RU2585532C2 - Plasmid for expression of human recombinant blood clotting factor ix, cho cell producing human recombinant blood clotting factor ix and method of producing said factor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, particularly to recombinant production of coagulation factors, and can be used for expression of human coagulation factor IX (hFIX). Method comprises constructing plasmid for expression of hFIX in CHO cells, consisting of field of replication start OF plasmid pUC, open reading frame of beta-lactamase (bla) and procariotic gene promoter bla; section of a terminal repetition EBVTR, functional promoter and first of elongation factor 1 alpha Chinese hamster, flanked 5' untranslated region of said gene; open reading frame gene coding hFIX; internal site IRES EMCV; open reading frame DHFR expressed in bicystronic mRNA together with gene coding hFIX; and functional terminator gene elongation factor 1 alpha Chinese hamster, flanked 3' untranslated region of said gene.

EFFECT: higher efficiency of product hFIX.

15 cl, 3 dwg, 3 tbl, 7 ex

Description

Application area

The invention relates to the field of biotechnology, and in particular to a technology for the production of biologically active substances (BAS) by genetic engineering methods, more specifically, to methods for producing human coagulation factor IX.

Relevance

Factor IX (FIX) is an element of the blood coagulation cascade, a proenzyme of serine protease, which in the presence of Ca ²⁺ and membrane phospholipids hydrolyzes the arginine-isoleucine bond in the factor X molecule to form activated factor X (FXa). The catalytic efficiency of activated factor IX (FIXa) increases dramatically when the cofactor of activated blood coagulation factor VIII (FVIIIa) is bound. The non-covalent complex of FIXa, FVIIIa and FX bound to the phospholipid membrane is called X -ase or tenase and is the main element of the positive feedback loop in the blood coagulation cascade.

FIX is synthesized in the liver as an inactive precursor protein, which is processed in the endoplasmic reticulum and Golgi apparatus, where it undergoes multiple post-translational modifications of various types and is secreted into the bloodstream after proteolytic cleavage of the propeptide from the N-terminus of the molecule. The circulating mature FIX has a molecular weight of about 57 kDa and an average plasma concentration of about 90 nM. In the coagulation cascade, FIX is activated after proteolytic cleavage by activated factor XI (internal pathway) or activated factor VII (external pathway) to form two polypeptide chains linked by a disulfide bond. Activated FIX is gradually inactivated, mainly by slow binding to antithrombin III, nexin-2, protein Z, a dependent protease inhibitor and hepatocyte endocytosis receptors, and also undergoes neutrophil elastase cleavage.

FIX consists of four structural domains - the Gla domain, two EGF-like domains (EGF - epidermal growth factor) and the C-terminal domain of the serine protease. The FIX N-terminal leader peptide is separated upon translocation of the polypeptide into the endoplasmic reticulum, the propeptide immediately preceding the Gla domain is separated upon secretion of the mature protein. The activation peptide located between the second EGF-like domain and the serine protease domain is specifically separated by factors XIa or VIIa upon activation of FIX.

The Gla domain located at the N-terminus of the mature FIX molecule provides binding of FIX and FIXa to the surface of endothelial cells, and this interaction is completely disrupted when γ-carboxylation of Asp residues in the domain is blocked. The first EGF-like domain of FIX contains a high-affinity calcium ion binding site, and also determines the interaction of FIX with Villa factor and with tissue factor (factor III). The second EGF-like FIX domain is involved in the formation of the FIXa-FVIIIa-FX complex. It binds to the serine protease domain with an activation peptide and a single disulfide bond. The FIX activation peptide contains most of the sites of post-translational modifications that affect the properties of FIX. The serine protease domain is about half the total mass of FIX, the active site contained in it is hidden by the activation peptide and exposed after its separation. Of all post-translational modifications, only γ-carboxylation in the Gla domain directly determines the procoagulant activity of FIX. The influence of other modifications on FIX functions is less pronounced; in some cases, it is absent or remains unknown.

Congenital absence of functional factor IX or its low level leads to the development of hemophilia B, an X-linked recessive genetic disease that requires constant replacement therapy with factor IX drugs. Hemophilia B occurs in one of 30 thousand people. Initially, hemophilia B therapy was limited to periodic transfusions of blood plasma, subsequently replaced by more effective concentrates of the prothrombin complex - a mixture of vitamin K-dependent coagulation factors IX, II, VII and X. The main limitation of such therapy was a significant risk of thrombotic episodes. FIX drugs with a higher degree of purity were obtained from fractions of blood plasma separated by the Cohn method using additional purification by ion exchange chromatography. The safety of FIX plasma concentrates is also limited by a significant level of impurity of activated FIX (FIXa) and residues of other coagulation factors sufficient to increase the risk of thrombotic episodes. Additional purification of FIX using immunoaffinity chromatography allows you to completely remove these impurities, however, as with other plasma processing products, the risk of viral or prion infection in patients cannot be completely eliminated.

Currently, only one recombinant FIX drug is registered - nonacog alpha (trade name Benefix), approved for use in the USA and EU countries in 1997. Nonacog alpha is obtained in CHO cells cultured in a nutrient medium containing no serum or other animal products . Isolation and purification of recombinant FIX is carried out using four chromatographic steps, without using immunoaffinity chromatography; potentially present viruses are removed by nanofiltration on a filter with a cutoff threshold of 70 kDa. Human albumin is not used in the nonacoga alpha formulation. Thus, in the process of obtaining a medicinal product, the use of substances of animal origin and donor plasma components is excluded.

State of the art

In the original studies describing the expression of FIX in CHO cells, it was found that secreted FIX contains a significant proportion of inactive molecules with an unprocessed propeptide. Complete or almost complete cleavage of the propeptide is achieved only by coexpression of the subtilisin / kexin-like PACE / furin convertase or PC5 convert homologous to it. The procoagulant activity of FIX depends on the level of γ-carboxylation of the Gla domain - in fully active FIX, the first 10 Glu residues must be converted to Gla residues, while the conversion level of the last two Glu residues does not affect the FIX properties. In the natural FIX molecule, all 12 Glu residues in the Gla domain are completely γ-carboxylated, while in the recombinant FIX obtained in CHO cells, the level of modification of the last two Glu residues is slightly reduced. The total number of Gla residues in recombinant FIX from CHO cells reaches 11.5 per protein molecule. The specific procoagulant activity of such FIX is not less than 200 IU / mg and does not differ from the specific activity of natural FIX.

The main methodological approach to creating eukaryotic producers of recombinant human factor IX in mammalian cells is described in US patent 4,770,999. The producers of recombinant factor IX on the basis of CHO cells were obtained using expression vector p91023-IX containing the SV40 virus enhancer, the open reading frame of factor IX under the control of the promoter region of the late AdMLP adenovirus genes, VA adenovirus genes, and mouse dihydrofolate reductase OPC region. During preparative transfection, the p91023-1X plasmid was co-transfected with the auxiliary plasmid pAd26SVpA3 encoding the dihydrofolate reductase gene under the control of the MLP promoter and the SV40 origin of replication. A population of stably transfected cells was cultured in the presence of increasing concentrations of methotrexate (up to 20 μM), while the concentration of secreted factor IX, measured by ELISA, increased from 0.015 mg / L to 43.4 mg / L. When vitamin K1 was added to the growth medium up to 10 μg / ml and then cultured in it the cells of the most productive population, the secretion rate of active factor IX was 0.268 IU / ml / day, thus, the specific activity of secreted factor IX was about 9% of normal.

The main reason for the lack of specific activity of recombinant factor IX secreted by CHO cells was the low level of propeptide processing by PACE / furin family proteases in the Golgi apparatus of CHO cells. When human PACE was co-expressed by factor IX producing cells, an increase in the activity of secreted factor IX was observed from 0.17 to 0.33 IU / ml (the concentration of all forms of factor IX was 30 mg / l, specific activity 11 IU / mg), according to immunoprecipitation, factor IX molecules with an unsplit propeptide were practically not detected (Wasley LC, Rehemtulla A., Bristol JA, Kaufman RJ // J. Biol. Chem. 1993. V.268. No. 12. P.8458-8465.). Similar data were obtained for a soluble shortened variant of human PACE (PACE) with a transmembrane domain removed.

The molecules of the recombinant factor IX with an undeleted propeptide can be specifically separated from the molecules of the mature protein by anion exchange chromatography as described in European patent application EP 0 705 901 A2, however, the degree of separation of these two forms of factor IX does not exceed 10.

