FI82074C - Process for the preparation of human leukocyte interferon alfa-2 - Google Patents

Description

1 82074

Method for preparing human leukocyte interferon alpha-2

The invention relates to the microbiological industry and in particular to the preparation and use in medicine of human leukocyte interferon alpha-2 having antiviral activity for the prevention and treatment of some widespread diseases in viral etiology.

Interferons are, in fact, proteins synthesized by specific human cells in response to viral infection. Depending on the type of cell producer and the specific structural and functional features of the interferons, they can be divided into three basic types, i.e., alpha or leukocyte interferon, which is produced by white blood cells; beta-, or 15 fibroblast interferon, produced by connective tissue cells; and gamma or immune interferon produced by T lymphocytes.

In addition to their antiviral effect, interferons also have an anti-proliferative effect as well as an effect on the immune response. This makes it possible to consider interferon-based drugs as a potentially effective adjunct in the treatment of some malignancies.

Highly pure human interferon preparations have now been prepared, which are in fact a group of 13 to 15 closely related proteins, each encoded by its own structural gene on chromosome 9.

However, isolating leukocyte interferon from the donor's blood does not produce enough of it for extensive clinical use, as the amount of interferon in 1 liter of blood is usually within 105 AU, whereas a simple effective dose of interferon for intravenous injection into a patient , is at least 106 AU based on experimental:: 35 clinical findings.

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Various methods are known in the art for the preparation of human leukocyte interferons, which methods are based on the use of numerous strains of microorganisms as producer strains to which the individual genes of human interferon 5 have been transferred by means of vector molecules. In particular, strains of the bacteria Escherichia coli, Bacillus subtilis, Methylophilus methy-lotrophus, etc. are used as producer strains (cf. EP patent publication 00062971-A2; published 1982; Int. Cl. C 12N 15/00).

10 Microorganisms containing a plasmid with an inserted human leukocyte interferon gene produce interferon when cultured under aerobic immersion conditions in nutrient media containing carbon and nitrogen sources, mineral salts, and growth factors.

A process for the preparation of human leukocyte interferon alpha-2 is known in the art (cf. GB Patent 2,079,291 A; published 1982; Cl. C 12 N 15/00), according to which E. coli K-12 strain 20,294 is used, obtained by inserting the recombinant plasmid pLeIFAtrp2.5, wherein the interferon alpha-2 gene is under the control of the regulatory regions of the E. coli tryptophan operon.

The above strain is cultured by immersion in nutrient media containing carbon and nitrogen sources, mineral salts and growth stimulants in the presence of an antibiotic.

The interferon yield for the above producer is 2.5 x 108 AU / liter of culture medium.

However, there are some disadvantages associated with the use of E. coli as an industrial producer. E. coli is conventionally considered to be a pathogenic microorganism, while various strains of this bacterium are permanently present in the human intestinal microflora. This fact places particularly stringent requirements on the purification of E. coli-based drugs and the complete separation of protein contaminants and endotoxins. This in turn li 3 82074 makes the interferon manufacturing process more expensive and adversely affects the yield of the pure final product. In addition, all hitherto known laboratory strains of E. coli are susceptible to phagolysis, with the number of specific phages raised for E. coli rising to many hundreds.

In addition, the above method is also burdened by the relatively low productivity of the culture, as a result of which the isolation of interferon from the cellular biomass and its subsequent purification until a homogeneous state is reached is a very laborious process.

It is a primary object of the present invention to provide human leukocyte interferon alpha-2 in higher yields and to provide a simplified method for obtaining a product using a new producer strain.

The above object is achieved by the process according to the invention for the production of human leukocyte interferon alfa-2 by submerging a producer strain containing a plasmid to which the human leukocyte interferon alpha-2 gene has been inserted in a nutrient medium containing carbon and nitrogen sources, mineral and growth factors, aerating in the presence of the antibiotic, followed by isolation and purification of the final product.

The method according to the invention is characterized in that the producer strain is Pseudomonas strain VG-84 with plasmid pVG3 and deposited in the microorganism collection of the USSR Antibiotics Research Institute under accession number 1742, and that the producer strain is cultured with antibiotics, i.e. in the presence of tetracycline or both. In the method, it is preferred that tetracycline is used in an amount of 30 to 50 mg / l and streptomycin in an amount of 50 to 150 mg / l.

