Solution for injection, recombinant plasmid dna psx50 encoding synthesis of human recombinant alpha-2b interferon, strain escherichia coli sx50 as industrial strain-producer of human recombinant alpha-2b interferon and method for industrial preparing interferon alpha-2b

FIELD: biotechnology, pharmacy.

SUBSTANCE: invention relates to creature of a interferon-base solution for injections and to recombinant strains of Escherichia coli (E. coli) and plasmids for its preparing. Invention proposes a novel recombinant multicopy plasmid DNA pSX50 that encodes synthesis of human leukocyte alpha-2b-interferon expression of that is under control of lactose and tryptophan promoters and transcription terminator. The strain E. coli SX50 as a producer of human recombinant leukocyte alpha-2b-interferon with productivity up to 0.901.0 g of alpha-2b interferon is obtained by transformation of the recipient strain E. coli BL21 cells with recombinant plasmid DNA pSX50. Method for preparing recombinant alpha-2b interferon is based on using the created recombinant strain E. coli SX50 and involving its submerged culturing on nutrient medium with decreased content of tryptophan and at continuous addition of nutrient substrates in the process of biosynthesis. The method involves mechanical destruction of microbial cells under high pressure, dissolving precipitated protein in the concentrated solution of guanidine hydrochloride followed by renaturation of interferon in physiological buffer solutions in the presence of chaotropic agents and its purification using three-step chromatography purification of interferon on resins of type Chelating Sepharose Fast Flow immobilized with Cu2+ ions, ion-exchange chromatography on ion-exchange resins of type CM Sepharose Fast Flow and gel-filtration chromatography on resins of type Superdex-75. Three-step chromatography purification of interferon provides preparing interferon substance of purity above 98% by data of electrophoresis under reducing and non-reducing conditions, and above 95% by data RF HPLC, and this interferon substance doesn't contain pyrogenes (LAL-test). The yield of the end product is 400 mg from 1 l of cultural medium, not less. Invention provides enhancing yield of interferon and stability of its solution.

EFFECT: improved preparing method of interferon.

12 cl, 5 tbl, 6 dwg, 2 ex

 

The invention relates to the pharmaceutical, biotechnology and concerns create a solution for injection interferon-based, as well as recombinant strains of Escherichia coli (E. coli) and plasmids for its receipt.

Known methods for producing leukocyte interferon man from leukocytes of donor human blood, induced by viruses and other inductors (SU 1713591, EN 2066188, EN 2080873).

The main drawback of these methods of receiving interferon is the likelihood of contamination of the final product by human viruses, such as hepatitis b and C, human immunodeficiency virus and other currently more promising recognized as the method of producing interferon by microbiological synthesis, which provides the ability to obtain the target product with a significantly higher yield from a relatively inexpensive starting material. The approaches allow to create optimal conditions for bacterial expression of variants of the structural gene and regulatory elements that control its expression.

Currently, as the source of microorganisms use different designs strains of Pichia pastoris, Pseudomonas putida and Escherichia coli. The disadvantage of using P.pastoris as a producer of interferon (J.N.Garcia, J.A.Aguiar et. al. // High level expression of human IFN-2b in Pichia pastoris. // Biotecnologia Aplicada., 12(3)., 152-155., 1995) are the edge of the e complex fermentation condition of this type of yeast, the need to strictly maintain the concentration of the inducer, in particular methanol, in the process of biosynthesis; the level of output of the target product is comparable to the level of the yield of the target product for recombinant strains of E.coli. The disadvantage of using strains Ps.putida (SU 1364343, SU 1640996, SU 1591484, EN 1616143, EN 2142508) is the complexity of the fermentation process with a low level of expression (10 mg interferon for 1 l culture medium). There are a large number of plasmids and created on their basis of E. coli strains expressing interferon: E. coli ATCC 31633 and 31644 with plasmids Z-pBR322 (Pst1) HclF-11-206 or Z-pBR 322(Pst1)/HclN SN 35-AHL6 (SU 1764515), strain of E.coli pINF-αA-P2 (SU 1312961), strain of E.coli pINF-αF-RA (AU 1312962), a strain of E. coli SG 20050 with plasmid p280/21FN (Kravchenko V.V. and other Bioorganic chemistry, 1987, Vol.13, No. 9, s-1193), a strain of E. coli SG 20050 with plasmid pINF14 (SU 1703691), a strain of E. coli SG 20050 with plasmid pINF16 (EN 2054041) and others. The lack of technology based on the use of these strains is their instability, and lack of expression of interferon.

The prototype of the proposed strain and plasmids strain is E. coli SS5 and the recombinant plasmid pSS5 containing three promoter: Plac, RT7and Ptrpand gene alpha-interferon with the introduced nucleotide substitutions (EN 2165455).

Expression of interferon by E. coli strain SS5 containing this plasmid is controlled by three promoters is: P lac, RT7and Ptrp.The expression level of interferon is about 800 mg per 1 l of cell suspension.

The disadvantages of this plasmid and strain on its basis are the use of plasmid strong unregulated promoter of T7 phage in E. coli strain BL21 (DE3), in which the gene for T7 RNA polymerase is under the promoter of the lac operon and which is always "flows". Therefore, the cell continuously, the synthesis of interferon, which leads to dissociation of plasmid and decreased viability of cells of strain and result in the reduction of the yield of interferon.

Along with specific strains of the efficiency of the process depends largely on the technology used in the isolation and purification of interferon.