Other known clonal lines producing recombinant factor IX are as follows:

1. Clone C6 described in European Patent EP 0 430 930 B1, obtained by transfecting the dog kidney cell line MDCK with plasmid pIJ5 / 9, including the OPC region of the human factor IX cDNA under the control of the SV40 promoter and a neomycin resistance marker. Clone C6 has a maximum productivity of 0.375 mg / L of factor IX at 8 days of cultivation (ELISA data, specific activity after purification 91% of normal, the estimated level of secretion of active factor IX is 0.094 IU / ml).

2. Clone GFSC-15 described in European patent application EP 2164971 A2, obtained by co-transfection of CHO cell line DG-44 with plasmid pMSG-FIXI containing the OPC region and a shortened first intron from human factor IX cDNA under the control of the SV40 promoter and association region with a nuclear matrix (MAR) from the human beta-globin gene, as well as a plasmid encoding mouse dihydrofolate reductase, followed by transfection of the obtained producer line with similar plasmids encoding auxiliary human furin and human gamma-carboxylase genes eka. The specific productivity of the best clonal producer line GFSC-15 was 4.4 μg / million cells / day (ELISA data), the maximum activity of secreted factor IX in the culture medium was about 2.3 IU / ml for 10 days of cultivation with additional nutrients on the 5th day.

3. The polyclonal population of Hek293 / FIX _D359V LXIN described in PCT patent application WO 2011011841 A1 and obtained by infection of human kidney line HEK 293 cells with replication defective retroviral particles obtained using the pLXIN vector. The described maximum level of mutein secretion of factor IX is 11.8 IU / ml. Mutation of factor IX B359V leads to a multiple increase in the level of secretion of factor IX by cells, however, the use of mutein as a drug for the treatment of hemophilia B is difficult due to the risk of an increase in the number of patients producing neutralizing antibodies.

4. The monoclonal line Huh7-CD4 described in European patent application EP 2496252 A1 and obtained by transfection of the line of immortalized human hepatocytes Huh7 with the plasmid pcDNAFIXwt encoding the OPC of human factor IX under the control of the immediate early promoter of cytomegalovirus (CMV), a selective marker of resistance gene. When the producer line was cultured in a serum-free medium in roller bottles, the specific productivity of about 0.8 μg / 2 million cells / day was achieved, the specific activity of the secreted product exceeded the specific activity of the factor IX standard by about one and a half times.

5. Clone # 6 described in US patent US 7375084 B2, obtained by transfection of the HEK 293 line with the plasmid pCMV-FIX-neo encoding factor IX under the control of the CMV promoter and containing the neomycin resistance gene, followed by transfection of the auxiliary plasmid pCMV-VKORC I - EDHpro encoding the OPC cofactor 1 of human vitamin K-oxidoreductase. The specific productivity of clone # 6 was 3.3 IU / million cells / day (26 μg / million cells / day according to ELISA). Despite the relatively high level of secretion obtained for producer lines based on the human cells HEK 293, the applicability of this expression system for the production of drugs of recombinant factor IX is limited, since cultured human cells are more likely to contain human viruses and human oncogenes that are residual amounts of which can contaminate the target product.

The closest in technical implementation analogue of the present invention is described in European patent application EP 1969127 A2 clone 130-17 and its analogues obtained by sequential transfection of cells of the Cho line with a plasmid encoding human factor IX OPC under the control of the promoter of the Chinese hamster elongation factor 1 gene (promoter CHEF-I or EEF1A), selection of a population of stably transfected cells, followed by transfection of plasmids encoding auxiliary genes for vitamin K dependent gamma carboxylase (VKGC) and vitamin K epox reductase (VKOR or VKORC1), selection of the most productive clones of stably transfected cells, re-transfection with a plasmid encoding VKOR and a final selection for clones producing. When culturing clone 130-17 thus obtained in stirred flasks without changing the medium for 18 days, the final activity of secreted factor IX was 7.75 IU / ml (31 mg / l of "active factor IX", the proportion of active factor IX was 64%) .

The system for expressing human factor IX in CHO cells described in patent application EP 1969127 A2 allows the target protein to be obtained in sufficiently large quantities for a very long cultivation period of 18 days, however, the target product may be contaminated with significant amounts of factor IX with an unprocessed propeptide due to the fact that obtaining clone 130-17 and related clones, the cells were not transfected with the human PACE / furin signaling protease genome. Data on the specific level of propeptide processing in secreted factor IX are not given in patent application EP 1969127 A2.

A brief description of the present invention

The technical problem solved by the authors was the creation of a highly productive technology for producing recombinant coagulation factor IX of a person for biopharmaceutical production.

The technical result was achieved by creating a new expression plasmid DNA p1.1-F9, encoding an open reading frame of a protein of human coagulation factor IX and creating a clonal producer cell line based on it.

The technology is based on the developed plasmid DNA p1.1-F9 with a length of 13299 bp (SEQ ID NO: 1, Figure 1), consisting of:

- DNA fragment, 1406 bp long, containing the Kozak synthetic sequence (ribosome binding site, SEQ ID NO: 1 6451 - 6459), providing translation initiation of mRNA, sequence 1383 bp (SEQ ID NO: 1 6460-842) encoding the human blood coagulation factor OPC IX and the stop codon block (SEQ ID NO: 1 7843-7848)

- fragment 11893 bp plasmids p1.1 (SEQ ID NO: 1 7852-6445) containing regulatory elements that provide expression of the target protein:

- 5 ′ and 3 ′ untranslated regions of the gene of the Chinese hamster alpha 1 elongation factor gene, respectively comprising a functional promoter (5 ′ NTO) and the terminator and polyadenylation signal (3 ′ NTO) of this gene, as well as flanking non-transcribed regions of this gene, providing euchromatization insertion sites and transcriptional activity of the genetic cassette in the genome of CHO;

- the sequence of the resistance factor of transfected cells to the effects of methotrexate - dihydrofolate reductase (DHFR) and regulatory elements for the expression of DHFR - the IRES sequence of encephalomyocarditis virus. (EMCV), which provides bicistronic expression in animal cells and a polyadenylation signal.

- the region of the beginning of replication of the plasmid pUC, the open reading frame of beta-lactamase (bla), and the prokaryotic promoter of the bla gene, which provides bacterial resistance to the antibiotic ampicillin, allowing for the preparative production of the plasmid in E. coli.

Plasmid p1.1-F9 contains unique recognition sites for restriction endonucleases AscI (16), PvuI (1409), FseI (5213), AbsI (6446), NheI (7852).

The plasmid was introduced into CHO DG-44 cells by liposomal transfection (Straus S.E. et al, J. Virol., 1981, Jul; 39 (1): 290-4). By culturing in a protein-free medium free of hypoxanthine and thymidine and additionally containing 200 nM DHFR inhibitor methotrexate (MTX), oligoclonal lines of stably transfected cells were obtained. For the selected oligoclonal line with high productivity, the cassette was amplified in the genome by successive cultivation in the presence of increasing concentrations of methotrexate. A polyclonal line of cells resistant to a high concentration of methotrexate and producing increased amounts of the target protein was obtained and used for cloning by the limiting dilution method. The obtained clones of producer cells were analyzed by enzyme-linked immunosorbent assay and coagulometry and clones were selected that gave the maximum expression level of the target protein. Amplification of the vector insert was performed with methotrexate at a maximum concentration of 4 μM. Additional plasmids p1.2-Zeo-CHO-VKORC1 and p1.2-Hygro- were sequentially introduced into the selected clone by electrotransfection (Straus SE et al, J. Virol., 1981, Jul; 39 (1): 290-4) FurVQ, encoding the genes of the shortened soluble variant of the human PACE / furin signaling protease and vitamin K epoxy reductase (VKOR or VKORC1) of the Chinese hamster, respectively, and providing a high level of post-translational processing of the molecules synthesized by factor IX cells to a biologically active form. Cells transfected with plasmid p1.2-Zeo-CHO-VKORC1 were cultured in the presence of 1 mg / ml zeocin until a cell viability of more than 85% was restored. Cells of this population were transfected with plasmid p1.2-Hygro-FurVQ and cultured in the presence of 750 μg / ml hygromycin until restoration of cell viability of more than 90%. A population of cells secreting more than 1.5 IU / million cells / day of active factor IX with a fraction of factor IX molecules with an unprocessed propeptide of less than 75% was obtained. The cells of this population were cloned by limiting dilution. The obtained clones of producer cells were analyzed by enzyme-linked immunosorbent assay and coagulometry. Clones were selected giving the maximum level of secretion of biologically active factor IX.

