RU2145610C1 - Method of purification of human recombinant erythropoietin - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method of purification of human erythropoietin involves immobilization of antibodies raised to erythropoietin on insoluble matrix, sorption of erythropoietin on immobilized antibodies and the following elution of erythropoietin. Monoclonal antibodies produced by cultured hybridoma clone taken among the group including clones PCE/6. PCE/10 and PCE/7 are used as antibodies. Erythropoietin is eluted with acid buffer solution, pH is less 4, and anion-exchange chromatography is used additionally for purification. Invention can be used for human erythropoietin preparing. EFFECT: enhanced effectiveness of process of human erythropoietin isolation. 5 cl, 6 dwg, 1 tbl, 7 ex

Description

 The invention relates to biotechnology, and in particular to a technology for producing human erythropoietin, and in particular to a method for its purification.

 Erythropoietin is a growth factor for red blood cell precursors in the bone marrow. In the human body, erythropoietin is synthesized mainly by the cells of the kidneys and liver; normally, the level of secretion of endogenous erythropoietin does not exceed several tens of international milliunits (mME). With a number of pathologies, the synthesis of endogenous erythropoietin decreases, which leads to the development of severe anemic conditions. The introduction of exogenous erythropoietin allows you to effectively restore erythropoiesis, normalizing the condition of patients.

 Widespread [see, for example, PCT applications 85/03079, 86/03520, EPO patents N 0148605, 1990, N 0255231, 1992] received technology for the production of exogenous erythropoietin using recombinant animal cell lines that can secrete human erythropoietin when cultured in vitro on synthetic culture media. Moreover, the level of erythropoietin production usually amounts to units or tens of micrograms per milliliter of nutrient medium, and the content of foreign protein in it is many times higher than the content of erythropoietin. In view of this, deep purification is required to obtain an erythropoietin substance suitable for the preparation of a medical preparation.

 A known method of fractionation of recombinant human erythropoietin at isoelectric points by preparative isoelectric focusing [Fuhr, G. et all., BBRS. 1981, v. 98, p. 930]. The specified method is highly efficient, but the separation process has a high cost, as a result of which it has not found wide application in biotechnology. In addition, the isolated erythropoietin requires additional purification from the solutions used in fractionation.

 Also known is a method for fractionating recombinant human erythropoietin by the amount of sialic acids [EPO application N 0428267, 1989, MKI C 07 K 07/10], according to which the forms are separated using ion exchange chromatography on a Q-Sepharose FF support in an acetic acid system (150-300 mM) - glycine (1 mM) - urea (6 M), followed by elution with a solution of sodium chloride. However, this method is only one (not the last) stage of a multi-stage cleaning scheme.

 Also known is a method for purification of recombinant human erythropoietin [EPO patent N 0209539, 1985, MKI C 07 K 07/10], comprising the steps of (1) precipitating a portion of the protein with ethanol, (2) fractionating on diethylaminoethyl agarose anion exchange resin, (3) separating on strong SP-Sephadex cation exchange resin, (4) gel filtration on Sephadex G-100, (5) chromatography on hydroxyapatite, (6) concentration by lyophilization and (7) reverse phase chromatography on Vydac С-4 in the water - trifluoroacetic acid - acetonitrile system. The specified method allows to obtain erythropoietin of a high degree of purity, however, the multi-stage and, consequently, relatively low yield of the target product make it ineffective for large-scale preparative processes. In addition, the use of trifluoroacetic acid for the last step, even capable of hydrolyzing the sugar portion of glycoproteins even in the concentration used, is highly undesirable.

The closest set of essential features to the claimed method is a method for the isolation of erythropoietin from biological fluids [EP 0116446, 1990, IPC ⁵ C 12 N 5/00], according to which monoclonal antibodies produced by a mouse hybridoma strain are immobilized on a solid substrate and a solution containing contact erythropoietin with immobilized antibodies and then erythropoietin bound to the immunosorbent, a solution of a 20-membered synthetic oligopeptide, is eluted. According to the prototype, the hybridoma strain was obtained by immunizing mice with the same 20-membered oligopeptide synthesized by the authors. EP 0116446 does not indicate the degree of purity of the erythropoietin isolated by this method. A significant drawback of the known method for the isolation of erythropoietin is the need to use a very expensive eluent - a solution of a synthetic 20-membered oligopeptide, caused by the low specificity of the monoclonal antibodies used in the process produced by the hybridoma obtained by the authors.

 The technical problem solved in the claimed method of purification is to simplify, reduce the cost and increase the efficiency of the process of allocation of human erythropoietin.