A known method of producing interferon, comprising culturing cells Ps.putida, the destruction of biomass processing polyethylenimine, fractionation sulfuric acid ammonium, hydrophobic chromatography on phenylsilane C-80, pH-fractionation of the lysate, its concentration and diafiltration, ion-exchange chromatography on cellulose DE-52, elution in a gradient of pH, ion-exchange chromatography eluent obtained on cellulose CM-52, concentration passing through the cartridge filters and gel-filtration on Sephadex G-100 (SU 1640996). The disadvantage of this method, in addition to complex multi-stage fermentation,is a multi-stage in obtaining the final product.

A known method of producing interferon, comprising the cultivation of a strain of E. coli SG 20050/pIF16, in LB-broth in flasks in a temperature-controlled shaker, centrifuged biomass, the washing buffer solution and treated with ultrasound for cell disruption. The resulting lysate centrifuged, washed with 3M solution of urea in a buffer, dissolved in the solution of guanidinate in the buffer, treated with ultrasound, centrifuged, conduct oxidative sulfites, dialyzed against 8 M urea, renaturation and final two-step chromatography on CM-52 cellulose and Sephadex G-50 (EN 2054041, EN 2118366, EN 2123010). The disadvantages of this method are its relatively low performance and low-tech main stages of the process of isolation and purification. This applies in particular to ultrasonic processing product, dialysis and oxidative sulfatase, which leads to instability of the output of interferon, as well as to the impossibility of using this method for the industrial production of interferon.

The prototype of the proposed method of producing interferon is a method for leukocyte interferon of man, which lies in the cultivation of recombinant strains of E.coli SS5, freezing the obtained biomass, the destruction of the cells of the microorganism with lysozyme, the destruction of DNA and RNA introduction to ISAT dnaase and purification of insoluble forms of interferon by washing with detergent, the dissolution of interferon in a solution of guanidine hydrochloride, renaturation and one-step purification on CM-52 cellulose (EN 2165455).

The disadvantage of this method is low manufacturability using enzymatic cell disruption, DNA and RNA of the microorganism and one-step chromatographic purification of interferon. This causes instability of the allocation process of interferon reduces its quality and limiting the use of this scheme for the industrial production of interferon.

When developing drugs based on interferon main problems faced by developers, are associated with the biological instability of interferon having a peptide structure.

In the manufacture and storage of drugs based on interferon may change their properties, proceeding at different speeds and degrees of manifestation. These changes affect the shelf life (storage).

As is known, the stability of drugs depends on many factors: temperature, pH, storage conditions, lighting conditions, composition of the surrounding atmosphere, method of preparation, auxiliary substances, types of dosage forms, especially its aggregate state, packaging, etc.

As a rule, with the aim of stabilization is commonly used as chemical methods - an Addendum is their stabilizers, antioxidants and preservatives, and physical protection from exposure to environmental factors, the use of highly purified drugs and excipients, and combinations of these methods.

In each case the use of stabilizers requires careful study to bring them in composition, especially in the case of injection solutions.

When creating dosage forms of interferon in the form of an aqueous solution attempts were made to use various stabilizers, including albumin, polymers, surfactants, buffer solutions, preservatives, polyols and carbohydrates.

In particular, the prior art aqueous solution of alpha-interferon containing nonionic surfactant, such as polyoxyethylene (20) sorbitan monooleate, benzyl alcohol, isotherwise agent, buffer based on ammonium acetate or sodium lactate, providing a pH of 4.5 to 5.5 (Patent EP 736303, publ. 09.10.1996, Hoffmann La Roche, AC 38/21, 9/08). However, this drug may lose its activity due to the introduction of benzyl alcohol, which, although it has an antimicrobial effect, can cause some aggregation of peptides.

Known liquid interferon Alfa for injection, remain stable, which contains as a stabilizer recombinant the th human albumin, the system buffer to control pH of 6.5-7.5 (Patent JP 2002-265383 priority from 14.03.2001, MITSUBISHI PHARMA CORPORATION). Introduction albumin is undesirable because of the possibility of allergic conditions.

Known water solution α-interferon for injection containing (0.1 to 100)×106IU/ml, phosphate buffer system that supports a pH of 4.4 to 7.1, helatoobrazovatel, such as the disodium salt of EDTA or citric acid, derived mono-9-octadecanoate poly(oxy-1,2-ethandiyl)-sorbitan, agent, providing the osmotic pressure in the solution, such as sodium chloride, preservative with antimicrobial activity, such as phenol, parabens and water for injection (Patent RU 2157236, publ. 10.10.200, SCHERING CORPORATION, AK 38/21, 47/00). The shelf life of freeze-dried form is up to 2 years, and in the form of a solution is much less. In addition, the introduction of preservatives can cause side effects while injecting.

Known stable aqueous interferon alpha, containing a buffer system, for example, including citric acid and diphosphate sodium, providing a pH of 4.5 and 9.0, a stabilizing agent, representing dextran or gidroksilirovanii starch, nonionic surfactant polyoxyethylene sorbitan monooleate, the regulator of osmotic pressure, such as sodium CHL the reed, mannitol or a mixture thereof, and water for injection (Patent EP 1250932, publ. 23.10.2002, TIANJIN NUALIDA BIOTECHNOLOGY, AK 38/21, 47/00). This solution can be specified as the closest analogue. The proposed arrangement does not allow to achieve optimal retention.

As you can see from the sources, characterizing the prior art, the last time in the world actively developed and patented pharmaceutical compositions on the basis of interferon, including interferon alpha-2. However, they are characterized by several disadvantages.

The task of the invention to provide a drug alpha interferon in the form of an aqueous solution for injection, with high activity and stability during storage and does not cause side effects.

To achieve the objectives of the proposed group of inventions, United by a common inventive concept.