Among them, clone P1.1-F9 / 3B12-86 was selected, when cultured in suspension culture in a serum-free medium in the absence of selection agents (methotrexate and antibiotics), the final activity of factor IX was 3.5 IU / ml (specific activity 190 IU / mg) at a cell concentration of 1.25 million cells / ml, the cultivation time is 3 days. The obtained cell line P1.1-F9 / 3B 12-86 was deposited in the Specialized Collection of Cultures of Vertebrate Cells of the Institute of Cytology of the Russian Academy of Sciences, Russian Collection of Cell Cultures 05.02.2013 under the number RKKK (P) 755D.

An object of the present invention is to provide a plasmid for expression of a recombinant human blood coagulation factor IX, in particular plasmid p1.1-F9. Another objective of the present invention is the provision of a monoclonal line of mammalian cells, in particular Chinese hamster ovary cells, P1.1-F9 / 3B 12-86 - producers of recombinant human coagulation factor IX containing multiple copies of the expression cassette corresponding to the linearized expression plasmid in the genome, in particular plasmids p1.1-F9. An illustrative example of this monoclonal cell line are Chinese hamster ovary cells of clone P1.1-F9 / 3B 12-86.

It is also an object of the present invention to provide a method for producing a recombinant human blood coagulation factor IX using said cells.

Detailed description of the present invention

To implement the present invention, the main technical task was to create a technology for the highly productive production of recombinant coagulation factor IX for biopharmaceutical production using cultured Chinese hamster ovary cells adapted for suspension cultivation in a protein-free medium containing multiple copies of the genetic cassette in the genome, which is a linearized expression plasmid containing a DNA fragment encoding coagulation factor cr vi IX under the control of a promoter and regulatory elements that function in a eukaryotic cell.

The term "expression plasmid" ("expression plasmid DNA") means a plasmid DNA containing all the necessary genetic elements for the expression of a gene embedded in it, for example, such as a promoter, terminator, polyadenylation signal. A specific example of the genetic elements necessary for the expression of recombinant coagulation factor IX in the expression cassette of the present invention is, but is not limited to, the promoter of the Chinese hamster alpha 1 elongation factor gene.

The DNA fragment encoding the recombinant coagulation factor IX according to the present invention is a gene encoding the OPC of the factor IX polypeptide, which can be obtained, for example, as described in Example 1. Also, this DNA fragment can be obtained, for example, using firm cloning technology Sloning BioTechnology described in PCT Application WO 2005071077.

The sequence of the gene encoding the OCR of the recombinant human blood coagulation factor IX according to the present invention is presented in the Sequence Listing under the number SEQ ID NO: 1 (nucleotides 6460-7842).

The amino acid sequence of the secreted coagulation factor IX of a person according to the present invention is presented in the Sequence Listing under the number SEQ ID NO: 2, and is the translation product of nucleotides 6460 - 7842 of the OPC sequence of SEQ ID NO: 1 without the first 18 amino acids of the secretion leader peptide and propeptide.

In order to ensure efficient translation of the gene in Chinese hamster cells, it is preferable that the OPC be preceded by a sequence for cap-dependent translation initiation (Kozak sequence), for example, synthetic.

DNA fragments that encode essentially the same protein can be obtained, for example, by modifying the nucleotide sequence of the DNA fragment (SEQ ID NO: 1 nucleotides 6460 - 7842), for example, by the method of site-directed mutagenesis, so that one or more amino acid residues at a particular site will be delegated, replaced, inserted or added. DNA fragments modified as described above can be obtained using traditional processing methods to obtain mutations. DNA fragments that encode essentially the same functional protein can be detected by expression of DNA fragments having the mutation described above in the corresponding cell and establishing the activity of the expressed product.

Coagulation factor IX (FIX) is a proenzyme of serine protease that, in the presence of Ca ²⁺ and membrane phospholipids, hydrolyzes the arginine-isoleucine bond in the factor X molecule to form activated factor X (FXa).

The natural coagulation factor of human IX in the bloodstream is mainly a glycoprotein with a molecular weight of about 57 kDa. Recombinant coagulation factor IX fully retains procoagulant (biological) activity, while its specific procoagulant activity, expressed in international units per milligram of pure protein, is 200-250 IU / mg.

Indicators of functional activity, in which it is believed that the obtained protein has the properties of a recombinant coagulation factor IX of a person, is determined by its ability to reduce the coagulation time (coagulation) of human blood plasma with removed factor IX in the activated partial thromboplastin time test (APTT). For example, the activity of recombinant human coagulability factor IX can be detected by reducing the time of clot formation in blood plasma with removed factor IX after mixing the test solution of factor IX, blood plasma with removed factor IX, contact activator of coagulation of ellagic acid, acid phospholipid micelles and solution calcium chloride. The clotting time of the plasma mixed with several dilutions of the test solution is determined and the measured clot formation times are compared with the clot formation times obtained for several dilutions of the standard of activity of factor IX — normal blood plasma or a solution of isolated factor IX. The concentration of human factor IX is determined using an enzyme-linked immunosorbent assay in comparison with the standard - blood plasma or a solution of purified factor IX with a known content. The specific activity of factor IX is defined as the ratio of procoagulant activity and concentration of factor IX and is expressed in IU / mg. It is believed that the protein variant has the properties of recombinant human blood coagulation factor IX, provided that the specific activity of this variant is not less than 10% of the specific activity of natural human factor IX, that is, at least 20 IU / mg.

The expression plasmid according to the present invention contains a DNA fragment encoding coagulation factor IX under the control of a promoter and regulatory elements that function in a eukaryotic cell. As the recombinant plasmid according to the present invention, various plasmids possessing the ability to be expressed in the recipient cell can be used, such as pcDNA3.1, pCMV-Myc, pDEF38 and the like, but the list of plasmids is not limited to them.

A specific embodiment of the present invention is a plasmid p1.1-F9 with a length of 13299 bp (SEQ ID NO 1), which contains the following functional elements listed in the order of their arrangement:

1. the region of the beginning of replication of the plasmid pUC (150-283), the open reading frame of beta-lactamase (Ba) and the prokaryotic promoter of the bla gene (965-1924) - allowing for the preparative production of the plasmid in E. coli.

2. fragment of the concatemer of terminal repeats of the Epstein-Barr virus (1960-2361) - providing an increase in the frequency of integration of the genetic cassette into the genome of CHO cells and an increase in the rate of amplification of the cassette in the genome.

3. a fragment of the sequence flanking the Chinese hamster elongation factor 1 alpha 1 upstream gene (2373-6443), including non-transcribed regions of this gene, the promoter of this gene, the first intron of this gene, which provides constitutive expression of the human factor IX gene in the genome of CHO cells ;

4. Kozak synthetic sequence (ribosome binding site) (6451-6459), providing cap-dependent initiation of mRNA translation in animal cells;

5. a sequence encoding an open reading frame of the coagulation factor IX of a person (6460-7842) and a block of stop codons (7843-7848)

6. the sequence of the internal binding site of the ribosome of the encephalomyocarditis virus EMCV (7872-8459), providing cap-independent initiation of translation of the second cistron of bicistronic RNA in animal cells;

7. the sequence encoding the open reading frame of dihydrofolate reductase (8472-9035) - a factor of resistance of transfected cells to methotrexate;

8. a fragment of the sequence flanking the Chinese hamster “1 downstream” elongation factor 1 alpha gene (9037 - 13295), including the terminator and polyadenylation signal, as well as non-transcribed regions of this gene, which provide constitutive expression of the human factor IX gene in the genome of CHO cells.

Plasmid p1.1-F9 contains unique recognition sites for restriction endonucleases Asci (16), Pvul (1409), Fsel (5213), Absl (6446), Nhel (7852). The structure of plasmid p1.1-F9 is shown in Figure 1.

Plasmid p1.1-F9 was constructed based on the expression plasmid p1.1, previously developed by the authors of the present invention and described in detail in RF patent application 2011146243, which is incorporated herein by reference in its entirety. Plasmid p1.1 contains the functional promoter and terminator of the 1 alpha Chinese hamster elongation factor gene, which provides constitutive expression of the target gene in CHO cells, flanked 5 ′ and 3 ′ UTR of this gene, and provides euchromatization of expression cassette integration sites in the CHO genome; plot for cloning the open reading frames of the target proteins; encephalomyocarditis virus ribosome internal binding site (EMCV IRES), which ensures translation initiation on bicistronic mRNA in mammalian cells and complete genetic linkage of target protein and DHFR production levels; Mouse OCR DHFR expressed in the bicistronic mRNA together with the target gene (IRES DHFR) and ensuring the stability of stably transfected cells against thymidine absence and dose-dependent resistance to methotrexate (MTX), which allows targeted selection of highly productive clones with multiple copies of the expression cassette in the genome of CHO; the Epstein-Barr human terminal terminal repeat (EBVTR) region, which provides an increased level of integration of cassettes into the genome of CHO cells and accelerates the amplification of the amplification of the target gene in the genome under the influence of MTX. Using plasmid p1.1 allows for high-frequency integration and accelerated amplification of the expression cassette in mammalian cells, as well as production lines with a high level of production of the target protein and high stability.

