RU2809355C1 - RECOMBINANT PLASMIDS pET32a-TNF-Thy, PROVIDING SYNTHESIS OF HYBRID PROTEIN α-TUMOR NECROSIS FACTOR - THYMOSIN ALPHA 1, BACTERIAL STRAIN ESCHERICHIA COLI BL21 (DE3)/pET32a-TNF-Thy - PRODUCER OF HYBRID PROTEIN TNF-Thy, METHOD OF OBTAINING TNF HYBRID PROTEIN-Thy AND MEDICINAL PRODUCT - Google Patents

Abstract

FIELD: biotechnology; genetic engineering.

SUBSTANCE: subject of the invention is a medicinal product which is a solution for parenteral administration, containing osmotic pressure regulators, a buffer system maintaining a pH of 7.0–7.8, and a highly purified TNF-Thy hybrid protein with the amino acid sequence SEQ ID NO 1, wherein the TNF-Thy hybrid protein was obtained by cultivating the Escherichia coli producer strain, BL21(DE3)/pET32a-TNF-Thy, where, after 3–5 hours of cultivation, biosynthesis induction in Escherichia coli cells BL21(DE3)/pET32a-TNF-Thy isopropyl-β -D-1-thiogalactopyranoside and incubation for 4–6 hours are performed to obtain cell biomass of the TNF-Thy protein-producing strain, isolation from the resulting cell biomass of the TNF-Thy protein-producing strain, subsequent purification of the isolated TNF-Thy hybrid protein by anion-exchange and gel permeation chromatography under denaturing conditions, renaturation of the TNF-Thy fusion protein by diluting a urea solution and purification of the renatured TNF-Thy fusion protein by anion exchange chromatography to obtain a fusion protein with a purity of at least 97%, and Escherichia coli strain BL 21(DE3)/pET32a -TNF-Thy — producer of the hybrid protein TNF-Thy — obtained by transforming the parent strain E. coli BL21(DE3) with the recombinant plasmid pET32a-TNF-Thy of 5929 base pairs (bp), providing the synthesis of a protein having the amino acid sequence SEQ ID NO 1 is 20.5 kDa in size and containing the following structural elements: the T7 promoter and the lacO operator, the synthetic nucleotide sequence SEQ ID NO 2 encoding the TNF-Thy hybrid protein, the ampicillin antibiotic resistance gene (AmpR) and the bacterial ampicillin resistance gene promoter (AmpR promoter) for selection of recombinant cells, the origin of replication of bacteriophage f1 (f1 ori), the lactose repressor gene LacI and the bacterial promoter of the lactose repressor gene (Laclpromoter) in the following ratio of components (wt.%): 0.0052–0.0064 of TNF-Thy with a specific activity of at least 1.7×10⁶ U/mg and purity of at least 97%; 1.8–2.9 of osmotic pressure regulators; 2.2–3.6 of buffer system maintaining pH 7.0–7.8; the rest is water.

EFFECT: invention makes it possible to obtain an effective agent for parenteral administration, which is a solution containing a purer chemically and biologically stabilized hybrid protein TNF-Thy, which increases the efficiency of action and reduces the side effects of the hybrid protein due to optimally selected composition of components and their quantity, and also allows to increase the shelf life of the medicinal product up to 3 years at the temperatures from 2 to 8°C.

3 cl, 4 dwg, 1 tbl, 12 ex

Description

The invention relates to the field of biotechnology, in particular to genetic engineering, and concerns a new strain of bacteria Escherichia coli BL 21/pET32a-TNF-Thy, which can be used to obtain a hybrid protein α -tumor necrosis factor - thymosin alpha 1 (TNF-Thy protein ), used in the medical industry to obtain a medicinal product.

Therapeutically significant proteins obtained based on recombinant DNA technology have become widespread in recent years for the treatment of a wide range of diseases. The most significant and widespread drugs of this class include drugs created on the basis of recombinant erythropoietin, granulocyte colony-stimulating factor and human cytokines. What is common to this class of substances is the practical impossibility and inexpediency of isolating them from natural sources and, as a consequence, obtaining them in artificially created prokaryotic (usually E. Coli) or eukaryotic (usually CHO) systems.

The search for effective and safe drugs for the treatment of diseases characterized by high mortality or leading to severe disability of patients is one of the most important tasks of medical science.

Today, in clinical practice, there are a large number of antitumor drugs, but the effectiveness of most of them is insufficient and therefore the issue of developing new, more active drugs remains relevant, as well as the search for substances that are effective against tumors with primary and acquired resistance to drug therapy. In this regard, the decision to promote a new drug with antitumor activity is made based on the following criteria: mechanism of action, high antitumor activity, selective cytotoxicity against certain cultures of tumor cells in vitro and human tumor xenografts, lack of cross-resistance with known substances.

There is a constant search for new drugs that could improve the quality of treatment for patients with cancer.

In the process of preparing dosage forms based on proteins of this class, it is necessary to solve problems associated with the stability of the drug. The most common approach at present is the introduction of serum albumin into the finished drug as a stabilizer and/or freeze-drying. However, both of these approaches are not free from drawbacks. The introduction of human serum albumin as a stabilizer increases the risk of viral contamination of the drug. Freeze drying not only reduces the activity of the drug, but also causes partial denaturation, which in turn sometimes leads to allergic reactions in patients.

Another approach to solving the problem of stability of recombinant proteins in finished dosage forms is the introduction of urea, a complex of amphoteric electrolytes (amino acids) and detergents into the composition, followed by freeze-drying, where it was possible to obtain a stable dosage form of recombinant human erythropoietin. (RU, 2043118, C1, A61K 38/22, 1987) At the same time, there are significant restrictions on the shelf life of the drug after its dissolution.

A known lyophilized drug with immunomodulatory and antiviral activity "Neoferon", including recombinant human interferon alpha-2, hybrid protein T-TNF-T, a stabilizing additive containing a plasma replacement solution polyglucin, a saline buffer system with a pH of 7.0-7.2 and water pyrogen-free (RU, 2129878, C1, A61K 39/395, 1996). It is recommended to store the drug at +4°C. The disadvantage of the drug is that the stabilizing composition does not ensure long-term storage of the drug in liquid form, as well as the need for storage at fairly low temperatures - no higher than +4°C.

Methods for obtaining liquid dosage forms based on recombinant proteins are also known (RU, 2180233, C1, A61K 38/00, 2001).

The closest analogue to the claimed invention, in relation to the plasmid, is recombinant plasmid DNA encoding the synthesis of the recombinant hybrid protein α-tumor necrosis factor-thymosin-α ₁ (RU, 2225443, C1, C12N 1/70, 07/25/2002). Recombinant plasmid DNA pThy316 for expression in Escherichia coli cells of the hybrid protein α-tumor necrosis factor-thymosin-α ₁ , having a size of 3.77 kb. and containing promoters A2 and A3 of phage T7, providing constitutive expression of the gene of the hybrid protein a-tumor necrosis factor-thymosin-α ₁ ; a ribosome binding site located downstream of the A3 promoter; a gene encoding a fusion protein a-tumor necrosis factor-thymosin-α ₁ , located between the XbaI and ClaI restriction sites and including a second ClaI restriction site; a terminator region following the genome of the hybrid protein and bounded by two XbaI restriction sites; ampicillin resistance gene; gene for resistance to aminoglycosides (kanamycin, neomycin and G-418), containing a third ClaI restriction site; as well as unique restriction sites: PstI in the β-lactamase gene; XhoI, HindIII and SmaI, located in the aminoglycoside phosphotransferase gene; Kpnl between the A3 promoter and the ribosome binding site; EcoRI between promoters A3 and A2.

The disadvantage of this is the use of plasmid pThy315 as a basis for creating a plasmid vector. It uses a tandem of early promoters A2 and A3 of bacteriophage T7 as a promoter. This tandem is not effective for the expression of recombinant proteins. In addition, it ensures constitutive expression of the protein, which creates additional metabolic pressure on the cells of the producer Escherichia coli. This, in turn, leads to them achieving lower densities compared to producers with inducible promoters and reduces productivity. As a result, the final productivity of this method for producing the TNF-Thy hybrid protein turns out to be low, which makes the production efficiency based on it quite low and the labor intensity high.

A plasmid-based strain for producing the TNF-Thy fusion protein is not known from the prior art.

A method for producing the TNF-Thy hybrid protein is known from the prior art, which involves culturing cells of the transformed Escherichia coli strain SG20050 by introducing the plasmid pThy315, encoding the synthesis of the specified protein, in a liquid nutrient medium, followed by isolation and purification of the protein (RU 2055896, C1, C12P 21/00 , 07/29/1992). In this case, cultivation is carried out at 36-38°C in a medium containing up to 1.4% peptone or tryptone, up to 1.6% yeast extract, up to 1.15% NaCl and 50 μg/ml ampicillin. In this case, freshly obtained transformants are used or those selected at the initial phase of exponential growth and stored at a temperature from minus 20 to minus 40 ° C in broth with 40% glycerol. After cell lysis, the sediment is washed with 1-3 M urea solutions and dissolved in a 7 M urea solution, the resulting solution is applied to an anion-exchange sorbent at pH 5.6-5.8 with elution with the same buffer with the addition of 150 mM NaCl, after which fractions with a hybrid protein is applied to a sorbent with an immobilized dye (blue), equilibrated with a buffer with 7 M urea at pH 5.5-5.8, and the protein is eluted with a buffer with a gradient of pH 7.0-8.5 in the presence of 7 M urea and 1.0 M NaCl, followed by dialysis at pH 7.2-7.4 against a solution containing 150 mM NaCl and 10 mM Na-phosphate.

The disadvantage of this production method is the use of a sorbent with an immobilized dye (blue) during chromatographic purification of the protein. This type of sorbent undergoes partial degradation during use, and the dye (blue) is separated from the sorbent and spontaneously elutes from the column in the form of a protein-bound complex. Such impurities are unacceptable for the preparation of medicines.

