RU2326944C2 - Method of production and preparation of analogue of human recombinant interferon gamma - Google Patents

Abstract

FIELD: technological processes; medicine.

SUBSTANCE: invention is related to preparation of recombinant analogues of human gamma-interferon and may be used in medicine for prophylactics and treatment of oncological diseases, neoplasms and inflammatory processes of humans. Highly pure genetically engineered analogue of human gamma-interferon - deltaferon with molecular mass of 16.2 kilo Daltons is produced by microbiological synthesis with further chromatographic purification on "KM"-sepharose - cationic-exchange sorbent with high linear speed of flow and suitable for preparative loads, at different pH values. On the basis of deltaferon, which contains by data of polyacrylamide gel-sodium dodecyl sulfate electrophoresis at least 98% of main substance in the form of monomer that possesses antiproliferative and anti-inflammatory activities inherent in gamma-interferon, medical product is prepared, which also includes low molecular polymer filler-stabiliser (reopolyglukine or polyvinylpyrrolidone) and salt buffer system with pH 7.0-7.1.

EFFECT: highly active and stable preparation of deltaferon is produced.

2 cl, 6 dwg, 1 tbl, 3 ex

Description

The invention relates to biotechnology, medicine. A method for producing a preparation based on a highly purified genetic engineering analogue of human gamma-interferon — deltaferon with a molecular weight of 16.2 kDa obtained by microbiological synthesis followed by chromatographic purification is proposed. Obtained by the proposed method, the analogue possesses antiproliferative and anti-inflammatory activities inherent in gamma interferon and contains, according to SDS page-SDS electrophoresis, at least 98% of the basic substance in the form of a monomer. The preparation incorporates a low molecular weight polymer filler-stabilizer (reopoliglyukin or polyvinylpyrrolidone) and a salt buffering system with a pH of 7.0-7.1. The drug has high activity, stability and can be used for the prevention and treatment of cancer, neoplasms and inflammatory processes in humans.

Recombinant human gamma-interferon (rhIFN-γ) is a protein with a molecular weight of about 17 kDa, which has significant antitumor, anti-inflammatory and antiviral effects, which makes its use in medicine very promising.

However, the widespread use of rhIFN-γ preparations in clinical practice is limited by a number of factors, among which the most significant is the increased sensitivity of this protein to the action of proteolytic enzymes both in vitro and in vivo [1-3]. In addition, upon receipt of rhIFN-γ, it is necessary to introduce the stages of protein denaturation and renaturation in the purification schemes, since rhIFN-γ accumulates in the form of insoluble aggregates during microbial synthesis, the so-called “Inclusion bodies”, which leads to a significant decrease in the yield of the target product, as well as an increase in the cost of the drug due to significant time and material costs.

Known methods for producing rhIFN-γ and its analogues from bacterial biomass [4, 5, 6, 7]. Common disadvantages of the presented methods were the use of high concentrations of chaotropic agents (urea and guanidine hydrochloride) for extraction and washing of rhIFN-γ from insoluble aggregates, as well as the low yield of the target protein - 1 mg per 1 g of biomass [6], the use of additional stages of sulfate reprecipitation rhIFN-γ [5] or the introduction of an additional stage of gel filtration [7].

In order to avoid such problems when obtaining protein, an analog of rhIFN-γ, deltaferon, was created, which has a deletion of 10 amino acid residues at the C-terminus of the molecule and replaces the KRKR cluster with KGSA (see Figure 1) [8]. As a result of this modification, a molecule of the rhIFN-γ analog, deltaferon, has a number of distinctive features, such as resistance to proteolysis and biosynthesis in prokaryotic cells in soluble form [9]. The developers of the described analogue conducted comparative studies of the biological functions of rhIFN-γ and deltaferon. The preservation of the antiproliferative activity characteristic of the initial rhIFN-γ in deltaferon was observed on human mononuclear cells, where it was shown that proteins reduced cell proliferation equally [10] (see Figure 2). In an experimental model of mouse adjuvant arthritis caused by intradermal administration of Freund's complete adjuvant (PAF), it was found that deltaferon did not significantly differ in level of anti-inflammatory activity from rhIFN-γ [10].

