO containing fraction to lectin-affinity chromatography to isolate an EPO containing glycoprotein; and (5) subjecting the EPO containing glycoprotein to gel filtration chromatography, wherein sf-21 cell is infected with a human EPO containing baculovirus; the cation exchange chromatography uses a pH 6.0 to 6.5 buffer solution; the cation exchange chromatography uses CM-sepharose, S-sepharose, CM-cellulose, S-cellulose or mono-s resin; the lectin-affinity column uses ConA sepharose; and the gel filtration chromatography uses superose 12, superdex 75, sephadex 75 or sephacryl S-100.

Description

Purification Method of Human Erythrocyte Growth Factor Expressed in Recombinant Insect Cells

The present invention relates to a method for purifying human erythrocyte growth factor (Erythropoietin; hereinafter referred to as "EPO") expressed by genetic recombination technology, and more specifically, multi-step chromatography of EPO expressed in insect cells using baculovirus. The present invention relates to a method for rapid and efficient purification while preserving physiological activity through a chromatography process.

EPO is a hormone that promotes the production and differentiation of red blood cells. It is a glycoprotein composed of 166 amino acids and several sugars. Erythrocytes are cells in the blood that supply oxygen to all tissues in the human body, and their amount is controlled according to the level of oxygen supply, and EPO acts as a bridge. That is, when the oxygen supply drops, specific cells in the kidneys detect this and increase the concentration of EPO. Increased EPO acts on systemic cells present in the bone marrow to promote the production and differentiation of red blood cells. As a result, the amount of red blood cells in the blood increases, thereby increasing the supply of oxygen in the human body. As such, EPO is an indispensable factor for the formation of red blood cells in the blood and is currently used for diagnosis and treatment of diseases caused by blood such as anemia.

EPO can not be purified from blood because there are very small amounts of 0.1 ng to 0.01 ng per ml of blood in normal human blood.In 1977, for the first time, EPO was purified from urine of patients with pernicious anemia. Miyakeet. al., J. Biol. Chem. 252, 558-64 (1977)). Human EPO genes were identified based on some amino acid sequences found from purified EPO (Jacobs K. et. Al., Nature, 313 , 806-810 (1985)), and these were determined using E. coli or Yeast (WO 8502610-A), insect cells (Korean Patent Application No. 93-16865) and mammalian cells (Lin FK et.al., Proc. Natl. Acad. Sci. USA, 82 , 7580-7584 (1985) ) To the expression.

EPO is currently obtained by purification from yona blood or transformed cells, the method of obtaining from yona blood first being a relatively complex purification consisting of seven steps as described in US Pat. Nos. 4397840, 4303650 and 3865801. It is reported that about 20% is obtained through the process, and the purified EPO also has a relatively low titer of about 70,000 units per mg, and the amount of EPO that can be obtained by the above method is reported to be extremely limited. The method for obtaining EPO from the culture supernatant of transformed cells is characterized by reversed phase liquid chromatography using an aqueous ethanol solution as described in Korean Patent Publication No. 93-3665.

EPO expressed in insect cells is about 26,000 Daltons in size, 4,000-6,000 Daltons less than EPO in blood or urine or EPO expressed in mammalian cells. This is a result of the glycoprotein EPO, the protein is composed of the same 166 amino acids, but the type and content of sugars are different. However, although the molecular weight of EPO expressed in insect cells is small, it shows the same antigenic antibody response as EPO in blood, and its physiological activity is reported to be the same or higher in vitro (Erslev et.al., Erytrhropoietion , Johns Hopkins University Press (1991).

It is an object of the present invention to provide a method for rapidly and efficiently purifying EPO expressed in insect cells while preserving physiological activity.

Accordingly, the present invention provides a method for purifying recombinant EPO expressed in an insect cell transformed with a microorganism containing human red blood cell growth factor (EPO), and culturing the insect cell transformed with the microorganism containing the EPO gene. The supernatant of the culture was concentrated, the concentrated culture was subjected to cation exchange resin chromatography to collect fractions containing EPO, and the collected EFO-containing fractions were collected by lectin affinity chromatography to separate the glycoproteins containing EPO and separated. Provided is a method of gel filtration chromatography of an EPO containing glycoprotein. In addition, the present invention provides a method for more quickly purifying by using an antigen-antibody response to EPO instead of measuring the physiological activity of EPO as a method for analyzing EPO during purification.

