EA027601B1 - Method of obtaining interleukin-2 preparation - Google Patents

Abstract

The invention relates to a method of obtaining interleukin-2 preparation, comprising cultivating yeast strain - interleukin-2 producent in nutrient medium for from 36 to 50 hours with aeration from 0.2 to 0.8 litres of air per minute per 100 litres of medium, obtaining sediment of yeast cells, mechanically destructing cells for from 6 to 40 minutes, purifying sediment from admixture yeast proteins and lipids and extracting target protein.

Description

The invention relates to the field of medicine and pharmacology, for the production of immunomodulating drugs by biotechnological methods. In particular, the present invention relates to a method for the preparation of interleukin-2, which can be used to treat diseases of various etiologies, accompanied by manifestations of secondary immunodeficiency, including infectious-inflammatory, septic and oncological diseases.

State of the art

The history of the discovery of interleukin-2 (IL-2) begins in the 1960s, when numerous studies of ίη νίίτο have shown that activated lymphocytes secrete a mediator that stimulates many of the immune responses ίη νίίτο and η νίνο. Only in 1976 was this mediator identified as a cell growth factor, which was called interleukin-2 in 1979. The developed technologies for obtaining it in a culture of activated human lymphocytes and purification made it possible to obtain the first IL-2 preparations for research and clinical purposes. These drugs are called lymphocytic or natural IL-2. However, these preparations containing human blood components are potentially dangerous. In addition, the possibility of scaling up such production is limited, which prevented its widespread use until 1983, when technologies for the production of recombinant proteins, in particular IL-2, were developed using genetic engineering and biotechnology.

Gene engineering methods for producing IL-2 are also known. Numerous firms (Csshz Sogr., Atdsp. 1ps., Neutap-ba Kosier, 1ps.) Used the methods of genetic engineering and biotechnology to obtain recombinant IL-2 as a drug for the treatment of various types of cancer.

In one of the methods for producing recombinant IL-2, E. coB (E. coli) is used as a producer. E. coli is a conditionally pathogenic flora. Therefore, IL-2 preparations from E. cob require thorough cleaning and can be toxic. Yeast cells are more attractive producers of heterologous IL-2.

Known (EP 0162898 A1, published 04.12.1985) a method for producing the drug IL-2 using a yeast producer. In this method, IL-2 is secreted into the culture medium and then must be isolated from the culture medium by chromatography, filtration, extraction, and the like. The disadvantage of this method is that the proteins secreted by the yeast are glycosylated, that is, yeast carbohydrate residues appear in their structure during secretion. Such glycoproteins are highly immunogenic for humans, which creates problems with their further use in therapy, for example, the accumulation of antibodies to such glycosylated proteins in humans.

Known (KI 2304586, priority date 03/27/2006, published 08/20/2007) a method for producing the IL-2 preparation, which provides for the destruction of the cells of the yeast producer IL-2, obtaining a precipitate of destroyed yeast cells, cleaning the sediment from water-soluble and lipid constituents, drying the precipitate with adding dry sodium dodecyl sulfate. To activate IL-2 and acquire biological activity, water is added to such a preparation. The specified method for the preparation of IL-2 does not provide for the extraction of IL-2 from solution. The preparation obtained in this way is characterized by a lower content of IL-2 (50% extractable IL-2 and 50% extractable yeast proteins) and a significant amount of water-insoluble components of the destroyed cells.

Known (EP 0152358, published 21.08.1985) is a method for producing an IL-2 preparation in which yeast culture cells are destroyed in which IL-2 synthesis was carried out, a precipitate of destroyed yeast cells is obtained by centrifugation, the precipitate is treated with sodium dodecyl sulfate solution, in order to in order to transfer IL-2 to the liquid phase, and IL-2 is extracted from the liquid phase. In this method, the extraction of IL-2 from the liquid phase is carried out without preliminary purification of the sediment of the destroyed yeast cells. The drug IL-2 is obtained in low yield and low purity of the target product (IL-2).