This task is solved in that a new composition the composition of the drug on the basis of high-purity alpha-interferon containing inert polymeric filler, representing dextran or reopoliglyukin with molecular weight dextran 30-50 KD, nonionic surfactant is tween 80, ethylenediaminetetraacetic acid or its disodium salt, a buffer system consisting of sodium acetate or sodium citrate to ensure a pH of 4.5 to 5.5, the three-is chloride as a regulator of osmotic pressure, and water for injection in the following ratio of components in mg/ml:

Interferon alpha (1-15)×106ME

Sodium acetate 1230-1640 or sodium citrate 1470-1960

EDTA or its disodium salt 60-80

Sodium chloride 5800-7500

Tween 80 100-150

Dextran or reopoliglyukin (in terms of dextran) 40000-100000

Water for injection up to 1 ml

As interferon alpha using highly purified preparations, preferably interferon Alfa-2 or even more preferably alpha-2b human recombinant interferon.

As the buffer is proposed to use acetate buffer including sodium acetate, or citrate buffer include sodium citrate. The use of buffer solutions to create a specific pH, as a rule, are of significant importance for the stabilization of solutions well known in pharmacy. However, as shown by biopharmaceutical research, their application is limited, as many of them react with medicinal substances. By the present invention it was found that the creation of the optimum pH of the solution with the help of acetate or sodium citrate significantly improves the stability of the drugs interferon and does not decrease the activity of the drug.

Tween 80 refers to a nonionic surfactant and has an inhibitory effect on the aggregation processes in solution and nonspecific adsorption on the surface of the storage containers.

EDTA and NTA is eevie salts are chelators, indirectly providing an antioxidant effect. This component binds unwanted impurities from the glass, technical equipment and used in the production of auxiliary substances, prevents oxidative reactions. Moreover, having emulsifying action on the system, which also increases the stability of the composition.

As one of the stabilizers are also included inert polymeric filler, representing dextran or reopoliglyukin. In an environment of high molecular weight polymers slows down oxidation. Introduction dekatron is also important to improve the condition when the intoxication accompanying viral diseases.

The proposed composition of the auxiliary components have allowed us to achieve significant improvement in the efficiency of stabilization caused by the use of a new combination of different mechanism of action of stabilizers, taken in certain proportions.

The resulting preparation is non-toxic and aerogenes in mice and rabbits, has controlled antiviral activity when tested in cultures of human cells. Not have any side effects.

The objective of the invention is also designing recombinant industrial producer strain E. coli with a high level of biosynthesis of interferon, and the development of ffektivnoe industrial technology for substance interferon medical devices, related to the quality of the European Pharmacopoeia for substance interferon Alfa-2b.

This problem was solved by creating a recombinant plasmid DNA pSX50 and Escherichia coli SX50 deposited in Russian national collection of industrial microorganisms (collection number At-8550).

Plasmid S50 has 3218 base pairs (BP) and is characterized by the presence of the following fragments:

sequence 1 nucleotide 176 nucleotide (n) includes the DNA fragment size 176 BP, containing the tryptophan promoter (Ptrp);

sequence with 177 N. in 194 BC, includes a synthetic DNA fragment with a size of 18 BP, containing the sequence Shine Delgarno responsible for initiating broadcast;

sequence with 195 N. in 695 ad includes the DNA fragment size 501 BP, containing the gene for interferon with the following nucleotide substitutions: 37 (A→ (C), 39 (G→T), 40 (a→ (C), 42 (G→T), 67 (A→ (C), 69 (G→T), 70 (a→ (C), 72 (A→T), 96 (G→A), 100 (→ (C), 102 (A→T), 114 (A→C), 120 (C→G), 126 (G→A), 129 (G→A), 330 (C→G), 339 (G→A), 342 (G→A), 487 (A→C), 489 (→T), 495 (G→A);

sequence with 696 N. in 713 ad includes a synthetic DNA fragment size 18 BP containing synthetic polylinker;

sequence with 714 N. in 1138 ad includes the DNA fragment of the plasmid RCC-3 4129 N. on 4553 N. the size of 425 BP, with the sequence containing a series of strict transcription terminator rrnBT 1T2;

sequence with 1139 N. in 1229 ad includes the DNA fragment of the plasmid pUC19 with 2487 N. on 2577 N. size 91 BP containing the promoter of the gene β-lactamase (resistance gene ampicillin - AmpR);

sequence with 1230 N. by 2045 N. includes DNA fragment from plasmid pUC4K with 720 N. at 1535 N. the size of 816 BP containing the structural region of the gene kan;

sequence with 2046 N. on 3218 N. includes the DNA fragment of the plasmid pUC19 1625 453 N. size 1173 BP containing the sequence responsible for replication of the plasmid (ori) and the lac promoter (Plac).

Figure 1 shows a diagram of the design vector plasmids pSX10, figure 2 - scheme of constructing recombinant plasmids pSX41, figure 3 - construction of recombinant plasmids RH and pSX45, figure 4 - scheme of constructing recombinant plasmids pSX50, figure 5 - physical map of the plasmid pSX50, figure 6 presents installed for plasmids pSX50 the complete nucleotide sequence (indicated by the position of the nucleotide bases, which made replacement).

Strain Escherlchia coli SX50 obtained by transformation of cells Eschenchia coli BL21 a plasmid pSX50 using conventional genetic engineering techniques. The strain of E.coli SX50 has the following features:

Cultural morphological traits

Cells small, straight, thickened rod-like forms, letters is catalinae, risperadone. Cells grow well on simple nutrient media. During growth on agar "Difco" formed round, smooth, convex, dull, shiny, grey colonies with smooth edges. With the growth in liquid medium (minimal medium with glucose or in LB-broth) intensive form a smooth suspension.

Fisico-biological signs

Aerobe. Temperature range for growth 4-42°when the pH optimum of 6.5-7.5. As the source of nitrogen used as a mineral salt in ammonium and nitrate forms, and organic compounds in the form of amino acids, peptone, tryptone, yeast extract, etc.