The present invention will be described in more detail as follows.

First, insect cells are infected with a baculovirus containing the EPO gene with a leader sequence. EPO expressed in infected insect cells is released into the culture by the leader sequence. After incubation for 5 days, the culture solution containing EPO is collected, centrifuged to remove insoluble precipitate in the culture solution, concentrated only by collecting the supernatant, and dialyzed in sodium phosphate buffer solution. The dialyzed culture is chromatographed with a cation exchange resin at pH 6.0 to pH 6.5, preferably pH 6.4, to effectively remove serum proteins in most cultures that are not adsorbed on the column. The adsorbed protein is eluted with increasing NaCl concentration and analyzed for the presence of EPO in each fraction by antigen-antibody reaction. Only the fraction containing EPO is collected. In this case, the cation exchange resin may be CM-sepharose, S-sepharose, CM-cellulose, s-cellulose or mono-s. Since the cation exchange resin can easily remove a large amount of serum proteins in the culture, it is preferable to carry out first.

The collected fractions are concentrated and adsorbed to glycoproteins by chromatography on a lectin affinity column and washed thoroughly with a buffer solution containing 0.5 to 2 M NaCl (preferably 1M) to protein and other substances other than glycoproteins. Remove them. Glycoproteins containing adsorbed EPO are eluted with methyl-D-mannoside, each fraction is analyzed by antigen antibody reaction, and only the fractions containing EPO are collected. In this case, Con-A Sepharose or Lintyl Lectin Sepharose may be used as the lectin column. By adsorbing only glycoproteins and removing other proteins and other substances with a lectin affinity column, an EPO solution with a purity of 80% or more can be obtained.

The EPO solution obtained above is concentrated again and chromatographed on a gel filtration column. In this case, as a gel, Super Rose 12, Superdex 75, Sephadex 75, Sephacryl S-100 or Sephacryl S-200 may be used. Proteins other than EPO, which are present at less than 20%, have a large difference in molecular weight from EPO, and can be removed using a gel-filtration column and converted into a desired buffer solution.

The physiological activity of EPO purified purely by the purification method described above can be measured by the increase rate of cell division by EPO (Krystal and Gerald, Esp. Hematol. 11 , 649-660 (1983)).

Hereinafter, the present invention will be described in more detail based on Examples. The following examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example 1: Purification of EPO

(Step 1)

Recombinant virus (ATCC VR2421; deposited on July 23, 1993) containing the EPO gene was infected with sf-21 cells (Invitrogen, SanDiego, USA, Cat. No. B821-50) as follows. First, sf-21 cells were added to 10% fetal calf serum (Sigma, Cat No. F-2138), 0.33% isolate (Yeastolate; Gibco-BRL, Cat No, 670-8190AG) and 0.33% lactoalbumin The culture was supplemented with a digested product (Gibco-BRL, Cat No. 670-8080AG) (hereinafter referred to as 'complete culture'). The cultured cells were collected, 3 × 10 ⁷ cells were added together with 60 ml of complete culture solution in a 175 cm 3 sized culture flask, and left at 27 ° C. for 2 hours to allow the cells to adhere to the bottom of the culture vessel. The supernatant was removed from the culture flask, and each culture flask was treated with 60 ml of a complete culture solution of Baculovirus 1.5 × 10 ⁸ pfu (plaque forming unit) containing the EPO gene prepared by the method described in Korean Patent Application No. 93-16865. Put it in. During incubation of the infected cells at 27 ° C., EPO was expressed in insect cells and secreted out of the cells. After 5 days, the culture solution was collected, centrifuged at 10,000 x g to remove suspended solids, and the supernatant was used for the next purification process.