The closest analogue to the claimed object of the invention, in relation to the method, is a method for obtaining a recombinant hybrid protein α-tumor necrosis factor-thymosin-α ₁ , in which the cultivation of a strain transformed by the introduction of the pThy316 plasmid is carried out at 37°C in a medium containing 1% peptone or tryptone, 1% yeast extract, 1% NaCl and 50 μg/ml kanamycin. (RU, 2225443, C1, C12N 1/70, 07/25/2002). After cell lysis, the lysate is centrifuged, the precipitate is washed with 1-2 M urea solutions and dissolved in a 7 M urea solution with 10 mM Tris-HCl, the resulting solution is applied to an anion-exchange sorbent at pH 7.5 with elution with the same buffer in a concentration gradient of 0-1 M NaCl, after which the fractions with the hybrid protein are applied to hydroxyapatite, equilibrated with 20 mM sodium phosphate buffer, pH 6.8, with 7 M urea, and the protein is eluted with a concentration gradient of 20-400 mM sodium phosphate buffer, pH 6.8, with 7 M urea. The resulting solution is applied to Sephadex G-50 sorbent, balanced with 10 mM sodium phosphate buffer pH 7.2 with 150 mM sodium chloride, chromatography is carried out to remove urea and renature the hybrid protein

The disadvantage of this method is the absence of a producer strain, and the preparation of a transformation mixture for fermentation does not allow the process to be carried out under strictly defined conditions, which affects the quality of the resulting hybrid protein. In addition, chromatography on a column with hydroxyapatite is not effective, as is renaturation under chromatographic conditions. Column chromatography does not allow obtaining a high level of renaturation of the hybrid protein. As studies have shown, the method described in the closest analogue, taking into account non-separation from oligomers, gives a low yield of renatured hybrid protein due to its aggregation (20-50%).

No analogues for the drug in the form of a solution based on the hybrid protein α-tumor necrosis factor-thymosin alpha 1 (TNF-Thy) have been identified from the prior art.

The objective of the invention is to develop a method for producing a liquid drug based on the hybrid protein tumor necrosis factor alpha-thymosin-alpha 1, which has high stable biological activity during storage and does not cause side effects, in the form of an aqueous solution for parenteral administration.

Also, the objective of the present invention is to create for the first time a strain producing the TNF-Thy hybrid protein based on a constructed plasmid that directs the synthesis of the TNF-Thy hybrid protein in order to increase the efficiency and reduce the labor intensity of obtaining the TNF-Thy hybrid protein, increasing the yield and purity of the TNF-Thy hybrid protein , as well as obtaining a more chemically and biologically pure and storage-stable drug in the form of an aqueous solution for parenteral administration, which is well tolerated, has minimal side effects and is highly active when used in the treatment of various diseases.

This task is achieved by creating a recombinant plasmid pET32a-TNF-Thy - 5929 base pairs (bp) in size, providing protein synthesis with the amino acid sequence SEQ ID NO 1 of 20.5 kDa in size and containing structural elements: T7 promoter and lacO operator, synthetic nucleotide sequence SEQ ID NO 2 encoding the hybrid protein TNF-Thy, the gene for resistance to the antibiotic ampicillin (AmpR) and the bacterial promoter of the gene for resistance to ampicillin (AmpR promoter) for selection of recombinant cells, the origin of replication of bacteriophage f1 (f1 ori), the lactose repressor gene LacI and the bacterial promoter of the lactose repressor gene (Laclpromoter),

The Escherichia coli strain BL 21/pET32a-TNF-Thy is also claimed to be a producer of the TNF-Thy hybrid protein obtained by transformation with the recombinant plasmid pET32a-TNF-Thy.

A method for producing a hybrid protein TNF-Thy having the amino acid sequence SEQ ID NO 1 is also claimed, including cultivating the producer strain Escherichia coli BL21(DE3)/ pET32a-TNF-Thy, and after 3-5 hours of cultivation the biosynthesis of the recombinant protein is induced cells of Escherichia coli BL21 (DE3)/pET32a-TNF-Thy and incubated for 4-6 hours, obtaining the cell biomass of the producer strain producing the TNF-Thy hybrid protein, isolating from the resulting cell biomass the strain-producer of the TNF-Thy hybrid protein, subsequent purification of the isolated TNF-Thy hybrid protein by anion exchange and gel permeation chromatography under denaturing conditions, renaturation of the TNF-Thy hybrid protein by diluting a urea solution and purification of the renatured TNF-Thy hybrid protein by anion exchange chromatography and obtaining a hybrid protein with a purity of at least 97%

In this case, the cultivation of Escherichia coli BL21 (DE3)/ pET32a-TNF-Thy cells is carried out in growth medium for at least 7 hours, but not more than 10 hours

And the induction of biosynthesis is preferably carried out by isopropyl-β-D-1-thiogalactopyranoside.

Isolation of the TNF-Thy fusion protein can be accomplished by separating cells from the culture fluid, disintegrating the cells, isolating inclusion bodies from the resulting disintegrate, and solubilizing the inclusion bodies.

Purification of the TNF-Thy hybrid protein is preferably carried out using anion exchange and gel permeation chromatography at a urea concentration of 7M-9M, pH 7.2-8.5

It is preferable to dilute the urea solution to a urea concentration of no more than 0.4 M

Another object of the claimed invention is a medicinal product, which is a solution for parenteral administration containing osmotic pressure regulators, a buffer system maintaining a pH of 7.0-7.8, and a highly purified TNF-Thy hybrid protein with the amino acid sequence SEQ ID NO 1, and The hybrid protein TNF-Thy was obtained by cultivating the producer strain of Escherichia coli, BL21(DE3)/ pET32a-TNF-Thy, where after 3-5 hours of cultivation, biosynthesis is induced in Escherichia coli cells BL21(DE3)/ pET32a-TNF-Thy isopropyl-β -D-1-thiogalactopyranoside and incubated for 4-6 hours, obtaining the cell biomass of the producer strain producing the TNF-Thy protein, isolating the TNF-Thy protein producer strain from the resulting cell biomass, subsequent purification of the isolated TNF-Thy hybrid protein by anion exchange and gel permeation chromatography under denaturing conditions, renaturation of the TNF-Thy hybrid protein by diluting a urea solution and purification of the renatured TNF-Thy hybrid protein by anion exchange chromatography to obtain a hybrid protein with a purity of at least 97%, and Escherichia coli strain BL 21(DE3) /pET32a-TNF-Thy is a producer of the TNF-Thy hybrid protein, obtained by transforming the parent strain E. coli BL21(DE3) with the recombinant plasmid pET32a-TNF-Thy of 5929 base pairs (bp), providing the synthesis of a protein having the amino acid sequence SEQ ID NO 1 is 20.5 kDa in size and contains the following structural elements: the T7 promoter and the lacO operator, the synthetic nucleotide sequence SEQ ID NO 2 encoding the TNF-Thy hybrid protein, the ampicillin antibiotic resistance gene (AmpR) and the bacterial promoter of the ampicillin resistance gene (AmpR promoter) for selection of recombinant cells, the origin of replication of bacteriophage f1 (f1 ori), the lactose repressor gene LacI and the bacterial promoter of the lactose repressor gene (Laclpromoter) with the following component ratio (mass%):

TNF-Thy with a specific activity of at least 1.7x 10 ⁶ U/mg and purity of at least 97% 0.0052-0.0064 Osmotic pressure regulators 1.8-2.9 Buffer system maintaining pH 7.0-7.8 2.2-3.6 Water rest

At the same time, it contains mannitol and sodium chloride as regulators of osmotic pressure. And as a buffer system maintaining a pH of 7.0 -7.8, it contains a phosphate buffer consisting of sodium phosphate disubstituted 12-aqueous and sodium phosphate disubstituted 2-aqueous.

Thus, the declared group of inventions is united by a single inventive concept aimed at creating for the first time a liquid medicinal product, which is obtained using the recombinant plasmid pET32a-TNF-Thy, providing the synthesis of the hybrid protein α-tumor necrosis factor -thymosin alpha1 by the first created bacterial strain Escherichia coli BL21 (DE3)/ pET32a-TNF-Thy hybrid protein producer, as well as a method for producing the TNF-Thy hybrid protein.

The method for obtaining the TNF-Thy protein from a newly created strain makes it possible to obtain a highly purified protein that does not have side effects, because it is free from the impurities inherent in protein obtained by other methods.

More specifically, the claimed invention relates to a composition for the first time containing the TNF-Thy protein in liquid form, having increased stability in solutions and high purity. This composition is also effective in terms of ease of manufacture and maximum concentration achievable per unit volume of dosage. According to this invention, the recombinant TNF-Thy protein is obtained from the first-created producer strain of Escherichia coli BL 21(DE3)/pET32a-TNF-Thy, into which the pET32a-TNF-Thy plasmid is inserted, carrying the recombinant gene for the tumor necrosis factor α hybrid protein -thymosin alpha1. The TNF-Thy protein has a purity of at least 97% and a specific biological activity of at least 1.7 x 10 ⁶ IU per 1 mg of protein.

The present invention relates to a TNF-Thy fusion protein having the amino acid sequence of SEQ ID NO 1, containing tumor necrosis factor α and thymosin-α1. The recombinant hybrid protein TNF-Thy, obtained from Escherichia coli strain BL21 (DE3)/pET32a-TNF-Thy, has the following parameters:

- The molecular weight of the monomer is 20.5±0.2 kDa.

- Protein impurities, oligomers, aggregates and monomers - no more than 3%;

- The content of cellular and vector DNA is no more than 100 pkg/10 ⁶ IU

- Molecular mass distribution of at least 97% trimer (61±2)kDa

- Bacterial endotoxins no more than 0.5 UE per 10 ⁴ units of activity

- Specific activity of at least 1.7×10 ⁶ U/mg protein

The inventive strain has the advantage that it was first obtained by transforming cells of the Escherichia coli strain BL 21(DE3) with recombinant plasmid DNA pET32a-TNF-Thy according to the present invention. It should be noted that the producer strain was obtained for the first time only through the original construction of the unique plasmid pET32a-TNF-Thy, which ensures effective gene expression, allowing the production of a significant amount of biomass containing the TNF-Thy hybrid protein.