Using the created plasmid according to the present invention, in particular plasmid p1.1-F9, it is possible to transform a eukaryotic cell, preferably an immortalized Chinese hamster cell. The choice of a particular cell line is not critical, since the methodology and methods of transformation are well known to those skilled in the art. After one or more rounds of selection and cloning of the transformed cell population, clonal producer lines of recombinant human coagulation factor IX can be obtained. Methodology and techniques for cell selection and cloning are known to those skilled in the art. Although the level of expression of coagulation factor IX of a person may vary depending on the type of cell and the conditions of cultivation and selection of transformants, the fact of transient expression of the target protein will take place subject to successful transformation of the recipient cell, the fact of stable expression of the target protein will occur if successful round selection of a population of transformant cells.

The procoagulant activity of recombinant FIX depends on the degree of gamma carboxylation of its Gla domain, and corresponds to the natural one upon carboxylation of the first 10 of 12 Gla glutamic acid residues of the Gla domain [McGraf D.M., Walsh G. Directory of therapeutic enzymes. NY 2005. 312pp.]. The specific activity of recombinant FIX can be increased by coexpression in production lines of the enzyme vitamin K-epoxide reductase (VKORC1), which restores vitamin K (electron donor for enzymatic gamma-carboxylation to its active form [Wajih N. Et al. Increased production of functional recombinant human clotting factor IX by baby hamster kidney cells engineered to overexpress VKORC1, the vitamin K 2,3-epoxide-reducing enzyme of the vitamin K cycle. Journal of Biological Chemistry 2005 280 (280), 31603-31607]. Illustrative plasmid variant for integration into the cell genome of additional copies of the vkorcl gene is the expression plasmid p1.2-Zeo-CH O-VK.ORC (SEQ ID NO: 20, FIG. 3) containing the vkorcl gene of the Chinese hamster under the control of a strong constitutive promoter.

The activity of recombinant FIX also depends on the presence or absence of a propeptide in its composition. To increase the activity of the target protein, it is preferable to co-express the recombinant FIX and the soluble truncated variant of the PACE / furin protease. An exemplary plasmid for the expression of the PACE / furin protease is plasmid p1.2-Hygro-FurVQ (SEQ ID NO: 18, FIG. 2).

"Transformation of a cell with a plasmid" means the introduction of a plasmid into a cell using methods well known to those skilled in the art. Transformation methods include any standard methods known to a person skilled in the art, for example electroporation, the use of transfection agents, for example the method described in the technical document of Invitrogen, Inc. "Lipofectamine® 2000 Reagent" Pub. No.MAN0000995 Rev. Date 20 July 2012.

According to the present invention, “cell line producing coagulation factor IX of a human IX” means a clonal mammalian cell line having the ability to produce and secretion of coagulation factor IX of a human when it is grown according to the present invention in said culture medium. As used herein, the term "cell line - producer of human coagulation factor IX” also means a cell line that is capable of secreting a functionally active coagulation factor of human IX with a productivity of at least 0.1 IU / million cells / day when cultured in suspension culture in serum free medium . The specified coagulation factor IX of a person is secreted by these cells into the cultivation medium.

Among mammalian cells, the use of Chinese hamster cells (Cricetulus griseus), preferably ovarian cells (CHO), is preferred. An example of a preferred Chinese hamster ovary cell is the CHO line DG44 (Invitrogen cGMP banked, USA; Mol Cell Biol 5, 1750-1759 Kaufman RJ et al. 1985). The range of CHO subline is not limited in any way, for example, CHO CHOZN DHFR subline, CHO DUKX B11 and the like can be used.

A specific example of a line for producing a producer of human coagulation factor IX in accordance with the present invention is the CHO line DG44, but the range of cell lines is not limited thereto.

The CHO DG44 line is characterized by the following cultural, morphological, physiological, biochemical, and genetic traits:

- Species data: Chinese hamster Cricetulus griseus, ovary.

- Control of species identity: karyological, isoenzymatic (LDH and G6FDG).

- Marker signs and methods for their assessment: immunological, cytogenetic, biochemical, physiological:

- Morphology: epithelial-like.

- Karyology: 2n = 22, the range of variation in the number of chromosomes is 10-28, the modal number of chromosomes is 22, pseudodiploid, there are microchromosomes, the number of polyploids is 9.0%.

- Biochemistry: Dihydrofolate reductase activity by resistance to methotrexate.

- Growing conditions: medium - serum-free, of a certain chemical composition, CD Opti CHO (Invitrogen, USA), temperature - + 37 ° С, СО ₂ concentration - 8%, mixing speed - 120-130 rpm.

- Cultural properties: cultivation method - suspension, sowing dose - 300 000 per ml, sieving ratio - 1: 2 - 1: 6, reseeding procedure - centrifugation of the suspension at a speed of 1200 rpm, 5 min, resuspension in a new growth medium. The optimal density is 1.0-1.2 × 10 ⁶ cells / ml. Transformation of CHO DG44 cell line by plasmid p1.1-F9, followed by selection and cloning, leads to the production of cell lines producing recombinant human factor IX. Subsequent transformation of the obtained cell lines with plasmids p1.2-Zeo-CHO-VKORC and p1.2-Hygro-FurVQ, followed by selection and cloning, results in cell lines that are efficient producers of highly active recombinant human factor IX. An example of a cell line that is an efficient producer of highly active recombinant human factor IX is the producer line P1.1-F9 / 3B 12-86.

The producer line P1.1-F9 / 3B12-86 provides the synthesis and secretion of recombinant human factor IX in an amount of 3.5 ME of the final activity of coagulation factor IX per 1 ml of medium at a cell concentration of 1.25 million cells / ml when cultured in suspension culture in serum-free medium. The producer line P1.1-F9 / 3B12-86 was deposited in the Russian Collection of Cultures of Vertebrate Cells of the Institute of Cytology RAS 02/05/2013, registration number RKKK (P) 755D.

It is also an object of the present invention to provide a method for producing recombinant human factor IX in mammalian cells, comprising culturing in a nutrient medium the above mammalian cells producing human recombinant factor IX, and isolating the obtained target recombinant protein from the culture fluid.

Cell growth, isolation and purification of the target protein from a culture or similar liquid can be carried out in a manner similar to traditional cultivation methods in which a recombinant protein is produced using mammalian cells.

The culture medium used for cultivation can be either synthetic or natural, provided that the medium contains sources of carbon, nitrogen, mineral additives and, if necessary, an appropriate amount of nutritional supplements necessary for cell growth. Various carbohydrates such as glucose or sucrose and other organic acids can be used as a carbon source. As the nitrogen source, various inorganic ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural sources of nitrogen, can be used. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. As vitamins, thiamine, yeast extract, and the like can be used.

To obtain a pharmaceutically significant recombinant human factor IX, it is preferable to use a serum-free culture medium for cultivation.

The cultivation can be carried out under aerobic conditions, preferably with a high content of CO ₂ (8%), such as mixing the culture fluid in the flasks, at a temperature in the range from 20 to 40 ° C, preferably in the range from 30 to 38 ° C. Usually, growing for 12 hours to 4 days leads to the accumulation of the target recombinant protein in the culture medium or in the cytoplasm of the cell.

After growth, solid residues, such as cells, can be removed from the culture fluid by centrifugation, and then the target protein can be isolated and purified by chromatography and / or concentration.

Features of the created expression plasmids, cell lines and the results of the practical application of the invention are shown in figures 1-3.

The implementation of the invention

Example 1. Obtaining a genetic construct p1.1-F9 encoding a protein of human coagulation factor IX, for transfection of the cell line CHO-DG-44.

A commercially available clone pCMV6-XL4 / NM_000133.2 (proprietary collection of Origene Inc, USA, number sc 126517, sequence verified to the IMAGE public collection) containing the complete sequence of the full length factor IX cDNA matching the cDNA gene was used as a cDNA source for fIX gene cloning. theoretically determined factor IX cDNA according to the Human Genome Organization. Using primers AD-9-AbsF and AD-9-NheR (SEQ ID NO: 3-4) and Origen sc 126517, a PCR product containing an open reading frame of human coagulation factor IX was obtained as a matrix. Oligonucleotides were synthesized by CJSC Evrogen, RF. PCR was performed using a Tersus polymerase mix (CJSC Evrogen, RF) according to the manufacturer's instructions on a RTS-100 Thermal Cycler device (MJ Reseach, USA).