(Step 2)

One liter of the culture supernatant collected in step 1 was placed in a concentrator (manufactured by Amicon) attached with a YM10 filtration membrane and concentrated 20 times or more under nitrogen gas pressure. The concentrated culture was dialyzed with Buffer A (10 mM sodium phosphate, pH 6.4 with 0.01% octyl D glucopyranoside). The precipitate formed during dialysis was removed by centrifugation at 10,000 x g for 20 minutes and then all of the supernatant (50 mL) was added to a CM-Sepharose column (1 cm x 10 cm) equilibrated with the same buffer A. Proteins and other substances not adsorbed on the column were washed out with 100 ml of buffer A, and the adsorbed material was eluted by linear gradient of 75 ml of the same buffer A and 75 ml of 0.4 M NaCl. At this time, the dissolution rate was 1 ml per minute, and the volume of each fraction was 5 ml. After confirming the presence of EPO in each fraction by antigen-antibody reaction, only fractions from which EPO was detected were collected from the 32nd to 40th fraction. The result of detecting the protein eluted from CM-Sepharose at the wavelength of 280 nm and the presence of EPO in each fraction by antigen antibody reaction are shown in FIG.

The process of detecting the fraction containing EPO by the antigen antibody reaction will be described in more detail as follows. 10 μl of each fraction was diluted in 190 μl of 50 mM sodium borate buffer solution at pH 9.0 and then adsorbed onto nitrocellulose membrane using a blotting device (Bio-Dot SF, Bio-Rad, Richmond, USA). After drying the protein-adsorbed membrane at room temperature, phosphate physiological saline containing 0.2% gelatin was shaken for 30 minutes to saturate the remaining protein adsorption site on the membrane. 1000-fold monoclonal antierythropoietin antibody (Genzyme, CAT.No.AE7A5, USA) in phosphate physiological saline containing 0.25% gelatin, 1% Triton X-100, ImM EDTA and 0.02% thimeral After diluting, the reaction was carried out at room temperature for 1 hour. The membrane was then washed four times with 5 minutes each of phosphate physiological saline containing 0.05% Tween 20. The washed membranes were horseradish peroxidase conjugated anti-mouse immunoglobin G antibodies diluted 1000-fold with physiological saline containing 10% serum, 1% picol, 0.05% Tween 20 and 0.02% thimerosal. -HRP, American Qualex, Cat.No.A206PS, USA) was added and reacted for 1 hour, followed by 4 washes with the wash solution, followed by 2 more washes with 50 mM Tris buffer. The membrane was developed by adding 50 mM Tris buffer containing 160 μg of 4-chloro-1 naphthol and 0.03% hydrogen peroxide.

(Step 3)

CaCl _2, MnCl ₂ and into a final concentration of MgCl ₂ to be respectively ImM, and then concentrated and YM10 membrane to a mounting which concentrator (Amicon Corp.) lectin on EPO solution obtained in Step 2 in the affinity column ConA column per minute Administration was at a rate of 0.5 ml. The non-adsorbed material was washed with 40 ml of buffer B (10 mM sodium phosphate containing 0.02% octyl D glucopyranoside, IM NaCl ImM CaCl ₂ , ImM MnCl ₂ and ImM MgCl ₂ , pH 6.4) followed by 300 mM methyl-D The adsorbed protein was eluted with Buffer B, which contained mannose, at a dissolution rate of 0.25 mL per minute and 4 mL of each fraction. 10 μl was taken from each fraction and analyzed by antigen antibody reaction as in Step 2, and only the 12th to 18th fractions from which EPO was detected were collected.

Figure 3 shows the results of detecting the protein from the lectin affinity at a wavelength of 280 nm and the presence or absence of EPO in each fraction by antigen antibody reaction. Arrow (↓) in FIG. 3 shows the time point of starting elution with buffer B containing 300 mM methyl-D-mannoside. As shown in FIG. 3, all of the EPO was adsorbed on the ConA column and eluted efficiently by methyl-D-mannoside.

