Interleukin-2 preparation and method for its preparing

FIELD: biotechnology, cytokines, pharmacy.

SUBSTANCE: invention relates to a biotechnological method for preparing medicinal agents. Proposed method involves destruction of cells of yeast producer of interleukin-2 (IL-2) wherein synthesis of required protein (IL-2) is carried out, but this producer doesn't secrete this protein, and the following separation of water-insoluble fraction. In the process for preparing water-insoluble components of disturbed cells are retained. The interleukin-2 preparation obtained by this method shows less cost, it is active in oral administration and shows higher activity in some cases as compared with IL-2 preparations purified from components of yeast cells fraction.

EFFECT: improved preparing method, improved and valuable properties of interleukin-2.

13 cl, 2 tbl, 10 ex

 

The invention relates to the field of production of immunomodulatory drugs biotechnological methods and can be used in the pharmaceutical industry. In particular, this invention relates to a method for the preparation of interleukin-2. This product can be used in order immunopotentiating therapy and, in particular, in the treatment of cancer, infectious, autoimmune and allergic diseases.

The history of the discovery of interleukin-2 (IL-2) starts with the 1960's, when numerous in vitro studies have shown that activated lymphocytes secrete mediators that stimulate a variety of immune responses in vitro and in vivo. Only in 1976 that the mediator identified as T-cell growth factor, obtained in 1979 the name IL-2. Developed technologies for its culture of activated human lymphocytes and purification led to the first preparations of IL-2 for research and clinical purposes. These drugs are called "lymphocytic" or "natural" IL-2. At the same time, the limited scale of its production did not allow its widespread use until 1983, when they were developed technology for production of recombinant proteins, in particular IL-2, using genetic engineering and biotechnology.

Known SPO is usually used for obtaining preparations of IL-2 from donor blood. However, the preparation containing the components of human blood, is potentially dangerous.

Also known gene-technological methods of obtaining IL-2. Numerous firms (Cetus Corp., Amgen, Inc., Hoffman-La Roche, Inc.) used methods for genetic engineering and biotechnology for producing recombinant IL-2 as a drug to treat various types of cancer.

One of the ways to obtain recombinant IL-2 as a producer using E.coli (Escherichia coli). E. coli is a conditionally pathogenic flora. Therefore, the preparations of IL-2 from E. coli require thorough cleaning and can be toxic.

Yeast cells are more attractive producers heterologous IL-2.

Known (EP 0162898 A1, published 04.12.1985) a method of producing a preparation of IL-2 using yeast producer. In this method, the IL-2 secreted into the culture medium and then must be isolated from the culture medium using chromatography, filtration, extraction, etc. the Disadvantage of this method is that secreted yeast proteins glycosylases, i.e. their structure in the process of secretion appear yeast carbohydrate residues. These glycoproteins are highly immunogenic for humans, which creates further problems when used in therapy, the example of the accumulation of human antibodies to these glycosylated proteins.

Also known (EP 0152358 published 21.08.1985) the method of producing drug IL-2, which destroy the cells of the yeast culture, which was carried out the synthesis of IL-2, centrifuged these destroyed cells treated precipitate obtained by centrifugation, the solution of sodium dodecyl sulfate to translate the IL-2 in the liquid phase, and extracted with IL-2 from the liquid phase. Extraction of IL-2, carried out in this way is costly and requires considerable investment of time, equipment and/or reagents.

In General, all known so far, the recombinant IL-2 is extremely expensive, and a significant contribution to their cost makes the technology of their separation from cells-producers.

The objective of the invention is to create a more technologically advanced method of obtaining drug IL-2, which would allow the drug is not inferior in activity and therapeutic features of known drugs, while providing the opportunity for large-scale production and not too high cost.

The task is solved in that a method of obtaining the drug interleukin-2, in which the security of the target protein destroy the cells of the yeast producer of IL-2, in which the synthesis of the target protein (IL-2) and not secretarye is this the target protein, and separating the water-insoluble fraction, and the product retain water-insoluble components of damaged cells.

In particular, in this way the yeast producer is a Saccharomyces cerevisiae strain deposited at the all-Union collection of industrial microorganisms under the number Y-791, which is the producer of the human interleukin-2.

Contrary to popular belief about the necessity of a thorough purification of recombinant protein the authors of the present invention unexpectedly discovered that the preservation of the remnants of cell walls of yeast recombinant IL-2 not only allows you to simplify its manufacturing process and to get a cheaper product, but also allows to obtain a very effective drug, therapeutic properties which in some situations superior to the properties of known drugs IL-2.

The implementation of the method of obtaining the drug without extraction (translation) IL-2 in the liquid phase and its subsequent extraction of the liquid phase leads to unexpected for such exclusion stage. By the way is not bad purified product, but an entirely new drug IL-2. Preserved in the drug remains insoluble fraction of yeast cell walls not only inhibit the effect of IL-2, but also serve as a kind of adjuvant, i.e accessories the m substance, stimulating the activation of T-cell immunity mammals. The transfer of water-insoluble IL-2 in the liquid phase with its subsequent extraction was considered prior to the inception of this invention is absolutely necessary and the efforts of the developers were previously focused on developing ways to enhance cleaning. After the creation of the present invention becomes clear that the preparation of the IL-2 should be obtained without separation from the water-insoluble fraction of yeast cell walls and effort can then be focused on improving the quality of the fraction of cell walls.