As the carbon source used amino acids, glycerol, carbohydrates.

Resistance to antibiotics. Cells are resistant to kanamycin (100 μg/ml). The strain Escherichia coli SX50 producing interferon.

The method, conditions and medium composition for the storage of strain

In L-agar with the addition of kanamycin to a concentration of 20 µg/ml under oil, in L-broth containing 15% glycerol and antibiotics as appropriate, in the ampoule at a temperature of minus 70°With, in dried condition - sealed at a temperature of 4°C.

The strain Escherichia coli SX50 identified by the Identifier of Berga (1974) as the type strain of Escherichia coli.

Feature of the proposed method is to develop technologies that allow the separation of interferon from nerator is my form accumulated during fermentation, which allows to significantly simplify the technological scheme of the process of selection and to increase the yield of the target product.

The method involves culturing in a culture medium of Escherichia coli SX50, the destruction of the cells of the microorganism in the cage mill type Gaulin at a pressure of 900 bar, the separation and purification of insoluble forms of interferon using detergents and subsequent centrifugation, dissolving the precipitate in a solution of guanidine hydrochloride and subsequent renaturation of interferon.

Received denaturirovannyj interferon purified using ion-exchange chromatography ion-exchange resins of the type CM Sephsrose Fast Flow and SP Sepharose Fast Flow, high performance liquid chromatography on reversed phase C18 and gel-filtration chromatography on resin type Superdex 75.

The optimal conditions of the individual stages receiving interferon are the following:

the fermentation was carried out under continuous addition of substrate throughout the entire process, resulting in high level expression of interferon;

- destruction of cells is carried out in the cage mill type Gaulin at a pressure of at least 700 bar;

- removal of soluble cellular components (DNA, RNA, proteins, lipopolysaccharides, and so on) produced by washing the insoluble form interf the Ron buffer solutions, containing denaturing agents and detergents (Triton X100, urea, etc.);

- obtained precipitate containing interferon, dissolved in a buffer solution containing 6 M guanidine hydrochloride;

- renaturation interferon is carried out in a physiological buffer solution containing chaotrope agents and detergents;

- the use of a pair of oxidized and reduced glutathione helps to ensure the correct formation of disulfide bonds in the molecule interferon;

- chetyrehstennoy the chromatographic purification of interferon carry out ion-exchange resins of the type CM Sephsrose Fast Flow and SP Sepharose Fast Flow, reversed phase 18 and gel-filtration chromatography on resin type Superdex 75;

after each chromatographic purification spend sterilizing filtration through a pyrogen-free filters with a pore size of 0.22 micron.

The output of interferon as a result of application of the described method is not less than 400 mg of interferon with 1 l of culture medium. The quality of the product meets the standards and requirements of the European Pharmacopoeia for substance alpha-2b interferon.

Significant differences of the proposed method from the prototype are:

- using strains with higher performance, allowing you to get in the biosynthesis of more interferon with 1 l of the cult of the social environment;

- the use of effective mechanical destruction of the cell biomass, which allows to obtain a more pure extract insoluble forms of interferon in a shorter time with fewer losses;

- using physiological buffer solutions at renaturation in the presence of chaotropic agents and detergents can improve the output correctly denaturirovannah forms of interferon;

- the use of a pair of oxidized and reduced glutathione helps to ensure the correct formation of disulfide bonds in the target product;

- chetyrehletija chromatographic purification of interferon allows you to get substance interferon with a purity of more than 99% according to electrophorese in reducing and nereguliruemyi conditions and more than 98% according to reversed-phase HPLC, does not contain pyrogens (LAL-test).

The nature and advantages of the claimed group of inventions is illustrated by the following examples.

Example 1. Construction of recombinant plasmids pSX50

Method of constructing plasmids pSX50 includes the following steps:

construction of a vector plasmid pSX10:

1) construction of plasmids pSX3 (2641 BP)

2) construction of vector plasmids pSX10 (2553 BP)

construction of recombinant plasmids pSX41 (3218 BP);

construction of recombinant plasmids pSX43 (3218 BP);

construction of recombinant plasmids pSX45 (3218 BP);

construction of recombinant plasmids pSX50 (3218 BP);

Construction of vector plasmids pSX10

Vector plasmid pSX10 is a vector pUC19 in which the coding sequence of the gene beta-lactamase, providing resistance to ampicillin, is replaced by the coding sequence of a gene kan, and contains a transcription terminator from plasmid RCC-3.

Construction of vector plasmids pSS10 is carried out in two stages:

- obtaining plasmids pSX3 (2641 BP), representing the plasmid pUC19 in which the coding region of the amp gene is replaced with the coding region of the gene kan;

- getting veternas plasmids pSX10 (2553 BP), which is a plasmid pSX3, in which the BamHI site inserted DNA fragment, encoding a transcription terminator rrBT1T2.

To obtain plasmids pSX3 spend five rounds of DNA amplification by PCR (polymerase chain reaction). During the first round, using DNA plasmids pUC19 as a template, perform the amplification of DNA fragment size 1828 BP (fragment PU1-PU2) using the primers:

PU1 5'-CCCGTTGAATATGGCTCATACTCTTCCTTTTTCAATATTATTG-3'

PU2 5'-ATGCTCGATGAGTTTTTCTAACTGTCAGACCAAGTTTAC-3'

This and subsequent PCR reaction is carried out in the following conditions: 20 mM Tis-HCl, pH 8.8, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgCl2, 0.1% Triton X100, 0.1 mg/m

BSA, 0.2 mM of each dNTP, of 1.25 units of Pfu DNA polymerase, 100 ng of DNA.

