RU2441914C1 - Method for producing a substance of the recombinant l-asparaginase from erwinia carotovora - Google Patents

Abstract

FIELD: biochemistry. ^ SUBSTANCE: invention relates to the area of pharmaceuticals industry and biochemistry. The proposed method for obtaining a substance of the recombinant L-asparaginase from Erwinia carotovora implies covalent modification with polyethyl glycol of the recombinant L-asparaginase split off fom the microbial mass of the genetically engineered producer strain Escherichia coli BL(DE3)/pACYS-LANS(KM) with the deleted gene of resistance to ampicillin in the pACYS-LANS plasmide. Such modification shall be performed by associating N-hydroxysuccinimide ester monometoxypolyethylene glycol- hemisuccinate (mPEG-suc-NHS) to the asparaginase lysine aminogroups. The obtained conjugate shall be subjected to chromatographic purification and freeze-drying. The method also provides for the stabilization of the modified and purified product. ^ EFFECT: obtaining of a new potent substance of PEGylated asparaginase based on Erwinia carotovora enzyme that may be used as antitumor drug. ^ 4 cl, 5 dwg, 5 tbl, 2 ex

Description

The invention relates to the field of pharmaceuticals and biotechnology and can be used to obtain the antitumor chemotherapeutic drug L-asparaginase Ervinia carotovora.

Class 2 bacterial asparaginases (K.F. 3.5.1.1) are periplasmic enzymes that hydrolyze L-asparagine to form L-aspartic acid and ammonium. Asparaginases also catalyze the hydrolysis of L-glutamine, but at a lower rate than L-asparagine. Bacterial asparaginases are used as antitumor drugs in combination chemotherapy for acute lymphoblastic leukemia, myeloid leukemia, Hodgkin’s disease and non-Hodgkin’s lymphoma, melanosarcoma and multiple myeloma. In leukemic cells, asparagine synthase activity is reduced or completely absent. In this case, protein synthesis and, accordingly, tumor cell growth depends on the level of exogenous asparagine. The introduction of the asparaginase drug leads to a decrease in the level of asparagine in the blood and tissues and, as a result, to selective inhibition of tumor cell proliferation. The use of asparaginase preparations in antitumor therapy is limited by various side effects. One of the established causes of asparaginase toxicity is the L-glutaminase activity of the enzyme. Therefore, the search for new bacterial asparaginases with low glutaminase activity is an important task of modern medical biotechnology.

Currently, there are two least toxic enzymes, namely the native preparation of L-asparaginase from Escherichia coli (EcA), as well as “pegylated” (“Oncaspar”) modified with polyethylene glycol and Erw, native enzyme (ErA). chrysanthemi, found application in oncotherapy (Umesh K. Narta, Shamsher S. Kanwar and Wamik Azmi, Pharmacological and clinical evaluation of l-asparaginase in the treatment of leukemia. Critical Reviews in Oncology / Hematology. 2007, 61 (3): 208- 221).

The least toxic drugs are those derived from Erw. chrysanthemi, which indicates the importance of the study of asparaginases of the genus Erwinia. Nevertheless, even asparaginases of Erwinia strains cause the development of immunological hypersensitivity to L-asparaginase, which manifests itself in the range from mild allergic reactions to anaphylactic shock.

Known methods for purification of native asparaginases from Erwinia strains, including extraction of the enzyme either from microbial cells at a pH of 11.3 to 11.5, followed by acidification of the extract to a pH of 4.8, or from acetone powder, followed by chromatographic purification on various sorbents (for example, US 5310670, 05/10/1994).

The disadvantages of such methods for the isolation of Erwinia asparaginases are the difficulties in processing large volumes of fractions at the initial stages of purification (alkaline lysis, obtaining acetone powder), the multi-stage purification process, and the relatively low yield of the purified target product.

Earlier, we developed the genetic engineering producer of asparaginase Erwinia carotovora - (ECAR-LANS), produced by the Escherichia coli strain BL (DE3) / pACYS_LANS, and developed a method for the isolation and purification of the enzyme (RU 2224797, February 27, 2004).

However, the process of isolation and purification takes considerable time, while the yield of the target product should be recognized not high enough, which leads to a high cost of the product.