Due to the use of a new, highly productive strain of Pseudomonas 35 VG-84, a higher yield of the final product is obtained by a mechanized and simpler method.

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Thus, according to the invention, a new strain of Pseudomo-nas species VG-84 into which the human leukocyte interferon alpha-2 gene has been cloned into the multicopy plasmid pVG3 under the control of the regulatory regions of gene D of bacteriophage 0X174 is used.

Plasmid pVG3 is a molecular hybrid of the broad-host multicopy vector plasmid pAYC34 and plasmid pIFN-α2-P2, which contains the replicon pBR 322 and the interferon alpha-2 gene, and is responsible for cellular resistance to tetracycline and ampicillin. The interferon alpha-2 gene of plasmid pIFN-α2-P2 is under the control of the regulatory regions of gene D of bacteriophage 0X174. The 9.4 thousand base pair vector plasmid pAYV34 is relative to the incompatibility group Inc p-4 / Q and confers cell resistance to strep-15 tomycin.

Plasmid pAYC34 and pIFN-α2-β2 DNA were treated with Pst I, after which E. coli strain C600 competent cells were transformed with this mixture. The transformants were then screened with complete medium containing antibiotics, i.e., ampicillin (50 μ / ml), streptomycin (100 μ / ml), and tetracycline (25 μ / ml). Plasmid DNA, designated pVG3, was found in the transformant clones and corresponded in size and restriction map to the hybrid of plasmids pAC34 and pIFN-α2-P2. Plasmid pVG3 confers cellular resistance to streptomycin, as does pAYC34, and to tetracycline and ampicillin such as pIFN-α2-P2. Plasmid pVG3 is 14.8 thousand base pairs in size and consists of the sum of plasmids pIFN-2242-P2 (5.4 thousand base pairs) and pAYC34 (9.4 thousand base pairs of nucleotides).

The broad host conjugate plasmid R 751 was inserted into one of E. coli strains C600 containing the hybrid plasmid pVG3. Through a series of conjugative crosses, plasmid pVG3 was transferred to bacteria of the genus Pseudomo-35 and some other gram-negative κ 82074 bacteria. The transconjugant bacteria were then isolated on a selective medium containing the above-mentioned antibiotics and tested for their ability to synthesize interferon.

Cultures obtained by the above method were then cultured under aerobic conditions at 30 ° C in L broth for 6 to 10 hours until a cell density of 2 to 5 x 10 9 cells / ml was reached. The bacteria were then harvested by centrifugation, lysed using ultrasound, and the in-10 terferon content of the cell extracts was determined by radioimmunoassay.

A total of 18 gram-negative bacterial strains belonging to the genera Escherichia, Salmonella, Methylomonas, Erwinia and Rhizobium were tested. The majority of the strains tested were found to produce active interferon. 15 Pseudomonas sp. The VG-84 strain gave the highest productivity.

Thus, under the conditions defined above, this strain was found to produce 5 x 109 to 1 x 10 10 AU of interferon per liter of culture medium.

Pseudomonas sp. The VG-84 strain has been deposited in the USSR Antibiotics Research Institute Microorganism Collection of Deposit-20 under accession number 1742. Pseudomonas sp. The characteristics of strain VG-84 are as follows: 25 Microscopic morphology of moving gram-negative rod cells 3 -5 pm

Morphology in different media: 30 meat extract agar forms colonies with irregular edges 3-4 mm in diameter with a rough surface and a light green color after 24 hours of culture at 25-35 at 30 ° C 6 82074 meat extract broth shows moderate growth after shock inoculation, mainly subcultured ) on the surface.

5 Grows weakly without aeration.

minimum nutrient medium, forms 2-3 in diameter

Ma with glucose mm round 10 gray colonies on the second day of cultivation

Physiological and bio-growth at 25-35 ° C, optimal chemical properties at 30 ° C at pH 15.0. Carbon sources: use Glu and arabinose. Nitrogen sources: connects well. Needs adeine. Resistant to streptomycin and tetracycline-20.

