RU2132386C1 - Method of human recombinant gamma-interferon preparing - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: human recombinant gamma-interferon is prepared from insoluble inclusion-bodies of E. coli cells by disintegration of the strain-producer cells in buffer solution in the presence of zinc and copper salts, solubilization of the product with a mixture of urea and lithium chloride followed by chromatography purification on cation-exchanger COOH-Biochrom K-1. Cation-exchanger is a copolymer of methacrylic acid and pentaerythritol dimethacrylate. Method ensures to carry out easily the process of the isolation and purification of this biologically active substance in large-scale quantity. EFFECT: improved method of preparing, high purity of the end product. 2 cl, 1 dwg, 3 tbl, 4 ex

Description

 The invention relates to biotechnology, in particular, to a method for producing recombinant immune interferon (interferon-gamma; INF-γ) of a person. The proposed method allows you to easily scale the process of isolation and purification of this biologically active substance.

 INF-γ is a semi-functional protein produced by cells of the immune system (immune interferon), has immunostimulating, antitumor and antiviral activities. The creation of microorganisms producing human INF-γ made it possible to obtain a biologically active recombinant protein in an amount necessary for both conducting broad structural and functional studies and for use in medical practice. Abroad, drugs based on recombinant INF-γ have been created for the treatment of immune, oncological and severe viral diseases (immunomax, immuneron, etc.). In this regard, the development of effective methods for producing recombinant INF-γ is relevant.

 Known methods [1-3] for producing recombinant human INF-γ, but they are all laborious, expensive, described for analytical amounts of protein and difficult to scale; in addition, the recombinant human immune interferon synthesized in Escherichia coli cells forms insoluble inclusion bodies during superproduction, which are precipitated by centrifugation of the destroyed bacterial cells together with cell wall debris. Ultrasound destruction in a medium without denaturing agents or enzymatic lysis of E. coli is accompanied by activation of a membrane-bound protease capable of limited hydrolysis of gamma interferon in a sequence containing four basic amino acids in a row at the end of the molecule [1,4].

Closest to the claimed method is the method [5; prototype], which allows to obtain an intact native INF-γ, which includes several stages: chemical lysis of cells of the producer strain E. coli and simultaneous solubilization of INF-γ from inclusion bodies with 7M guanidine chloride solution; removal of insoluble components by centrifugation; 10-fold dilution of the target fraction with pH 9.5 buffer and separation of the resulting protein precipitate by centrifugation; silica gel fractionation of proteins (NuGel-952 AC); and finally, affinity chromatography on a sorbent with monoclonal antibodies. The resulting product is in a solution of 20 mm sodium phosphate buffer containing 1M NaCl and 1M guanidine chloride (or 50% ethylene glycol), and is a protein with a molecular weight of 18000, homogeneous by electrophoresis. The yield of 25 g of cells is 8 mg (25-32% of the content in the cell extract). The biological activity of the drug in inhibiting the cytopathic effect is 10 ⁷ units / mg protein.

 The disadvantage of the prototype method is that the simultaneous destruction of cells and the solubilization of INF-γ does not allow one of the advantages of the formation of inclusion bodies, which consists in the easy separation of the ballast water-soluble proteins of the bacterial cytoplasm from the insoluble target product by simple centrifugation. The use of an immune sorbent for protein purification is very expensive, low-tech, and difficult to scale. An additional complex analysis of the final product is required for the absence of the oncogenic material of the parent myeloma cell used in the preparation of monoclonal antibodies.

 The technical task of the invention is the development of technological easily scalable method for producing highly purified unfragmented INF-γ person.

 To preserve the possibility of removing water-soluble proteins and preserve the intact recombinant human immune interferon, the producer of zinc and copper salts, which, as shown in [4], being present in the medium at low concentrations, inhibit destructive gamma interferon membrane-bound protease of E. coli.

 The solubilizing properties of guanidine chloride, one of the most powerful and often used for dissolving inclusion bodies of denaturing agents, can be modeled by mixtures of urea and lithium chloride [6], which, unlike guanidine, can be used for more selective extraction of the product. In addition, renaturation from urea solutions occurs more efficiently than from guanidine solutions [6].