Also known (AO 87/00056, published 01/15/1987) a method for producing hydrophobic recombinant proteins, such as, for example, IL-2, in which the stages of destruction of producer cells are carried out, the precipitate of destroyed yeast cells is separated by centrifugation, protein solubilization by treatment with dodecyl sulfate solution sodium and subsequent extraction of the target protein. The protein preparation is obtained in low yield and low purity. To increase the protein yield, it is additionally oxidized, followed by additional chromatographic purification to increase the purity of the protein preparation.

Thus, there still remains a need for a simple method for the preparation of IL-2, which provides a high yield and purity of the target product, as well as its high biological activity.

SUMMARY OF THE INVENTION

The objective of the present invention is to develop a highly efficient technological method for producing the drug IL-2, which is characterized by high yield of the target protein

- 1 027601 (IL-2), its biological activity and the purity of the drug.

This problem is solved by the fact that the proposed method for producing the drug interleukin-2, including cultivating a strain of the yeast-producer of interleukin-2 in a nutrient medium for 36-50 hours with aeration from 0.2 to 0.8 liters of air per minute per 100 liters of medium obtaining a yeast cell pellet, mechanically destroying the cells while preserving the target protein for 6 to 40 minutes, purifying the pellet from impurity yeast lipids and proteins, and extracting the target product (IL2).

The combination of these parameters when implementing the method according to the invention unexpectedly provides a high level of yield, purity and biological activity of the target protein (IL-2).

The present invention also relates to the preparation of interleukin-2, which is obtained by the above method. Using the method of the invention, an IL-2 preparation is obtained with high purity, yield and biological activity of the target protein (IL-2).

The present invention also relates to a pharmaceutical preparation containing the preparation of IL-2, obtained by the method according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

At the present stage of the development of biotechnology and genetic engineering, the most common yeast producer is §assBatotuss8 ssgstMas. In addition, the producers can be any other non-pathogenic yeast, for example 8assbatotuss8 LiuAtF or yeast of the genus RyuLa, for example RyuLaPaYoGe, genus K1iuusgotuss8, for example K1iuusgotuss88aca8, of the genus Naschepuya. for example, Nap8spi1a ro1utogrba, and the like. The technology for producing recombinant plasmids containing the IL-2 gene, in particular human IL-2, is well known. Techniques for producing yeast strains containing such recombinant plasmids are also well known.

In particular, an example of a yeast producer of human IL-2, which can be used in the method according to the invention, is the yeast strain §assBatotuss8 ssgsu181ac, deposited in the All-Union collection of industrial microorganisms under the number 79-791 (RF, 117545, Moscow, 1st Road passage, 1) (patent §I No. 1770359). This strain is a producer of human IL-2. The design of the expression vector introduced into the yeast strain is such that the cells do not secrete the target protein. Any other yeast strain producing heterologous IL-2 can also be used.

To obtain a culture of a strain of yeast producing IL-2, you can use culture media based on well-known and widely used, which can be modified in accordance with the genotype of the cultured strain. Such media compositions are known and are within the competence of a specialist.

So, in particular, for strain Υ-791, you can use the minimum nutrient medium 1 (M1) and the minimum nutrient medium 2 (M2). The culture of strain Υ-791 is first grown in M1 medium to obtain a fresh strain culture, and then this culture is inoculated into M2 medium.

The composition of the nutrient medium M1.

- 2 027601

The composition of the nutrient medium M2.

Environment component Quantity per 1 liter Histidine-HC1xH ₂ O 0.07 g (1CHN ₄ ) ₂ citrate 7.6 g MdZO ₄ x 7 N ₂ O 1.02 g No. C1 0.1 g CaCl ₂ x2H ₂ O 0.2 g KC1 1.5 g KN ₂ RO ₄ 0.007 g Glucose 20 g H ₃ WHO 0.5 mg Cu5O ₄ x5H ₂ O 0.063 mg ΚΙ 0.1 mg GeS1 ₃ x6H ₂ O 0.335 mg Mp8O ₄ 0.4 mg №ΜοΟ ₄ χ2Η ₂ Ο 0.25 mg Ζπ3Ο ₄ χ7Η ₂ Ο 0.72 mg Biotin 0.025 mg Calcium pantothenate 2.5 mg Folic acid 0.025 mg Inositol 12.5 mg A nicotinic acid 5.0 mg P-aminobenzoic acid 0.25 mg Pyridoxine hydrochloride 0.625 mg Riboflavin 0.25 mg Thiamine hydrochloride 0.625 mg