It should also be noted that the effectiveness of asparaginases in antitumor therapy is limited by the rapid proteolytic degradation of the enzyme in the blood. In this regard, it is relevant to obtain modified forms of asparaginases of Erwinia strains that are resistant to proteolytic degradation in the blood, have low immunogenicity while maintaining high enzymatic activity.

Known drug PEG-L-asparaginase Escherichia coli, in which asparaginase is covalently linked to monomethoxy-PEG (Keating M.J., Holmes R., Lerner S. But D.H. (1993) Leuk. Lymphoma 10 Suppl. 153-157).

Compared to native E. coli asparaginase, PEG-L-asparaginase has lower immunogenicity and a 5-fold longer half-life from the body.

However, in the literature there is no data on the use for chemotherapy of acute leukemia with PEGylated asparaginase, obtained on the basis of an enzyme from Erwinia strains.

An object of the present invention is to provide a method for producing a pegylated asparaginase (PEG asparaginase) substance based on an enzyme from Erwinia carotovora, which is suitable for industrial scaling, and the resulting product can be used for chemotherapy of leukemia.

The problem is solved by the described method for the preparation of a substance of recombinant L-asparaginase Erwinia carotovora, which involves the modification of L-asparaginase with polyethylene glycol, while the modification is subjected to recombinant L-asparaginase isolated from the microbial mass of the genetically engineered producer strain Escherichia coli p BLACLANS3) KM), in which the ampicillin resistance gene has been deleted in plasmid p / ACYS_LANS, modification is carried out by addition of the monomethoxypolyethylene glycol hemisuccinate N-hydroxysuccinimide ester (mPEG-suc-NHS ) to the amino groups of asparaginase lysine, after modification, the obtained conjugate of the recombinant L-asparaginase Erwinia carotovora with polyethylene glycol (PEG asparaginase) is chromatographed and lyophilized.

Preferably, the purified PEG asparaginase is stabilized using a compound selected from the range of proteins, amino acids, polyvinyl pyrrolidones, poly- or monosaccharides, polyalkylene oxides, amino sugar, and salts as stabilizer.

The method involves the use of the microbial mass of the strain deposited in VKMP under collection number No. B-10370 as a source of L-asparaginase, wherein the isolation of L-asparaginase is carried out by destroying the cells on the French Press, followed by salting out of the target product with ammonium sulfate and chromatographic purification.

In the proposed method, a recombinant Erwinia carotovora L-asparaginase is used, the nucleotide sequence of which lanS gene, as well as the amino acid sequence of L-asparaginase are shown in FIG.

Using for modification of recombinant L-asparaginase obtained using the producer strain Escherichia coli BL (DE3) / pACYS_LANS (XM), provides the microbial mass of the producer strain, in the cells of which the asparaginase content reaches 20% of the total protein, which allows further use highly effective methods of cell destruction at the French Press with salting out of the target product from a cell-free extract, which, in turn, intensifies the process of isolation and further purification of L-asparaginase. All these techniques can increase the yield of the enzyme and reduce the cost of the resulting substance asparaginase.

As for the choice of the covalent modification method, it is based on the following.

As is known, for the modification of protein molecules, the most suitable and frequently used derivative of polyethylene glycol is polyethylene glycol monomethyl ether (mPEG), which can be used either as a linear molecule with a preferred molecular weight of from 2000 to 5000, or as a branched molecule of high molecular weight ( see e.g. Ton GN, Fineb JP, Kwonc GS Methoxypoly (ethylene glycol) -conjugated carboxypeptidase A for solid tumor targeting Part I: Synthesis and characterization Journal of Controlled Release 2005, vol. 104, pp. 129-139, or Alexandre Learth Scares, Gledson Manso Guimarães, Bronislaw Polakiewicz, Ronaldo Nogueira de Moraes Pitombo and Jose Abrahão-Neto. Effects of polyethylene glycol attachment on physicochemical and biological stability of E. coli L-asparaginase International Journal of Pharmaceutics, 2002, vol. 237 (1-2), pp. 163-170). The use of monomethoxy-PEG-p-nitrophenylcarbamate (mPEG-NPh) for the preparation of PEG asparaginase is not possible due to their high cost.