In the product obtained in this way, IL-2 is in an inactive form that ensures its long-term storage. To activate IL-2 to the drug you want to add a solubilizer and water.

It is advisable to destroy cells in the presence of protease inhibitors as protease released during the destruction of the yeast cells, can cause destruction of the target protein.

It is preferable to destroy cells using a high-speed grinder with glass beads in the presence of protease inhibitors, as in this case, achieving a good degree of destruction of cells and does not occur the destruction of the target product.

In the preferred case, after separation of the water-insoluble fraction of it washed the La more complete removal of water soluble components. Thus there is a more complete removal of water soluble proteases, other yeast proteins, RNA, chromosomal and plasmid DNA, ETC.

Preferably the separated water-insoluble fraction is dried. Drying can be carried out immediately after separation or after washing.

More practicelink case, after separation of the water-insoluble fraction of it is treated with acetone, followed by removal of the acetone fraction. This destroyed yeast cells are dehydrated. This stage also allows you to get rid of the lipid components of the water-insoluble fraction, which leads to a drug with improved physical properties, it is easy to handle and more enjoyable therapeutic application.

When the specified processing acetone preparing a suspension of water-insoluble fraction in water, this suspension is poured to the acetone at the ratio of the volume of the suspension and acetone from 1:5 to 1:20, preferably 1:10, to obtain a suspension in acetone, filtering the resulting suspension and the remaining on the filter residue wyszukiwanie processing acetone insoluble fraction may be washed, that is, the washing is carried out after the separation of water-insoluble fractions before preparing its suspension in water.

In the preferred embodiment of the method, after the stage of drying water-insoluble FR the options to this dried fractions add dry the solubilizer. Thus obtained product in the form of the composition of the dried water-insoluble fraction and a solubilizer. In the product obtained in this way, IL-2 is also in an inactive form that ensures its long-term storage. To activate IL-2 to this drug you need to add water. With this addition, the concentration of the solubilizer in the solution becomes optimal, which ensures the production of the proper conformation of the protein and a drug that has biological activity.

It is advisable as a solubilizer to use sodium dodecyl sulphate.

In one of the embodiments of the method, upon receipt of the preparation of IL-2 after adding a solubilizer add water. In this way we obtain a preparation in which IL-2 is active, is in an optimal conformation and is ready for immediate use.

According to the invention also offers the drug interleukin-2 obtained as described above in any of its possible variants. The drug has a lower cost and can be used in oral therapy that has always, from the point of view of the patient, is more preferable. Unexpectedly also showed that this drug exerts therapeutic effects beyond the effects of the drug based on purified And the -2, obtained by known methods.

At the present stage of development of biotechnology and genetic engineering of yeast producers of IL-2 is known, can be obtained from depositories or re-created.

Although the most common yeast producer is Saccharomyces cerevisiae, such producers may be any other non-pathogenic yeast, for example Saccharomyces bulardi or yeast of the genus Pichia, such as Pichia pastoris, the genus Kluyveromyces, such as Kluyveromyces lactis, the genus Hansenula, such as Hansenula polymorpha, and the like. The technology of recombinant plasmids containing the gene for IL-2, in particular, human IL-2, are well known. A well-known technology for strains containing recombinant plasmids.

In particular, the example yeast producer of human IL-2 is a strain of the yeast Saccharomyces cerevisiae deposited in the all-Union collection of industrial microorganisms under the number Y-791. This strain is a product of human IL-2. The design of the expression vector introduced into a strain of yeast, such that cells do not secrete the target protein. Can be used for any other strain, producing heterologous IL-2.

For each producer strain known optimal conditions, which will be a synthesis of IL-2. The synthesis of IL-2 may be constitutive or require induction or derep is ASCII, that is determined by the genetic structure of the producer strain. In General, taking into account characteristics of the selected strain, the expert is able to choose the cultivation conditions, which will result in a culture of yeast cells, in which the synthesized IL-2.

In particular, strain Y-791 first grow with suppression of the synthesis of IL-2 on minimal mineral medium Sd1 with histidine to achieve the optical density OD600from 2.5 to 4.0, preferably OD600=3.0V. The synthesis of IL-2 is inhibited by the presence in the environment of the cultivation of phosphate. Then subcultured received the inoculum in a greater amount of the second medium for cultivation, in which the synthesis of IL-2 derepression, and cultured until reaching phase, early stationary, that is, until the optical density OD600from 6.0 to 9.0, preferably OD600=7,5. As a second medium for cultivation, you can use the environment Sd2 or the environment, PEP (peptone 20 g/l, glucose 20 g/l).