(Step 4)

The EPO solution obtained in step 3 was concentrated to 0.5 ml and then administered to a Superrose 12 column equilibrated with physiological saline. Fractions were received in increments of 1 ml with physiological saline at a rate of 0.25 ml per minute. 10 μl was taken from each fraction and analyzed by antigen antibody reaction as in step 2, and only the 13th and 14th fractions in which EPO were detected were collected.

The result of detecting the protein from gel-filtration at the wavelength of 280 nm and the presence or absence of EPO in each fraction by antigen antibody reaction are shown in FIG.

Example 2: Confirmation of Physiological Activity and Purity of EPO

In Example 1, after measuring the protein concentration of the collected EPO solution after each chromatography in the purification process, 1 μg, 0.1 μg, 0.01 μg and 0.001 μg were added to the nitrogen in the same manner as the antigen-antibody reaction described in step 2 above. The increase in purity according to the purification process was analyzed by adsorbing onto the cellulose membrane and comparing each other with the minimum amount that the EPO antibody can detect as shown in FIG. In the same manner, the protein in the cell culture supernatant was not detected up to 10 μg, and the EPO solution after CM-sepharose chromatography was also detected up to 0.01 μg (FIG. 5, CM). It can be seen that more than 1000 times purified. As shown in FIG. 4, it was purified by ConA affinity about 10 times again (FIG. 5, ConA), and it was confirmed that the purity was also slightly increased by the gel-filtration column (FIG. 5, S-12). Meanwhile, 100, 10, 1, and 0,1 units of EPO purchased from the US R & D System were adsorbed onto the membrane in the same manner for comparative analysis (FIG. 5, EPO). Since 0.01 μg of purified EPO responded more than twice as much as 1 unit, it was confirmed by the present invention that the physiological activity of the purified EPO was at least 200,000 units per mg.

The purity of EPO according to the purification process was analyzed by electrophoresis and western blotting, and the results are shown in Figures A and B, respectively. The culture supernatants were difficult to fractionate by electrophoresis because of too much serum protein, but most of them were detected by CM-sepharose, the first purification process, since trace amounts of serum proteins remained after CM-sepharose (Figure 6, A, 1). It was found that serum proteins were effectively removed. EPO having a purity of 80% or more was obtained by ConA affinity chromatography (FIG. 6, A, 2). EPO having a purity of 98% or more was removed by gel filtration chromatography because proteins other than the remaining EPO had a high molecular weight. Was obtained (Fig. 6, A, 3). As shown in FIG. 6B, no protein other than EPO reacted with the EPO antibody during purification.

Example 3: Determination of Physiological Activity of Purified EPO

The physiological activity of purified EPO was measured by the increase rate of cell division when splenocytes of mice injected with phenylhadazine were treated with EPO (Krystal and Gerald, Esp. Hematol. 11 , 649-660 (1983)). That is, as cell division is increased by EPO and the number of cells increases, the amount of ³ H-thymidine contained in total cells increases in proportion to this. This will be described in more detail as follows. First generation hybrid F1 (C57BL / 6 × C3H) obtained by crossing mice C57BL / 6 and C3H was purchased from Simonsen's Lab, CA, USA. Phenylhydrazine was injected into the abdominal cavity of the mice at a rate of 60 mg / kg body weight. Phenylhydrazine was dissolved in physiological saline and sterilized with a filter. After 24 hours the same amount of phenylhadrazine was injected and three days later the mice were dissected to remove the spleen. Cells were released out of the tissue by injecting α-MEM medium (Gibco-BRL, Cat No, 410-1900EB, USA) with 20% fetal calf serum and 0.01 mM beta-mercaptoethanol. The cells that came out of the tissues were collected in a 50 ml tube and allowed to stand for 5 minutes to remove the clumped cells. The supernatant was discarded by centrifugation at 250 × g for 5 minutes, and the precipitated cells were resuspended in α-MEM medium containing 20% fetal calf serum and 0.01 mM beta-mercaptoethanol. After measuring the number of cells with a hematocytometer, each well of the 24-well culture plate and the same number of cells (2 × 10 ⁵ ) and different concentrations of EPO (1000, 500, 250, 125, 62.5, 31.3 15.6, 7,8 and 3.9 pg) were added together with 0.4 ml of the culture solution and incubated in a 37 ° C. incubator under 7% CO ₂ supply. After 20 hours of incubation, 2 uCi of ³ H-thymidine (Amersham, Cat. No. TRK686, 68 Ci / mmol) was added to each well, and further incubated for 4 hours. The cultured cells were attached to a glass fiber filter using a cell harvester (12Well Cell Harvester, Millipore, Cat No 2702550), and then the culture medium containing ³ H-thymidine was removed and once with 1 ml of distilled water, and 1 ml of After washing once with methanol, the mixture was left at 60 DEG C for 30 minutes to dry the filter. The dried filter was placed in a scintillation vial together with 2 ml of scintillation cocktail (Packard, Cat No. 6013324) and left at room temperature for 30 minutes, and then radioactivity contained in cells was measured by a scintillation counter (Packard). On the other hand, after purchasing the EPO (4,000 units per ml) commercially available from the United States R & D System (R & D System) and experimented in the same way for several units of EPO, the physiological activity of the purified EPO in the present invention is as follows. Calculated together. First, the standard curves as shown in A and B of FIG. 7 were obtained based on the radioactive weight of ³ H-thymidine contained in the cells. Since the concentrations of 72.5 pg and 33.4 milliunits, which are half the maximum values in the curve, each had the same physiological activity, the physiological activity of the purified EPO in the present invention was calculated to be 450,000 units per mg.