Culturing conditions, such as, for example, the temperature and speed of rotation of the stirrer, are also well known and commonly used to obtain cultures of the corresponding yeast strains. For example, a temperature of from 25 to 34 ° C, preferably from 28 to 32 ° C, most preferably 30 ° C, can be used. The speed of rotation of the mixer may be from 100 to 400 rpm, preferably 200 rpm

The synthesis of IL-2 can be constitutive or require induction or derepression, which is determined by the genetic construct contained in the producer strain.

In general, taking into account the characteristics of the selected producer strain, the specialist is able to select the medium and the above-mentioned well-known conditions for the cultivation of the strain, which will lead to a culture of yeast cells in which IL-2 is synthesized.

In the method according to the invention, the cultivation of the producer strain of IL-2 must be carried out for from about 36 to about 50 hours, which means any period of time included in this interval (for example, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 hours exactly or approximately). The level of aeration of the culture should be from about 0.2 to about 0.8 liters of air per minute per 100 liters of medium, which means 0.2, 0.3, 0.4, 0.5, 0.6, 0, 7 and 0.8 liters of air per minute per 100 liters of medium. Such cultivation conditions (time and rate of aeration of the culture) unexpectedly make it possible to obtain a yeast culture from which it is much easier to obtain the IL-2 preparation with a high yield, purity and high biological activity, which, among other things, is due to a decrease in the amount of undesirable concomitant yeast proteins formed.

The optical density ΘΌ _{600 of the} resulting yeast cell suspension depends, first of all, on the particular strain used. For strain Υ-791 in a medium, Μ1 ΘΌ ₆₀₀ can be from 2.0 to 4.5, preferably from 2.5 to 4.0, most preferably 3.0, and in a medium of Μ2, from 6.0 to 10.0 preferably from 4.0 to 9.0, most preferably 8.0. When culturing strain Υ-791, as a rule, from 8 to 14 g of cells from 1 l of culture medium are obtained.

At the end of the cultivation, the cells are separated from the culture medium using, for example, centrifugation or filtration, which are well-known operations when working with yeast cultures. So get a yeast cell pellet.

All further manipulations are carried out with the cells, and the culture medium is discarded. The resulting biomass can be used immediately or frozen for storage with a view to further use in the method according to the invention.

The destruction of yeast cells is carried out by any known methods, which are mechanical destruction, in particular, using a high-speed grinder with glass balls. An example of a high-speed grinder is the Eupo-MSH disintegrator.

Mechanical destruction of the yeast cells is carried out for 6 to 40 minutes, preferably 10 to 30 minutes, more preferably 15 to 25 minutes, more preferably 20 to 25 minutes, most preferably 25 minutes This time used in the method according to the invention, allows you to effectively destroy the yeast cells of the producer strain of IL-2 and to provide an effective protein yield.

The destruction is preferably carried out at temperatures from 2 to 10 ° C, more preferably from 3 to 8 ° C, more preferably from 4 to 6 ° C, most preferably at 4 ° C; screw speeds from 2000 to 6000 rpm, preferably from 3000 to 5000 rpm, most preferably at 3000 rpm. The size of the glass balls used for mechanical destruction can vary, for example, from 0.4 to 0.6 mm.

It is possible, but not necessary, to add protease inhibitors to the suspension of destructible cells to ensure the preservation of the target protein. Phenylmethylsulfonyl fluoride (PMSF), ethylene diamine tetraacetate (EDTA) are traditionally used as a yeast protease inhibitor, but any other inhibitor or mixture of protease inhibitors can be used. It is sufficient to use PMSF at a concentration of 1-3 mM and EDTA at a concentration of 2 mM, however, another suitable amount of a protease inhibitor can be used.