After culturing cells can be separated from culture medium by centrifugation or filtration. All further manipulations are performed with cells and culture medium drop. When using strain Y-791 receive, usually from 8 to 12 g of cells per 1 liter of nutrient medium.

The resulting biomass can also be used immediately or freeze to use in gave the largest predlozenom method.

It is noted that a formula containing intact cells, has only trace activity of IL-2 compared with the drug from the destroyed cells.

The destruction of the yeast cells can be accomplished by any known means, for ensuring the conservation of the target protein. In particular, physical methods, for example, using a high speed grinder with glass beads, using high pressure, the method of freezing and thawing. While securing the desired product is achieved by adding to the suspension of the destroyed cells of protease inhibitors to proteases released during the destruction of the yeast cells are not destroyed by IL-2. Enzymatic destruction method of yeast cells is also possible, if you can avoid proteolytic action used by the agent to the target protein.

The security of the target product is obtained by carrying out the method at low temperatures, for example at temperatures from 2 to 8°With, in particular if 4°C.

An example of a high-speed grinder with glass balls is disintegrator DynoMill. Destruction is preferable to lead at a temperature of from 2 to 10°and speed snake from 4000 to 6000 Rev/min

As an inhibitor of yeast proteases traditionally used by fenil Tralfamadore (PMSF), however, it can be used for any other inhibitor or a cocktail of protease inhibitors. It is enough to use FMF in quantities of 1 mm, but can be used otherwise appropriate amount of protease inhibitor, which can be set according to the results of monitoring the content of IL-2 in the preparation method of polyacrylamide gel electrophoresis.

The separation of the water-insoluble fraction is carried out by centrifugation. Almost all water-soluble components of the yeast cells pass into the supernatant (supernatant), which is discarded, and the sediment remain water-insoluble components. When centrifugation is removed proteases, soluble proteins, RNA, chromosomal and plasmid DNA, etc. Separating water-insoluble fraction can be carried out and other available methods, in particular by filtering.

Centrifugation is advisable to carry out at low temperatures, for example at 4°S, and the rotor speed from 4,000 to 10,000 rpm

The proposed method can perform additional processing of the water-insoluble fraction for a more complete removal of water soluble components, namely flushed the buffer solution. As such a buffer solution can be used in 0.005 M Tris-HCl buffer solution, pH 7,2.

Drug a is according to dry. Drying of the preparation is carried out to enable its storage until further use. In the dried drug are the remains of yeast membranes together with the target protein. In this way excluded the stage of extraction of the target protein from the insoluble fraction, which is unexpected decision in the art, as is usually the effort is concentrated precisely at this stage. All preserved in the preparation of water-insoluble components are not only inhibiting drug in certain conditions their target properties, but sometimes also potentiate these properties.

Commonly used drying by lyophilization, but you can also use any other suitable methods.

The proposed method can also perform additional processing of the water-insoluble fraction with acetone. Before processing the acetone insoluble fraction can be pre-rinsed with the buffer solution. Treatment with acetone is the following. Of the water-insoluble fraction of preparing a suspension in water and mix it with acetone by injection of a suspension in acetone under stirring. The ratio of the volume of suspension in water and acetone is from 1:5 to 1:20, mostly 1:10. The suspension should have a sufficient thickness, approximately 60-80%, preferably 75%. Treatment with acetone Prov is car Ried out at a temperature of -20° C. Separating the dehydrated thus destroyed the yeast cells from acetone by vacuum filtration or by centrifugation at a temperature of from -10 to -30°and revs from 2000 to 5000 Rev/min the precipitate destroyed yeast cells may be washed two to ten times (preferably five) volume of acetone and again separated from the acetone by vacuum filtration or by centrifugation at a temperature of from -10 to -30°and revs from 2000 to 5000 Rev/min the Precipitate is dried in a fume hood at room temperature for 20-30 hours. Acetone dehydrates the preparation of the cells, therefore, to obtain a dry preparation is not required freeze drying.

In one of the embodiments of the method, the drug is prepared as a composition of a dried water-insoluble fraction of damaged cells and dry solubilizer.

The solubilizer is a substance that helps dissolve in water water-insoluble substances. Recombinant IL-2, synthesized inside yeast cells, is water-insoluble and associated with the cell wall. It was found that the solubilizer also allows you to separate the IL-2 from the cell wall. As a solubilizer may be used sodium dodecyl sulphate, laurylsarcosine, dezoksiholatom, urea, Triton-X100. If as a solubilizer is used dodecyl sulphate NAT the Oia, it is added in quantities of from 4.0 to 20.0% by weight of dry water-insoluble fraction, preferably 12%.

When the preparation of the IL-2 is presented in the form of the composition of the dried water-insoluble fraction of damaged cells and a solubilizer, it can be prepared for use by adding water. Enough to 100 mg of the drug to add from 1 to 10 ml, preferably 5 ml of water.

The lyophilized product can be stored at a temperature from +4 to -20°With 2 years without adding a special stabilizing or preservative components. Adding water to the drug should be carried out immediately before use.