We have proposed a different approach to the preparation of PEG asparaginase. The synthesis of N-hydroxysuccinimide ester of monomethoxypolyethylene glycol hemisuccinate (mPEG-suc-NHS) is preliminarily carried out, and then it is attached to the amino groups of asparaginase. This reagent compares favorably with polyethylene glycol p-nitromethyl carbamate monomethyl ether in that the ester bond connecting succinic anhydride and polyethylene glycol is more stable than the ureidyl bond connecting the carboxyl group and polyethylene oxide in mPEG-NPh.

The list of drawings illustrating the invention:

figure 1 - the nucleotide sequence of the lanS gene and the amino acid sequence of L-asparaginase Erwinia carotovora;

figure 2 - electrophoretic analysis of asparaginase samples under reducing conditions after staining the gel with Kumasi dye - 250;

figure 3 - electrophoretic analysis of asparaginase samples under reducing conditions after staining the gel with silver at a load per well of 5 μg of protein;

figure 4 is a chromatogram of the products of the pegylation reaction;

figure 5 (a, b, c) - antitumor cytotoxic activity of substances of L-asparaginases.

Below are examples illustrating the implementation of the invention and the technical result achieved by its use.

Example 1

Stage 1 - Growing producer Escherichia coli BL (DE3) / pACYS_LANS (KM) in a 300 liter fermenter

The producer strain is obtained by transforming competent cells of the recipient strain of E. coli BL21 (DE3), (Novagen, Cat. No. 69387-3) with the plasmid pACYC_LANS (KM).

To create a cell bank, individual clones of E. coli BL21 (DE3) / pACYC_LANS (ICM) transformants obtained on LB agar with kanamycin are used. For laying for storage, a producer colony is grown in LB broth until the middle of the logarithmic growth phase and the resulting suspension is introduced into cryovials containing a 30% glycerol solution in a 1: 1 ratio. The mixture is thoroughly mixed and quickly frozen at a temperature of -70 ° C.

To develop the methodology for preparing seed for fermentation in a 300-liter fermenter, a scheme is used that provides optimal temporal characteristics of the cultivation process in combination with a minimum number of producer passages at the scaling stage. As a starting material for fermentation, a producer’s night culture grown on a medium with glucose and kanamycin is used, which, after reseeding into a fresh nutrient medium, is grown until the middle of the exponential growth phase and introduced into a 30-liter inoculator, after cultivation in which the producer enters a 300-liter fermenter. Cultivation is carried out in a nutrient medium based on LB-M9 broth, at a temperature of 37 ° C, pH 7.0, with aeration with air at a flow rate of 150-70 l / min. At the end of the cultivation, the culture fluid is cooled to a temperature of 14-15 ° C with running tap water, concentrated for 3 hours on a membrane unit (pore size of the membranes 0.25 μm) to a volume of 30 l and separated on a separator ASG-3M at 9000 rpm to obtain wet biomass. The level of asparaginase activity is determined by direct non-sclerization.

Table 1 shows the results of growing the producer strain on the flasks.

Table 1 Producer growing conditions on flasks Induction time OD 560 nm Activity ME / mg * (%) asparaginase in the cell Option No. 1 40 ml LB, 180 rpm 5 hour 4.8 6.7 7.4 21 hours 4.2 1.7 6.6 Option No. 2 160 ml LB, 90 rpm 5 hour 2.7 6.5 9.0 21 hours 4.0 35.0 20.3 25 hour 3.8 25.0 18.2 * the content of asparaginase in the cell (% of total protein)

Table 2 shows the results of the cultivation of the strain in a 300-liter fermenter.

table 2 The results of the cultivation of the strain in a 300-liter fermenter The volume of culture medium (l) Biomass yield (g) Asparaginase Activity Level (IU / ml) 260 2500 140 260 1900 150

Stage 2 - Purification of rec-ECAR_LANS from biomass grown in the fermenter and enzyme characterization

First, cell-free extract is obtained.

The destruction of biomass is carried out on a French Press in a lysis buffer (10 mM EDTA, pH 9.5) at a cell to lysis buffer ratio of 1: 4 and a working pressure of 1580-1600 bar. The yield of enzyme activity at the stage of cell-free extract is 80-93%.

Stage 3 - Ammonium sulfate fractionation and desalination

The process of salt fractionation is carried out at a temperature of + 4 ° C. When saturating the cell-free extract with ammonium sulfate up to 20%, it is possible to get rid of part of the ballast proteins by centrifugation, with further saturation of the supernatant with ammonium sulfate in the range from 20 to 60%, a precipitate forms that contains almost all asparaginase activity.