The prototype method of the present invention is patent RU 2105813 [11, prototype], which describes a laboratory method for producing deltaferon by ion exchange chromatography on a Mono S HR5 / 5 column (Amersham Pharmacia Biotech). The method is based on the use of E. coli strain MH-1 / pIFN D10 (deposited in the collection of the All-Russian Research Institute of Genetics under the number VKPM B-6663). When implementing this method, 1 g of producer biomass is suspended in 5 ml of a buffer solution (0.01 M Tris-HCl pH 8.5) and sonicated (5 times for 30 seconds at 0 ° C at minute intervals). The cell debris is separated by centrifugation for 30 minutes at 17,000 rpm and T = 4 ° C. The supernatant was applied to a Mono S HR5 / 5 column, equilibrated with the same buffer, after which the sorbed proteins were eluted with a NaCl gradient from 0 to 1 M, pH 8.5. Fractions containing an analog of rhIFN-γ are detected by gel electrophoresis under completely denaturing conditions. The rhIFN-γ analogue-containing material was dialyzed overnight against 0.01 M ammonium acetate, pH 6.0. The dialyzed protein is then loaded onto a Mono S HR5 / 5 column, equilibrated with 0.01 M ammonium acetate, pH 6.0, and eluted with a linear NaCl gradient (0 to 1 M) in the same buffer. Detection of the rhIFN-γ analogue in fractions is carried out as described above. The fractions containing the target protein are combined and dialyzed against 0.01 M ammonium acetate, pH 6.0, sterile filtered and stored until use at 4 ° C.

According to the authors, this method provides high protein purity (at least 99%), but it is proposed for processing small amounts of producer biomass (1 g of biomass) and cannot be used for preparative volumes. The Mono S HR5 / 5 cation exchange column used in the prototype method is used in analytical high performance liquid chromatography (HPLC) and is not designed for preparative loading. Also, the prototype method does not provide data on the yield of the target protein and there are no characteristics on the presence of impurities of DNA, lipopolysaccharides (LPS) and proteins of the producer strain in the final preparation. While the presence of these cellular components in recombinant protein preparations for medicine is extremely undesirable and strictly regulated [12] - DNA is not more than 100 pg / mg protein, impurity proteins are not more than 200 ng / mg protein, LPS is not more than 200 ng / mg protein.

The purpose of the present invention is to obtain a highly purified preparation of an analogue of rhIFN-γ - deltaferon with antiproliferative and anti-inflammatory activities, and to develop a preparative method for its preparation, which would guarantee a high yield and a high degree of purity of the drug necessary for its medical use, as well as its stability in the process storage.

The goal is achieved by increasing the amount of synthesized target protein in cells and its most complete extraction, together with protein purification by ultrasonic disintegration of cells, separation of cell debris by centrifugation and 2 subsequent chromatography of the cell extract on KM-sepharose - cation exchange sorbent, with a high linear flow rate and suitable for preparative loads at different pH values.

The essence of the method is as follows.

The biomass of E. coli MH-1 / pIFN D10 cells grown on M9 medium with the addition of casamino acids was suspended in a disruption buffer (50 mM Tris-HCl, 1 mM CuCl ₂ , 1 mM ZnCl ₂ , pH 8.3) until homogeneous condition. The suspension is subjected to sonication to reduce the optical density at a wavelength of 550 nm to 50-60% of the original value. The cell debris after centrifugation is removed, and the supernatant is applied to a KM-Sepharose column equilibrated with buffer A (50 mM Tris-HCl, pH 8.3). The load on the column is 6-10 mg of protein per 1 ml of sorbent. The column is washed with buffer A until the optical density in the effluent is reduced at a wavelength of 280 nm to the initial value and the proteins are eluted with a linear gradient of buffers of the following composition: 50 mM Tris-HCl, pH 8.3 (buffer A) - 50 mM sodium citrate, pH 6 5 s 0.5 M NaCl. Fractions containing deltaferon are pooled and dialyzed against 2 L of buffer B (50 mM Na citrate, pH 6.5) for 12 hours at 6 ° C. The tertiary treatment of deltaferon is carried out by rechromatography on KM-Sepharose, previously equilibrated with buffer B. The pH of the protein solution is adjusted to pH 6.5 before application, adding a 0.1 M hydrochloric acid solution dropwise. After application, the column is washed with buffer B to reduce the optical density in the resulting solution at a wavelength of 280 nm to the original value. Protein elution is carried out by an exponential gradient of the following composition: buffer B (50 mM Na citrate, pH 6.5) - buffer A (50 mM Tris-HCl, pH 8.3 with 1 M NaCl). Fractions of the substance of deltaferon are combined and dialyzed against 1 l of buffer B (10 mM KH ₂ PO ₄ , 0.15 M NaCl, pH 7.0) for 12 hours at 6 ° C. After dialysis, the substance of deltaferon is sterilized by ultrafiltration using a membrane filter with a pore size of 0.22 μm. The electrophoretic purity of the final preparation is more than 98%.