The amplification process comprises the following steps: heating at 95°5 min, 35 cycles of PCR (30 s 95°s, 30 s 56°C, 2 min 72° (C) and incubation of 10 min at 72°C. After amplification (and after further amplification) DNA fragment purified by electrophoresis in 1% agarose gel. In the second and third rounds, using DNA plasmid pUC4K as a matrix, perform the amplification of DNA fragment size 555 BP (fragment KM1-KM2) using the primers:

KM1 - 5'-CAATAATATTGAAAAAGGAAGAGTATGAGCCATATTCAACGGG-3'

KM2 - 5'-CCGGTGAGAATGGCAAGAGTTTATGCATTTC-3'

and amplification of DNA fragment size 258 BP (KM3 KM4) using primers

KM3 - 5'-GAAATGCATAAACTCTTGCCATTCTCACCGG-3'

KM4 - 5'-GTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAGCAT-3'

Further fragments (KM1-KM2) and (KM3 KM4) unite in the fourth round of amplification, using primers KM1 and KM4, get the DNA fragment size 813 BP (KM1-KM4), encoding the structural part of the gene kan.

In the fifth round of PCR performed combining fragments (PU1-PU2) and (KM1-KM4) under the following conditions: heating at 95°5 min, 5 cycles of PCR (30 s 95°s, 30 s 56°C, 10 min 72° (C) and incubation of 10 min at 72°C. DNA obtained after the last PCR, directly transform into the cells of the strain E. coli DH5 and plated on Wednesday LA containing 20 μg/ml kanamycin. After incubation for 12 h at 37°With clones Tsevaot, videreutdanning DNA and carry out restriction analysis. The result is the plasmid pSX3 size 2641 BP

To obtain the vector plasmid pSX10 conduct three rounds of DNA amplification by PCR. During the first round, using DNA plasmids pSX3 as a matrix, perform the amplification of DNA fragment size 2025 BP (fragment 10.1-10.2) using the primers:

10.1 - 5'-CCGCCAAAACAGCTGCAGGTCGACGGATCCTCTAGAGTCGACCTGCAGGC-3'

10.2 - 5'-AATAATATTGAAAAAGGAAGAGTATGAGCCATATTCAACGGG-3'

During the second round, using DNA plasmids RCC-3 as a matrix, perform the amplification of DNA fragment size 528 BP (fragment KK1-KK2) using the primers:

KK1 - 5'-GCCTGCAGGTCGACTCTAGAGGATCCGTCGACCTGCAGCTGTTTTGGCGG-3'

KK2 - 5'-CCCGTTGAATATGGCTCATACTCTTCCTTTTTCAATATTATT-3'

In the third round panorama spend the joining of fragments (10.1-10.2) and (KK1-KK2) under the following conditions: heating at 95°5 min, 5 cycles of PCR (30 s 95°s, 30 s 56°C, 10 min 72° (C) and incubation of 10 min at 72°C. DNA obtained after the last NDP, directly transform into the cells of the strain E. coli DH5 and plated on Wednesday LA containing 20 μg/ml kanamycin. After incubation for 12 h at 37°With clones Tsevaot, isolated plasmid DNA and carry out restriction analysis. The result is the plasmid pSX10 size 2553 BP

Construction of recombinant plasmids pSX41

Recombinant plasmid pSX41 is a Hind III - BamHI DNA fragment of the vector plasmid pSX3 (2529 BP), Hind III - EcoRI DNA fragment size 168 p. the., encoding the promoter of tryptophan operon of E. coli (Ptrp), EcoRI-XbaI synthetic DNA fragment with a size of 20 BP, encoding SD sequence (Shine-Dalgarno) and XbaI-BamHI DNA fragment size 501 BP, encoding gene alpha 2b interferon person.

To obtain the Hind III - BamHI DNA fragment of the vector plasmid pSX3 (2529 BP) DNA plasmids pSX3 treated with restriction enzymes Hind III and BamHI, followed by electrophoretic cleaned in 1% agarose gel.

Hind III - EcoRI DNA fragment size 168 BP, encoding a promoter of tryptophan operon (Ptrp)get method NDP, using total DNA of E. coli as the template and primers TRP1 and PRP2, with subsequent processing of the amplified fragment restrictase Hind III and EcoRI:

TRP1 - 5'-CCCAAGCTTCGTAAATCACTGCATAATTCGTG-3'

TRP2 - 5'-CCGGAATTCCTGGCGATACCCTTTTTACG-3'

To obtain the EcoRI-XbaI synthetic DNA fragment with a size of 20 BP, encoding SD sequence (Shine-Dalgarno), synthesized following complementary oligonucleotides:

SD1 - 5'-AATTCAGGAGGCCT-3'

SD2 - 5'-CTAGAGGCCTCCTG-3'

XbaI-BamHI DNA fragment size 501 BP, encoding gene alpha 2b interferon man, produced by PCR, using total human DNA as template and primers IFN1 and IFN2, with subsequent processing of the amplified fragment restrictase XbaI and BamHI:

IFN1 - 5'-TGCTCTAGATGTGTGATCTGCCTCAAAC-3'

IFN2 - 5'-CGCGGATCCTCATTCTTTACTACGTAAACTTTCTTGC-3'

Then e is operations purified fragments unite, are ligated enzyme ligase of phage T4 DNA transformed into the cells of the strain E. coli DH5 and plated on Wednesday LA containing 20 μg/ml kanamycin. After incubation for 12 h at 37°With clones Tsevaot, isolated plasmid DNA, perform restriction analysis and determine the primary structure of DNA. The result is the plasmid pSX41 size 3218 BP Then hold stepwise mutagenesis of a gene of interferon to increase the expression level of the target product. Mutagenesis of the gene of interferon is to replace rare in E. coli triplets encoding the corresponding amino acids, often found in E. coli triplets that encode the same amino acids. Mutagenesis of the DNA of the gene of interferon carried out by PCR method.