For desalination, a 5.5 L (14.0 x 35.5 cm) Sephadex G-50 column (coarse) was used in 20 mM potassium phosphate buffer containing 1 mM glycine and 1 mM EDTA, pH 5.6. Desalination is carried out at flow rates of about 30 ml / min.

Stage 4 - Ion Exchange Chromatography on CM-Sepharose FF

The desalted fraction of the ammonium sulfate precipitate was diluted in half with the same buffer and applied to a column of CM-Sepharose FF (10 × 3.2 cm), equilibrated with 20 mM potassium phosphate buffer containing 1 mM glycine and 1 mM EDTA, pH 5.6 at a rate of about 400 ml / hour. The slip is controlled by determining the enzymatic activity qualitatively (micromethod) using Nessler's reagent. After applying the sample, the column is washed with 20 mM potassium phosphate buffer, pH 6.3, to remove ballast proteins. The elution of the target substance is carried out with 20 mm potassium phosphate buffer, pH 7.0 and 7.6. The final stage of chromatographic purification of the prefabricated asparaginase is a stage of sample concentration, which is simultaneously accompanied by a deeper degree of purification.

Stage 5 - Preparation of the drug for lyophilization

Concentration is carried out on a column of CM-Sepharose FF (2.5 × 10.0 cm) balanced with 20 mM potassium phosphate buffer, pH 6.3. A column with a DEAE sorbent CnC-DEAE-Bio (5 × 6 cm) was used as a pre-column. Columns with a DEAE sorbent are also equilibrated with 20 mM potassium phosphate buffer, pH 6.3.

The target fraction obtained from a 250 ml CM-Sepharose FF column was diluted with an equal volume of 20 mM KH ₂ PO ₄ , while the pH of the fraction was reduced from 7.0 to 6.3-6.4. The sample is applied through a pre-column with a DEAE sorbent to the main column with CM-Sepharose FF with a flow rate of 8 ml / min. After applying the sample, the column is washed thoroughly with 20 mM potassium phosphate buffer, pH 6.3 to the baseline, and then the protein is eluted with 20 mM potassium phosphate buffer, pH 7.6.

The yield of the target substance at this stage is 95-97%.

As a stabilizing additive, glucose is used, which is added to the preparation before lyophilization to a final concentration of 0.5%.

Stage 6 - lyophilization.

Lyophilization is carried out on an Alpha freeze dryer ("Christ", Switzerland). In some experiments, concentrates containing 0.5% glucose are lyophilized on a shelf at -10 ° C for 2 days, then the shelf is heated to a temperature of 30 ° C. In others, lyophilization is carried out in flasks at room temperature. Loss of asparaginase activity during lyophilization in both cases is from 5 to 10%.

Thus, an intermediate product is obtained - the substance of asparagase, not subjected to modification by polyethylene glycol.

Characteristics of the obtained substance asparaginase

The protocol for isolating a sample of the substance is presented in table 3.

Table 3 Asparaginase Isolation Protocol Cleaning stage Common protein Asparaginase Activity Exit, % General (ME) Specific (IU / mg protein) Cell suspension - 971 960 - one hundred Cell-free extract - 894,800 - 92 Ammonium sulfate fraction (20-60%) + Desalination (103 g sulfate precipitate) 7437 855 324 115 88 Chromatography on CM-Sepharose 1339 641,493 479 66 Concentration on CM-Sepharose at pH 6.3 1308 612 334 468 63 Lyophilization 1277.6 544,297 426 56 * Biomass amount - 128 g (984 718 ME). Lysis buffer 10 mM EDTA, pH 9.5 Biomass ratio: lysis buffer 1: 4 Operating pressure 1580-1600 bar

The enzyme yield at the final stage of purification was 56%, which is approximately 20% lower than when using the laboratory method, which is associated with a scaling of the process of isolation of the target product and an increase in the number of purification steps.

Determination of the purity, homogeneity and molecular weight of the preparation of recombinant asparaginase Erwinia carotovora

- According to the electrophoresis in SDS-PAGE with a protein load of 10 or 40 μg per well and gel staining with Kumasi R-250 dye, asparaginase preparations are presented mainly in one strip (Fig. 2). In the case of silver staining of the gel with a protein load of 5 μg per well, one main band with a molecular weight of 34-35 kDa and several minor bands with a lower molecular weight (from 15 to 30 kDa) are also detected (Fig. 3), as well as in an asparaginase solution E.coli produced by the German company Medac. These low molecular weight components, the amount of which increases during storage of the enzyme, are apparently products of the degradation of asparaginase.