If desired, the agent can be added stabilizers, antioxidants, fillers or other substances that will make the product suitable pharmaceutical form.

Thus, the method according to the invention can be, in particular, carried out in one of the following: the destruction of the yeast cells, the separation of the water-insoluble fraction and drying; or the destruction of the yeast cells, the separation of the water-insoluble fraction, washing and drying of this faction; or the destruction of the yeast cells, the separation of the water-insoluble fraction, perhaps her washing, treatment with acetone to remove the acetone fractions and drying of the residue; in either case, preferably after in the sesivany to prepare the drug in the form of a composition with a dry solubilizer.

Evaluation of biological activity of the drug received IL-2 can be held in the international standard test culture with IL-2-dependent tumor-specific cytotoxic T-lymphocytes mouse line CTLL-2 (Hammerling U. et al. "Development and validation bioassay for interleukin-2". // J. Pharmaceut. & Biochem. Analysis - 1992. - vol.10. - p.547. A.J.H. Gearing and Thorpe R. "The international standard for human interleukin-2. Calibration by international collaborative study." // J.Immunol. Methods. - 1984. - vol.74. - p.39). Biologically active recombinant IL-2 should stimulate proliferation of these cells. Determination of biological activity performed using the colorimetric method with tetrazolium dye MTT. Viable cells stimulated with IL-2, absorb the dye and accumulate in the cytoplasm of the water-insoluble dark blue crystals formazan (restored MTT). After removing the culture medium and the crystals are completely dissolved in dimethyl sulfoxide measure the optical density of the resulting solution on computerized photometer at a wavelength of 530 and 690 nm. The deep blue colour of the solution in the samples containing IL-2, should be dependent on the concentration of IL-2, introduced into the culture medium. Comparing the optical density of the sample and standard activity of IL-2, determine the biological activity of IL-2 in the incubation sample.

The number of IL-2 can be determined by any standard method the distribution of the target protein, for example, by the method of polyacrylamide gel electrophoresis (Laemmli U. "Cleavage of structural proteins during the assembly of the head of bacteriophage T4". // Nature. - 1970. - 227. - p.680-685) or enzyme immunoassay (Gehman L.O. and Robb R.J. "An ELISA-based assay for quantitation of human interleukin-2." // J. Immunol. Methods. - 1984. - vol.74. - p.39), and others

In General, the method according to the invention alternatively can include, or consist essentially be composed of any suitable stages, disclosed herein, and such method according to the invention can additionally or alternatively to exclude any stage or object, which is used in the method known from the prior art, or that is not necessary to obtain a technical result of the present invention.

The same applies to the preparation according to the invention, which, in principle, an alternative may include, or consist essentially consist of any suitable components disclosed in this description, and such a preparation according to the invention may additionally or alternatively be prepared so that it excludes any component, material, ingredient, or object, which is used in the product are known from the prior art, or that is not necessary to obtain a technical result of the present invention.

The invention is further illustrated by the following examples which do not limit the data the invention.

Example 1. Obtaining the drug interleukin-2

1.1. Obtaining yeast biomass producer of IL-2, in which the synthesis of the target protein (IL-2)

Take an aliquot of the strain of the yeast Saccharomyces cerevisiae Y-791 stored at -70°With glycerol, and inoculated into a nutrient medium Sd1 containing 50 mg/l histidine. The composition of the medium Sd1; Of 0.67% Yeast nitrogen base ("Difco Laboratories, Detroit, Ml), 2% glucose. The ratio of the mass of cells to medium volume Sd1 approximately 0.2 g to 1 litre.

Cultivation in the environment Sd1 lead to values of optical density OD600from 2.5 to 4.0, preferably OD600=3,0.

Upon reaching the required value of optical density transfer thus obtained inoculum in a tenfold greater volume environment Sd2 and lead cultivation to the value OD600=7,5.

At the end of the cultivation environment Sd2 cells separated from the culture medium by centrifugation. Get 10 g of cells from 1 liter of nutrient medium. All further manipulations are performed with cells and culture medium drop.

The composition of the nutrient medium Sd2
Component environmentQuantity per 1 l
Histidine0.05 g
(NH4)-citrate (anhydrous)7.6 g
MgSO4 0.5 g
NaCl0.1 g
CaCl2×6H2O0.1 g
KCl1.5 g
KN2PO40.03 g
Glucose20 g
H3IN30.5 mg
CuSO4×5H2O0.04 mg
KI0.1 mg
FeCl3×6N2O0.2 mg
MnSO40.4 mg
NaMoO4×2H2O0.2 mg
ZnSO4×7H2O0.4 mg
Biotin0.02 mg
Calcium Pantothenate2 mg
Folic acid0.002 mg
Inositol10 mg
Nicotinic acid0.4 mg
P-aminobenzoic acid0.2 mg
Pyridoxine hydrochloride0.4 mg
Riboflavin0.2 mg
Thiamine0.4 mg

1.2. The destruction of cells yeast producer of IL-2, in which the synthesis of the target protein (IL-2)

Prepared in 50%cell suspension in buffer solution And adding PMSF (phenylmethylsulfonyl) up to the final concentration of 1 mM. A solution is a 0.005 M Tris-HCl buffer solution, pH of 7.2. Spend the destruction of the yeast cells on the cage mill Dyno Mill at a temperature of from 2 to 10°and speed of screw rotation 3000 rpm/min, the Suspension of the destroyed cells are collected in the cups for centrifugation.