 Thus, the goal is achieved in that the destruction of producer cells containing recombinant INF-γ in the form of inclusion bodies is carried out in a buffer solution in the presence of zinc and copper salts that inhibit the membrane-bound protease that destroys INF-γ, and the product is solubilized with a mixture of urea and chloride lithium, and chromatography is performed on a CO-OH-Biochrom K-1 cation exchanger with an increased exchange capacity, which is a copolymer of methacrylic acid and pentaerythritol dimethacrylate.

 The destruction of cells in a buffer solution allows simple centrifugation of the cell extract to separate a large mass of E. coli ballast cell proteins from the insoluble target product located in the inclusion bodies, while zinc and copper salts prevent the proteolytic destruction of the target protein. The use of the denaturing agent urea, which is less powerful than guanidine chloride, for the solubilization of INF-γ from Taurus provides a more efficient protein renaturation [6]. The use of COOH-Biochrom K-1 sorbent for chromatographic purification significantly simplifies and reduces the cost of the method compared to the use of an immune sorbent. In addition, this sorbent compares favorably with KM cellulose proteins widely used in preparative chromatography for their high exchange capacity and ability to withstand high pressures (up to 70 atm). Moreover, the use of COOH-Biochrome K-1 allows the use of low and medium pressure chromatography equipment, and therefore does not significantly increase the cost of the chromatography step.

The proposed method for the preparation and purification of recombinant human immune interferon, which forms insoluble inclusion bodies in E. coli cells, consists of several successive steps:
1. Producer cells are destroyed by ultrasound in a buffer containing salts of copper and zinc, inhibiting the membrane-bound protease capable of degrading gamma interferon. As a producer, any genetically transformed E. coli strain that accumulates immune interferon in the form of inclusion bodies can be used. In this case, the isolation is carried out from the E. coli MH1 strain [pIFN-γ-trp2], in which the expression of the gamma interferon gene is controlled by the promoter of the tryptophan operon of E. coli. The level of biosynthesis of immune interferon during constitutive synthesis is about 10% of the total protein of the cell and about 50% during the induction of tryptophan promoter [7].

 2. The insoluble form of gamma-interferon is collected by centrifugation of a suspension of cells destroyed by ultrasound and washed with 4M urea solution to remove impurity proteins.

 3. The immune interferon is solubilized in 8M urea containing lithium chloride at a concentration of greater than or equal to 5M in a slightly alkaline medium (preferably at pH 9.5).

 4. Dissolved rembinant protein is desalted by gel filtration in 8M urea, pH 9.5, or dialysis against 8M urea, pH 9.5.

 5. A solution of desalted protein is applied to a column with a COOH-Biochrom K-1 cation exchanger with a high ion-exchange capacity and high protein sorption capacity, balanced with 8M urea buffer with a pH of 9.5 (optimal pH value for chromatographic purification of gamma-interferon). The load on the sorbent can reach 100 mg of total protein per ml of carrier. Immune interferon having an isoelectric point above 9.5 is well retained on the COOH-Biochrom K-1 sorbent, while the bulk of the impurities are found in the breakthrough.

 6. Sorbed proteins elute in a concentration gradient of sodium chloride (from 0 to 1M). Fractions containing the target protein by gel electrophoresis are combined and dialyzed against 8M urea, pH 6.0.

 7. A dialyzed solution of immune interferon is applied to a COOH-Biochrom K-1 column balanced with 8M urea, pH 6.0. Sorbed proteins elute in a gradient of sodium chloride (from 0 to 1M). Fractions containing gamma-interferon are combined and dialyzed against 2M urea in sodium acetate, pH 6.0, then against a buffer without urea at pH 6.0.

The purity of the product, determined by gel electrophoresis in a polyarylamide gel under completely denaturing conditions, is at least 98%. Protein after electrophoresis is detected in the form of a single band with a molecular weight of about 17 kD without low molecular weight fragments. This protein gives a positive reaction with monoclonal antibodies to human immune interferon in Western blot. The product elutes at one peak by reverse phase chromatography on Octadecyl = Si100 Polyol in an acetonitrile gradient in trifluoroacetic acid. Antiviral activity, determined by suppressing the cytopathic effect of the encephalomyocarditis virus in a monolayer culture of human lung fibroblasts L68, is 2 • 10 ⁷ units / mg. The yield of the finished product is about 12 mg from 20 g of cells (22-25% of the content in the original extract).