The method according to the invention is carried out as follows:

Pseudomonas sp. The VG-84 strain is cultured in agar-medium at 28-30 ° C for 8-12 hours, after which it is re-inoculated into liquid medium and the inoculum is cultured with vigorous stirring and aeration for 3-6 hours at 28 ° C. The fermentation is carried out in a fermenter in a liquid nutrient medium suitable for the purpose and containing carbon and nitrogen sources, mineral salts and growth factors, etc .; e.g. in a nutrient medium containing glucose, yeast extract or autolysate or tryptone or peptone or casein or yeast hydrolysates, or mixtures thereof. 35 The fermentation is carried out in the presence of antibiotics, i.e. streptomycin or tetracycline or a mixture of both li 7 82074. It is preferred that tetracycline is present in an amount of 30 to 50 mg / l and streptomycin 50 to 150 mg / l. The duration of the fermentation process is 6 to 10 hours at 28 to 32 ° C and the pH is between 6.7 and 7.1; under intense aeration. During the fermentation, the optical density A550 of the bacterial suspension is measured, the optical density being 5 or 7 optical units at the end of the fermentation process.

Under the culture conditions described above, the yield of interferon 10, as determined by radioimmunoassay or by determining antiviral activity in human fibroblast cell culture, is between 5 x 10 9 and 1.5 x 10 10 AU / liter of culture medium.

After completion of the fermentation process and reaching the aforementioned optical density, the bacterial cells are collected by centrifugation, resuspended in a suitable buffer and lysed by sonication or other methods resulting in cell lysis or cell membrane rupture, e.g. by lysozyme treatment using ballistic methods, - or the use of metal balls, using a French press, etc.

The organic cellular debris is then separated by centrifugation, after which the interferon is separated from the supernatant, which is in fact a cell extract, using known protein fractionation and purification methods. The yield of homogeneous interferon, as shown by various physicochemical methods, is 20-60%, with a purification ratio of 200-500-fold and the specific activity of the final product thus purified is 4 x 10® AU.

The course of purification is monitored by determining the antiviral activity of interferon in human fibroblast cell culture using vesicular stomatitis-35 virus as an irritant and by RIA or ELISA. The purity of the interferon preparations obtained from the purification is tested by immunological, β 82074, electrophoretic, gel filtration and sedimentation methods, as well as by determining the amino acid composition and N-terminal amino acid sequence of the isolated final product.

The homogeneous preparations of human leukocyte-5 interferon alpha-2 thus obtained have all the structural and functional properties specific to this type of interferon.

The method according to the invention is advantageous compared to the known method in that a high level of activity can be achieved due to the use of a new highly productive strain. Thus, the same amount of interferon can be obtained from a smaller amount of biomass, which contributes to the reduced cost of isolating and decomposing the biomass, as well as for all subsequent work steps associated with the production of a homogeneous product.

According to data from an automated Edman N-terminal sequencing, Pseudomonas sp. Homogeneous interferon isolated from the biomass of the VG-84 producer strain has the N-terminal amino acid sequence Cys-Asp-Leu-

Pro-Glu-Thr-His-Ser-, and does not contain an N-terminal methionine residue, thus being completely similar to interferon alpha-2 produced by human leukocytes.

To clarify the invention, the following examples are provided for the preparation of human leukocyte interferon alpha-2.

Example 1

Pseudomonas sp. Strain VG-84 is cultured at 28 ° C for 12 hours on a solid agar medium having the following composition: tryptone 10 g / l; yeast extract 5 g / l; adenine 80 mg / l; glucose 10 g / l; streptomycin 150 mg / l; tetracycline 50 mg / l; distilled water to make up 1 liter, and agar-agar 15 g / l.

35 Biomass grown on the above agar medium is used for inoculum production. At this point

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9 82074 Biomass cells are transferred to 750 ml Erlenmeyer flasks containing 100 ml of culture medium as defined above (agar-free) and cultured on a shaker for 6 hours with vigorous stirring (249 rpm) at 28 ° C. The optical density of the inoculum-5 culture is 2 optical units.