1.3. Department of water-insoluble fractions

The suspension obtained in stage 1.2., centrifuged at a temperature of 4°and a rotor speed of 10,000 rpm for 45 minutes. The supernatant liquid discarded.

1.4. Drying water-insoluble fractions

The precipitate is dried in the apparatus for freeze drying for 24 hours. From 1 gram grown cells receive 100 mg plate glassy and fragile freeze-dried ecru color.

Example 2. Obtaining the drug interleukin-2

Carry out stage 1.1.-1.3 example 1. Next, the precipitate washed with 0.005 M Tris-HCl buffer solution, pH of 7.2, and then again centrifuged at a temperature of 4°and a rotor speed of 10,000 rpm the Supernatant discarded. Next, carry out stage, as described in 1.4.

Example 3. Obtaining the drug interleukin-2

Carry out stage 1.1.-1.3 example 1. The precipitate was washed with 0.005 M Tris-HCl buffer solution, pH of 7.2, and then again centrifuged at a temperature of 4°and a rotor speed of 10,000 rpm the Supernatant fluid is any drop.

Prepare 75% suspension of the resulting sludge destroyed cells cooled to 4°With water, for example, to 1 g of sediment add 0.3 ml of water. Preparation of the suspension is carried out in an ice bath.

For further manipulations all utensils and acetone cooled to a temperature of -10 to -30°C. the Ratio of the volume of the suspension and acetone is 1:10, for example, 1 ml of suspension required 10 ml of acetone.

The suspension is destroyed yeast cells slowly poured into acetone and homogenized for 5 minutes.

Then separate destroyed the yeast cells from the acetone by filtration at a temperature of from -10 to -30°C.

Wash the precipitate destroyed yeast cells five times the volume of acetone and again filtered at a temperature of from -10 to -30°C.

The precipitate is transferred into enamel container and dried in a fume hood at room temperature for 18 hours.

As a result of 10 g of wet yeast cells receive 1 g of dried sediment destroyed yeast cells in the form of a thin uniform whitish-beige powder.

Example 4. Adding a solubilizer

To 1 g of the powder obtained in example 3, add 120 mg of sodium dodecyl sulfate (LTOs) and stirred. The product obtained at this stage, the application requires the addition of water.

Example 5. Add water

To 100 mg CR is parathas, obtained in example 4, add 5 ml of water and stirred to obtain a suspension.

Example 6. Preparation of the suspension to determine the number and activity of IL-2

The suspension obtained in example 5, centrifuged for 5 minutes at 10,000 rpm and select the supernatant, i.e. the extract. The precipitate discarded. For analysis of the obtained extract on the specific activity and the quantitative content of RIL-2 take away his aliquot, e.g. 10 ál.

The presence and quantity of IL-2 in the obtained extract was confirmed by electrophoresis, and its biological activity in a standard test cell line CTLL-2.

Example 7. Electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate

In addition to the sample extract to determine the concentration of IL-2 in the gel also make the test solutions of bovine serum albumin to construct a calibration straight in quantities of 1, 2, 4, 6 and 8 µg / well, as well as markers of molecular masses of proteins 94, 67, 43, 30, 20.1 and 14.4 kDa.

Analysis of electrophoregram confirms the presence in the sample of IL-2 with a molecular weight of 15.3 kDa (compared with markers of molecular masses of proteins) and allows to determine the amount of IL-2 using direct calibration by bovine serum albumin.

Example 8. The biological activity of the drug received IL-2

As a standard for ODA the fission activity test IL-2 in the extract, obtained in example 6, using calibrated to international reference reagent sample IL-2, and stored at -70°and thawed only once.

All wells with the standard and the samples containing 10 µl of the extract obtained in example 6 add 100 ál of the washed medium RPMI 1640 cell CTLL-2 (1×105living cells per ml) and close the tablet leaky cap. Incubated at 37°C for 42 hours. In samples containing IL-2, cell proliferation CTLL-2. After incubation, each well contribute 10 μl of MTT solution and incubated for another 6 hours. In viable cells, stimulated IL-2, form water-insoluble dark blue crystals formazan.

Tablets centrifuged for 5 min at 2000 rpm and remove the supernatant from the wells sharply strjahivaniem fluid. To each well add 100 ál of DMSO and shaken tablets on the shaker to dissolve the crystals formazan (about 15 min).