Sorbent COOH-Biochrom K-1, convenient for scaling the chromatographic stage of protein purification, including recombinant human immune interferon, is obtained by copolymerization of methacrylic acid and pentaerythritol dimethacrylate in n-butanol in the presence of dinitrilazobisisobutyric acid as an initiator. To obtain a granular sorbent, the reaction mixture is introduced at room temperature into an aqueous solution of sodium chloride in the presence of polyvinyl alcohol with vigorous stirring, then the mixture is heated at 70 ^o C for 5 hours. After the reaction, the washed granules are scattered on a sieve, isolating a fraction with a size of 60 - 120 microns, and determine the characteristics of the sorbent by conventional methods. The cation exchanger synthesized by the described method, called COOH-Biochrom K-1, has a dextran exclusion limit of 5 • 10 ⁵ D, a total exchange capacity of 0.5 mEq / ml, an adsorption capacity for bovine serum albumin of 110 mg / ml, volume pore space 0.5 - 0.6, pK of acid groups from 5 to 6, maximum working pressure - up to 70 atm. Sorbent COOH-Biochrom K-1 has a certificate for use in the purification and isolation of medical products, including drugs.

 New in comparison with the prototype method is the separation of the stages of destruction of the cells of the producer strain and the solubilization of the target recombinant protein synthesized in inclusion bodies, which allows a simple centrifugation of the suspension of destroyed cells to separate the bulk of soluble intracellular proteins from the target product and then use easily scalable for cleaning chromatography on a cation exchanger instead of an expensive sorbent with monoclonal antibodies. The use of low concentrations (20 mM) of zinc and copper salts in the cell disruption medium avoids significant proteolytic degradation of the protein molecule INF-γ and, together with the effective process of renaturation of recombinant INF-γ from a solution of urea, weaker denatured alcohol than guanidine chloride, provides a high yield undegraded target product.

The present invention is illustrated by the figure of a graphic image, which presents an electrophoretic analysis of proteins in a 12.5% polyacrylamide gel under denaturing conditions during the purification of recombinant interferon-gamma:
lane 1 - protein marker, mol. mass 14400;
lane 2 — cell extract of the producer strain E. coli MH-1 (pIFN-γ-trp2);
lane 3 - supernatant after centrifugation of the cell extract;
lane 4 - precipitate after centrifugation of the cell extract (inclusion body);
lane 5 - washing Taurus inclusion 4M urea;
lane 6 - solution of inclusion bodies in 8M urea with 5M lithium chloride, pH 9.5;
lane 7 — desalted solution of inclusion bodies;
lane 8 — preparation after the first chromatographic purification;
lane 9 - preparation after the second chromatographic purification.

 The essence of the invention is disclosed in the following examples.

 Example 1. Obtaining a carboxyl cation exchanger COOH-Biochrom K-1.

In a 500 ml glass container, a solution of methacrylic acid (23 h) (70 mol%) and pentaerythritol dimethacrylate (32 h) (30 mol%) in n-butyl alcohol (200 h) is prepared. The initiator, dinitrilazobisisobutyric acid (1.7 h), is introduced into the solution. A distilled water solution (700 h) containing polyvinyl alcohol (5 h) and sodium chloride (90 h) was placed in a temperature-controlled reactor equipped with a mechanical stirrer, a refrigerator, and a tube for bubbling argon, and the reaction mixture prepared in a glass container was introduced at continuous stirring at a speed of 300 rev / min for 15 minutes at room temperature, further include heating the reactor to 70 ^o C. copolymerization process is carried out for 5 hours. The pellets were washed with hot distilled water from the polyvinyl alcohol, ATEM isopropanol - from monomers and solvents and distilled water from isopropanol. The granules are dispersed on sieves, separating a fraction with a size of 60-120 microns. The product yield is 96% of the amount of methacrylic acid taken into the reaction.

Cation exchange resin is a white spherical granule and has the following characteristics:
- dextran exclusion limit 500 • 10 ³ D;
- full exchange capacity of 0.5 mEq / ml;
- adsorption capacity for bovine serum albumin 110 mg / ml;
- the volume of the pore space of 0.5 - 0.6;
- pK acid groups from 5 to 6;
- maximum working pressure of 70 atm.

 The cation exchange resin is named COOH-Biochrom K-1.