Escherichia coli strain 21(DE3)/pET32a-TNF-Thy was deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures “GKPM-Obolensk” under number B-10277.

The present invention relates to a TNF-Thy fusion protein having the amino acid sequence SEQ ID NO1:

Below is the nucleotide sequence of the synthetic fragment encoding the TNF-Thy hybrid protein SEQ ID NO2

The invention is illustrated by the following graphic figures:

In FIG. 1 shows a Plasmid Map containing:

fl ori - origin of replication of bacteriophage f1,

AmpR - ampicillin resistance gene (β-lactamase gene),

ori - colE1 origin of replication,

T7 promoter - promoter of bacteriophage T7,

TNF-Thy - TNF-Thy hybrid protein gene,

T7 terminator - T7 bacteriophage terminator,

LacI - lactose repressor gene LacI.

In FIG. Figure 2 shows a photograph of the electrophoretic separation of a total cell lysate:

1 - before the start of induction

2 - at the moment of induction

3 - 2 hours after induction

4 - 4 hours after induction

5 - 5 hours after induction

6 - molecular weight standards, Pierce Unstained Protein MW Marker, Thermo, cat # 26610

Figure 3 shows a chromatogram of the hybrid protein α-tumor necrosis factor - thymosin-α1, obtained by the method described in the closest analogue (RU 2225443).

In FIG. Figure 4 shows a chromatogram of the proposed hybrid protein α-tumor necrosis factor - thymosin-α1 (TNF-Thy), obtained from Escherichia coli strain BL21 (DE3)/pET32a-TNF-Thy.

In this case, shown in Fig. 3, Fig. 4 results were obtained by high-performance exclusion chromatography. The method is used to determine the quality of protein according to the “Purity” indicator. Related compounds and extraneous impurities."

In the claimed technical solution, the problem is solved by creating a recombinant plasmid pET32a-TNF-Thy for the expression of the specified hybrid protein, first obtained by the highly productive bacterial producer strain Escherichia coli BL21(DE3)/pET32a-TNF-Thy, as well as a method for producing the hybrid protein TNF- Thy, which makes it possible to obtain the TNF-Thy hybrid protein in high yield and high purity. This makes it possible to use the hybrid protein α-tumor necrosis factor - thymosin-α1 in a medicinal product that has a long shelf life while maintaining biological activity in the absence of side effects on the human body.

The strain and purification method proposed for the first time have a number of significant advantages compared to those known from the prior art, incl. previously mentioned sources.

Because hybrid protein is used in the production of medicinal products, it must be taken into account that in order to ensure the consistency of the properties of the medicinal product containing acceptable impurities within a certain range, a number of requirements must be met during fermentation, protein expression and purification.

Obtaining a transformation mixture for fermentation, as in the closest analogue, and not a strain, does not allow the process to be carried out under strictly defined conditions described in standard operating procedures, which affects the quality of the resulting hybrid protein, and accordingly this affects the medicinal product. The creation of the Escherichia coli BL21(DE3)/pET32a-TNF-Thy producer strain for the first time makes it possible to obtain a reference culture, store a working culture of strain cells, and carry out the fermentation process under standardized conditions. This is currently one of the requirements of Good Manufacturing Practice (GMP). In the claimed invention, the yield of biomass from 1 liter of culture liquid ranges from 50 to 80 g. And in the above patent, a biomass of 8 g/l is obtained. Accordingly, the fermentation efficiency increased by 6-10 times.

It should be noted that chromatography on a column with SepraPrep™Q sorbent is more effective compared to DEAE cellulose, because allows to significantly reduce the time of the stage.

A method of chromatography on Sephadex G-100 under denaturing conditions was also announced for the first time. Dissolved and even purified of foreign impurities, inclusion bodies produce the TNF-Thy hybrid protein in various oligomeric forms. The hydroxylapatite chromatography described in the closest analogue does not allow the separation of these forms and is not a method that significantly increases the purity of the hybrid protein. The inventive method using urea chromatography on Sephadex G-100 made it possible to separate TNF-Thy oligomers and the trimeric active form of the hybrid protein. In addition, during this stage, purification from foreign high-molecular impurities occurs. The yield of the active trimeric form is 25-30% of the total protein. The purity at this stage is 90%, which allows the next important stage of the process to be carried out efficiently - obtaining a correctly folded TNF-Thy hybrid protein

Subsequent chromatography described in the closest analogue is carried out on a Sephadex G-50 column, equilibrated with 10 mM sodium phosphate buffer pH 7.2 with 150 mM sodium chloride, to remove urea and renature the hybrid protein. However, column chromatography does not allow obtaining a high level of renaturation of the hybrid protein. As studies have shown, the method described in the closest analogue, taking into account non-separation from oligomers, gives a low yield of renatured hybrid protein due to its aggregation (20-50%). A more accurate and simplified method for renaturation of a hybrid protein is claimed, by diluting a urea solution, without the use of expensive sorbents, while reducing the process time, which allows increasing the yield of correctly folded hybrid protein to 90-100%, while the purity of the hybrid protein is 97%.

Thus, a recombinant TNF-Thy fusion protein with a purity of at least 97% is claimed.

Also, the claimed TNF-Thy hybrid protein corresponds to the parameters presented above and is stable at pH from 7 to 8.5, while the specific activity is at least 1.7 x 10 ⁶ IU per 1 mg of protein.

For the first time, a drug based on the α-tumor necrosis factor, thymosin-al, was obtained according to this invention in liquid form. This drug is non-toxic and non-pyrogenic when tested for acute and chronic toxicity and pyrogenicity in mice and rabbits, and has controlled activity when tested in cell cultures. Has no side effects. As studies have shown, this is due to the absence of protein impurities and endotoxins.

In FIG. Figure 3 shows the results of the analysis of the hybrid protein α-tumor necrosis factor - thymosin-α1, obtained from the closest analogue. From the presented chromatogram it follows that the TNF-Thy hybrid protein solution contains protein impurities, the amount of which is 6.6%. Thus, the purity of the target protein is 93.4%.

The results of the analysis of the tumor necrosis factor α-thymosin-α1 fusion protein obtained from the claimed Escherichia coli strain BL21(DE3)/pET32a-TNF-Thy are shown in FIG. 4. As can be seen from the chromatogram, there are practically no impurities in this sample and the purity is 100%.

It is known that any medicinal substance does not enter the body in its pure form. Therefore, the main requirement for medicines is their chemical and biological purity. The presence of even small amounts of organic and/or inorganic impurities can cause unwanted side effects in patients.

When creating a drug, it is important not only the purity of the protein to reduce side effects, but also the preservation of the recombinant hybrid protein α-tumor necrosis factor - thymosin-α1 in an active form for an extended period of time.

The main problem associated with any protein compositions is overcoming the physical instability of the proteins. Physical instability does not cause changes in covalent bonds in proteins. Physical instability rather involves a change in the structure of higher order proteins, such as secondary structure. Such changes include denaturation, adsorption to surfaces, aggregation and precipitation.

Buffer solutions containing phosphates were used as a buffer. The use of buffer solutions to create a specific pH, which is usually essential for stabilizing solutions, is well known in pharmacy. However, their use is limited, since many of them react with drugs. The present invention has found that creating an optimal pH value of the solution using phosphates significantly improves the stability of interferon preparations and does not reduce the activity of the drug. The implementation of the invention determined that the buffer solution should be used in the pH range between 7.0 and 7.8, preferably 7.4

As agents that regulate osmotic pressure, it is preferable to use sodium chloride and mannitol in an amount sufficient to obtain an isotonic solution.

It should be noted that due to the production of highly purified TNF-Thy protein and due to the experimentally selected components: mannitol, sodium chloride, sodium phosphate disubstituted 12-aqueous and sodium phosphate disubstituted 2-aqueous, the claimed drug has no side effects and is isotonic, physiological . This fact is confirmed by the studies; none of the subjects experienced any side effects (itching, burning, inflammation at the injection site, allergic reactions, etc.), which is confirmed by the examples below.

The technical result of the claimed invention is the creation for the first time of a liquid medicinal product for parenteral administration, which is a solution containing a purer chemically and biologically stabilized hybrid protein TNF-Thy, which made it possible to increase the efficiency of action and reduce the side effects of the hybrid protein, due to the optimal composition of the components , and their quantity, and also made it possible to increase the shelf life of the drug to 2 years at a temperature of 2 to 8°C. In addition, a cheaper and safer drug that does not contain toxic components is obtained.

Also, the technical result of the claimed invention is to increase the efficiency of obtaining the hybrid protein TNF-Thy, namely, to increase the yield of protein from the biomass of the strain with a purity of the final product of at least 97%, due to the creation of a recombinant strain of Escherichia coli - a producer of the hybrid protein α-tumor necrosis factor - thymosin-α1, obtained on the basis of a constructed recombinant plasmid, which provides an increased yield of TNF-Thy, due to an increase in the amount of biomass obtained during cultivation by 6-10 times, while maintaining the content of total protein and the proportion of the TNF-Thy hybrid protein in the total protein cells.

This technical result is achieved by creating for the first time a strain based on the constructed recombinant plasmid pET32a-TNF-Thy and a method for producing protein using such a strain.

Also, the specified technical result is achieved by creating a medicinal product based on highly purified TNF-Thy hybrid protein in liquid form, while the TNF-Thy hybrid protein obtained according to this invention practically does not contain endotoxins, covalent oligomers, monomers and aggregates, but also According to the stated set of objects of the invention, during the production process it was possible not only to obtain the hybrid protein TNF-Thy biologically and chemically pure, but also to stabilize it due to optimally selected components.

The results of the stability study of the drug obtained according to Example No. 8 are presented in Table 1.

When implementing this preparation method, a preparation is obtained that ensures its storage at a temperature of 2 to 8 ° C for at least 2 years in sealed packaging.