As is clear from the above description, EPO having high physiological activity can be efficiently purified from the culture medium of insect cells transformed with a gene containing EPO by the method of the present invention.

Figure 1 schematically shows the purification of human erythrocyte growth factor from recombinant insect cells,

2 shows the result of detecting the chromatogram of human erythrocyte growth factor expressed in recombinant insect cells by cation exchange resin column and the fraction containing human erythrocyte growth factor by antigen antibody reaction.

3 is a result of detection of a chromatogram separated by a lectin-affinity column and a fraction containing human erythrocyte growth factor by antigen antibody reaction.

4 is a result of detecting the chromatogram separated by the gel filtration column and the fraction containing human erythrocyte growth factor by antigen antibody reaction,

5 is a result of comparing and analyzing the amount of human hematopoietic factor in the solution obtained from each process in the purification process according to the present invention by antigen antibody reaction,

6 is a result of analyzing the purity of human erythrocyte growth factor purified according to the method of the present invention by electrophoresis (A) and Western blotting (B),

7 is a result of measuring the physiological activity of human erythrocyte growth factor purified according to the method of the present invention.

Claims (7)

In the method for purifying recombinant EPO expressed in insect cells transformed with microorganisms containing human red blood cell growth factor (EPO), 1) culturing the insect cells transformed with the microorganism containing the EPO gene, 2) concentrating the supernatant of the culture obtained in step 1) by filtration, dialysis and centrifugation; 3) collecting the fraction containing EPO by performing cation exchange resin chromatography on the culture medium concentrated in step 2); 4) separating the EPO-containing glycoprotein by lectin affinity chromatography on the EPO-containing fraction collected in step 3), 5) gel filtration chromatography of the EPO-containing glycoprotein separated in step 4) Method comprising a. The method of claim 1, Infecting sf-21 cells with baculovirus containing the human red blood cell growth factor (EPO) Way. The method of claim 1, cation exchange chromatography with a buffer ranging from pH 6.0 to pH 6.5 Way. The method of claim 1, Characterized in that the cation exchange chromatography uses CM-sepharose, S-pepharose, CM-cellulose, S-cellulose or mono-S resin. Way. The method of claim 1, The lectin-affinity column is characterized by using a ConA Sepharose or Lintyl Lectin Sepharose resin Way. The method of claim 1, The gel filtration column uses a resin such as Superrose 12, Superdex 75, Sephadex 75 or Sephacryl S-100. Way. The method according to any one of claims 1 to 6, After each chromatography, the fraction containing EPO is quickly detected by using an antibody antibody reaction. Way.