 The problem is solved in that in a method for purifying human erythropoietin, which includes immobilizing antibodies to erythropoietin on an insoluble matrix, sorption of erythropoietin on immobilized antibodies and subsequent elution of erythropoietin, monoclonal antibodies produced by a clone of a cultured PC group including selected clones are selected as antibodies / F6, PCE / D10 and PCE / D7, moreover, the elution of erythropoietin is carried out with an acidic buffer solution with a pH of less than 4.

 These clones have not been previously described, obtained by us for the first time, their properties and the production method are described in example 1.

 Antibodies to recombinant human erythropoietin are immobilized on a biocompatible insoluble matrix, for example, Sepharose FF from Pharmacia Biotech activated by bromine cyan. The resulting immunosorbent is placed in a column, balancing it with phosphate buffer. A pre-clarified microfiltration culture fluid containing recombinant erythropoietin is passed through an immunosorbent column. Erythropoietin, adsorbed on an immunosorbent, is eluted with an acidic buffer solution, for example, 0.1 M citric acid solution, 0.1 M glycine buffer pH 3.5, etc. The eluent and washing solutions may contain 0.01 - 0.05 wt.% Non-ionic surfactant (detergent). As a detergent, you can use Tween-20 detergent brand Merck, Nonidet P-40 company Pharmacia and others.

 The purity of the drug, achieved in one stage of immunoaffinity chromatography in the present method, is more than 95%. If a higher degree of purification of erythropoietin is required, the eluate obtained at this stage can be further sorbed on strong anion exchange resin with stepwise elution with increasing ionic strength and / or lowering the pH of the eluent or diafiltration (dialysis) against Tris buffer and sorption on anion exchange resin also with stepwise elution of erythropoietin with eluents of increasing ionic strength and / or lower pH. With additional cleaning, the purity of the drug exceeds 98%.

 The method is further illustrated by examples.

 Example 1. Obtaining and characterization of clones of cultured hybridoma of mice.

To obtain a collection of hybridomas producing monoclonal antibodies to human erythropoietin, almost homogeneous, not containing noticeable impurities of oligomers (see Figs. 1 and 2), erythropoietin was immobilized on a nitrocellulose membrane, type HAHY (Millipore, USA), the membrane was homogenized to dimethyl the suspension was administered to Balb / c mice in the pads of the hind legs. After 30 days, the mice were boosted intraperitoneally with an erythropoietin solution in an amount of 10 μg per mouse. After 4 days, the mice were sacrificed and lymphocytes from the spleen and lymph nodes were used for subsequent hybridization. The fusion of lymphocytes from immune mice was carried out according to the standard procedure using polyethylene glycol. Myeloma partner Px63.Ag8.6.5.3 was used as a myeloma partner during fusion. After fusion, cells were transferred to 96-cell microplates at a concentration of 10 ⁵ -10 ⁶ cells per well in a selective nutrient medium containing HAT. After 10 days, the microplates were microscopically monitored for the presence of growing cell colonies in the cells. Samples of culture fluid were taken from cells with growing colonies in order to identify colonies producing antibodies that react with human erythropoietin.

Colonies producing antibodies against human erythropoietin were screened by enzyme-linked immunosorbent assay for erythropoietin immobilized on the surface of microplate cells for enzyme-linked immunosorbent assay (Costar, Holland, high-bond), according to the standard procedure (Engvall, E. and Perlmann, P., J. Immunology, 109, 129, 1972). Cells from positive cells with growing colonies were subjected to cloning by transfer to new microplates at a concentration of 1 to 5 cells per cell. After 10-12 days, the presence of growing clones in the microplates was microscopically monitored and culture fluid samples were taken from cells with growing colonies in order to identify clones producing antibodies to human erythropoietin. Positive clones were used for further cloning and propagated for culture deposition. Of the 112 initially positive colonies, as a result of the cloning and screening procedure, 3 clones were selected, named PCE / D7, PCE / D10 and PCE / F6, respectively. These clones were characterized by a high growth rate, high stability in the production of antibodies that specifically react with human erythropoietin, and high levels of antibody production reaching 50 μg / ml when cultured under stationary conditions. Antibodies produced by the resulting hybridomas were further characterized by immunological and physicochemical procedures. For this purpose, monoclonal antibodies were isolated from culture liquids obtained by cumulative cultivation of PCE / D7, PCE / D10 and PCE / F6 hybridomas on serum-free culture media by gel permeation and ion exchange chromatography, which were characterized by the following parameters:
- an isotype of the immunoglobulin heavy chains was established using the Calbiochem enzyme immunoassay.

- using enzyme immunoassay tested the ability of monoclonal antibodies to bind human erythropoietin from solution;
- using the enzyme immunoassay and immunofluorescence analysis, the ability of monoclonal antibodies to bind to human serum components, bovine serum components, to the proteins of cells of the recipient strain of CHO used to obtain the cell line producing erythropoietin, and the ability of monoclonal antibodies to bind to spleen cell proteins was tested;
- cross-specificity of monoclonal antibodies was verified using enzyme-linked immunosorbent assay;
- analyzed the isoelectric focus spectrum of monoclonal antibodies.