The resulting drug is non-toxic and pyrogen-free when tested for acute and chronic toxicity and pyrogenicity in mice and rabbits, and has controlled antiviral activity when tested on human cell cultures, which is confirmed by the presented chromatogram of the claimed hybrid protein. In addition, the claimed drug does not have side effects.

The technical result is also achieved by creating a recombinant plasmid pET32a-TNF-Thy for the expression of a hybrid protein having the amino acid sequence SEQ ID NO 1, having a length of 5929 base pairs and consisting of the following key genetic elements:

- T7 promoter and lacO operator;

- a synthetic nucleotide sequence presented in SEQ ID NO 2, encoding a TNF-Thy fusion protein;

- the gene for resistance to the antibiotic ampicillin (AmpR) and the bacterial promoter of the gene for resistance to ampicillin (AmpRpromoter);

- the lactose repressor gene LacI and the bacterial promoter of the lactose repressor gene (Laclpromoter);

- origin of replication of bacteriophage f1 (f1 ori).

Plasmid pET32a was chosen as an expression vector. To obtain a plasmid according to the invention, the sequence SEQ ID NO 2, obtained by complete nucleotide synthesis, is inserted into the plasmid vector pET-32a at the XhoI and NdeI restriction sites.

This plasmid, hereinafter referred to as pET32a-TNF-Thy, consists of the following key genetic elements arranged in accordance with FIG. 1:

- the gene for resistance to the antibiotic ampicillin (AmpR) and the bacterial promoter of the gene for resistance to ampicillin (AmpRpromoter);

- origin of replication of bacteriophage f1 (f1 ori);

- T7 promoter and lacO operator;

- the lactose repressor gene LacI and the bacterial promoter of the lactose repressor gene (Laclpromoter);

- a synthetic nucleotide sequence presented in SEQ ID NO 2, encoding a Tnf-Thy hybrid protein (SEQ ID NO 1), including tumor necrosis factor α and thymosin-α ₁ .

The structure of said plasmid DNA (plasmid), consisting of said key genetic elements, is shown in FIG. 1.

In the above plasmid, the AmpR gene is intended for the selection of stable Escherichia coli cells. The bacterial ampicillin resistance gene promoter (AmpRpromoter) is designed for its expression.

The origin of replication of bacteriophage ƒ1 /Analysis ofGenesandGenomes, JohnWiley&Sons, 2004, S. 140/ is widely used to create expression vectors.

The translation product of the synthetic sequence SEQ ID NO 2 is a polypeptide of the sequence SEQ ID NO 1, including tumor necrosis factor α and thymosin-α ₁ .

Also, the specified technical result is achieved by creating a strain based on the constructed recombinant plasmid pET32a-TNF-Thy and a method for producing protein using such a strain.

After cloning the synthesized sequence as part of the vector, the resulting recombinant plasmid was transformed into Escherichia coli BL 21(DE3) bacterial cells.

The starting material for creating the producer strain according to the invention is the E. coli strain BL21 (DE3), genotype E. colistx, known from the prior art. B F ^- /HaeyoungJeong, ValérieBarbe, ChoongHoonLee, DavidVallenet, DongSuYu, Sang-HaengChoi, ArnaudCouloux, Seung-WonLee, SungHoYoon, LaurenceCattolico, Cheol-GooHur, Hong-SeogPark, BéatriceSegurens, SunChangKim, TaeKwangOh, RichardE. Lenski, F. William Studier, Patrick Daegelen, Jihyun F. Kim, Genome Sequences of Escherichia coli BstrainsREL606 and BL21(DE3), Journal of Molecular Biology, Volume 394, Issue 4, 2009, 644-652/.

For a better understanding of the essence of the claimed group of inventions, examples of implementation are given below.

Example 1: Construction of an expression plasmid

Chemical synthesis of oligonucleotides is performed by the solid-phase phosphoamidite method on an ASM-102U DNA synthesizer (BIOSSET, Novosibirsk) with the extension of the oligonucleotide chain in the direction from the 3'-end to the 5'-end using protected phosphoamidites - 5'-dimethoxytrityl-N-acyl-2 '-deoxynucleoside-3'-O-(β-cyanoethyl-diisopropylamino)-phosphites activated by tetrazole. The synthesis is carried out on a scale of 0.5-0.7 µmol, using porous glass (pore size 500 Å) as a carrier, to which the first nucleoside unit is attached via a 3'-succinate bond (load 20-30 µmol/g). The synthetic cycle of the standard phosphoamidite method is used. To prepare the vector, pET32a plasmid DNA (3 μg, 1 pmol) is treated in 40 μl of buffer 20 mM Tris acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 100 μg/ml BSA with restriction enzyme XhoI (10 act units), and then - in 40 μl of buffer 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl ₂ , 100 μg/ml BSA with restriction enzyme NdeI (10 act units) for 1 hour at 37°C. The vector fragment after electrophoresis in a 15% agarose gel is cut out from the gel and transferred to 200 μl of NT buffer, dissolved at 50°C for 5-10 minutes and applied to a NucleoSpin ExtractII column. Wash with NT 3 buffer and elute with 50 µl NE buffer.

The oligonucleotide sequence SEQIDNO 2, encoding the fusion protein SEQ ID NO 1, is obtained by total nucleotide synthesis. The resulting synthetic sequence SEQ ID NO 2 is treated in 40 μl of buffer with 20 mM Tris acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 100 μg/ml BSA with restriction enzyme XhoI (10 act units), and then in 40 μl of buffer 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl ₂ , 100 μg/ml BSA with NdeI restriction enzyme (10 units) for 1 hour at 37°C. After electrophoresis in a 15% agarose gel, the synthetic fragment is cut out from the gel and transferred to 200 μl of NT buffer, dissolved at 50°C for 5-10 minutes and applied to a NucleoSpinExtractII column. Wash with NT 3 buffer and elute with 50 µl NE buffer.

The resulting synthetic fragment described above in an amount of 2 pmol is added to a solution of 1 μg obtained from the DNA of the pET32a plasmid of the vector fragment described above in 10 μl of buffer (20 mM Tris-HCl, pH 7.56, 10 mM MgCl ₂ , 0.2 mM rATP, 10 mM dithiothreitol) and ligated with 10 units of act. T4-DNA ligase for 12 hours at 10°C.

Place tubes with competent cells (XL1-Blue, Evrogen) on ice until the contents are completely thawed at the rate of one tube per transformation. Gently mix the cell suspension with gentle shaking. Add an aliquot of the reaction mixture obtained after treatment with T4-DNA ligase to each test tube, and gently mix the contents with gentle shaking. Incubate the tubes in ice for 20-30 minutes. Transfer the tubes to a water bath (plus 42°C) for 30-45 seconds. Quickly transfer the tubes from the water bath to ice and incubate for 3-5 minutes. Add at least 3 volumes in advance and incubate at 37°C for 40-60 minutes in an orbital shaker-incubator (Multitron, Infors) at a speed of 225-250 rpm. Sow the contents of the tubes onto Petri dishes with a diameter of 60 mm.

An aliquot of the resulting plasmid DNA is used to transform competent E. coli BL21 (DE3) cells. Transformants are plated on plates with LB agar medium, to which ampicillin is added to a final ampicillin concentration of 50 μg/ml. The pET32a-TNF-Thy plasmid DNA is isolated from the clones. Recombinants are screened using sequencing.

Example 2. Obtaining the producer strain E. coli BL21 (DE3)/ pET32a-TNF-Thy and characterizing its productivity

The producer strain E. coliBL21 (DE3)/pET32a-TNF-Thy is obtained by transforming competent E. coli BL21 (DE3) cells with a plasmid, the preparation of which is described in Example 1.

Preferably (without limitation), for the introduction of the specified plasmid into the cells of the E. coli strain BL21 (DE3) using the electroporation method known from the prior art /Kleber-Janke, Wolf-Meinhard Becker, Use of Modified BL21(DE3) Escherichia coli Cells for High-Level Expression of Recombinant Peanut Allergens Affected by Poor Codon Usage, Protein Expression and Purification, Volume 19, Issue 3,2000, 419-424/ and incorporated herein by reference.

The producer strain E. coli BL21 (DE3)/pET32a-TNF-Thy is cultivated at 37°C in 100 ml of liquid nutrient medium LB with the addition of ampicillin to a final ampicillin concentration of 100 μg/ml for 3 hours in Erlenmeyer flasks (1 l, Corning) in an orbital shaker-incubator with a rotation speed of 220 rpm until the optical density of the culture liquid at a wavelength of 600 nm reaches 0.7-0.8 units. Then, the biosynthesis of the recombinant protein is induced by adding isopropyl-β-D-1-thiogalactopyranoside to a final concentration of isopropyl-β-D-1-thiogalactopyranoside 0.5 mM and incubated for 4 hours. A sample of 2 ml is taken every hour, the amount of culture is corresponding to 1 ml, centrifuge for 10 minutes at 6000 rpm. The precipitated cells are transferred to 100 μl of lysis buffer with bromophenol blue dye, with the addition of 2-mercaptoethanol, incubated for 5 minutes at 98°C, 3 μl aliquots are used for electrophoresis in 14% SDS-PAGE. The gel is stained by adding 0.1% Coomassie R-250 solution and scanned using a Shimadzu CS-930 densitometer. The results are presented in Fig. 2.: 5 - cell lysate, 6 - molecular weight standards. The yield of the TNF-Thy hybrid protein, according to the results of densitometric analysis, is at least 20% relative to the total cell protein.

The proposed producer strain Escherichia coli BL21(DE3)/pET32a-TNF-Thy is characterized by the following characteristics:

a) Morphological characteristics. The cells are rod-shaped, gram-negative, non-spore-bearing.

b) Cultural characteristics. Cells grow well on simple nutrient media. When grown on agar medium “LB” (per 1 liter 10 g peptone, 5 g yeast extract, 10 g NaCl, 20 g agar) the colonies are grayish, slightly convex, moist, shiny, with a pasty consistency.

c) Physical and biological characteristics. Cells grow at temperatures from 4°C to 40°C with an optimal pH value of 6.8 to 7.5. Both mineral salts in ammonium form and organic compounds, including peptone, tryptone, yeast extract, amino acids, etc., are used as a source of nitrogen. Amino acids, glycerol, and carbohydrates are used as a carbon source.

d) Antibiotic resistance. Cells exhibit resistance to penicillin antibiotics (up to 500 μg/ml).