- Under denaturing conditions, according to the results of electrophoresis in SDS-PAGE, asparaginase is represented by a monomer with a molecular weight of 34 ± 2 kDa.

- Under native conditions, asparaginase has a molecular weight of 67.6 ± 3 kDa according to gel filtration on a Superose ^R 12 column.

The purified substance rec-ECAR-LANS fully meets the requirements for immunobiological recombinant preparations (the content of bacterial impurity proteins E.coli and endotoxins) (see table 4).

Table 4 Characterization of the Prefabricated L-Asparaginase Options Values Concentration (mg / ml) 36.8 The monomer content according to SDS-EF (%) 92 The homogeneity of L-asparaginase under native conditions according to gel filtration chromatography 98.10 The content of endotoxins (eE / mg) 1,63 E.coli protein content (ng / mg) 58 Specific Specific Activity (IU / mg protein) 386.9

Rec-ECAR-LANS was obtained in a close to homogeneous state, with a specific activity corresponding to the bacterial asparaginase Erwinia carotovora. L-glutaminase activity of rec-ECAR-LANS is approximately 5% of L-asparaginase, which will minimize toxic side effects when using substances associated with the hydrolysis of L-glutamine.

Stage 7 - Obtaining starting reagents for the synthesis of pegylated forms rec-ECAR_LANS

1) Synthesis of monomethoxy-PEG5000-hemisuccinate. To obtain PEG-hemisuccinate, 50 g of monomethoxy-PEG (mPEG) is dissolved in absolute benzene and the benzene is carefully evaporated in a vacuum of a water-jet pump. Due to the ability of benzene to form an azeotropic mixture with water, this removes traces of water from the polymer. This procedure is repeated three times, after which the polymer is dissolved in absolute chloroform and a 10-fold molar excess of succinic anhydride and absolute triethylamine is added. The reaction mixture was kept for 3 days at 60 ° C, after which it was filtered through a glass filter and chloroform was evaporated on a rotary evaporator. Next, the resulting carboxylated mPEG is dissolved in 30 ml of 1 M hydrochloric acid, washed with diethyl ether and the modified mPEG is extracted into chloroform (300 ml). The chloroform layer is washed several times with 1 M HCl to remove succinic acid. Then the chloroform polymer solution is dried over anhydrous sodium sulfate and evaporated on a rotary evaporator. At the last stage, the resulting product is dissolved in water and dialyzed against water using benzoylated membranes that do not allow substances with a molecular weight of more than 2 kDa. The resulting solution was freeze-dried. The resulting product is an odorless white powder. The product yield is 50%.

2) To obtain the N-hydroxysuccinimide ester of PEG-hemisuccinate, 50 g of the preparation of polyethylene glycol-hemisuccinate are dissolved in 100 ml of absolute benzene and evaporated in vacuo. The procedure is repeated three times to remove traces of water in the PEG preparation. Next, the resulting preparation was dissolved in 200 ml of freshly distilled thionyl chloride, boiled for 4 hours under reflux, kept for another 20 hours at room temperature, after which the thionyl chloride was distilled off on a rotary evaporator. The resulting product is mPEG-suc acid chloride, mPEG-suc-Cl, which is immediately used in the synthesis of N-hydroxysuccinimide ester. The drug is dissolved in 300 ml of absolute methylene chloride and placed in a dropping funnel attached to a three-necked flask. A solution of 1.5 g (8.7 mmol) of N-hydroxysuccinimide and 1 ml (7 mmol, 0.7 g) of absolute triethylamine in methylene chloride was placed in the flask and cooled in an ice-salt bath to -10 ° C. The mPEG-suc-Cl solution was carefully added to the N-hydroxysuccinimide solution with vigorous stirring, making sure that the temperature did not rise above -5 ° C. After the addition of the total acid chloride, the reaction mixture was allowed to stir at room temperature for another 18 hours, after which the product was precipitated with hexane and the precipitate was isolated in a centrifuge. Next, the product is washed three times with hexane and dried in a vacuum desiccator in a vacuum of a water-jet pump over alkali. The product is stored in a desiccator over alkali to avoid contact with water vapor.