Construction of recombinant plasmids RH

To obtain recombinant plasmids pSX43 conduct one round of DNA amplification by PCR using DNA of plasmid pSX41 as template and the primers and IFN3 IFN4:

IFN3 - 5'-TAGCCGTCGTACCTTGATGCTCCTGGCACAGATGCGTCGTATCT-3'

IFN4 - 5'-AGATACGACGCATCTGTGCCAGGAGCATCAAGGTACGACGGCTA-3'

PCR is carried out in the following conditions: heating at 95°5 min, 20 cycles Poland (30 s 95°s, 30 s 56°C, 10 min 72° (C) and incubation of 20 min at 72°C. DNA obtained after PCR, directly transform cells of E. coli strain DH5 and plated on Wednesday LA containing 20 μg/ml kanamycin. After incubation for 12 h at 37°With clones of sewout, allocate plasmid DNA, perform restriction analysis and determine the primary structure of DNA. The result is the plasmid pSX43 size 3218 BP

Construction of recombinant plasmids pSX45

To obtain recombinant plasmids pSX45 conduct one round of DNA amplification method NDP, using DNA plasmids pSX43 as template and the primers IFN5 and IFN6:

IFN5 - 5'-CTTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGC-3'

IFN6 - 5'-GCCAAACTCCTCCTGGGGAAATCCAAAGTCATGTCTGTCCTTCAAG-3'

Poland is carried out in the following conditions: heating at 95°5 min, 20 cycles Poland (30 s 95°s, 30 s 56°C, 10 min 72° (C) and incubation of 20 min at 72°C. DNA obtained after Poland, directly transform cells of E. coli strain DH5 and plated on Wednesday LA containing 20 μg/ml kanamycin. After incubation for 12 h at 37°With clones Tsevaot, isolated plasmid DNA, perform restriction analysis and determine the primary structure of DNA. The result is the plasmid pSX45 size 3218 BP

Construction of recombinant plasmids pSX50

To obtain recombinant plasmids pSX50 conduct one round of DNA amplification method NDP, using DNA plasmids pSX45 as template and the primers IFN7 and IFN8:

IFN7 - 5'-CAGAGACTCCGCTGATGAAAGAAGACTCCATTC-3'

IFN8 - 5'-GAATGGAGTCTTCTTTCATCAGCGGAGTCTCTG-3'

PCR is carried out in the following conditions: heating at 95°5 min, 20 cycles of PCR (30 s 95°s, 30 s 56°C, 10 min 72° (C) and Incubus is of 20 min at 72° C. DNA obtained after PCR, directly transform into the cells of the strain E. coli DH5 and plated on Wednesday LA containing 20 μg/ml kanamycin. After incubation for 12 h at 37°With clones Tsevaot, isolated plasmid DNA, perform restriction analysis and determine the primary structure of DNA. The result is the plasmid pSX50 size 3218 BP

Example 2. Obtaining strain E. coli SX50 - producer of interferon

Producing strains of E. coli interferon SX50 is obtained by transformation of cells of the strain E. coli BL21 recombinant plasmid pSX50. Producing strains of interferon grown in a 30 l fermenter until the optical density 25,0-30,0 PU in the M9 medium containing 1% acid hydrolyzed casein (Difco), 1% glucose, 40 μg/ml kanamycin, at a temperature of 38-39°C. In the fermentation process carried out continuous addition of nutrient substrate using gravimetric controller.

The content of interferon in the biomass of cells obtained from 1 l of culture, is based on a series of 0.9-1.0 g interferon.

Example 3. The method of selection of interferon from strain E. coli SX50

Receiving interferon was performed in 4 stages:

stage 1. Cultivation of a strain of E. coli SX50

stage 2. Isolation and purification of insoluble forms of interferon

stage 3. Dissolution and resaturate interferon

stage 4. Chromatographic purification of interferon

stage 1. Stump the licensing strain of E.coli SX50

Grown seed strain of E. coli SX50 in volume of 3 l in rich LB medium for 12 h at 26°filled contribute in a fermenter containing 27 l of sterile medium containing M9, 1% acid hydrolysate of casein, 1% glucose, 1 mm MgCl2, 0.1 mM CaCl2, 40 mg/ml kanamycin. Culturing in the fermenter is carried out at a temperature of 38-39°maintaining a pH of 7±0,15 by automatically Podarok 40%sodium hydroxide solution. The concentration of dissolved oxygen in the range (50±10)% of the saturation support by changing the rpm of the stirrer from 100 to 800 rpm and air flow from 1 to 15 l/min the Concentration of substrates, in particular glucose, measured during fermentation and maintain their concentration by changing the feeding speed of concentrated solutions through peristaltic pumps using gravimetric controller.

The accumulation of interferon in the form of insoluble forms - Taurus inclusions" - controlled by phase-contrast microscopy. The fermentation is stopped after reaching the maximum optical density (˜25-30 PU) and the emergence of Mature Taurus inclusions". At the end of the fermentation culture liquid sephirot by centrifugation in running the rotor at rotation speed 5000-10000 rpm Biomass Packed in plastic bags and frozen at is the temperature of minus 70° C.

stage 2. Isolation and purification of insoluble forms of interferon

100-150 grams of frozen biomass of the strain E. coli SX50 suspended in 3000 ml of buffer 1 (20 mm Tris-HCl, pH8.0, 10 mm EDTA, 0.1% Triton X100). The suspension is passed through a flow homogenizer type Gaulin support pressure 900 bar and centrifuged in running the rotor at 15000 rpm the precipitate is washed under similar conditions sequentially with buffer 2 (20 mm Tris-HCl, pH8.0, 1 mm EDTA, 3 M urea) and buffer 3 (20 mm Tris-HCl, pH 8.0, 1 mm EDTA) and finally the precipitated interferon suspended in 200 ml of buffer 3. At the same time, the isolation and purification of insoluble forms of interferon is not more than 5 hours.

stage 3. Dissolution and resaturate interferon

To the obtained in the previous step suspensions of insoluble forms of interferon add dry guanidine hydrochloride to a concentration of 6 M urea to a concentration of 8 M add solutions of Tris-HCl, pH 8.0 to a concentration of 20 mm EDTA to a concentration of 5 mm and dithiothreitol to a concentration of 50 mm and incubated at room temperature for 2 hours, the Insoluble material is separated by centrifugation with subsequent sterilizing by filtration through a membrane with pore diameters 0.22 micron.