PCR products were purified using the Wizard SV Gel and PCR Clean-Up System reagent kit (Promega, USA) according to the manufacturer's instructions, and cloned into the pAL-TA (Eurogen) vector by T4 DNA ligase (Fermentas, Lithuania) according to methodology of the enzyme manufacturer. The obtained ligase mixture was used to transform E. coli cells of strain TOP10 (Invitrogen, USA) with the genotype: F ^- mcrA Δ (mrr-hsdRMS-mcrBC) φ80lacZAΔM15 ΔlacX74 nupG recAl araD139 A (ara-leu) 7697 galElrr15. For this, 5 μl of the ligase mixture was added to 100 μl of a frozen suspension of E. coli cells, incubated on ice for 30 minutes to sorb plasmid DNA, heated to 42 ° C for 45 seconds, and incubated on ice for 2 minutes. Then 800 μl of SOC nutrient broth (20 g / l tryptone, 5 g / l yeast extract, 0.5 g / l NaCl, 250 mM KCl, 10 mM MgC ₂ ) was added and incubated at 37 ° C for 60 minutes. After incubation, the suspension was transferred to a Petri dish with solid agar medium 2xYT-arap (16 g / l tryptone, 10 g / l yeast extract, 10 g / l NaCl, 18 g / l agar) containing ampicillin at a concentration of 100 μg / ml and placed in a thermostat for 18 hours at 37 ° C. Individual transformant clones were inoculated in 5 ml LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, increased for 18 hours at 37 ° C, and plasmid DNA was isolated using Wizard Plus SV Minipreps reagents (Promega, USA) according to the manufacturer's protocol . The insertion regions of the obtained plasmids were completely sequenced using primers for the T7prom and SP6 vector sequences (SEQ ID NO: 5-6), as well as a specific primer for the human factor IX gene sequence 9SQf (SEQ ID NO: 7) using the BigDye Terminator v kit. 3.1 cycle sequencing kit (Applied Biosystems, USA) and ABI PRISM 3730 genetic analyzer capillary sequencer (Applied Biosystems, USA); data analysis was performed using Chromas 1.45 software (Technelysium Pty Ltd, Australia).

After the detection of clones with the correct nucleotide sequence of the insertion region, the PAL-F9 plasmid was restricted with AbsI and NheI endonucleases and the fragment containing OPC F9 with the Kozak consensus sequence section and stop codon block was transferred into vector p1.1 restricted by AbsI and NheI endonucleases to form expression plasmid p1 .1-F9 (SEQ ID NO: 1). Ligation, transformation and isolation of plasmid DNA was performed as described above.

The open FIX reading frame, as well as the regions of the main functional elements of the expression construct, were sequenced as described above. For sequencing of the OPC region and the functional elements of the expression plasmid used a specific primer? to the sequence of the human factor IX gene 9SQf (SEQ ID NO: 7), as well as primers to the sequences of the vector SQ-5CH6-F, IRESArev (SEQ ID NO: 8-9). The results were compared with the theoretically predicted sequences of the human coagulation factor FIX gene and corresponding regulatory elements; no nucleotide substitutions were found.

The resulting expression design of coagulation factor IX of a person in vector p1.1-p1.1-F9 (Figure 1) has a size of 13299 bp and contains unique recognition sites for restriction endonucleases AscI (16), PvuI (1409), FseI (5213), AbsI (6446), NheI (7852). This design is intended for transfection of eukaryotic cells with the human coagulability factor genome IX, followed by selection and amplification according to the linked DHFR ⁺ trait ^. Tells recipient cells the following phenotypic traits: during transformation of prokaryotes, resistance to ampicillin, carbenicillin; with transfection of eukaryotes - limited resistance to methotrexate, increasing with selective selection, expression of recombinant human coagulability factor FIX. The created genetic construct was used to obtain a producer line.

Example 2. Obtaining the expression genetic construct p1.2-Hygro-FurVQ encoding the human PACE / furin protease for transfection of the cell line DG-1.1-F9.

Using primers AD-FUR-AbsF and AD-FUR-XbaR (SEQ ID NO: 10-11) and Origene SC118550, a PCR product containing an open reading frame of a soluble deletion variant of the human PACE / furin protease with a deletion of two amino acids was obtained as a matrix (Vq). The polymerase chain reaction was carried out as described above, the product was cloned into the pAL-TA vector as described above, the resulting pAL-Fur plasmid was sequenced using T7prom and SP6 primers (SEQ ID NO: 5-6), as well as specific primers SQ-FUR639 -F, SQ-FUR1228-F, SQ-FUR1563-R (SEQ ID NO: 12-14).

To introduce the six missing nucleotides into the open PACE / furin reading frame in accordance with NM_002569, site-directed mutagenesis of the pAL-Fur plasmid was performed by inverted PCR using IP-fVQ-F primers (SEQ ID NO: 15) and IP-fVQ-R (SEQ ID NO: 16). Inverted PCR site-directed mutagenesis was performed according to [Michael P. Weiner, Tim Gackstetter, Gina L. Costa, John C. Bauer, and Keith A. Kretz. Site-directed Mutagenesis using PCR in Molecular Biology: Current Innovations and Future Trends. Eds. AM Griffin and HG Griffin. ISBN 1-898486-01-8 1995 Horizon Scientific Press, PO Box 1, Wymondham, Norfolk, UK] with modifications. Each primer oligonucleotide was phosphorylated separately. The reaction was carried out in Tris-HCl buffer, pH 7.5, containing 10 mM MgCl ₂ , 50 mM dithiothreitol, 1 mM ATP and 100 pM oligonucleotide, 1 unit. polynucleotide kinase of phage T4 (Sibenzym, Russia) for 30 minutes at 37 ° C. After the reaction, the enzyme was inactivated at 65 ° C for 10 min. Then the reaction mixture was used to conduct inverted PCR in an amount of 20 pM per reaction. PCR was performed using the Encyclo PCR kit reagent kit (ZAO Evrogen, Russia) according to the manufacturer's instructions. PCR was carried out according to the following scheme: 1 cycle: 4 min 94 ° C, 2 min 50 ° C, 2 min 72 ° C; then 11 cycles of 11 min - 94 ° C, 1 min - 55 ° C, 2 min 72 ° C. After that, DpnI endonuclease buffer was doubled with a single buffer, DpnI endonuclease (10 U) was added and incubated at 37 ° С for 30 minutes, then Pfu polymerase (2.5U) was added and transferred to 72 ° С and incubated for another 30 minutes. The resulting mixture was purified using a set of "Wizard SV Gel and PCR Clean-Up System"("Promega", USA) according to the manufacturer's protocol. After that, the purified inverted PCR product was ligated using T4 phage DNA ligase and standard buffer solution (Fermentas, Lithuania) for 1 hour at room temperature. The obtained ligase mixture was used to transform E. coli cells of the TOPU strain (Invitrogen, USA) as described above. E. coli colonies were analyzed by clone PCR using T7prom and SP6 primer oligonucleotides (SEQ ID NO: 5-6) and a specific oligonucleotide SQ-fVQ-R (SEQ ID NO: 17) per mutant.

4 clones were grown in 5 ml of 2xYT-Amp nutrient broth and plasmid DNA was isolated using the GeneJET Plasmid Miniprep Kit (Fermentas, Lithuania) according to the manufacturer's protocol. For the obtained genetic constructs, the nucleotide sequence was determined by PCR sequencing using T7prom and SP6 oligonucleotides (SEQ ID NO: 5-6), as well as specific primers SQ-FUR639-F, SQ-FUR1228-F, SQ-FUR1563-R (SEQ IDNO: 12-14).

After detection of clones with the correct nucleotide sequence of the insertion region, the PAL-FurVQ plasmid was restricted with AbsI and XbaI endonucleases and the fragment containing OPC PACE / furin with the Kozak consensus sequence section and stop codon block was transferred into the p1.2-Hygro vector restricted with AbsI and NheI endonucleases the formation of the expression plasmid p1.2-Hygro-FurVQ (SEQ ID NO: 18). Ligation, transformation and isolation of plasmid DNA was performed as described above. For sequencing, specific primers SQ-FUR639-F, SQ-FUR1228-F, SQ-FUR1563-R (SEQ ID NO: 12-14), as well as primers for the sequences of the vector SQ-5CH6-F (SEQ ID NO: 8) were used. 3CH1-Rev (SEQ ID NO: 19).

Example 3. Preparative isolation of plasmid DNA for transfection and obtaining primary polyclonal cell lines of CHO-DG44 stably transfected with plasmid p1.1-F9.