Example 3. Method for producing TNF-Thy fusion protein

Cultivation of the producer strain

A 20-liter fermenter is used under conditions according to Example 2. After the end of cultivation, the cells of the recombinant protein producer (biomass) are separated by centrifugation (5000 g, 20 min, 4°C).

100 g of biomass is suspended in 10 l of 0.05 M phosphate buffer pH 7.2 with 0.15 M sodium chloride and cooled to plus 8°C. The cell suspension is passed twice through a Ranie disintegrator spinneret at a pressure of 300 and 600 bar. The suspension of destroyed cells is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at a temperature from plus 4 to 8°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 2 mM EDTA, 0.15 M NaCl, 1% Triton X-100, pH 7.0. The sediments are suspended and incubated for 30 minutes at a temperature from plus 4 to 8°C. The suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30. 400 ml of buffer 20 mM Tris, 2 mM EDTA, 0.15 M NaCl, 0.5% Triton X- are added to the sediments in centrifuge beakers. 100, pH 8.0. The sediments are suspended and incubated for 30 min at 8°C. Then the suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at a temperature from plus 4 to 8°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 2 mM EDTA, 1 M urea, pH 6.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. The suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 5 mM EDTA, 2 M urea, pH 6.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. The suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

The precipitates are dissolved by adding a buffer of 20 mM Tris, 5 mM EDTA, 7 M urea, pH 7.0 and incubated for 18 hours at (6±2)°C. The resulting protein solution is clarified by centrifugation in a JA-14 rotor at 13,000 rpm for 60 minutes at plus (6±2)°C. Next, purification is carried out on a 2-liter column packed with SepraPrep Q sorbent and equilibrated with a buffer of 20 mM Tris⋅HCl, 7 M urea, pH 6.8. After applying the protein, the column is washed with the same buffer and passed through 4 liters of buffer 20 mM Tris⋅HCl, 20 mM NaCl, 7 M urea, pH 6.8, then 20 mM Tris⋅HCl, 50 mM NaCl, 7 M urea, pH 6.8 and 20 mM Tris⋅HCl, 200 mM NaCl, 7 M urea pH 6.8. In the fraction obtained after elution with a buffer of 20 mM Tris⋅HCl, 200 mM NaCl, 7 M urea pH 6.8, increase the concentration of urea from 7 M to 9 M and maintain at plus 16°C for 18 hours. Next, the process of concentrating the protein solution is carried out TNF-Thy on “hollow fibers” in the AR-1-15PS apparatus, reducing the volume to 0.5 l of protein concentrate.

Chromatographic purification of TNF-Thy protein on Sephadex G-100

Apply 500 ml of TNF-Thy protein concentrate to a PSC 200/1000 column with 28.3 L of Sephadex G-100 and elute using gel filtration buffer 20 mM Tris⋅HCl, 150 mM NaCl, 9 M urea, pH 7, 2 at a rate of 3.0 ml/min. In the collected fractions, _A280 and _A260 are determined spectrophotometrically. The presence of TNF-Thy is confirmed using 17.5% sodium dodecyl sulfate (SDS) PAGE. Fractions containing, according to electrophoresis in 17.5% SDS-PAGE, more than 90% TNF-Thy, are combined. In the combined fraction of the highly purified preparation, TNF-Thy is determined spectrophotometrically: A ₂₈₀ , A ₂₅₄ , protein. The purity of the TNF-Thy preparation is determined by electrophoresis in 17.5% SDS-PAGE, as well as by HPLC in a C ₁₈ silica gel column.

The combined fraction containing TNF-Thy with a purity greater than 90% is transferred to the renaturation stage.

Renaturation of TNF-Thy protein

To carry out renaturation, a glass reactor from Simax with a mixing device and a cooling system is used. 50 liters of phosphate buffer with sodium chloride with pH 7.3 are loaded into the cleaned reactor and cooled to plus (12±1) C. Through a filter with pores of 0.22 microns, the cooled buffer is fed into the reactor at a speed of (50±5) ml/ min 2 l of purified TNF-Thy protein solution. After adding the entire protein solution, incubation is carried out with the stirrer running. During the renaturation process, the solution temperature is maintained at (12±1)°C using a cooling system. Duration of the process (18±1) hours.

Chromatographic purification of renatured TNF-Thy hybrid protein on DEAE Sepharose Fast Flow sorbent

A 2 L DEAESepharose FastFlow column is used. Equilibrate with 20 mM phosphate buffer, pH 7.3. A solution of the renatured fusion protein in phosphate buffer is then applied and washed with equilibration buffer.

20 mM phosphate buffer, 80 mM NaCl, pH 7.3 and 20 mM phosphate buffer, 300 mM NaCl, pH 7.3 are successively passed through the column with the sorbent. All eluted proteins are collected in 0.4 L fractions and analyzed by electrophoresis in 17.5% SDS-PAGE. Fractions containing at least 95% TNF-Thy according to electrophoresis data of 17.5%) on SDS-PAGE are combined. After combining, the TNF-Thy solution is transferred to sterilizing filtration and the parameters are controlled. A sample is also taken to determine biological activity. The resulting TNF-Thy solution is stored at minus (20±2)°C.

Activity Definition

To study the antitumor activity of the protein, we used a strain of diploid cells of mouse subcutaneous connective tissue (NCTCclone 929 [Lcell, L-929, derivative of StrainL], ATCC® CCL-1™). The ampoule with cells is transferred from liquid nitrogen to a water bath at a temperature of (40±0.1)°C. After thawing, the cells are aseptically transferred to a plastic mattress with a capacity of 500 ml and growth medium is added to it at intervals of 1.5-2 minutes (DMEM medium - 90%, fetal calf serum liquid - 10%) in an amount of 0.25; 0.25; 0.5; 0.5; 0.75; 1.0; 1.0; 2.0; 2.0; 10.0 ml. After counting the number of cells, the concentration of cells in the suspension is adjusted to the inoculum by adding growth medium with the addition of antibiotics (kanamycin 100 U/ml, gentamicin 80-160 U/ml). Cells form a monolayer with typical morphology on the 4th day.

To determine the activity of the drug, control the number of cell culture passages, which should not exceed 30 passages.

The cell monolayer obtained in a 500 ml mattress is removed from the glass with a 0.125% trypsin solution and suspended in 40 ml of growth medium and the number of cells in the Goryaev chamber is counted. The suspension is diluted with growth medium so that 1.0 ml contains 200 thousand cells. 0.1 ml of cell suspension is added to the wells of microplates. The prepared tablet is incubated for 48-72 hours at a temperature of (37±1.0)°C and a CO ₂ concentration of (5.0±0.5)%.

To determine activity, first prepare ten-fold dilutions: 1:10, 1:100, 1:1000 and 1:10000, and then successive two-fold dilutions: 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, 1:640000, 1:1280000, 1:2560000 of the test sample in the growth medium. The medium is removed from the plate with a monolayer of cells into a waste glass and 100 μl of serial dilutions of the sample and 100 μl of maintenance medium with actinomycin D at a concentration of 2 μg/ml are added to the wells. For each dilution, use at least 4 wells of cell culture. 4 wells with culture are left as controls. An additional 100 μl of maintenance medium with actinomycin D is added to the control wells. The prepared plate is incubated for 48 hours at a temperature of (37 ± 1.0) ° C and a CO ₂ concentration of (5.0 ± 0.5)%.

A preliminary assessment of the state of the monolayer is carried out after 24 hours. Further recording of TNF-Thy activity is carried out if there are no signs of degeneration in the control culture. The final count is carried out after 48 hours. To do this, the monolayer is washed from dead cells with a sterile saline solution. To do this, first remove the growth medium from the wells, add 200 μl of saline solution, which is also removed. Repeat 2-3 times. The contents of the wells are then stained with a 0.2% crystal violet solution. To do this, after removing the growth medium, add 100 μl of 96% ethyl alcohol into the wells and incubate for 10 minutes. The alcohol is removed and the wells are washed with purified water, adding 100 µl per well and removing it by shaking off. The washing procedure is repeated three times. The plates are then air dried for 30 minutes. Then add 200 μl of a 0.2% crystal violet solution to each well and keep the plate for 20 minutes with vigorous shaking. The dye is removed by shaking off and the plates are washed with purified water. To remove bound dye, 100 μl of 10% acetic acid is added to the wells, which is then removed by shaking off. The results are recorded visually (using an inverted microscope) or automatically, using a tablet spectrophotometer, at a wavelength of 540 nm.

The activity of the test sample is taken to be the reciprocal of the dilution at which 50±5% cell death occurs.

Specific activity is calculated using the formula

where P is the dilution of the sample at which 50% damage to the monolayer is observed (a decrease in optical density by 50% of the control).

Calculation of specific activity:

where A is the specific activity of the drug sample, units;

B - amount of protein in the drug sample, mg

A hybrid protein is obtained with a purity of 97.5% and a specific biological activity of 1.9 x 10 ⁶ IU per 1 mg of protein.

Example 4. Method for producing TNF-Thy fusion protein

Cultivation of the producer strain

A 20-liter fermenter is used under the conditions of Example 2, but after inducing the biosynthesis of the recombinant protein by adding isopropyl-β-D-1-thiogalactoprianoside to a final concentration of isopropyl-β-D-1-thiogalactoprianoside 0.5 mM, the culture liquid is incubated for 6 hours. After the end of cultivation, the recombinant protein producer cells (biomass) are separated by centrifugation (5000 g, 20 min, 4°C).