Renaturation interferon carried out by fast dilution of the resulting solution 100-fold with buffer 7 (20 mm Tris-HCl, pH 8.0, 35 mm NaCl, 0,8M urea, 0.1% of the CHAPS Ilarion X-114, 1 mm oxidized and reduced glutathione or 25 μm copper sulfate), then renaturation the mixture incubated under stirring for 12-15 hours at a temperature of 12-16°C. Next, the aggregate material removed sterilizing by filtration through a membrane filter with a pore diameter of 0.22 micron.

stage 4. Chromatographic purification of interferon

The chromatographic purification of interferon carried out in four stages.

1. Received denaturirovannyj interferon in the first stage purified using cation-exchange chromatography on resin type CM Sepharose Fast Flow and SP Sepharose Fast Flow (GE Healthcare) and interferon elute the gradient of 0.0-0.5 M) of NaCl in buffer 50 mm Na(CH3MEO) pH 5.0. At this stage is reached the purification of interferon from components renaturation buffer, proteins and DNA of the host cell.

2. In the second phase chromatographic purification solution interferon subjected obetovannoi chromatography (Vydac). Impurities incorrectly folded and oxidized isoforms interferon separated using a gradient of acetonitrile (30%→80%) 0.2% triperoxonane acid.

3. Cleaning of residue from the organic solvent with simultaneous preconcentration is performed using cation-exchange chromatography on resin type SP Sepharose Fast Flow or CM Sepharose Fast Flow (GE Healthcare) and interferon elute the gradient of 0.0-0.5 M) NCl buffer 50 mm Na(CH 3MEO) pH 5.0.

4. Purification of Monomeric forms of interferon from the remnants of polymeric forms of interferon spend at the fourth stage of purification of interferon - gel filtration resin type Superdex 75 (Amersham Biosciences). Chromatography is carried out in a buffer of 25 mm sodium acetate, sodium citrate or sodium phosphate pH 5.0-7.0, containing 0.15 M NaCl.

Described the isolation and purification of interferon allows you to get 4-5 g of purified interferon for one cycle of selection within 7-10 days from biomass obtained from 10 liters of culture medium. The quality of interferon fully complies with the requirements of the European Pharmacopoeia for substance interferon Alfa-2b, namely:

A. The concentration of interferon for at least 2×108IU/ml

b. The specific activity of interferon is not less than 1.4×108IU/mg.

C. Electrophoretically purity of the drug is not less than 98.4% in reducing and not reducing conditions when staining the gels with silver;

d. The content correctly denaturirovannah form of interferon is not less than 95% according to reversed-phase HPLC;

that is the Isoelectric point of the selected interferon is in the region of pH 5,8-6,3;

f. Peptide map of selected interferon not fundamentally different from the peptide map for European standard interferon Alfa 2b CRS;

g. Bacterial endotoxin content not Bo is its 100 ME 1 mg of interferon.

As follows from the above examples, the claimed group of inventions allows you to receive interferon Alfa-2b with high yield in a relatively simple and reliable technology.

The possibility of carrying out the invention can be illustrated by the following examples:

Example 1. The composition of the drug per dose:

Interferon alpha-3×106ME

Sodium acetate 1640

EDTA disodium salt 60-80

Sodium chloride 5800

Twin 80 100

Dextran 40 50000

Water for injection up to 1 ml

Method of preparation:

To prepare the specified solution is mixed with 200 ml of 1 M solution of sodium acetate, 100 ml of 1 M sodium chloride and 10 ml of 0,02M solution of ethylenediaminetetraacetic acid disodium salt, prepared with water for injection. The volume was adjusted to 400 ml with water for injection. To the solution add 10 ml of 1% solution of tween 80 and 500 ml of 10% solution of dextran 40. If necessary, bring the pH to 4.5 to 5.5 0.1 M acetic acid. The solution is cooled to 4°With, then add to it a solution of the substance interferon Alfa-2b to a concentration of 3.6 million IU/ml in a liter of final solution. The resulting solution is brought to 1 l with water for injection, is subjected to sterilizing filtration on a membrane filter of 0.22 μm and poured into ampoules of 1 ml or vials 2 ml

The resulting solution is stable when stored at 2-8°With over 30 who nazev.

Example 2. The study of the stability methods, "accelerated aging"

Samples of the solutions proposed drug on the basis of human interferon Alfa-2b recombinant laid on storage at a temperature of 37°With defined experimental retention period, in which was preserved the quality indicators presented in accordance with the regulations (ND) and was calculated equivalent shelf life under normal conditions.

Dates are calculated according to the following formula:

C=Ce×And[(txp-tFS)/10],

where Ceexperimental retention period (in days)

With the period of storage at the temperature specified in FS, in days

A - temperature coefficient of the rate of the chemical reaction, adopted A=2

txp- storage temperature method "accelerated aging", °

tFS- storage temperature for ND in °

The results of stability studies of the drug Interferon Alfa-2b human recombinant, solution for injection are presented in tables 2-5.