The expression plasmid p1.1-F9 encoding human factor IX was constructed according to the procedure described in Example 1, grown in preparative quantities in the E. coli strain TORU (Invitrogen, Ltd., USA), purified using the EndoFree Plasmid MaxiKit kit, (Qiagen, USA), linearized with Pvul restriction endonuclease (Fermentas, Lithuania) with the destruction of the beta-lactamase gene and sterilized by filtration.

CHO-DG-44 cells (Life Technologies, USA) were cultured in Lonza Pro-CHO 5 protein-free medium (Lonza AG, Switzerland) with 8 mM L-glutamine. To support the growth of cells defective in the dhfr gene, nucleotide additives, γ-hypoxanthin YumM and thymidine 1.6 mM, were added to the medium. Cells were grown in sterile disposable Erlenmeyer flasks (30 ml of suspension in flasks with a capacity of 125 ml) on an orbital shaker at 130 rpm in an atmosphere containing 5% carbon dioxide at a temperature of 37 ° C. Calculation of the density of the cell suspension and the proportion of living cells is carried out in the counting chamber of Goryaev, when stained with trypan blue. Passaging of the culture was carried out upon reaching a density of 1.2 million cells / ml, inoculum concentration of 0.3 million cells / ml. 48 hours and 24 hours before transfection, the cells were additionally passaged to a seeding concentration of 0.5 million cells / ml. Transfection of the linearized plasmid p1.1-F9 was performed using FuGENE HD liposome reagent (Promega, USA) in serum-free CHO-S-SFM2 medium (Life Technologies, USA) using 1.5 × 10 ⁷ cells in 30 ml of medium in an Erlenmeyer flask , 60 μg of plasmid DNA and 80 μl of transfection agent. The mixing of DNA and transfection reagent was performed according to the method of the manufacturer of the agent. The transfection efficiency for registration of the fluorescent product of a transiently transfected (5% of the total DNA mass) plasmid pEGFP-C2 (Clontech, USA) after 48 hours was 8.2%. After 48 hours after transfection, the medium was completely replaced by centrifugation on Lonza PRO-CHO 5 with 8 mM glutamine, but not containing hypoxanthine and thymidine. In this case, the cell mass was divided into three equal parts and subsequently cultivated separately under the suspension conditions described above in the presence of a specific dihydrofolate reductase inhibitor (DHFR) methotrexate (MTX) at concentrations of 50 nM, SWONM and 200 nM. Cultures were reseeded after 5 days with complete replacement of the medium by centrifugation, or upon reaching a density of 1.2 million cells / ml by dilution to a seed density of 3 × 10 ⁵ cells / ml. Cultures were considered stable when the proportion of living cells reached more than 90% by trypan blue staining. Stable polyclonal lines were generated in 6 passages and took from 27 to 30 days. For the polyclonal cultures obtained in this way, the FIX secretion level was determined by enzyme-linked immunosorbent assay (ELISA) after reaching a transfer density of 1.2 million cells / ml. The level of secretion in three consecutive passages was 0.69 ± 0.04 μg / ml, 1.05 ± 0.05 μg / ml and 1.83 ± 0.24 μg / ml with 50 nM, 100 nM and 200 nM MTX, respectively, while the doubling time The resulting cultures were defined as 29.5 hours, 26.8 hours and 28.1 hours. The polyclonal line obtained by 200 nM MTX was cloned by limiting dilution in serum-free ex-cell Cloning medium (Sigma-Aldrich, USA) with 8 mM L-glutamine, 20 mM hypoxanthine and 3.2 mM thymidine without MTX selection pressure. The cloning efficiency was 41%. The best FIX secretion clonal cultures were returned to suspension culture without nucleotide additions and selective pressure. The FIX secretion level for the three best monoclonal lines obtained was 11.9 ± 0.4 μg / ml, 12.3 ± 0.4 μg / ml, 9.9 ± 0.3 μg / ml after 3 days of cultivation. The monoclonal line p1.1-F9-T2 / S, chosen for further work, showed in three successive passages a specific productivity of 2.99 ± 0.06 pg / cell / day, which was more than 6 times higher than the polyclonal cell pool with a reduced doubling time cell culture 22.5 hours. Moreover, the copy number of the vector insert in the genome, determined by the dhfr gene, as described in Example 8, was 14 ± 7 copies of the target gene per haploid genome, which turned out to be less than 20 ± 4 for the parent polyclonal population copies.

Example 4. Amplification of the target gene under the influence of methotrexate and obtaining clonal lines producing FIX.

Received, as described in Example No. 3, the monoclonal cell line p1.1-F9-T2 / S was amplified by a vector insert in the genome. For this, the initial monoclonal cell line was cultured under suspension conditions as described in Example 3, using Lonza PRO-CHO 5 nutrient medium with 8 mM glutamine and 20 μg / ml water-soluble vitamin KZ (menadione bisulfite) but not containing hypoxanthine and thymidine. At each amplification step, increasing concentrations of MTX 1 μM, 2 μM, 4 μM, 8 μM were added to the cell suspension. Cultures were reseeded after 5 days with complete replacement of the medium by centrifugation, or upon reaching a density of 1.2 × 10 ⁶ cells / ml by dilution to an inoculum density of 3 × 10 ⁵ cells / ml. Cultures were considered stable when reaching a proportion of living cells of more than 90% by trepan blue staining. A stable culture was frozen in Lonza PRO-CHO 5 medium with the addition of 10% dimethyl sulfoxide (DMSO) in the form of 1 ml samples in which cells were concentrated by centrifugation at 300 g to a density of 10 ⁷ cells / ml. For stable cultures thawed under ordinary conditions of suspension cultivation under the action of MTX generation concentration, the level of secretion of recombinant FIX, the cell doubling time of the culture (according to the methods of Example 3) and the copy number of the vector insert into the genome (according to the method of Example 8) were determined. Successfully thawed culture was also transferred to the next step of amplification with the following concentration of MTX. Moreover, the use of MTX at a concentration of 8 μM did not allow to obtain a stable culture, thus, the maximum selective pressure was 4 μM MTX.

Table 1 Indicators of producers during amplification of a transgene under the influence of methotrexate. Culture Code and Clonality MTX Concentration ELISA specific productivity, pg GC factor / cell / day Dhfr genome insert copy number, copies / genome Cell Doubling Time, hours p1.1-F9 / 200-T2 / S monoclonal - 2.99 ± 0.09 14 ± 7 20.9 p1.1-F9.T2 / 1k polyclonal 1 μM 3.53 ± 0.12 23 ± 4 19.8 p1.1-F9.T2 / 2k polyclonal 2 μm 4.03 ± 0.31 39 ± 2 22.5 p1.1-F9.T2 / 4k polyclonal 4 μm 5.97 ± 0.18 81 ± 7 24.2 p1.1-F9.T2 / 8k polyclonal 8 μM not determined 1.1-F9-T2 / 4k-3B12 monoclonal - 10.72 ± 0.43 75 ± 4 20.1

The resulting polyclonal culture p1.1-F9-T2 / 4k was cloned by limiting dilution in serum-free ex-cell Cloning medium with 8 mM L-glutamine, 20 mM hypoxanthine and 3.2 mM thymidine without MTX selection pressure. The cloning efficiency was 34.6%. A total of 432 monoclonal colonies were obtained, for which the concentration of factor IX accumulated in the medium was determined by ELISA on 21 days of growth under attached conditions. The best secretion of factor IX clonal cultures (24 clones) re-adapted to suspension culture without nucleotide additives and selective pressure, while half of the monoclonal cultures lost their productivity and / or ability to grow in suspension (less than 0.5 pcg / cell / day or time of doubling more than 48 hours).

The best of 12 successfully adapted to suspension cultivation of monoclonal cell cultures p1.1-F9-T2 / 4k-3B12 in three consecutive passages showed a specific productivity of 10.72 ± 0.43 pg factor IX / cell / day, which is 2 times higher than polyclonal cell pool at a time of doubling the cell culture of 20.2 hours (determined by the method of Example 3). In this case, the copy number of the vector insert in the genome decreased to 75 ± 4 (determined by the method of Example 8).