The culturing process is carried out in a fermentation apparatus equipped with temperature, pH, agitation rate and aeration control systems and a pressure sensor to measure the partial pressure of dissolved oxygen.

10 For this purpose, the inoculum is placed in a 5% ratio in a fermenter containing a nutrient substrate having the above composition (agar-free) and cultured for 6 hours at 28 ± 0.5 ° C at a pH of 6.7 to 6.9 under strong aeration and se-15 agitation. The aeration and mixing conditions are chosen so that the concentration of dissolved oxygen does not restrict growth, for which purpose the partial pressure of oxygen is kept at a level of 5 to 10% of saturation. Liquid ammonia is used to stabilize the pH.

Biomass growth is monitored by measuring the optical density of the culture medium, which is determined as often as every hour. Fermentation ends when the optical density A550 reaches 5 optical units.

When the fermentation process is complete, the human leukocyte interferon alfa-2 concentration is 1.5 x 1010 AU lit

per broth. To determine the concentration of interferon thus obtained, a solid phase variant of the RIA is used, comprising the use of anti-interferon-rabbit polyclonal antibodies as well as monoclonal murine antibodies labeled with the 125 I isotope. In addition, the antiviral activity of interferon is determined by the protective effect of interferon against the cytopathic effect of vesicular stomatitis virus induced by cultures of human diploid fibroblasts. The interferon standard MRC is used as Ver-35 tailings

B69 / 19 (English).

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Cells harvested by centrifugation after fermentation are used as a source of interferon. They are suspended in 0.1 M Tris buffer with a pH of 7.5 (1 g of cells per 5-10 ml of buffer) and digested in an ultrasonic digester. The organic cellular debris is then separated by centrifugation at 30,000 g and homogeneous human leukocyte interferon alpha-2 is isolated from the supernatant (cell extract).

Subsequent purification results in an electrophoretically homogeneous preparation of human leukocyte interferon alpha-2 with a purification ratio of 210 and a specific activity of 4.0 x 10® AU in a yield of 41% (calculated as activity).

The accompanying drawing shows values from an electrophoretic analysis of human leukocyte interferon alpha-2, in which Ailia shows the interferon preparation before purification and B the interferon preparation after purification, while the molecular weight (in kD) of the reference proteins is shown in numbers.

Electrophoresis is performed on a 12.5% polyacrylamide gel in the presence of sodium dodecyl sulfate. In terms of specific activity, molecular weight (18.4 kD), amino acid composition and other properties, the human leukocyte interferon alfa-2 2 thus isolated does not differ in any way from the human leukocyte interface Roni alfa-2 isolated from other bacterial products. -jakannoista.

Example 2

The method is carried out in the same manner as in Example 1. The producer is grown in the presence of antibiotics, i.e. streptomycin at 50 mg / l and tetracycline at 30 mg / l. After a 6-hour fermentation process, the optical density of the bacterial suspension is 4.2 optical units, while one liter of culture medium contains 7.5 x 109 AU of 35 human leukocyte interferon alfa-2.

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Example 3

The method is carried out as described in Example 1, in which the producer is cultured in the presence of an antibiotic, i.e. tetracycline, in an amount of 50 mg / l. After 5.5 hours of fermentation process, the optical density of the bacterial suspension is 5.5 optical units, while the interferon concentration per liter of culture medium is 9.5 x 109 AU.

Claims (2)

12 82074 A method for producing human leukocyte interferon alpha-2 by immersion culture of a producer strain containing a plasmid to which the human leukocyte interferon alpha-2 gene has been inserted in a nutrient medium containing carbon and nitrogen sources, mineral salts and growth factors, growth factors , followed by isolation and purification of the final product, characterized in that the producer strain is Pseudomonas species VG-84 with plasmid pVG3 and deposited in the USSR Antibiotics Research Insitute microorganism collection under accession number 1742, and that the producer strain is cultured with antibiotics , i.e. in the presence of streptomycin 15 or tetracycline or a mixture thereof. Process according to Claim 1, characterized in that tetracycline is used in an amount of 30 to 50 mg / l and streptomycin in an amount of 50 to 150 mg / l. Il ia 82074