100 g of biomass is suspended in 10 l of 0.05 M phosphate buffer pH 7.2 with 0.15 M sodium chloride and cooled to plus (6±2)°C. The cell suspension is passed twice through a Ranie disintegrator spinneret at a pressure of 300 and 600 bar. The suspension of destroyed cells is centrifuged in a J A-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 2 mM EDTA-0.15 M NaCl, 1% Triton X-100, pH 7.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. The suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C. To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 2 mM EDTA, 0.15 M NaCl, 0.5% Triton X-100, pH 8.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. Then the suspension of “inclusion bodies” is centrifuged in a J A-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 2 mM EDTA, 1 M urea, pH 6.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. The suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 5 mM EDTA, 2 M urea, pH 6.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. The suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

The precipitates are dissolved by adding a buffer of 20 mM Tris, 5 mM EDTA, 7 M urea, pH 7.0 and incubated for 18 hours at (6±2)°C. The resulting protein solution is clarified by centrifugation in a JA-14 rotor at 13,000 rpm for 60 minutes at plus (6±2)°C. Next, purification is carried out on a 2 L column packed with SepraPrep Q sorbent and equilibrated with a buffer of 20 mM Tris-HCl, 7 M urea, pH 6.8. After applying the protein, the column is washed with the same buffer and passed through 4 l of buffer 20 mM Tris-HCl, 20 mM NaCl, 7 M urea, pH 6.8, then 20 mM Tris-HCl, 50 mM NaCl, 7 M urea, pH 6.8 and 20 mM Tris-HCl, 200 mM NaCl, 7 M urea pH 6.8. In the fraction obtained after elution with a buffer of 20 mM Tris-HCl, 200 mM NaCl, 7 M urea pH 6.8, increase the concentration of urea from 7 M to 9 M and maintain at plus 16°C for 18 hours. Next, the process of concentrating the protein solution is carried out TNF-Thy on “hollow fibers” in the AR-1-15PS apparatus, reducing the volume to 0.5 l of protein concentrate.

Chromatographic purification of TNF-Thy protein on Sephadex G-100

Apply 500 ml of TNF-Thy protein concentrate to a PSC 200/1000 column with 28.3 l of SephadexG-100 and elute using gel filtration buffer 20 mM Tris-HCl, 150 mM NaCl, 9 M urea, pH 7.2 at a rate of 3.0 ml/min.

In the collected fractions, _A280 and _A260 are determined spectrophotometrically. The presence of TNF-Thy is confirmed using 17.5% sodium dodecyl sulfate (SDS) PAGE. Fractions containing, according to electrophoresis in 17.5% SDS-PAGE, more than 90% TNF-Thy, are combined.

In the combined fraction of the highly purified preparation, TNF-Thy is determined spectrophotometrically: A ₂₈₀ , A ₂₈₀ , protein. The purity of the TNF-Thy preparation is determined by electrophoresis in 17.5% SDS-PAGE, as well as by HPLC in a C ₁₈ silica gel column. The combined fraction containing TNF-Thy with a purity greater than 90% is transferred to the renaturation stage.

Renaturation of TNF-Thy protein

To carry out renaturation, a glass reactor from Simax with a mixing device and a cooling system is used. 50 liters of phosphate buffer with sodium chloride with pH 7.3 are loaded into the cleaned reactor and cooled to plus (12±1)°C. Through a filter with pores of 0.22 μm, 2 liters of a solution of purified TNF-Thy protein is fed into the reactor with a cooled buffer at a speed of (50±5) ml/min. After adding the entire protein solution, incubation is carried out with the stirrer running. During the renaturation process, the solution temperature is maintained at (12±1)°C using a cooling system. Duration of the process (18±1) hours.

Chromatographic purification of renatured TNF-Thy hybrid protein on DEAE Sepharose Fast Flow sorbent

A 2 L DEAESepharose Fast Flow column is used. Equilibrate with 20 mM phosphate buffer, pH 7.3. Then the protein solution from the renaturation step in phosphate buffer is applied and washed with equilibration buffer.

20 mM phosphate buffer, 80 mM NaCl, pH 7.3 and 20 mM phosphate buffer, 300 mM NaCl, pH 7.3 are successively passed through the column with the sorbent. All eluted proteins are collected in 0.4 L fractions and analyzed by electrophoresis in 17.5% SDS-PAGE. Fractions containing at least 95% TNF-Thy according to electrophoresis data of 17.5%) on SDS-PAGE are combined. After combining, the TNF-Thy solution is transferred to sterilizing filtration and the parameters are controlled. A sample is also taken to determine biological activity. The resulting TNF-Thy solution is stored at minus (20±2)°C.

Determination of activity is carried out as described in Example 3.

A hybrid protein is obtained with a purity of 98%, a specific activity of 1.25 x 10 ⁶ IU/ml and a specific biological activity of 2.0 x 10 ⁶ IU per 1 mg of protein.

Example 5. Method for producing TNF-Thy fusion protein

Cultivation of the producer strain

A 20-liter fermenter is used under conditions according to Example 2. After the end of cultivation, the cells of the recombinant protein producer (biomass) are separated by centrifugation (5000 g, 20 min, 4°C).

100 g of biomass is suspended in 10 l of 0.05 M phosphate buffer pH 7.2 with 0.15 M sodium chloride and cooled to plus (6±2)°C. The cell suspension is passed twice through a Ranie disintegrator spinneret at a pressure of 300 and 600 bar. The suspension of destroyed cells is centrifuged in a J A-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 2 mM EDTA, 0.15 M NaCl, 1% Triton X-100, pH 7.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. The suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C. To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 2 mM EDTA, 0.15 M NaCl, 0.5% Triton X-100, pH 8.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. Then the suspension of “inclusion bodies” is centrifuged in a J A-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 2 mM EDTA, 1 M urea, pH 6.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. The suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 5 mM EDTA, 2 M urea, pH 6.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. The suspension of “inclusion bodies” is centrifuged in a J A-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

The precipitates are dissolved by adding a buffer of 20 mM Tris, 5 mM EDTA, 7 M urea, pH 7.0 and incubated for 18 hours at (6±2)°C. The resulting protein solution is clarified by centrifugation in a JA-14 rotor at 13,000 rpm for 60 minutes at plus (6±2)°C. Next, purification is carried out on a 2-liter column packed with SepraPrep Q sorbent and equilibrated with a buffer of 20 mM Tris⋅HCl, 7 M urea, pH 6.8. After applying the protein, the column is washed with the same buffer and passed through 4 liters of buffer 20 mM Tris⋅HCl, 20 mM NaCl, 7 M urea, pH 6.8, then 20 mM Tris⋅HCl, 50 mM NaCl, 7 M urea, pH 6.8 and 20 mM Tris⋅HCl, 200 mM NaCl, 7 M urea pH 6.8. In the fraction obtained after elution with a buffer of 20 mM Tris⋅HCl, 200 mM NaCl, 7 M urea pH 6.8, increase the concentration of urea from 7 M to 9 M and maintain at plus 16°C for 18 hours. Next, the process of concentrating the protein solution is carried out TNF-Thy on “hollow fibers” in the AR-1-15PS apparatus, reducing the volume to 0.5 l of protein concentrate.

Chromatographic purification of TNF-Thy protein on Sephadex G-100

Apply 500 ml of TNF-Thy protein concentrate to a PSC 200/1000 column with 28.3 l of Sephadex G-100 and perform elution using a gel filtration buffer of 20 mM Tris⋅HCl, 150 mM NaCl, 7 M urea, pH 8, 5 at a rate of 3.0 ml/min.

In the collected fractions, _A280 and _A260 are determined spectrophotometrically. The presence of TNF-Thy is confirmed using 17.5% sodium dodecyl sulfate (SDS) PAGE. Fractions containing, according to electrophoresis in 17.5% SDS-PAGE, more than 90% TNF-Thy, are combined.

In the combined fraction of the highly purified preparation, TNF-Thy is determined spectrophotometrically: A ₂₈₀ , A ₂₆₀ , protein. The purity of the TNF-Thy preparation is determined by electrophoresis in 17.5% SDS-PAGE, as well as by HPLC data in a C ₁₈ silica gel column. The combined fraction containing TNF-Thy with a purity greater than 90% is transferred to the renaturation stage.

Renaturation of TNF-Thy protein

To carry out renaturation, a glass reactor from Simax with a mixing device and a cooling system is used. 50 liters of phosphate buffer with sodium chloride with pH 7.3 are loaded into the cleaned reactor and cooled to plus (12±1)°C. Through a filter with pores of 0.22 μm, 2 liters of a solution of purified TNF-Thy protein is fed into the reactor with a cooled buffer at a speed of (50±5) ml/min. After adding the entire protein solution, incubation is carried out with the stirrer running. During the renaturation process, the solution temperature is maintained at (12±1)°C using a cooling system. Duration of the process (18±1) hours.

Chromatographic purification of renatured TNF-Thy hybrid protein on DEAE Sepharose Fast Flow sorbent

A 2 L DEAESepharose Fast Flow column is used. Equilibrate with 20 mM phosphate buffer, pH 7.3. Then a solution of the protein obtained at the renaturation stage in phosphate buffer is applied and washed with equilibration buffer. 20 mM phosphate buffer, 80 mM NaCl, pH 7.3 and 20 mM phosphate buffer, 300 mM NaCl, pH 7.3 are successively passed through the column with the sorbent.

All eluted proteins are collected in 0.4 L fractions and analyzed by electrophoresis in 17.5% SDS-PAGE. Fractions containing at least 95% TNF-Thy according to electrophoresis in 17.5% SDS-PAGE are combined. After combining, the TNF-Thy solution is transferred to sterilizing filtration and the parameters are controlled (protein, e/f, HPLC). A sample is also taken to determine biological activity. The resulting TNF-Thy solution is stored at minus (20±2)°C.

Determination of activity is carried out as described in Example 3.

A hybrid protein is obtained with a purity of 98.5%, a specific activity of 1.3 x 10 ⁶ IU/ml and a specific biological activity of 2.0 x 10 ⁶ IU per 1 mg of protein.