Example 5. Obtaining a cell line stably transfected with plasmids p1.1-F9 and p1.2-Zeo-CHO-VKORCL

The monoclonal cell line p1.1-F9-T2 / 4k-3B12 when determining its specific productivity (as described in Example 4) on three-day cultivation cycles with a density at the time of reseeding of 1.2 × 10 ⁶ cells / ml accumulated no more than 0.22 IU / ml of active factor IX at a total concentration of factor IX, determined by ELISA, 59 μg / ml, which corresponded to a specific activity of secreted human factor IX of 1.83% (total specific activity is 200 IU / mg). The procoagulant activity of recombinant FIX depends on the degree of gamma-carboxylation of its Gla domain, and corresponds to the natural one upon carboxylation of the first 10 of 12 Gla-domain glutamic acid residues [McGraf DM, Walsh G. Directory of therapeutic enzymes. NY 2005. 312 pp.]. The specific activity of recombinant FIX can be increased by co-expression in the production lines of the vitamin K-epoxide reductase enzyme (VKORC1), which restores vitamin K (electron donor for enzymatic gamma-carboxylation to its active form [Wajih N. Et al. Increased production of functional recombinant human clotting factor IX by baby hamster kidney cells engineered to overexpress VKORC1, the vitamin K 2,3-epoxide-reducing enzyme of the vitamin K cycle. Journal of Biological Chemistry 2005 280 (280), 31603-31607].

For integration into the genome of the monoclonal cell line p1.1-F9-T2 / 4k-3B12 additional copies of the Chinese hamster vkorcl gene under the control of a strong constitutive promoter, the expression plasmid p1.2-Zeo-CHO-VKORC (SEQ ID NO: 20 , Fig. 3), obtained analogously to that described in Example 2. The plasmid was introduced into the cells by the method of electrotransfection with a single rectangular pulse of 20 ms duration at a constant voltage of 200 V and a cell gap of 4 mm, 30 μg of plasmid DNA p1.2-Zeo-CHO-VKORC was used linearized restriction enzyme PvuI. The transfection efficiency was 12.4%. 48 hours after transfection, the selection antibiotic zeocin (Life Technologies, USA) at a concentration of 1 mg / ml was added to the culture. Passaging of the culture was carried out upon reaching a density of 1.2 million cells / ml, but no later than 5 days after the previous passage. 30 days after transfection, a stable culture was obtained with a proportion of live cells of 87%. The level of factor IX secretion in the obtained culture was 6.87 ± 0.39 pg / cell / day with a cell culture doubling time of 22.5 hours (determined by the methods of Example 3), and the copy number of the vkorcl gene insert was 3.24 ± 0.53 copies on the haploid genome after canceling the selection pressure of zeocin (according to the method of example No. 8).

Example 6. Obtaining a cell line stably transfected with plasmids p1.1-F9, p1.2-Zeo-CHO-VKORCl and p1.2-Hygro-FurVQ.

During the secretion of the mature form of coagulation factor IX, which can exhibit procoagulant activity, the propeptide is separated from it. The same post-translational modification can be performed in the culture medium by co-expression of recombinant FIX and a soluble shortened variant of the PACE / furin protease [McGraf D.M., Walsh G. Directory of therapeutic enzymes. NY 2005. 312 pp.]. The monoclonal cell culture p1.1-F9-T2 / 4k-3B12 obtained in Example No. 4 secreted recombinant factor IX with a low specific activity of 1.8% of the standard, with more than 97% of factor IX molecules containing an unseparated propeptide according to ELISA data using antibodies to the propeptide factor IX.

To increase the activity of the target protein, the polyclonal cell population p1.1-F9-T2 / 4k-3B12-VKOR / Zeo obtained in Example 5 was transfected with expression plasmid pl.2-Hygro-FurVQ (SEQ ID NO: 18, Figure 2) obtained as described in Example 2. Transfection was performed by electroporation according to the procedure described in Example 5, but Hygromycin B (Sigma-Aldrich, USA) at a concentration of 750 μg / ml was used as a selection antibiotic. The transfection efficiency was 20.2%. The generation of a stably transfected polyclonal culture with a living cell fraction of more than 90% lasted 21 days. The resulting polyclonal population p1.1-F9-T2 / 4k-3B12-VKOR / Zeo-FurVQ / Hyg had a factor IX secretion level of 5.83 ± 0.42 pg / cell / day, a fraction of factor IX molecules with an unseparated propeptide of 3.1 %, the specific procoagulant activity of the product in the culture medium is 27% of the standard, the copy number of the insert of the fix gene in the genome is 40.6 ± 1.0 copies per haploid genome (according to the method of Example 8).

The specific productivity of the p1.1-F9-T2 / 4k-3B12-FurVQ / Hyg population by biologically active factor IX was 1.6 ± 0.03 IU / million cells / day at a cell doubling rate of 23.25 hours.

In order to obtain monoclonal production lines of recombinant factor IX suitable for pharmaceutical production, the polyclonal population p1.1-F9-T2 / 4k-3B12-VKOR / Zeo-FurVQ / Hyg was cloned by limiting dilution in serum-free ex-cell cloning with 8 mM L-glutamine, 20 mM hypoxanthine and 3.2 mM thymidine without MTX selection pressure or antibiotics. The cloning efficiency was 25.9%. A total of 199 monoclonal colonies were obtained, for which the total level of factor IX secretion was determined by ELISA on 21 days of growth under attached conditions. According to the results of the initial screening, 80 clones were collected that accumulated more than 12.5 μg / ml of the target protein. The selected clones were transferred to semi-adhesive cultivation without stirring in Lonza PRO-CHO 5 medium with 8 mM glutamine and 20 μg / ml vitamin KZ, but not containing hypoxanthine, thymidine, and selection components. After 5 days of cultivation in an air-conditioned environment using a Fusion-Alpha FP HT 4 fluorimeter (PerkinElmer, Inc, USA) with a pair of 400 nm / 460 nm filters, the peptidase activity against the fluorogenic PACE / furin proteinase substrate was measured kinetically. A total of 24 monoclones were selected, for which the relative peptidase activity in the conditioned medium exceeded more than 100 times the peptidase activity in the conditioned medium of the parent line 1.1-F9-T2 / 4k-3B12, not transfected with the PACE / furin gene. For all selected clones, the fraction of factor IX molecules containing unseparated propetid was less than 3.5% (i.e., it was below the limit of determination for the ELISA method used). For further suspension cultivation with stirring, 12 monoclonal lines were selected that secreted more than 14.5 μg / ml of factor IX over 3 days of cultivation. Only 5 of the 12 selected monoclonal lines retained the ability to divide under normal conditions of suspension cultivation in Lonza PRO-CHO 5 medium with 8 mM glutamine and 20 μg / ml vitamin K3. The specific activity of recombinant factor IX secreted by all five selected monoclonal producer lines was more than 185 IU / mg. Other characteristics of the obtained cell lines are summarized in Table 2.

table 2 The main properties of monoclonal producer lines. Line code, clonal Factor IX secretion, PCG / cell / day (ELISA) Specific productivity, IU / million cells / day (coagulometry) The proportion of active secreted factor IX The proportion of factor IX molecules with propeptide The number of inserts in the genome by dhfr, copies / hapl. genome Cell doubling time, h p1.1-F9 / 200-T2 / S monoclonal 2.99 ± 0.09 but - 96% 14 ± 7 20.9 p1.1-F9.T2 / 1k polyclonal 3.53 ± 0.12 but - 97% 23 ± 4 19.8 p1.1-F9-T2 / 2k polyclonal 4.03 ± 0.31 but - 96% 39 ± 2 22.5 p1.1-F9-T2 / 4k polyclonal 5.97 ± 0.18 0.02 ± 0.005 1.1% 99% 81 ± 7 24.2 1.1-F9-T2 / 4k-3B12 monoclonal 10.72 ± 0.43 0.03 ± 0.001 1.8% 97% 75 ± 4 20.1 p1.1-F9.T2 / 4k-3B12-FurVQ / Hyg polyclonal 5.83 ± 0.42 1.6 ± 0.029 27% 3.03% 40 ± 3 23.1 p1.1-F9 / 3B 12-05 monoclonal 7.03 ± 0.21 1.21 ± 0.008 89% 1.21% 21 ± 1 19.6 p1.1-F9 / 3B12-78 monoclonal 10.10 ± 0.16 1.71 ± 0.004 84% 2.65% 9 ± 1 20.9 p1.1-F9 / 3B 12-86 monoclonal 11.19 ± 0.27 2.08 ± 0.003 95% 1.41% 22 ± 2 25.71

Example 7. Measurement of the number of copies of expression plasmids introduced into the genome of cells and levels of human FIX mRNA in cells.