Purification of the precipitate and extraction of IL-2 from the resulting suspension is carried out using any of the well-known methods (see, for example, New in DNA cloning. Methods // Edited by D. Glover, Moscow, Mir, 1989). For example, centrifugation and washing, treatment with sodium dodecyl sulfate are used to transfer IL-2 to the liquid phase, followed by chromatographic purification by reverse phase HPLC and / or gel filtration.

Purification from impurity yeast lipids and proteins begins with centrifugation of the suspension obtained after mechanical destruction of yeast cells, which can be carried out at low temperatures, for example, from 2 to 8 ° C, preferably from 3 to 6 ° C, most preferably at 4 ° C, and rotor speeds of 4,000 to 12,000 rpm, preferably 6,000 to 11,000 rpm, more preferably 8,000 to 10,000 rpm. The precipitate of the destroyed yeast cells is freed from concomitant yeast proteins and lipids by washing it with buffer solution and nbutanol.

From the washed pellet, IL-2 cells are transferred to the liquid phase by treating the pellet with a solution containing sodium dodecyl sulfate in a concentration of from 1 to 5 wt.%, Preferably from 2 to 3 wt.%, Most preferably 2 wt.%. A solution containing solubilized IL2 is subjected to gel filtration, resulting in the preparation of IL-2.

The invention also provides an IL-2 preparation obtained by the above method in any of its possible variants.

The biological activity of the obtained IL-2 preparation can be evaluated in an international standard test with a culture of IL-2-dependent tumor-specific cytotoxic T lymphocytes.

- 4,027,601 mice of the STL-2 line (Nuttsgkpd i.s1 a1., EsuPortsSh apy wakyop Yoazzau Gog s1sg1siksh-2 // I. Rkagtassik & Vyuskst. Apa1u818, - 1992. - yo1. 10. - p. A. 7.ag 1.N. apy Tkogrs K. Tks 1n1sgpayopa1 81apyagy Gog kitap 1i1sg1sik1i-2. Saakkakop LU t1sgpa0op1 sokakogakus 81uyu // 11ttypo1. Ms1kOi5. Biologically active recombinant IL-2 should stimulate the proliferation of these cells. The determination of biological activity is carried out using the colorimetric method with tetrazolium dye MTT. Viable cells stimulated by IL-2 absorb the dye and accumulate in the cytoplasm water-insoluble dark blue crystals of formazan (reduced MTT). After removal of the culture medium and complete dissolution of the crystals in dimethyl sulfoxide, the optical density of the resulting solution was measured on a computer photometer at a wavelength of 530 and 690 nm. The dark blue staining of the solution in 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 the activity standard of IL-2, determine the biological activity of IL-2 in the incubation sample.

The amount of IL-2 can be determined by any standard method for determining the target protein, for example, by polyacrylamide gel electrophoresis (Lastk and Clausaugts 81gislig1 rg1ssh8 yiggd 1x azzstuy 1x ksai ghaskgurkads T4. // Ba19gg. 680-685) or using an enzyme-linked immunosorbent assay (Osktai L.O. apy Kob K. 1. Ap Ebyba-zazz azzau Gog diakopak oG kitap t1sg1sikt-2 .// T 1ttypo1. MsShoyz. -1984. -Yuo. 74, - p. 39) and others.

Obtained in this way, the drug IL-2 is used for inclusion in pharmaceutical preparations with immunomodulating activity and intended for the treatment of diseases of various etiologies, accompanied by manifestations of secondary immunodeficiency, which are used for intravenous and subcutaneous administration, as well as in the form of for oral, rectal and / or vaginal administration: gelatin capsules (hard and soft), suppositories (with lipophilic, or hydrophilic, or lipophilic-hydrophilic base), as well as re cetal pipettes (rectiols). These pharmaceutical preparations are also suitable for the treatment of severe conditions in mammals, such as oncological, infectious and purulent-inflammatory diseases.