 Example 2. Obtaining highly purified recombinant human immune interferon.

All procedures are carried out at 4 ^o C.

20 g of biomass of E. coli MH1 [pIFN-γ-trp2] with the content of immune interferon (in the form of insoluble inclusion bodies) in the amount of 10% of the total cellular protein is suspended in 40 ml of 50 mM Tris-HCl, pH 7.5, 2 mM ZnCl ₂ , 2 mm CuCl ₂ (buffer A) to obtain a homogeneous suspension. Cells are destroyed by ultrasound at full emitter power (UZDN-2T) for 25-20 s (5 times with 2-3 minute intervals between scoring) to reduce the optical density of the suspension at 600 nm by at least 2 times. An equal volume of buffer A is added to the mixture, homogenized by sounding, and centrifuged for 30-60 minutes at 10,000 rpm in a J2-21 centrifuge in a JA-14 rotor. After centrifugation, the supernatant is discarded, and the pellet containing immune interferon is poured into 100 ml of 4M urea, 50 mM Tris-HCl, pH 9.5, and the mixture is homogenized by sonication for 25-30 s. The suspension is centrifuged at 10,000 rpm for 30-40 minutes and a precipitate containing recombinant gamma interferon is collected.

 Immune interferon is extracted from the precipitate with 100 ml of 8M urea in 50 mM Tris-HCl, pH 9.5, 5M LiCl by homogenizing the mixture by sonication for 20-30 s and subsequent stirring overnight. The suspension is centrifuged for 40 min at 18,000 rpm, the supernatant containing the extracted proteins is collected and subjected to desalination on Biogel P2 (Bio-Rad, USA; column size 5 x 75 cm) in 8 M urea, 50 mM Tris-HCl, pH 9, 5. Fractions with recombinant immune interferon, having an electrical conductivity of less than 1 mS, are combined and applied to a column with COOH Biochrom K-1 (2 x 4 cm, volume 12.6 ml), balanced with 8 M urea, 50 mm Tris-HCl, pH 9, 5. The column is washed until the optical density of the eluate at 280 nm is reduced to the initial value, then sorbed proteins are eluted in a linear gradient of sodium chloride concentration from 0 to 1 M. The fractions are analyzed for the content of recombinant immune interferon by gel electrophoresis of proteins in 12.5% polyacrylamide gel under completely denaturing conditions according to [8]. Fractions containing interferon gamma were combined and dialyzed against 8M urea, 50 mM sodium acetate, pH 6.0, overnight. After dialysis, the solution was applied to a COOH-Biochrom K-1 column (2 x 4 cm), equilibrated with 8M urea, 50 mM sodium acetate, pH 6.0 and chromatography was performed as described above, but at pH 6.0. The collected product is dialyzed sequentially against 2M urea, 50 mM sodium acetate, pH 6.0, for 16 hours, then against 50 mM sodium acetate, pH 6.0, for 16 hours. The resulting product is analyzed by conventional methods. The material balance of the process is presented in table. 1 (tab. 1-3 see at the end of the description).

The product yield is 615 μg / g biomass, purity - not less than 98%, antiviral activity, determined by suppressing the cytopathic effect of the encephalomyocarditis virus in a monolayer culture of human L68 cells, 2 • 10 ⁷ units / mg. During gel electrophoresis under completely denaturing conditions, the protein appears as a single band with a molecular weight of about 17000 D, reacting with monoclonal antibodies to human immune interferon in the Western blot.

 Example 3. The effect of the concentration of lithium chloride on the solubilization of recombinant immune interferon in urea solutions.

 The preparation and washing of the inclusion bodies of the recombinant immune interferon is carried out as described in Example 2. The precipitate of the washed inclusion bodies obtained from 10 g of the biomass of the producer strain is suspended in 50 ml of distilled water, 0.5 ml samples are taken from the suspension in microcentrifugation tubes and the precipitate is collected by centrifugation for 2 minutes at 12,000 rpm. The precipitate is dissolved in 1 ml of urea and lithium chloride solutions of various concentrations (see table 2) at a pH of 9.5 for 30 minutes at room temperature. The extracted proteins are subjected to electrophoresis on a 12.5% polyacrylamide gel under completely denaturing conditions, the relative content of recombinant human immune interferon is determined by scanning the gel after staining the Coomassie protein with bright blue G250. The protein concentration in the extracts is determined by the Lowry method.