When scaling the production of a recombinant product, the reliability of the purification technology of the target product is extremely important. Chromatography on an ion exchanger at various pHs allows one to achieve additional separation of the target protein and impurity components using the same chromatographic carrier.

The specific specific activity of the substance deltaferon obtained by the proposed method, determined by the effect of suppressing the cytopathic effect of the encephalomyocarditis virus on a culture of human diploid fibroblasts [15], corresponded to the activity of the drug obtained by the prototype method. The content of E. coli impurity proteins determined by enzyme-linked immunosorbent assay [16] is 40 ng / mg of deltaferon, the content of DNA impurities determined by molecular hybridization [17] is 80 pg / mg of the drug, and the content of LPS determined by the enzyme-chemical method [18] - 30 ng / mg of the drug. The values of the presented impurities in the deltaferon substance obtained by the proposed method are much lower than their maximum permissible values [12].

The invention also includes a dry injection preparation consisting of a highly purified analog of rhIFN-γ - deltaferon obtained by the proposed method, a low molecular weight polymer filler-stabilizer (reopoliglyukin or polyvinylpyrrolidone) and a buffered salt system. The quantitative composition of one therapeutic dose of a drug with a volume of 1 ml is as follows:

- an analogue of rhIFN-γ - deltaferon - 50 μg / ml (50,000 IU / ml);

- reopoliglyukin (or polyvinylpyrrolidone) dry - 50,000 μg / ml;

- NaCl - 8.78 μg / ml;

- KH ₂ PO ₄ - 1.36 μg / ml, (pH = 7.0 ± 0.1).

The invention is illustrated by the following graphic materials.

Figure 1. Amino acid sequences of the C-terminus of recombinant human interferon gamma and deltaferon molecules.

Figure 2. Diagram of the effect of recombinant human gamma interferon and deltaferon on the proliferative activity of donor mononuclear cells (confidence interval calculated at p <0.05).

Figure 3. Profile of the first chromatography of deltaferon on KM-Sepharose; electrophoretic track number corresponds to the fraction number.

Figure 4. Repeat chromatography profile of deltaferon on KM Sepharose; electrophoretic track number corresponds to the fraction number.

Figure 5. Purification of deltaferon by stages of isolation, where:

1 - cell lysate after destruction by ultrasound (5 μl),

2 - supernatant after centrifugation (cell extract 5 μl),

3 - sediment after centrifugation,

4 - deltaferon after the first chromatography (20 μg),

5 - deltaferon after rechromatography (20 μg).

6. Electrophoretic analysis of the substance of deltaferon, where lanes 1 and 3 are deltaferon (25 and 45 μg), and lane 2 - proteins - molecular weight markers.

For a better understanding of the invention, the following are examples of its specific implementation.

Example 1. Obtaining transformed cells, selecting highly productive clones and growing them in a liquid selective medium.

Transformation of competent cells of E. coli MH-1 plasmid DNA pIFN D10 is carried out by the calcium method, as described previously [17]. The cell suspension is seeded in plates with agar medium containing tetracycline at a concentration of 15 μg / ml. The grown colonies of transformed cells are scattered in sectors on plates with agar medium containing tetracycline at a concentration of 15 μg / ml. Part of the cells from the sectors was selected by a microbiological loop and analyzed by electrophoresis in a 15% polyacrylamide gel under denaturing conditions [13] for the quantitative content of the target protein. The calculation of the relative content of the target protein is carried out by processing electrophoregrams in the program Gel-pro analyzer, Ver. 3.1 "(Media Cybernetics Inc. USA). Clones with the highest content of the target protein are used to produce the inoculum. They are grown in liquid nutrient medium M9 with casamic acids (2%) containing tetracycline at a concentration of 10 μg / ml, at 32 ° C until the middle of the logarithmic growth phase (D ₅₅₀ = 0.5-0.6).

Fermentation is carried out in an Ultraferm fermenter with a working volume of 4 l at a temperature of 31-32 ° C. Nutrient medium - M9 with casamic acids (2%) containing tetracycline at a concentration of 10 μg / ml. The rotation speed of the mixer in the fermenter is from 100 to 200 rpm, the air supply is 0.5-1.0 l / min per 1 liter of medium. For sowing using seed, obtained as described above. The seed volume is 1% (v / v) of the volume of the medium for fermentation. The fermentation time is 9-10 hours. Fermentation is completed at the end of the logarithmic growth phase. The culture fluid is cooled to 5-10 ° C, the cells are collected by centrifugation and stored at minus 70 ° C.