Stage 8 - Obtaining pegylated forms of asparaginase

Dry lyophilized powder of the asparaginase preparation is placed in a vessel equipped with a magnetic stirrer. To an enzyme preparation with a concentration of 35 mg / ml in 20 mM K ₂ PO ₄ add 0.5 M borate pH 9.5 (1/5 by volume) to bring the pH of the medium to 8.5 (optimal for modification with N-hydroxysuccinimide esters), after which the solution the enzyme is added to dry mPEG-suc-NHS. The solution was stirred vigorously for 30 minutes at room temperature until the drug was completely dissolved, then cooled to + 4 ° C and continued incubation in the cold with constant stirring for 4 hours. After this, the enzyme is separated from PEG using gel chromatography on a column of Sephadex G-50.

Stage 9 - Removal of the components of the reaction mixture to obtain purified PEG asparaginase

PEG asparaginase is purified in three stages: after the pegylation reaction, the reaction mixture is dialyzed through membranes with an excluded molecular weight of 100 kDa against potassium phosphate buffer, pH 7.2, for a day, after which the resulting preparation is subjected to two-stage chromatographic purification on a column with DEAE-Sepharose FF and on a column with Diasphere ST-150.

Column chromatography with DEAE-Sepharose FF: after dialysis, a 41 ml sample containing about 500 mg of PEG asparaginase is applied to a 25 × 20 mm column (10 ml volume), equilibrated with 20 mM potassium phosphate buffer, pH 7.2 . Flow rate 1 ml / min. After passing the sample, elution of the PEG asparaginase is carried out with the same buffer. The target substance is determined in the slip and makes up a fraction of 46 ml.

Chromatography on a CT-150 Diasphere column: a 42 ml sample was applied to a 25 × 70 mm column (37 ml volume), equilibrated with 20 mM potassium phosphate buffer, pH 7.2. Flow rate 3 ml / min. After applying the sample, elution is carried out with the same buffer. The target substance is determined in the slip and constitutes a 38 ml fraction with a protein concentration of 6.8 mg / ml and a specific activity of PEG asparaginase of 350 IU / mg.

Thus, the yield of the target substance after the chromatographic purification step is about 50%.

Example 2 - Stabilization of purified PEG asparaginase using various stabilizers

The combined fractions containing purified PEG asparaginase are dialyzed against 20 volumes of 20 mM sodium phosphate buffer, pH 7.2 at a temperature of 6 ± 3 ° C. The resulting dialysate is placed in plastic or glass vials with a siliconized surface, tween-80 or tween-20 is added to a final concentration of 0.002% and stabilizers.

As stabilizers, proteins, amino acids, polyvinylpyrrolidones, polysaccharides, polyalkylene oxides, monosaccharides, amino sugar, and salts were tested (the data are summarized in Table 5).

In specific examples, the following substances were used. As polyvinylpyrrolidone use polyvinylpyrrolidone-12000 at a concentration of 3-6%, as a protein - human serum albumin at a concentration of 1%, as an amino acid use L-arginine at a concentration of 2-6%, polyethylene glycol 1500 is used as a polyalkylene oxide at a concentration of 2- 5%, dextran-60 at a concentration of 3-6% is used as a polysaccharide, mannitol at a concentration of 4-7% is used as a monosaccharide, glucosamine is used at a concentration of 6-10% as amino sugars, sodium chloride is used as salt concentration of 0.9%.

The stability of the obtained compositions was studied by accelerated storage at a temperature of (36 ± 2) ° C for 2 weeks. After 2 weeks, aliquots were selected and the biological activity of PEG asparaginase was analyzed. The duration of storage at a temperature of (36 ± 2) ° C is calculated as the duration of storage at a temperature of (6 ± 2) ° C according to the formula

T _x = T _exp xA ^{(texp-tk) / 10} ,

where T _x is the estimated shelf life (weeks) at a temperature of (6 ± 2) ° C;

T _exp is the storage period in the experiment (36 ± 2) ° С;

t _exp is the storage temperature in the experiment (36 ± 2) ° С;

t _k - storage temperature (6 ± 2) ° С;

A is a constant equal to 3.