 The results of the experiment are presented in table 2.

 According to the results of the analysis for practical use, a solution of 6M LiCl in 6-8M urea was selected, because at the indicated salt concentration, the highest yield of the target product and its highest content in the extract are achieved.

 Example 4. The effect of pH on the chromatography of recombinant human immune interferon on cation exchange resin COOH-Biochrom K-1.

 The procedure for isolating recombinant human immune interferon to the first chromatography step is carried out as described in Example 2. Then, 5 samples of desalted INF-γ with a protein content of 50 mg (target protein content of 10%, i.e. 5 mg) are taken and the pH of the solution of individual samples is adjusted by adding a solution of alkali or acid to a pH value of 10.5 to 6.5. The prepared samples are applied to a COOH-Biochrom K-1 column (0.9 x 1.5 cm, V = 1 ml), balanced with each sample with a buffer solution with 8M urea. The column is washed until the optical density of the eluate at 280 nm is reduced to the initial value, then sorbed proteins are eluted in a linear gradient of sodium chloride concentration from 0 to 1 M in a buffer with 8 M urea and the corresponding pH. The analysis of fractions for the content of the target protein is carried out by gel electrophoresis of proteins in a 12.5% polyacrylamide gel under completely denaturing conditions.

 The results of the experiments are presented in table 3.

 From the data of table 3 it is seen that at pH 9.5 the best (7 times) purification of the target protein occurs and with a large yield (42.7%). At pH values above and below 9.5, the content of interferon -γ in the eluate decreases, and the degree of its purification during chromatography on COOH-Biochrome K-1 also decreases.

 Thus, the destruction of the cells of the producer strain containing the recombinant target block in inclusion bodies in an aqueous medium and the solubilization of the target protein after preliminary removal of soluble cell proteins makes it possible to use a simple, well reproducible, and easily scalable 2-stage chromatographic chromatography to obtain highly purified INF-γ cleaning on a COOH-Biochrom K-1 cation exchanger. The addition of small concentrations (20 mM) of zinc and copper salts to the cell destruction buffer avoids significant proteolytic degradation of INF-γ and, together with the effective process of renaturation of the recombinant protein from urea, a weaker denaturing agent than guanidine chloride, provides a high yield of non-degraded target product. The use of an affordable domestic denaturing reagent and an easily synthesized cation exchanger in the method allows to successfully scale the process.

Literature
1. US patent 4604284, CL. A 61 K 37/02, publ. 08/05/86.

 2. US patent 4751078, cl. A 61 K 45/02, publ. 06/14/88.

 3. US patent 4617378, cl. C 07 K 15/26, publ. 10/14/86.

 4. K. Sugumura, N. Higashi. // J. Bacteriol. 1988, v. 170, N8, p. 3650-3654.

 5. US patent 4681930, CL C 07 K 15/26, publ. 07.21.87.

 6. M. Matsubara, D. Nohara, T. Sakai // Chem. Pharm.Bull., 1992, v. 40, N2, p. 550-552.

 7. E. D. Sverdlov, S. A. Tsarev, R. A. Krikbaev, I. P. Chernov, V. M. Rostapshov // FEBS Lett., 1987, v. 212, p. 233-236.

 8. V.K. Laemmli // Nature (London), 1970, v. 227, p. 680-685.1

Claims (2)

 1. The method of producing recombinant human interferon-gamma from transformed Escherichia coli cells containing recombinant immune interferon in the form of insoluble inclusion bodies, which includes cell destruction, extraction of interferon-gamma, centrifugation and chromatographic purification followed by isolation of the target product, characterized in that the cells destroy by ultrasound in the presence of zinc and copper salts, insoluble inclusion bodies are separated by centrifugation, interferon-gamma extraction is carried out a mixture of urea and lithium chloride, and chromatographic purification is carried out in two stages on a COOH-Biochrom K-1 cation exchanger, which is a granular copolymer of methacrylic acid and pentaerythritol dimethacrylate, the elution being carried out at pH 9.5 in the first stage and at the second pH 6.0.  2. The method according to claim 1, characterized in that the extraction is carried out with 6-8M urea in the presence of lithium chloride at a concentration of 6M.

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