 The results obtained are presented in table 1 and in FIG. 3.

 As follows from the table, all three established hybridomas produce monoclonal antibodies that are able to recognize human erythropoietin in solution, have a heavy chain IgG1 isotype, do not compete with each other for binding to the erythropoietin molecule, and, therefore, independent antigenic determinants are recognized in its structure. do not have non-specific immunoreactivity with human serum proteins, bovine serum proteins and the recipient CHO cell line used to produce the erythro producer Poetina, and also do not react with the proteins of the cells of the human spleen. As follows from FIG. 3, monoclonal antibodies produced by established hybridomas are characterized by isoelectric focusing by the presence of isoforms ranging in the pI range from 5.3 to 6.0. Thus, monoclonal antibodies produced by established hybridomas have the ability to specifically bind human erythropoietin by independent antigenic determinants both in the immobilized state of the solid phase and from solution, do not have nonspecific immunoreactivity with proteins of human and bovine serum, CHO and human cell proteins and can be used to purify human erythropoietin. The established hybridomas were deposited in the Specialized collection of transplantable somatic cells of vertebrates of the All-Russian collection of cell cultures under the numbers VSCK / P / 634D (strain PCE / D7), VSCK / P / 635D (strain PCE / D10) and VSCC / P / 636D (strain PCE / F6 )

Example 2. Purification of erythropoietin using an immunosorbent based on PCE / D7 monoclonal antibodies
The immunosorbent was prepared based on monoclonal antibodies produced by the PC / ED7 clone. For this, 2500 mg of monoclonal antibodies isolated from the ascites fluids of F1 Balb / C / DBA mice were immobilized in 250 ml of CNBr-activated Sepharose FF according to the matrix manufacturer's technique and packed in a K50 / 30 column (sorbent column height 12.7 cm). The sorbent was balanced with phosphate buffer (PBS) containing 0.02% Tween-20.

28 liters of culture fluid containing 300 mg of rEPO were frontally applied to the column. The sorbent was washed sequentially with 4 volumes of PBS containing 0.02% Tween-20, 3 volumes of phosphate buffer containing 1 M NaCl and 0.02% Tween-20, and 2 volumes of PBS containing 0.02% Tween-20. Elution was carried out with 0.1 M citric acid solution. The eluate (160 ml, 285 mg, 95% yield) was immediately neutralized and analyzed. According to electrophoresis and ELISA, the eluted preparation is erythropoietin with a characteristic IEF spectrum and protein purity of more than 95% (Fig. 4). The biological activity of the drug, determined according to example 1, was 1 • 10 ⁵ ME / mg protein.

Example 3. Purification of erythropoietin using an immunosorbent based on PCE / F6 monoclonal antibodies
Prepared as in Example 2, an immunosorbent based on PC / EF6 monoclonal antibodies isolated from the culture fluid in an amount of 50 ml was placed in a K25 / 30 column and equilibrated with PBS containing 0.02% Tween-80.

The 5.2 L clarified microfiltered culture liquid containing 50 mg of rEPO was concentrated 10 times using a hollow fiber module with a permeability of 10 KD and applied frontally to the column. The column was washed and eluted as in Example 2, with the difference that 0.02% Tween-80 was used as a detergent. The eluate (60 ml, 47 mg, 94% yield) was immediately neutralized and analyzed. According to electrophoresis and ELISA, the eluted preparation is erythropoietin with a characteristic IEF spectrum and protein purity of more than 95% (Fig. 4). The biological activity of the drug, defined as in example 1, was 1 • 10 ⁵ ME / mg protein.

Example 4. Purification of erythropoietin using an immunosorbent based on monoclonal antibodies PCE / D10
Prepared as in Example 2, an immunosorbent based on PC / ED10 monoclonal antibodies isolated from the culture fluid in an amount of 60 ml was placed in a K25 / 30 column and equilibrated with PBS containing 0.05% Nonidet P-40.

The microfiltered clarified culture fluid in an amount of 5.2 L, containing 50 mg of rEPO, was concentrated 10 times using a flat module with a permeability of 10 KD and applied frontally to the column. The column was washed in the same manner as in Example 3, with the difference that Nonidet P-40 (0.05 wt.%) Was used as a detergent. The eluate (75 ml, 46 mg, yield 92%) was immediately neutralized and analyzed. According to electrophoresis and ELISA, the eluted preparation is erythropoietin with a characteristic IEF spectrum and protein purity of more than 95% (Fig. 4). The biological activity of the drug, determined according to example 1, was 1 • 10 ⁵ ME / mg protein.