On the basis of spectrophotometric data for the standard and test samples calculate the activity of IL-2 in the extract, calculate its activity at 100 mg of powder, which is on average 4-5 million

Example 9. Treatment of food allergies preparation of example 3

The product of example 4 in the amount of 100 mg was diluted in 5 ml of water and given to the patient suffering of all what rgiya on a green Apple. The course of treatment was 5 days according to the scheme: the contact with the allergen was performed every other day, with the time of occurrence of allergic symptoms, the patient took the composition according to the invention. Contact with green Apple and the drug according to the invention was carried out 3 times.

On the 15th day after the beginning of the test PRICK-test showed no reaction to green Apple. Control after 6 months gave the same results.

Thus, the preparation obtained by the method according to the invention has proved successful in the treatment of disease, which before he could not heal completely, as all known so far, the drugs would offer only symptomatic treatment. Treatment of food allergies some famous still drugs based on recombinant IL-2 also previously was not done because it was assumed that it would either ineffective or the side effects typical of these drugs will exceed the expected benefits.

Example 10. Monotherapy of HIV infection using the product from example 4

The first two courses were conducted with the drug "Roncoleukin®dry injection (biotech, Russia), is a purified protein, the latter two with the preparation obtained by the method according to the invention, taken orally.

The dynamics of CD4+ and HIV viral load
IndexBefore treatmentAfter two courses of Roncoleukin®After 3 courses of the drug according to the inventionAfter the 4th course of the preparation according to the invention
CD4+(u/µl)4184807561066
HIV (copies/ml)41000200001500950

Dynamics as the first stage of treatment (Ronkoleykin®)and the second (with the preparation according to the invention) is good.

A significant improvement on the dynamics of the content of CD4+ if the drug is obtained by the method according to the invention, compared to a net IL-2 for injection may be caused by the fact that the drug according to the invention has the property not only to increase the proliferation of cells of the immune system, but also has the ability to activate them, because it is the absence of activating factor sometimes results in a lack of effect of IL-2.

Thus, the preparation obtained by the method according to the invention, along with biologically active interleukin-2 also contains a set of adjuvant substances of the type that allow you to use this drug in the treatment failure with use of pure IL-2.

When this drug acquire the technological way, the method of its production does not require excessive time and special equipment. Received the drug is active by oral administration.

1. The method of obtaining the drug interleukin-2 (IL-2), in which the security of the target protein destroy the cells of the yeast producer of IL-2, in which the synthesis of the target protein (IL-2) and which does not secretes this target protein, and separating the water-insoluble fraction, characterized in that the drug remain water-insoluble components of damaged cells.

2. The method according to claim 1, characterized in that the yeast producer is a Saccharomyces cerevisiae strain deposited at the all-Union collection of industrial microorganisms under the number Y-791, which is the producer of the human interleukin-2.

3. The method according to claim 1, characterized in that destroy cells in the presence of inhibitors of yeast proteases.

4. The method according to claim 1, characterized in that the cells destroy using a high-speed grinder with glass beads.

5. The method according to claim 1, characterized in that the separated water-insoluble fraction is washed for a more complete removal of water soluble components.

6. The method according to claim 1, characterized in that the separated water-insoluble fraction is dried.

7. The method according to claim 1, characterized in that separated vodorastvorimoe the fraction is treated with acetone, followed by removal of the acetone fraction.

8. The method according to claim 7, characterized in that after separation of the water-insoluble fraction prepared its suspension in water, this suspension is poured to the acetone at the ratio of the volume of the suspension and acetone from 1:5 to 1:20 with obtaining a suspension in acetone, filtered received acetone slurry and remaining on the filter residue is dried.

9. The method according to claim 8, characterized in that after separation of the water-insoluble fraction before preparing its suspension in water, it is washed for a more complete removal of water soluble components.

10. The method according to any one of claims 1 to 9, characterized in that after drying the separated water-insoluble fraction to the dry residue add dry the solubilizer.

11. The method according to claim 10, wherein the solubilizer is sodium dodecyl sulphate.

12. The method according to claim 10, characterized in that after the addition of a solubilizer add water.

13. The drug interleukin-2, obtained by the method according to any one of claims 1 to 12.



 

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1 tbl

FIELD: biotechnology.

SUBSTANCE: invention provides preparation containing microorganism strains Bacillus mycoides var "V.A.VNIISKhM No D138" and Azotobacter vinelandii var. "NP VNIISKhM No D24" with cell titer (0.8-1.2)·108 cl/mL taken at ratio (1.8-2.2):1, and further containing humates with space elements so that total composition is as follows: microorganism strains 25-30%, humates 8-12%, space elements 0.05-0.08%, and water the balance.

EFFECT: increased activity against various diseases and imparted growth stimulation effect.

5 ex

FIELD: biotechnology.

SUBSTANCE: invention provides preparation containing microorganism strains Bacillus mycoides var "V.A.VNIISKhM No D138" and Azotobacter vinelandii var. "NP VNIISKhM No D24" with cell titer (0.8-1.2)·108 cl/mL taken at ratio (1.8-2.2):1, and further containing humates with space elements so that total composition is as follows: microorganism strains 25-30%, humates 8-12%, space elements 0.05-0.08%, and water the balance.