Example 2. Obtaining a highly purified substance deltaferon

30 g of the biomass of E. coli MH-1 / pIFN D10 cells are suspended in 300 ml of disruption buffer (50 mM Tris-HCl, 1 mM CuCl ₂ , 1 mM ZnCl ₂ pH 8.3) until homogeneous. Then the suspension is placed in an ice bath and subjected to ultrasound treatment (UZDN-2T unit) at a frequency of 22 kHz in sessions of 30 seconds with an interval of 1 minute, to cool the mixture (the temperature in the suspension should not exceed 8 ± 2 ° C). Sound scoring continues until the optical density decreases at a wavelength of 550 nm to 50-60% of the initial value. Cell debris was removed by centrifugation at 12,000 rpm for 60 min (JA-21, JA-14 rotor, t = 4 ± 2 ° C), after which the supernatant containing the target protein was collected and applied to a KM-Sepharose column, equilibrated with buffer A (50 mM Tris-HCl, pH 8.3). The load on the column is 6-10 mg of protein per 1 ml of sorbent. The column is washed with buffer A to reduce the optical density in the resulting solution at a wavelength of 280 nm to the original value. The deltaferon is eluted from the column by a linear gradient of buffers of the following composition: buffer A — 50 mM Na citrate, pH 6.5 with 0.5 M NaCl. The effluent solution is collected in fractions, which are then analyzed by electrophoresis in 15% PAAG gel under denaturing Lemmley conditions [13]. The calculation of the relative content of the target protein is carried out by processing electrophoregrams in the program package Gel-pro analyzer Ver. 3.1 "(Media Cybernetics Inc. USA). The chromatography profile and electrophoretic analysis of the collected fractions are presented in figure 3. The analysis of fractions showed that at this stage it was possible to achieve 80% purity of the target protein. Fractions containing deltaferon are pooled and dialyzed against 2 L of buffer B (50 mM Na citrate, pH 6.5) for 12 hours at 6 ° C. The tertiary treatment of deltaferon is carried out by rechromatography on KM-Sepharose, previously equilibrated with buffer B. The pH of the protein solution is adjusted to pH 6.5 before application by adding a 0.1 M hydrochloric acid solution dropwise. After application, the column is washed with buffer B to reduce the optical density in the resulting solution at a wavelength of 280 nm to the original value. Protein elution is carried out by an exponential gradient of the following composition: buffer B - buffer A with 1 M NaCl. The profile of the second chromatography is presented in figure 4. The collected fractions are analyzed by electrophoresis as described above. Fractions containing one protein band with a molecular weight of 16.2 kDa in the analysis are combined and dialyzed against 1 L of buffer B (10 mM KH ₂ PO ₄ , 0.15 M NaCl, pH 7.0) for 12 hours at a temperature of 6 ° C. After dialysis, the protein concentration is determined by the method [14] and then the deltaferon substance is sterilized by ultrafiltration using a membrane filter with a pore size of 0.22 μm. The relative yield of deltaferon is 27.3% of its content in the cell extract, 185 mg of 30 g of biomass (6.1 mg of 1 g of biomass). Data on the isolation and purification of protein are presented in the table.

Data on the isolation and purification of deltaferon from 30 g of cells with an initial content of the target protein of 17.7% Purification stage Volume ml Total protein Deltaferon Exit, % mg / ml mg % mg Cell extract 510 7.5 3825 17.7 677 one hundred 1 chromatography on KM-sepharose 70 3.67 256.9 90 231.21 34 Rechromatography on KM-Sepharose 69 2.75 189 98 185 27.3

The picture of the cleaning stages is presented in figure 5. The substance is electrophoretically homogeneous at a load of 45 μg per well (Fig.6 lane 3). The specific specific activity of the substance deltaferon, determined by the effect of suppressing the cytopathic effect of the encephalomyocarditis virus on a culture of human diploid fibroblasts, is at least 1 × 10 ⁶ U / mg protein [15]. The content of E. coli impurity proteins determined by enzyme-linked immunosorbent assay [16] is 40 ng / mg of deltaferon, the content of DNA impurities determined by molecular hybridization [17] is 80 pg / mg of the drug, and the content of LPS determined by the enzyme-chemical method [18] - 30 ng / mg of the drug.