Introduced into the body with these drugs, IL-2 provides an adequate and targeted medical correction of immune dysfunctions, replenishes the deficiency of endogenous regulatory molecules and fully reproduces their effects. High immunocorrecting effectiveness, predictability and selectivity of the action of drugs based on IL-2 are due to the presence of specific receptors on the cells, and the existence of natural mechanisms for its elimination. Pharmaceutical preparations based on IL-2 are powerful means of pathogenetic immuno-oriented therapy and have both a direct replacement effect and have various inductive effects.

In addition to the interleukin-2 preparation obtained by the method according to the invention as an active ingredient, the pharmaceutical preparation contains pharmaceutically acceptable excipients, such as, in particular, a stabilizer, a solubilizer, an antioxidant agent, a buffer, a carrier, which can be realized in the required dosage forms that are specifically known for such drugs (e.g. KI 2140283).

The preparation of pharmaceutical preparations containing IL-2 is well known in the art (see KI 2140283).

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

Examples

Example 1. Obtaining the drug IL-2 (according to the invention)

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

The cells of the yeast strain Zasskagotussz ssgsu131ac Υ-791, stored at -70 ° C in glycerin, are introduced into M1 medium so that the ratio of cell mass to volume of M1 medium is approximately 0.6 g to 1 liter. Cultivation is carried out in M1 medium and the resulting inoculum is transferred to a tenfold larger volume of M2 medium. Cultivation in an M2 medium is carried out at a temperature of 30 ° C, a stirrer rotation speed of 200 rpm, with an aeration level of 0.2 liters of air per minute per 100 liters of medium, for 36 hours

At the end of cultivation in M2 medium, the cells are separated from the culture medium by centrifugation and 12 g of cells are obtained from 1 L of culture medium.

1.2. The destruction of the cells of the yeast producer strain of IL-2

From the obtained biomass, a 50% suspension of cells in 5 mM Tris-HCl buffer solution with the addition of 2 mM EDTA and 2 mM PMSF, pH 7.2 (solution A) is prepared. Next, mechanical destruction of the yeast cells is carried out using glass balls from 0.4 to 0.6 mm in size in a high-speed Eupo MSH disintegrator at a temperature of 4 ° C and a screw rotation speed of 3000 rpm for 6 minutes. A suspension of disrupted cells is collected and centrifuged.

1.3. Purification and extraction of the target protein

- 5,027,601

The suspension obtained in step 1.2 is centrifuged at a temperature of + 4 ° C and a speed of 10,000 rpm. The precipitate is washed many times to remove yeast proteins with solution A, and to remove lipids with n-butanol. After processing the precipitate with n-butanol, the last washing of the precipitate is carried out with the above solution A.

IL-2 was extracted from the treated precipitate with a buffer having the following composition: 0.1 M ammonium hydrogen carbonate, 2% sodium dodecyl sulfate, 2 mM mercaptoethanol, 2 mM EDTA, 2 mM PMSF, pH 8.0 (solution B). The extract obtained is applied to a Sephacryl 8-300 column balanced with solution B. Gel filtration is carried out and protein fractions are collected, in which the optical density is measured at a wavelength of 280 nm, and an elution profile is obtained. The quality of the fractions with IL-2 is assessed by electrophoresis under denaturing conditions, comparing with the standard protein IL-2. Based on the results obtained, a conclusion is drawn on the selection of the purest protein fractions containing IL-2. Combine fractions containing IL-2 with a minimum amount of impurity proteins. For immunomodulatory drugs from non-pathogenic sources, the minimum amount of impurity proteins should not exceed 5%. The purity and amount of IL-2 in the combined eluate was determined by electrophoresis under denaturing conditions, as well as the biological activity of the CTb-2 cell culture assay.

Get the drug with a biological activity of 1.3 million IU / mg and a purity of IL-2 95%. The yield of the drug is 78%.

Example 2 (according to the invention).

The preparation of IL-2 according to Example 1 is carried out, except that the cultivation time is 50 hours. A preparation is obtained with a purity of IL-2 of 95% and a biological activity of 1.2 million IU / mg. The yield of the drug is 80%.