Example 6. Method for producing TNF-Thy fusion protein

Cultivation of the producer strain

A 20-liter fermenter is used under conditions according to Example 2. After the end of cultivation, the cells of the recombinant protein producer (biomass) are separated by centrifugation (5000 g, 20 min, 4°C).

100 g of biomass is suspended in 10 l of 0.05 M phosphate buffer pH 7.2 with 0.15 M sodium chloride and cooled to plus (6±2)°C. The cell suspension is passed twice through a Ranie disintegrator spinneret at a pressure of 300 and 600 bar. The suspension of destroyed cells is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 2 mM EDTA, 0.15 M NaCl, 1% Triton X-100, pH 7.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. The suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C. To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 2 mM EDTA, 0.15 M NaCl, 0.5% Triton X-100, pH 8.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. Then the suspension of “inclusion bodies” is centrifuged in a J A-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 2 mM EDTA, 1 M urea, pH 6.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. The suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

To the sediments in centrifuge beakers, add 400 ml of buffer 20 mM Tris, 5 mM EDTA, 2 M urea, pH 6.0. The sediments are suspended and incubated for 30 minutes at (6±2)°C. The suspension of “inclusion bodies” is centrifuged in a JA-10 rotor at 9000 rpm for 30 minutes at plus (6±2)°C.

The precipitates are dissolved by adding a buffer of 20 mM Tris, 5 mM EDTA, 7 M urea, pH 7.0 and incubated for 18 hours at (6±2)°C. The resulting protein solution is clarified by centrifugation in a JA-14 rotor at 13,000 rpm for 60 minutes at plus (6±2)°C. Next, purification is carried out on a 2-liter column packed with SepraPrep Q sorbent and equilibrated with a buffer of 20 mM Tris⋅HCl, 7 M urea, pH 6.8. After applying the protein, the column is washed with the same buffer and passed through 4 liters of buffer 20 mM Tris⋅HCl, 20 mM NaCl, 7 M urea, pH 6.8, then 20 mM Tris⋅HCl, 50 mM NaCl, 7 M urea, pH 6.8 and 20 mM Tris⋅HCl, 200 mM NaCl, 7 M urea pH 6.8. In the fraction obtained after elution with a buffer of 20 mM Tris⋅HCl, 200 mM NaCl, 7 M urea pH 6.8, increase the concentration of urea from 7 M to 9 M and maintain at plus 16°C for 18 hours. Next, the process of concentrating the protein solution is carried out TNF-Thy on “hollow fibers” in the AR-1-15PS apparatus, reducing the volume to 0.5 l of protein concentrate.

Chromatographic purification of TNF-Thy protein on Sephadex G-100

Apply 500 ml of TNF-Thy protein concentrate to a PSC 200/1000 column with 28.3 L of Sephadex G-100 and elute using gel filtration buffer 20 mM Tris⋅HCl, 150 mM NaCl, 9 M urea, pH 7, 2 at a rate of 3.0 ml/min.

In the collected fractions, _A280 and _A260 are determined spectrophotometrically. The presence of TNF-Thy is confirmed using 17.5% sodium dodecyl sulfate (SDS) PAGE.

Fractions containing, according to electrophoresis in 17.5% SDS-PAGE, more than 90% TNF-Thy, are combined.

In the combined fraction of the highly purified preparation, TNF-Thy is determined spectrophotometrically: A280, A260, protein. The purity of the TNF-Thy preparation is determined by electrophoresis in 17.5% SDS-PAGE, as well as by HPLC data in a C ₁₈ silica gel column. The combined fraction containing TNF-Thy with a purity greater than 90% is transferred to the renaturation stage.

Renaturation of TNF-Thy protein

To carry out renaturation, a glass reactor from Simax with a mixing device and a cooling system is used. 90 liters of phosphate buffer with sodium chloride with pH (7.3±0.1) are loaded into the cleaned reactor and cooled to plus (12±1)°C. Through a filter with pores of 0.22 μm, 2 liters of a solution of purified TNF-Thy protein is fed into the reactor with a cooled buffer at a speed of (50±5) ml/min. After adding the entire protein solution, incubation is carried out with the stirrer running. During the renaturation process, the solution temperature is maintained at (12±1)°C using a cooling system. Duration of the process (18±1) hours.

Chromatographic purification of renatured TNF-Thy hybrid protein on DEAE Sepharose Fast Flow sorbent

A 2 L DEAESepharose Fast Flow column is used. Equilibrate with 20 mM phosphate buffer, pH 7.3. Then a solution of the protein obtained at the renaturation stage in phosphate buffer is applied and washed with equilibration buffer.

20 mM phosphate buffer, 80 mM NaCl, pH 7.3 and 20 mM phosphate buffer, 300 mM NaCl, pH 7.3 are successively passed through the column with the sorbent.

All eluted proteins are collected in 0.4 L fractions and analyzed by electrophoresis in 17.5% SDS-PAGE. Fractions containing at least 95% TNF-Thy according to electrophoresis in 17.5% SDS-PAGE are combined. After combining, the TNF-Thy solution is transferred to sterilizing filtration and the parameters are monitored. A sample is also taken to determine biological activity. The resulting TNF-Thy solution is stored at minus (20±2)°C.

Determination of activity is carried out as described in Example 3.

A hybrid protein is obtained with a purity of 97%, a specific activity of 1.2 x 10 ⁶ IU / ml and a specific biological activity of 1.85 x 10 ⁶ IU per 1 mg of protein

Example 7. Preparation of a drug with an activity of 90,000 units/vial

Composition: hybrid protein obtained from Escherichia coli strain BL21 (DE3)/pET32a-TNF-Thy, according to example 4 - 7.2 ml, mannitol -1.5 g, sodium chloride -0.88 g, sodium phosphate disubstituted 12-water -2.9 g, sodium phosphate monosubstituted 2-water -0.3 g, water for injection up to 100 ml.

All components, with the exception of TNF-Thy, were weighed and dissolved in water for injection. The pH of the solution was checked and, if necessary, adjusted to 7.4. Then the thawed TNF-Thy solution obtained according to example 4 was added

The solution was filtered under sterile conditions. The bottles were aseptically filled with 1 ml of the resulting composition and rolled. The product was stored at a temperature of 2 to 8°C.

The activity of the drug was determined

The drug should have a cytotoxic effect in L-929 cell culture.

A diploid mouse subcutaneous connective tissue cell strain (NCTCclone 929 [Lcell, L-929, derivativeofStrainL], ATCC® CCL-1™) is stored as a frozen suspension. One cryovial with test culture cells from a Dewar flask with liquid nitrogen is placed in a water bath until the suspension is completely thawed (for no more than 2 minutes) at a temperature of (37±2)°C. In a laminar flow hood under aseptic conditions, the contents of the test tube are transferred to a disposable sterile a 15 ml centrifuge tube containing 6 ml of maintenance medium (see Note 3). After centrifugation at 2000 rpm for 10 minutes, the supernatant is discarded, 10 ml of growth medium is added to the sediment (see Note 2), the sediment is resuspended and the suspension is transferred to a 25 cm flask for cultivation. The bottle is placed in a CO ₂ incubator for cultivation under standard conditions (at a temperature of (37±2)°C and (5.0±0.5)% carbon dioxide) for 48-72 hours until a monolayer is formed.

After the formation of a monolayer on the surface of the bottom of the culture flask, the cell passaging procedure is carried out, i.e. transferring part of the monolayer cells into a second culture flask with growth medium. Cell transfer must be done without allowing the formation of a dense layer to ensure that the cells are in the logarithmic growth phase.

The culture medium is removed from the bottle with the formed cell monolayer and washed three times with 5-6 ml of 0.25% Trypsin-EDTA solution heated to a temperature of (37±2)°C. The last portion of the solution added to the bottle is not completely removed, but about 1 ml is left and distributed evenly over the entire surface of the monolayer. The vial with cells is placed in a CO ₂ incubator until the cells are completely detached from the bottom of the culture vial. Add 2 ml of growth medium, suspend the cells by pipetting, transfer them to a Petri dish and take a sample to count the number of cells in the Goryaev chamber. Transfer 1-1.5 ml of cell suspension into a new vial and add 5-6 ml of growth medium until the seeding cell concentration is 100 thousand cells/ml. After transferring the detached cells to a new vial with growth medium, they are both placed in standard culture conditions for 24-48 hours. When a monolayer is formed at the bottom of the bottles, one bottle is used for repeated transfer of cells and determination of specific activity, and with the other the procedure for removing cells is repeated for placing in a working cell bank.

Before each procedure of working with cells, the morphology of the cells and the purity of the culture in the culture flask are assessed using an inverted microscope. In the absence of foreign microflora and normal cell morphology, the culture is determined to be suitable for use.

Next, prepare the test sample. Prepare three tenfold dilutions. To do this, 900 μl of a supporting medium with actinomycin D is added to Eppendorf tubes. 100 μl of a solution of the test drug is added to test tube No. 1 and mixed thoroughly. Transfer 100 μl of solution from test tube No. 1 into test tube No. 2 and mix thoroughly. Transfer 100 μl of the solution from test tube No. 2 into test tube No. 3 and mix thoroughly.

Double working dilutions of the test sample are prepared from test tube No. 3. To do this, add 500 µl of the supporting medium with actinomycin D into 8 Eppendorf tubes. Transfer 500 µl from tube No. 3 with the solution of the test drug to tube No. 4 and mix thoroughly. From test tube No. 4, transfer 500 μl of the solution to test tube No. 5 and mix thoroughly. Repeat the procedure for test tubes No. 6-11. The following dilutions are obtained: 1:2×10 ³ , 1:4×10 ³ , 1:8×10 ³ , 1:16×10 ³ , 1:32×10 ³ , 1:64×10 ³ , 1:128× 10 ³ , 1:256×10 ³ . Dilutions in test tubes No. 4-11 are prepared for adding to the plate.

To determine the suitability of the previously obtained cell monolayer, at the end of incubation, its condition is visually assessed using a microscope. The cells must have a typical morphology and there must be no foreign microflora.

The culture medium is removed from the resulting cell monolayer. To do this, turn the tablet over a container with a working solution of hydrogen peroxide.