EFFECT: increased activity against various diseases and imparted growth stimulation effect.

5 ex

FIELD: virology.

SUBSTANCE: invention proposes avian influenza virus strain, subtype H5N1. The strain is deposited in the Specialized virus collection of Virology center NII microbiology in Russian Federation defense Ministry at № 1141. Invention can be used in viral, serodiagnosis, immunological and molecular-biological methods of investigation.

EFFECT: valuable properties of strain.

1 tbl

FIELD: medicine, in particular drug screening for tuberculosis treatment.

SUBSTANCE: diagnosis is carried out by determination of tuberculosis mycobacteria sensitivity to isoniaside based on mutation detection in katG, inhA, oxyR/ahpC genes by conformational polymorphism of single-strain fragments and detection mutation in katA gene, which is responsible for abovementioned preparation resistance additionally in 10 % experiences. Also disclosed are primers for DNA of tested katA gene, amplification conditions and electrophoresis conditions. Nucleotide sequences of primer pair are disclosed in specification. Buffer containing in 30 mul Tris-HCl 10 mM, pH 8.8; KCl 50 mM; 0.5 % of Twin 20, formamide 5 %; MgCl 2.0 mM MgCl2; Tag-polymerase 1.5 U; each mucleoside triphosphate 200 muM; each oligonucleotide primer 0.1-0.15 muM; and sample 3 mul is used for amplification.

EFFECT: improved method for diagnosis of mycobacterium tuberculosis strain sensitivity to isoniaside.

FIELD: biotechnology; methods of purification of the oil polluted bleaching soil.

SUBSTANCE: the invention is pertaining to the biotechnology, in particular, to purification of the bleaching earth from pollution with the oil products. The method provides for introduction into the soil of the biomass of the consortium of petroleum oxidizing strains - Bacillus brevis IB DT 5-1 and Arthrobacter species IB DT 5-3 and the biomasses of the strain of bacteria Azotobacter vinelandii IB 4 at the mass ratio of the biomasses equal to 1:1. The invention allows to activate the process of the oil products biodegradation, at that the degree of the degradation for 5 months compounds 81 %.

EFFECT: the invention ensures activation of the process of the oil products biodegradation with the degree of the degradation for 5 months making 81 percent.

5 tbl, 1 ex

FIELD: biotechnology; methods of purification of the oil polluted bleaching soil.

SUBSTANCE: the invention is pertaining to the biotechnology, in particular, to purification of the bleaching earth from pollution with the oil products. The method provides for introduction into the soil of the biomass of the consortium of petroleum oxidizing strains - Bacillus brevis IB DT 5-1 and Arthrobacter species IB DT 5-3 and the biomasses of the strain of bacteria Azotobacter vinelandii IB 4 at the mass ratio of the biomasses equal to 1:1. The invention allows to activate the process of the oil products biodegradation, at that the degree of the degradation for 5 months compounds 81 %.

EFFECT: the invention ensures activation of the process of the oil products biodegradation with the degree of the degradation for 5 months making 81 percent.

5 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to method for production of L-threonine using bacterium belonging to genus Escherichia with inactivated ItaE gene.

EFFECT: high effective method for production of L-threonine.

2 cl, 2 dwg, 2 ex

FIELD: biotechnology.

SUBSTANCE: Specific bacterium strain Rhodococcus rhodochrous has ferment system including nitrilehydrase and amidase enzymes for hydrolysis of nitriles and amines. Claimed method includes application of said strain as biocatalyst in hydrolysis process both acrylonitrile and acrylamide. Method of present invention makes it possible to produce mixed solutions of acrylamide and ammonium acrylate monomers in industrial scale.

EFFECT: improved method for large scale production of acrylamide and ammonium acrylate monomers.

2 cl, 1 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: Specific bacterium strain Rhodococcus rhodochrous has ferment system including nitrilehydrase and amidase enzymes for hydrolysis of nitriles and amines. Claimed method includes application of said strain as biocatalyst in hydrolysis process both acrylonitrile and acrylamide. Method of present invention makes it possible to produce mixed solutions of acrylamide and ammonium acrylate monomers in industrial scale.

EFFECT: improved method for large scale production of acrylamide and ammonium acrylate monomers.

2 cl, 1 tbl, 6 ex

FIELD: biotechnology, genetic engineering, microbiology.

SUBSTANCE: invention represents nucleic acid molecules isolated from Corynebacterium glutamicum that encode polypeptide with the protein activity providing the resistance to lincomycin. Also, invention relates to recombinant expressing vectors comprising such nucleic acids molecules, and to host-cells wherein these expressing vectors have been inserted. This invention relates to a method for preparing amino acids by culturing above indicated cells.

EFFECT: expanded assortment of enzymes, enhanced effectiveness in preparing amino acids.

36 cl, 4 tbl, 14 ex

FIELD: organic chemistry, polymers, chemical technology, biotechnology.