As follows from the presented test data, developed a new composition of the solution for injection has a high stability, keeping the indicators corresponding to the requirements of normative documents, for a long time.

Developed new composition p is eparate allows you to increase the shelf life at its high activity and no side effects.

The preparation can be stored for a long time without the need for lyophilization, requiring additional energy consumption and could lead to reduced activity of the drug.

1. Solution for injection, including interferon Alfa-2b human recombinant, inert polymeric stabilizing agent, nonionic surfactant, a buffer system, a regulator of osmotic pressure, and water for injection containing ethylenediaminetetraacetic acid or its disodium salt, as the polymeric stabilizing agent is dextran or reopoliglyukin with molecular weight dextran 30-50 KD, as a non-ionic surfactant is tween 80, as the buffer system comprises sodium acetate or sodium citrate to ensure a pH of 4.5-5.5, and as a regulator of osmotic pressure of sodium chloride in the following ratio of components µg/ml:

Interferon Alfa(1-15)·106ME
Sodium acetate or1230-1640
sodium citrate1470-1960
EDTA or its disodium salt60-80
Sodium chloride5800-7500
Tween 80100-150
Dextran or reopoliglyukin (in terms of
on dextran)40000-100000
Water for injectionTo 1 ml

2. The solution according to claim 1, wherein the alpha interferon is an interferon Alfa-2b, recombinant human.

3. Recombinant plasmid DNA pSX50 encoding the synthesis of recombinant human alpha-2b interferon according to claim 1, has 3218 base pairs (BP), and characterized by the presence of the following DNA fragments: the sequence with 1 nucleotide 176 to nucleotide (n) includes the DNA fragment size 176 BP, containing the tryptophan promoter (Ptrp), the sequence with 177 N. in 194 BC, includes a synthetic DNA fragment with a size of 18 BP, containing the sequence Shine Delgarno responsible for the initiation of translation, the sequence with 195 N. in 695 ad includes the DNA fragment size 501 BP, containing the gene for interferon with nucleotide substitutions: 37 (A→ (C), 39 (G→T), 40 (a→ (C), 42 (G→T), 67 (A→ (C), 69 (G→T), 70 (a→ (C), 72 (A→T), 96 (G→A), 100 (→ (C), 102 (A→T), 114 (A→C), 120 (→G), 126 (G→A), 129 (G→A), 330 (With→G), 339 (G→A), 342 (G→A), 487 (A→C), 489 (→T), 495 (G→A), the sequence with 696 N. in 713 ad includes a synthetic DNA fragment with a size of 18 BP, sod is Rashi synthetic polylinker, the sequence with 714 N. in 1138 ad includes the DNA fragment of the plasmid RCC-3 4129 N. on 4553 N. the size of 425 BP, containing a sequence of strict transcription terminator rrnBT1T2the sequence with 1139 N. in 1229 ad includes the DNA fragment of the plasmid pUC19 with 2487 N. on 2577 N. size 91 BP containing the promoter of the gene β-lactamase (resistance gene ampicillin - AmpR), the sequence with 1230 N. by 2045 N. includes DNA fragment from plasmid pUC4K with 720 N. at 1535 N. the size of 816 BP containing the structural region of the gene kan, a sequence with 2046 N. on 3218 N. includes the DNA fragment of the plasmid pUC19 1625 453 N. size 1173 BP containing the sequence responsible for replication of the plasmid (ori) and the lac promoter (Plac).

4. The strain of bacteria Escenchia coli SX50 containing plasmid pSX50 according to claim 3, deposited in Russian national collection of industrial microorganisms (collection number At-8550) - producer of recombinant leukocyte alpha-2b interferon person.

5. The method of producing interferon Alfa-2b human, characterized in that a strain of Escherichia coil SX50 according to claim 4, cultivated in a nutrient medium, with the constant addition of substrates in the process of biosynthesis, the cells of the microorganism mechanically destroy at high pressure, separating the soluble cellular components from insoluble interferon, then dissolve and terferon in 6M buffer solution of guanidine hydrochloride or 8 M urea, then spend his renaturation physiological buffer solution in the presence of chaotropic agents and/or detergents and forth chetyrehstennoy the chromatographic purification of interferon.

6. The method according to claim 5, where the use of a new strain allows you to get in the biosynthesis of more interferon with 1 l of culture medium.

7. The method according to claim 5, where the deep cultivation carried out in a nutrient medium with reduced levels of tryptophan with continuous addition of nutrient substrates.

8. The method according to claim 5, where the destruction of cell biomass is carried out mechanically at high pressure.

9. The method according to claim 5, where the separation of the soluble cellular components (DNA, RNA, proteins, lipopolysaccharides) from insoluble forms of interferon carried out by washing the insoluble forms of interferon buffer solutions containing detergents and chaotropes agents (Triton X100, urea) at high pressure.

10. The method according to claim 5, where the use of physiological buffer solutions at renaturation in the presence of chaotropic agents and/or detergents can improve the output correctly denaturirovannah forms of interferon.

11. The method according to claim 5, which used a controlled oxidation of interferon by means of pairs of oxidized and reduced glutathione, sulphate of copper and oxygen or peroxide, bodoro whom and under constant control of the redox potential allows to ensure the correct formation of disulfide bonds in the protein.

12. The method according to claim 5, where they chetyrehstennoy the chromatographic purification of interferon, a substance interferon more than 99% purity according to electrophorese in reducing and nereguliruemyi conditions and more than 98% according to HPLC RF and does not contain pyrogens (LAL-test).



 

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The invention relates to biotechnology, in particular to a technology for production of recombinant proteins, and can be used to produce recombinant alpha-2b-interferon person

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