At the end of incubation, the state of the cell monolayer is visually assessed using a microscope.

The activity of the standard and test samples is taken into account in the absence of signs of degeneration in the wells with control cells (Cc) that were not exposed to drugs.

The result is assessed using an inverted microscope.

The activity of the standard and test samples is taken to be the reciprocal of the dilution that causes damage to 50% of L-929 mouse adenocarcinoma cells.

Calculation of activity (X) of the standard and test sample is carried out according to the formula:

where P is the dilution of the drug sample at which 50% cell damage occurred.

The resulting drug has specific activity when titrated in cell culture L-929 90,000 units in a vial

Example 8. Preparation of a drug with an activity of 100,000 units/vial

Composition: hybrid protein obtained from Escherichia coli strain BL21 (DE3)/pET32a-TNF-Thy, according to example 5 - 7.7 ml, mannitol -1.0 g, sodium chloride -0.87 g, sodium phosphate disubstituted 12-water -2.1 g, sodium phosphate monosubstituted 2-water - 0.6 g, water for injection up to 100 ml.

All components, with the exception of TNF-Thy, were weighed and dissolved in water for injection. The pH of the solution was checked and, if necessary, adjusted to 7.0. Then the thawed TNF-Thy solution obtained according to example 5 was added.

The solution was filtered under sterile conditions. The bottles were aseptically filled with 1 ml of the resulting composition and rolled. The product was stored at a temperature of 2 to 8°C. The activity of the drug was determined as described in example 7. The resulting drug has specific activity when titrated in L-929 cell culture with 100,000 units in a vial

Example 9. Preparation of a drug with an activity of 110,000 U/vial Composition: hybrid protein obtained from Escherichia coli strain BL21 (DE3)/pET32a-TNF-Thy, with an activity of 1.2 x 10 ⁶ U/ml - 9.1 ml , mannitol -2 g, sodium chloride -0.89 g, sodium phosphate disubstituted 12-water - 3.3 g, sodium phosphate disubstituted 2-water - 0.1 g, water for injection up to 100 ml.

All components, with the exception of TNF-Thy, were weighed and dissolved in water for injection. The pH of the solution was checked and, if necessary, adjusted to 7.8. Then the thawed TNF-Thy solution obtained according to example 6 was added.

The solution was filtered under sterile conditions. The bottles were aseptically filled with 1 ml of the resulting composition and rolled. The product was stored at a temperature of 2 to 8°C.

The activity of the drug was determined. The resulting drug has specific activity when titrated in cell culture L-929 110,000 units in a vial

Example 10. Comparative results of the clinical use of the drug obtained according to example 8 and the drug "REFNOT".

Due to the fact that for the first time a drug for the recombinant hybrid protein α-tumor necrosis factor-thymosin-α1 is being announced in liquid form, for comparison the drug “Refnot” was taken in lyophilized form, which was dissolved in 1 ml of water for injection before use

The drug obtained according to example 8 and the drug "REFNOT" were used in groups of 200 patients aged 20-80 years with cancer of various locations, various stages (I-IV), with metastases of various locations or without metastases.

The drug obtained according to example 8 and the drug "REFNOT" were prescribed against the background of basic chemotherapy once a day in a dosage of 100,000 units to 400,000 units according to various schemes, according to the instructions for medical use of the drug recombinant hybrid protein α-tumor necrosis factor- Thymosin-α1.

All patients were observed for about two months. Therapy with a lyophilized drug obtained according to example 8 and a drug of the recombinant hybrid protein α-tumor necrosis factor-thymosin-α1, obtained according to the closest analogue, showed comparable effectiveness: positive clinical dynamics, stabilization of the dynamics according to RECIST, stabilization of the dynamics of metastases were noted.

In patients treated with REFNOT, the most common adverse event (in 12% of patients) was fever. When using the drug obtained according to example 8, an increase in temperature was observed in 1% of patients.

Example 11. Clinical study of a medicinal product obtained according to example 9.

Due to the fact that for the first time a drug for the recombinant hybrid protein α-tumor necrosis factor-thymosin-α1 is being announced in liquid form, for comparison the drug “Refnot” was taken in lyophilized form, which was dissolved in 1 ml of water for injection before use

The drug declared in example 9 was used in a clinical study in which 80 patients with a clinical diagnosis of breast cancer participated. The patients were divided into two treatment groups.

For group I: 1 ml of the drug obtained according to example 9 was injected subcutaneously: 10 injections of 100,000 units daily. Therapy began 2 days before chemotherapy, days of chemotherapy were skipped. The average number of courses was 2 (range 1 to 8). For group II: The drug “REFNOT” dissolved in 1 ml of water for injection was injected subcutaneously: 10 injections of 100,000 units daily. Therapy began 2 days before chemotherapy, days of chemotherapy were skipped. The average number of courses was 2 (range 1 to 8).

The objective response rate in group I was 47%. Control of tumor growth was recorded in 25% of cases. One patient had complete tumor regression in response to therapy. Median progression-free survival was 107 months (95% CI 25-189 months). Median overall survival was 115 months (95% CI 47-183 months).

The objective response rate in group II was 43%.” Control of tumor growth was recorded in 13% of cases. Median progression-free survival was 80 months (95% CI 38-128 months). Median overall survival was 67 months (95% CI 32-102 months).

In group I, there was a tendency towards an increase in relapse-free and overall survival of patients (approximately 1.5 times). Upon further observation, it was found that in group I the total number of patients with metastases following treatment is slightly smaller, with damage to 1 or 2 organs more often observed, while in group II patients with metastases in 2 or more organs predominate.

The number of adverse events in group I was lower than in group II. There were no serious adverse events observed in either group. All adverse events that occurred did not require discontinuation of therapy.

Example 12. Clinical study of the medicinal product obtained according to example 7. Due to the fact that for the first time a medicinal product of the recombinant hybrid protein α-tumor necrosis factor-thymosin-α1 in liquid form is being declared, for comparison the drug “Refnot” was taken in lyophilized form, which when used, dissolved in 1 ml of water for injection

The drug declared in example 7 was used in a clinical study, which involved 200 patients with various forms of malignant neoplasms, predominantly with locally advanced forms of the disease (stages 2A-3B), who completed the main course of antitumor treatment. The patients were divided into two treatment groups.

For group I: 1 ml of the drug obtained according to example 7 was administered subcutaneously: 100,000 units per day subcutaneously every other day, from days 1 to 19. Then a break of 10 days. There are 10 introductions in total for 1 course. A total of 6 courses of therapy.

For group II: The drug “REFNOT” dissolved in 1 ml of water for injection was administered subcutaneously: 100,000 units per day subcutaneously every other day, from days 1 to 19. Then a break of 10 days. There are 10 introductions in total for 1 course. A total of 6 courses of therapy.

Relapse-free survival was statistically significantly higher (p<0.05) among patients who completed the main course of antitumor treatment and then received a lyophilized drug obtained according to example 7 for 6 months. Secondary malignant neoplasms occurred in 2 and 5 patients in groups I and II, respectively. Progression-free survival was significantly higher in patients receiving the drug obtained according to example 7.

There were no serious adverse events reported in either group. The total number of patients who reported adverse events was slightly higher in group II. Patients complained of increased temperature during therapy, weakness and arthralgia. All adverse events were mild in severity and resolved without requiring discontinuation of therapy.

Claims (4)

1. A drug that has the activity of the hybrid protein tumor necrosis factor alpha - thymosin alpha 1 (TNF-Thy), which is a solution for parenteral administration containing osmotic pressure regulators, a buffer system maintaining a pH of 7.0-7.8, and highly purified TNF-Thy hybrid protein with the amino acid sequence SEQ ID NO 1, wherein the TNF-Thy hybrid protein was obtained by cultivating the Escherichia coli producer strain, BL21(DE3)/pET32a-TNF-Thy, where after 3-5 hours of cultivation, biosynthesis is induced in Escherichia coli BL21(DE3)/pET32a-TNF-Thy cells with isopropyl-β-D-1-thiogalactopyranoside and incubated for 4-6 hours to obtain the cell biomass of the producer strain producing the TNF-Thy protein, isolation from the resulting cell biomass of the producer strain TNF-Thy protein, subsequent purification of the isolated TNF-Thy fusion protein by anion exchange and gel permeation chromatography under denaturing conditions, renaturation of the TNF-Thy fusion protein by dilution of a urea solution and purification of the renatured TNF-Thy fusion protein by anion exchange chromatography to obtain a fusion protein with purity not less than 97%, and the Escherichia coli strain BL 21 (DE3)/pET32a-TNF-Thy is a producer of the TNF-Thy hybrid protein - obtained by transforming the parent strain E. coli BL21(DE3) with the recombinant plasmid pET32a-TNF-Thy with a size of 5929 base pairs (bp), providing the synthesis of a protein having the amino acid sequence SEQ ID NO 1 of 20.5 kDa in size, and containing the structural elements: T7 promoter and lacO operator, synthetic nucleotide sequence SEQ ID NO 2, encoding the TNF-Thy hybrid protein, the ampicillin antibiotic resistance gene (AmpR) and the bacterial promoter of the ampicillin resistance gene (AmpR promoter) for selection of recombinant cells, the origin of replication of bacteriophage f1 (f1 ori), the lactose repressor gene LacI and the bacterial promoter of the lactose repressor gene (LacIpromoter) with the following ratio components (mass%): TNF-Thy with a specific activity of at least 1.7×10 ⁶ U/mg and purity of at least 97% 0.0052-0.0064 Osmotic pressure regulators 1.8-2.9 Buffer system maintaining pH 7.0-7.8 2.2-3.6 Water rest 2. The drug according to claim 1, characterized in that it contains mannitol and sodium chloride as osmotic pressure regulators. 3. The drug according to claim 1, characterized in that as a buffer system maintaining a pH of 7.0-7.8, it contains a phosphate buffer consisting of sodium phosphate disubstituted 12-aqueous and sodium phosphate disubstituted 2-aqueous.