From the formula it follows that 2 weeks of storage at a temperature of (36 ± 2) ° C correspond to 54 weeks (about a year) of storage at a temperature of (6 ± 2) ° C.

Table 5 shows the data on the storage of preparations of stabilized PEG asparaginase with different stabilizers.

Table 5 The effect of various substances on the stability of the PEG asparaginase preparation in 20 mM Na-phosphate buffer containing 0.002% Tween-80 when stored for 2 weeks at a temperature of (36 ± 2) ° С. Initial PEG Asparaginase Activity, Series 251109 (IU / mg) Additive Concentration (%) (W / V) Activity after storage (%) 320 NaCl 0.9 350 Dextran 60 3-6 400 Polyethylene glycol 1500 2-5 293 Polyvinylpyrrolidone 12000 3-6 376 Mannitol 4-7 370 Glucosamine 6-10 239 Human Serum Albumin 1-2 260 L-arginine 2-6 320

The target product we obtained was investigated for the possibility of its use for chemotherapy.

The antitumor cytotoxic activity of the substance PEGylated recombinant asparaginase against eukaryotic cells was evaluated.

In the experiments, the human leukemia cell lines K-562 (chronic myeloid leukemia), Jurkat and Molt-4 (acute lymphoblastic leukemia) were used. When determining the antitumor activity of the PEG-asparaginase and L-asparaginase Erwima carotovora preparation, the comparison drug is E. coli L-asparaginase (Medac, Germany).

In the experiments, PEG asparaginase was used, obtained at a PEG / protein molar ratio of 1:25, purified on a Sephadex G-50 column. The specific activity of the substance is 314 IU / mg protein. The protein concentration is 1.38 mg / ml.

The antitumor activity of L-asparaginases is determined after 72 hours of incubation of K-562, Jurkat and Molt-4 cells with the asparaginase substance. The results presented in Fig. 5 (a, b, c) show that the antitumor activity of PEG asparaginase is slightly lower than the antitumor activity of other L-asparaginases. A significant decrease in the number of viable K-562 and Jurkat cells is already observed at an Erwinia carotovora and E. coli enzyme concentration of 0.5 IU / ml, while PEG asparaginase causes a similar inhibition of cell growth at a concentration of 5.0 IU / ml for K cell line -562 and 2.0 IU / ml for Jurkat. The Molt-4 cell line was less sensitive to the action of the PEG asparaginase preparation compared to K-562 and Jurkat.

From the presented data it follows that the Erwinia carotovora PEG asparaginase obtained by the claimed method has antitumor cytotoxic activity against the studied human leukemia cell lines.

The effectiveness of the substance obtained by the claimed method allows us to recommend it as the basis for the creation of anticancer drugs, while the method we developed is simple and suitable for industrial scaling.

Claims (4)

1. A method of obtaining a substance of recombinant L-asparaginase Erwinia carotovora, which consists in the fact that carry out covalent modification of L-asparaginase with polyethylene glycol, and modification is subjected to recombinant L-asparaginase isolated from the microbial mass of the genetically engineered producer strain Escherichia coli BL (DE3) pACYS-LANS (KM), in which the ampicillin resistance gene has been deleted in the p / ACYS-LANS plasmid, the modification is carried out by the addition of monomethoxypolyethylene glycol hemisuccinate (mPEG-suc-NHS) N-hydroxysuccinimide ester to amino groups lysine asparaginase, chromatographic purification of the resulting conjugate of recombinant L-asparaginase Erwinia carotovora with polyethylene glycol (PEG-asparaginase) and lyophilization are performed. 2. The method according to claim 1, characterized in that the purified PEG asparaginase is additionally subjected to stabilization using a compound selected from the range of proteins, amino acids, polyvinylpyrrolidones, poly- or monosaccharides, polyalkylene oxides, amino sugar, and salts as stabilizer. 3. The method according to claim 1, characterized in that the microbial mass of the strain deposited in VKMP under collection number No. B-10370 is used as the source of L-asparaginase, while the isolation of L-asparaginase is carried out by destroying the cells on a French Press followed by salting out the target product with ammonium sulfate and chromatographic purification. 4. The method according to claim 1, characterized in that the recombinant L-asparaginase of Erwinia carotovora, the nucleotide sequence of the gene lanS and the amino acid sequence of L-asparaginase are shown in FIG. 1, is covalently modified with polyethylene glycol.

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