Example 5. Purification of erythropoietin with the additional use of gradient ion-exchange chromatography
In order to further improve the quality and purity of the preparation, 45 mg of erythropoietin purified according to Example 2 was dialyzed against three shifts of 5 liters each of 0.01 M Tris buffer pH 7.2 containing 0.02% Tween-20 and applied to a K16 / 30 column containing 45 ml Q-Sepharose FF equilibrated with the same buffer. The column was washed with the same buffer and the drug was eluted with a linear NaCl gradient in the same buffer from 0 to 0.3 M. The rEPO peak was observed in the range of NaCl concentrations from 0.2 to 0.25 M. According to the analysis (HPLC, IEF, SDS-PAGE), the degree of purity of the preparation exceeded 98%, the preparation contained 8 bands in zones pI 3.5-5.5. The yield of the drug was 34 mg (75%) (Fig. 5). The biological activity of the drug, determined according to example 1, was 1.1 • 10 ⁵ ME / mg protein.

Example 6. Purification of erythropoietin with the additional use of gradient ion-exchange chromatography
Erythropoietin, purified as in Example 2, in an amount of 43 mg was dialyzed against three shifts of 5 liters each 0.01 M Tris buffer pH 6.8 containing 0.02% Tween-80 and applied to a K16 / 30 column containing 45 ml of Q-Sepharose FF balanced by the same buffer. The column was washed with the same buffer and the drug was eluted with a linear NaCl gradient in the same buffer from 0 to 0.3 M. The rEPO peak was observed in the range of NaCl concentrations from 0.15 to 0.20 M. According to the analysis (HPLC, lEF, SDS-PAGE), the purity of the preparation exceeded 98%, the preparation contained 8 bands in zones pI 3.5-5.5. The yield of the drug was 31 mg (72%) (Fig. 5). The biological activity of the drug, defined as in example 1, was 0.95 • 10 ⁵ ME / mg protein.

Example 7. Purification of erythropoietin with the additional use of ion exchange chromatography and diafiltration
To obtain a preparation of erythropoietin enriched in more acidic forms and depleted in alkaline, and thereby increase its quality, as well as to increase the yield, the preparation obtained in Example 2 was subjected to additional purification. For this, rEPO in an amount of 280 mg was placed in a hollow fiber module with a permeability of 10 kD and diafiltered against 12 volumes of 0.02 M Tris buffer pH 7.2 containing 0.02% Tween-20. The resulting preparation was frontally applied to a K24 / 30 column filled with 120 ml of Q-Sepharose FF, equilibrated with 0.02 M Tris buffer pH 7.2, containing 0.02% Tween-20. The slip was discarded. At the end of sorption, the column was washed with the same buffer until the absorption level of the eluate stabilized at a wavelength of 280 nm (800 ml). The alkaline forms of rEPO were eluted with 0.01 M acetate buffer pH 4.0. A pronounced peak was observed. At the end of elution with alkaline forms, the column was again equilibrated with 0.02 M Tris buffer pH 7.2 containing 0.02% Tween-20. The acid forms of rEPO were eluted with 0.02 M Tris buffer, pH 7.2, containing 0.25 M NaCl and 0.02% Tween-20. An acute protein peak was observed. According to the analysis (HPLC, IEF, SDS-PAGE), the degree of purity of the preparation exceeded 98%, the preparation contained 4 major bands in zones pI 3.5-4.5. The yield of the drug was 224 mg (80%) at a concentration of 5 mg / ml (Fig. 5, 6). The biological activity of the drug, determined according to example 1, was 1.38 • 10 ⁵ ME / mg protein.

 The inventive method allows to obtain a preparation of human erythropoietin, characterized by a high degree of purity, in almost one stage of immunoaffinity chromatography. The inclusion of anion exchange chromatography stage in the process allows to obtain erythropoietin in an almost homogeneous state.

Claims (5)

 1. The method of purification of recombinant human erythropoietin by immunoaffinity chromatography, comprising immobilizing antibodies to erythropoietin on an insoluble matrix, sorption of erythropoietin on immobilized antibodies and subsequent elution of erythropoietin, characterized in that monoclonal antibodies are used as antibodies, which are cultured by including clones PCE / F6, PCE / D10 and PCE / D7, and the elution of erythropoietin is carried out with acidic buffer solution with a pH of less than 4.  2. The method according to claim 1, characterized in that the eluents used in immunoaffinity chromatography contain from 0.01 to 0.05 wt.% Non-ionic surfactants.  3. The method according to claim 1 or 2, characterized in that the elution is carried out with glycine buffer.  4. The method according to p. 1 or 2, characterized in that the elution is carried out with a solution of citric acid.  5. The method according to p. 1 or 2, characterized in that the eluate of immunoaffinity chromatography is additionally subjected to sorption on anion exchange resin with stepwise elution of erythropoietin with decreasing pH and increasing the ionic strength of the eluent.

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