SUBSTANCE: method involves carrying out the oxidative polymerization reaction of aniline for a single step. Laccase that uses air molecular oxygen as a substrate is used a s catalyst for reaction wherein in process of this reaction oxygen is reduced to water. After carrying out the enzymatic polymerization reaction a formed polyaniline precipitate is separated from reaction solution, washed out with water and, if necessary, the dedoping process of emeraldin conducting polyaniline salt with ammonia aqueous solution is carried out. Advantage of method involves it's a single step in carrying out the process and ecological purity of the end product. Invention can be used in creature of bio- and chemosensors and separation of optically active compounds.

EFFECT: improved method of synthesis.

5 ex

FIELD: biotechnology, amino acids.

SUBSTANCE: invention relates to a method for preparing L-amino acids by culturing bacterium of genus Bacillus that shows ability for producing L-amino acids in medium containing methanol as a carbon source followed by collection of L-amino acid from medium or cells of bacterium said. As a bacterium method involves using bacterium comprising the inserted gene encoding enzyme methanol dehydrogenase and enhancing activity of enzymes hexulosophosphate synthase and phosphohexulosoisomerase. Invention provides L-amino acids with the high effectiveness degree.

EFFECT: improved preparing method.

6 cl, 1 dwg, 2 tbl, 6 ex

FIELD: food industry.

SUBSTANCE: mixture of D-pantotenic acid and its salts is prepared via culturing of D-pantotenic acid-producing microorganism, wherein pantotenic acid (pan) and/or isoleucine/valine (ilv) biosynthesis control is spoiled and which, in the fermentation process in culturing medium, produces at least 2 g/L D-pantotenic acid salt, while no free β-alanine and/or β-alanine salt is added to the culturing medium. Fermentation solution containing D-pantotenate is then passed through one or several ion-selective membrane under action of electric field resulting in that low-molecular weight ions are removed from D-pantotenate-containing solution and wherein content of D-pantotenic acid salts in the form of monovalent cations is lowered to concentration below or equal to 1 g/kg. PH value of solution containing free D-pantotenic acid is adjusted to 3-10 by adding calcium and/or magnesium bases and thus obtained solution containing calcium and/or magnesium pantotenate is dried and formatted. Thus obtained formulation is useful as feed additive.

EFFECT: simplified process and reduced amount of undesired impurities.

23 cl, 1 tbl, 3 ex

FIELD: biotechnology, fertilizers.

SUBSTANCE: invention proposes an organic nitrogen-containing composition comprising enzymatic mother solution prepared by culturing microorganism of genus Enterobacter that is able to produce L-glutamic acid in liquid medium. Culturing is carried out at pH value providing precipitation of L-glutamic acid under condition that L-glutamic acid is produced and accumulated with accompanying precipitation and the following separation of L-glutamic acid from medium. Microorganism can metabolize carbon source in liquid medium containing L-glutamic acid in the saturation concentration and carbon source at the definite pH value and possesses capacity to accumulate L-glutamic acid in the amount exceeding the saturating concentration of L-glutamic acid in liquid medium at this pH value. Prepared composition is used as a component of fertilizer. The claimed invention provides expanding assortment of fertilizers.

EFFECT: improved preparing method.

5 cl, 9 dwg, 2 tbl, 1 ex

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to synthesis of organic compounds. Acrylonitrile is hydrated in aqueous solution in the presence of a biocatalyst. Control for carrying out the reaction is carried out by measurements of parameters of the technological process in "on-line" regimen by infrared spectroscopy method with Fourier-transformation. The reaction is carried out in reactor fitted by circulation contour wherein part of the reaction mixture is pumped in closed cycle. Device comprises infrared spectrometer with Fourier-transformation providing carrying out measurements in "on-line" regimen rapidly and to retain activity of biocatalyst at the practically constant level in the reaction course and to produce larger amount of the more pure product.

EFFECT: improved method of synthesis.

16 cl, 1 dwg

FIELD: biotechnology.

SUBSTANCE: claimed method includes interaction of mixtures of natural phosphatides or components thereof, for instance, soybean or egg lecithin or animal phospholipids, or synthetic phosphatides, by reaction thereof with D phospholipase having transphosphatidylase activity in water medium.

EFFECT: simplified method for isolation of pure phosphatides of improved purity with increased yield.

28 cl, 13 ex

FIELD: food processing industry, in particular canned soup for space feeding.

SUBSTANCE: claimed method includes carrot and onion cutting, roasting in melt butter and chopping; potato blanching, and chopping; fresh white cabbage shredding, freezing, and chopping; marrow cutting and chopping; scallop fillet and parsley green chopping; green pease blanching and chopping. Abovementioned components are blended tomato paste, table salt, CO2-extract from biomass of specific micromycete specie, black pepper, bay leaf, and drinking water. Obtained mixture is pre-packed in aluminum tubes, sealed and sterilized.

EFFECT: canned goods of increased digestion value.

41 cl

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