Example 3 (according to the invention).

The preparation of IL-2 is carried out according to Example 1, except that aeration is 0.8 L of air per minute per 100 L of medium. A preparation is obtained with a purity of IL-2 of 95% and a biological activity of 1.2 million IU / mg. The yield of the drug is 79%.

Example 4 (according to the invention).

The preparation of IL-2 is carried out according to Example 1, except that the destruction time of yeast cells is 40 minutes. Get the drug with a purity of IL-2 95% and a biological activity of 1.1 million IU / mg. The yield of the drug is 80%.

Example 5 (according to the invention).

The preparation of IL-2 is carried out according to Example 1, except that the cultivation time is 43 hours, aeration is 0.4 L of air per minute per 100 L of medium, and the destruction time of yeast cells is 25 minutes. A preparation is obtained with a purity of IL-2 of 96.5% and a biological activity of 1.5 million IU / mg. The yield of the drug is 85%.

Example 6 (comparative).

The preparation of IL-2 according to Example 1 is carried out, except that the cultivation time is 30 hours. A preparation is obtained with a purity of IL-2 of 80% and a biological activity of 0.95 million IU / mg. The yield of the drug is 40%.

Example 7 (comparative).

The preparation of IL-2 according to Example 1 is carried out, except that the cultivation time is 60 hours. A preparation is obtained with a purity of IL-2 of 85% and a biological activity of 0.9 million IU / mg. The drug yield is 60%.

Example 8 (comparative).

The preparation of IL-2 is carried out according to Example 1, except that aeration is 0.1 L of air per minute per 100 L of medium. Get the drug with a purity of IL-2 80% and a biological activity of 0.95 million IU / mg. The yield of the drug is 61%.

Example 9 (comparative).

The preparation of IL-2 is carried out according to Example 1, except that aeration is 0.9 L of air per minute per 100 L of medium. Get the drug with a purity of IL-2 78% and a biological activity of 0.9 million IU / mg. The yield of the drug is 50%.

Example 10 (comparative).

The preparation of IL-2 is carried out according to Example 1, except that the destruction time of yeast cells is 4 minutes. Get the drug with a purity of IL-2 80% and a biological activity of 0.95 million IU / mg. The yield of the drug is 40%.

Example 11 (comparative).

The preparation of IL-2 is carried out according to Example 1, except that the destruction time of yeast cells is 50 minutes. A preparation is obtained with a purity of IL-2 of 75% and a biological activity of 0.8 million IU / mg. The yield of the drug is 35%.

Thus, the method of obtaining the preparation of IL-2 according to the invention allows to obtain an effective preparation of IL-2 with the optimal combination of parameters of purity, quantity and biological activity.

Claims (8)

CLAIM 1. A method for producing an interleukin-2 preparation, including culturing strain 8assbagotussk ssgstias - producer of interleukin-2 in a nutrient medium for 36 to 50 hours with aeration from 0.2 to 0.8 liters of air per minute per 100 liters of medium, obtaining a precipitate yeast cells, mechanical destruction of cells within 15 to 40 min, purification of the precipitate from impurity yeast lipids and proteins, including washing with n-butanol, extraction of the target protein and chromatographic purification of the extract. 2. The method according to claim 1, where the yeast producer of human interleukin-2 is a strain of yeast §assLagoshuss8 segOmas VKPM Υ-791. 3. The method according to claim 1, where the cultivation is carried out for 43 hours 4. The method according to claim 1, where the cultivation is carried out by aeration of 0.4 l of air per minute per 100 l of medium. 5. The method according to claim 1, where the mechanical destruction of the cells is carried out for 25 minutes 6. The method according to claim 1, where the yeast cell pellet is obtained by centrifugation or filtration. 7. The method according to claim 1, where the cells are destroyed in the presence of yeast protease inhibitors. 8. The method according to claim 1, where the mechanical destruction of the cells is performed using a high-speed grinder with glass balls.