Example 3. Obtaining an injection form of the drug deltaferon

The sterile substance of deltaferon is used to prepare a dry, storage-stable injection drug. The drug is prepared by mixing a concentrated solution of the substance deltaferon in buffer B (10 mM KH ₂ PO ₄ , 0.15 M NaCl, pH 7.0 ± 0.1) with a 10% sterile solution of a low molecular weight polymer filler-stabilizer (reopoliglukin or polyvinylpyrrolidone ) to a final concentration of deltaferon of 50 μg / ml (50,000 IU per therapeutic dose) and a content of reopoliglukin (or polyvinylpyrrolidone) of 5%. The resulting solution is poured into ampoules or vials of 1 ml and frozen at a temperature of minus 40 ° C. The lyophilization regime includes drying at a temperature of minus 40 ° C for 3 hours, followed by a gradual increase in temperature to 30 ° C. Upon reaching this temperature, the drug is dried for 12 hours to a residual moisture content of 2-5%. The resulting dry injectable preparation is a white or slightly yellowish tablet that dissolves in water for injection within a few seconds to form a clear or yellowish solution. Checking antiproliferative and anti-inflammatory activities, as described above, showed their complete safety from the original, when stored for 12 months at a temperature of 6-8 ° C.

Thus, a preparative method for producing a highly purified analog of rhIFN-γ, with antiproliferative and anti-inflammatory activities, which provides a high yield of the drug as well as its stability during storage, is proposed. The proposed method in comparison with the prototype allows you to:

- to achieve an increased yield of the target product (from 6 mg and above with 1 g of biomass);

- receive highly purified protein that meets all the indicators required for medical genetic engineering preparations;

- use increased amounts of biomass (30 g or more), it is easy to scale the process;

- use at the stages of chromatographic purification a commercially available sorbent with a high linear flow rate (KM-sepharose);

- provide high stability and preservation of the drug during storage.

Literature

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2. Dobeli H., Gents R., Jucker W., Garotta G., Hartmann D.W., Hochuli E. "Role of the carboxy-terminal sequence of the biological activity of human immune interferon". // J. Biotechnology, 1988, v. 7, p. 199-216.

3. Rutenfranz I., Bauer A., Kirchner H. "Pharmacokinetic study f liposome-encapsulated human interferon-g after intravenous and intramuscular injection in mice". // J. lEN. Res., 1990, v. 10, p. 337-341.

4. RF patent No. 2214832, cl. A61K 38/21, publ. 10.27.2003

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10. Tatkov S.I., Smirnova O.Yu., Tsivkovsky R.Yu., Kochneva G.V., Kuzmicheva G.A., Khristoforov VS, Kosarev I.S., Chernykh E.R., Khonina N. A., Lebedev L.R., Danilenko E.D., Fadina V.A., Pustoshilova N.M., Masycheva V.I., Sandakhchiev L.S. Human mutant gamma interferon with a shortened C-terminus and its properties // Dokl. RAS. - 2000. - T.372, No. 6. - S. 833-835.

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Claims (3)

1. A method of producing an analog of recombinant human gamma interferon, including culturing the producer strain Escherichia coli MH-1 / pIFD10, destroying cells with ultrasound, removing cell debris and chromatography on a sorbent column, characterized in that highly productive clones are used to obtain the inoculum, which are previously selected by electrophoretic analysis in a 15% polyacrylamide gel under denaturing conditions for the relative content in the transformed cells of the target protein, chromatographic the source of the product is carried out on KM-sepharose, first in a linear gradient of 50 mM Tris-HCl buffers, pH 8.3-50 mM sodium citrate, pH 6.5 with 0.5 M NaCl, and then in an exponential gradient of 50 mM sodium citrate, pH 6 5-50 mM Tris-HCl, pH 8.3 with 1 M NaCl. 2. The preparation of the analogue of recombinant human gamma interferon, containing the following components per one therapeutic dose of 1 ml: the recombinant human gamma interferon analog obtained according to claim 1, 50 μg / ml (50,000 IU / ml); low molecular weight polymer filler - stabilizer 50,000 microns / ml; NaCl 8.78 μg / ml; KH ₂ PO ₄ 1.36 μg / ml, (pH 7.0 ± 0.1). 3. The preparation of the analogue of recombinant human gamma interferon according to claim 2, characterized in that reopoliglukin or polyvinylpyrrolidone (dry) is used as a low molecular weight polymer stabilizer filler.

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