Quantitative analysis of the copy number of the expression cassette in the genome and the mRNA level was carried out by real-time PCR (PCR-RV) using the iCycler iQ amplifier (Bio-Rad, USA). For PCR, a ready-made 5-step hot-start reaction mixture containing intercalating dye SYBR Green I, qPCRmix-HS SYBR (Eurogen, Russia) was used. Each reaction was repeated 3 times in a volume of 25 μl, in 3-5 repetitions. Genomic DNA was isolated from 0.5-5 million cells using the Wizard SV Genomic DNA Purification System kit (Promega, USA) according to the manufacturer's instructions. Total RNA was isolated from 0.5-5 million cells using the RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's instructions. To prepare cDNA samples, we used 1 μg of total RNA and a set of Mint reagents (Eurogen, Russia). To determine the copy number of the expression cassette in the genome, genomic DNA and oligonucleotide primers not homologous to the genomic sequences of the Chinese hamster — to the human FIX gene — RT-F9-f and RT-F9-r (SEQ ID NO: 21-22) and to the IRES-DHFR region RT-ID-F and RT-ID-R (SEQ ID NO: 23-24). To analyze the copy number of auxiliary cassettes in the genomic DNA of cells, a pair of primers for the hygromycin resistance gene, RT-Hyg-F and RT-Hyg-R (SEQ ID NO: 25-26), was used; a pair of primers for the zeocin resistance gene RT-Zeo-F and RT-Zeo-R (SEQ ID NO: 27-28); a pair of primers to the C-terminal region of VKORC1 of a Chinese hamster and 3 ′ NTO plasmids p1.2-Zeo-CHO-VKORC - RT-cVKOspC -F and RT-cVKOspC-R (SEQ ID NO: 29-30), a pair of primers to C the terminal region of human furin RT-FURC-F and RT-FURC-R (SEQ ID NO: 31-32).

To determine the level of mRNA expression, cDNA and unique oligonucleotide primers not homologous to the sequences of the Chinese hamster were used as matrices. Primers RT-cVKOspN-F and RT-cVKOspN-R (SEQ ID NO: 33-34) were used to determine the level of VKORC1 expression.

For amplification of the reference genes, in the case of PCR with genomic DNA, primers were used for the peptidyl-prolyl isomerase B gene region (cyclophilin B), presumably unique to the CHO genome, RT-PPIB-F and RT-PPIB-R (SEQ ID NO: 35 -36), and in the case of PCR with cDNA, beta-actin CHO primers, RT-bACT-F and RT-bACT-R (SEQ ID NO: 37-38), were used.

The copy number of the expression cassette was determined from a calibration curve constructed for serial dilution of expression plasmids p1.1-F9, p1.2-Zeo-CHO-VKORC1, p1.2-Hygro-FurVQ. PCR results were compared with the results for the control amplicon presented in the genome of CHO cells only once by searching for BLAST algorithm from the NCBI Nucleotide Collection database. When analyzing the levels of mRNA expression, it was found that the PCR efficiency is at least 99% for all used primer pairs; the relative mRNA concentrations were calculated using the formula C = 2 ^ (- CT), where CT is the threshold amplification cycle.

Although the invention has been described in detail with reference to Examples, it will be apparent to those skilled in the art that various changes can be made and equivalent replacements made, and such changes and replacements are not outside the scope of the present invention.

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16. Michael P. Weiner, Tim Gackstetter, Gina L. Costa, John C. Bauer, and Keith A. Kretz. Site-directed Mutagenesis using PCR in Molecular Biology: Current Innovations and Future Trends. Eds. A.M. Griffin and H.G. Griffm. ISBN 1-898486-01-8 1995 Horizon Scientific Press, PO Box 1, Wymondham, Norfolk, U.K.

Short Description of Figures.

Figure 1 shows a map of the expression plasmid p1.1-F9. The following notation is used: pUC origin - region of the beginning of replication of plasmid pUC; bla - an open reading frame of beta-lactamase providing resistance to ampicillin; bla promoter - prokaryotic promoter of the bla gene; EBV TR - plot of the terminal repeat of the Epstein-Barr virus person; CHO EEF1A UFR functional promoter of the elongation factor 1 gene of the Chinese hamster alpha, 5 'untranslated region of this gene and untranscribed region flanking this gene; transcription start - transcription start point, TATA - TATA box, CHO EEF1A intron I - first intron of the Chinese hamster alpha 1 elongation factor gene, EMCV IRES - internal ribosome binding site of encephalomyocarditis virus (EMCV); DHFR - open reading frame of mouse dihydrofolate reductase for selective selection and amplification in eukaryotic cells; CHO EEF1A DFR functional I terminator and polyadenylation signal of the elongation factor gene 1 Chinese hamster alpha, 3 ′ untranslated region of this gene and untranscribed region flanking this gene; Kozak - region encoding the Kozak sequence for cap-dependent translation initiation, F9 ORF - ORS of human blood coagulation factor IX, stop - block of stop codons. The arrows indicate the directions of gene transcription, the numbers of the first and last nucleotides of the fragments are indicated in parentheses. The recognition sites of restriction endonucleases are in italics, and the numbers of nucleotides at the cutting points are indicated in parentheses.

Figure 2 shows a map of the expression plasmid p1.2-Hygro-FurVQ. Designations are used similarly to Figure 1, as well as Fur ORF - ORS proteas of human PACE / furin, SV40 promoter - region of the SV40 virus promoter; SV40pA, terminator - terminator and signal for polyadenylation of the SV40 virus; hygroB is the OPC of the hygromycin resistance gene, VQ is the insertion point of a pair of codons encoding amino acids V and.

Figure 3 shows a map of the expression plasmid p1.2-Zeo-CHO-VKORC1. Designations are used similarly to Figure 1, as well as CHO VKORC1 ORF - OPC VKORC1 of a Chinese hamster, SV40 promoter - region of the SV40 virus promoter; SV40pA, terminator - terminator and signal for polyadenylation of the SV40 virus; ZeoR - OPC of the zeocin resistance gene.

Claims (15)

1. Plasmid for expression of recombinant human coagulability factor IX in cultured cells of the Chinese hamster CHO line, in the following sequence essentially consisting of the region of the beginning of replication of the pUC plasmid, open reading frame of beta-lactamase (bla) and the prokaryotic promoter of the bla gene; plot of the terminal repeat of the Epstein-Barr virus of a person (EBVTR), a functional promoter and the first intron of the gene for the elongation factor 1 alpha of the Chinese hamster flanked by a 5 ′ untranslated region of this gene; open reading frame of a gene encoding human blood coagulation factor IX; encephalomyocarditis virus (EMCV) internal ribosome binding site (IRES); open reading frame of dihydrofolate reductase (DHFR), expressed as a part of bicistronic mRNA together with a gene encoding human coagulation factor IX; and a functional terminator of the gene for the elongation factor 1 alpha of the Chinese hamster flanked by a 3 ′ untranslated region of this gene. 2. The plasmid according to claim 1, characterized in that the human blood coagulation factor IX gene encodes a human blood coagulation factor polypeptide IX, the amino acid sequence of which is shown in SEQ ID NO: 2. 3. The plasmid according to claim 1, characterized in that the plot of the terminal repeat of the Epstein-Barr virus of a person (EBVTR) is a fragment of the concatemer of the terminal repeat of EBVTR. 4. The plasmid according to claim 1, characterized in that an open reading frame of mouse dihydrofolate reductase (DHFR) is used. 5. The plasmid according to claim 1, characterized in that said plasmid further comprises a Kozak consensus sequence before the sequence encoding the open reading frame of human coagulation factor IX. 6. The plasmid according to claim 1, characterized in that the plasmid is plasmid p1.1-F9, presented in the List of sequences under the number SEQ ID NO: 1. 7. Chinese hamster ovary cell (CHO) - producer of recombinant human coagulability factor IX, transformed with a plasmid according to paragraphs. 1-6. 8. The cell of claim 7, further transformed with a plasmid expressing vitamin K-epoxide reductase (VKORC1). 9. The cell according to claim 8, characterized in that the plasmid is plasmid p1.2-Zeo-CHO-VKORC1, presented in the List of sequences under the number SEQ ID NO: 20. 10. The cell of claim 8, further transformed with a plasmid expressing a soluble truncated shortened version of the PACE / furin protease. 11. The cell according to p. 10, characterized in that the plasmid is plasmid p1.2-Hygro-FurVQ, presented in the List of sequences under the number SEQ ID NO: 18. 12. The cell according to claim 11, characterized in that said cell is an ovary cell of a Chinese hamster of the monoclonal line P1.1-F9 / 3B12-86 deposited in the Russian Culture Collection of Vertebrate Cells of the Institute of Cytology RAS under registration number RACC (P) 755D. 13. A method of obtaining a recombinant coagulation factor IX of a person, comprising the following stages:
- cultivation in a nutrient medium of cells according to paragraphs. 7, 8 or 10; and
- isolation of the resulting recombinant protein from the culture fluid. 14. The method according to p. 13, characterized in that the ovary cells of the Chinese hamster of the Chinese hamster of the monoclonal line P1.1-F9 / 3B12-86, deposited in the Russian Culture Collection of Vertebrate Cells of the Institute of Cytology RAS, registration number RKKK (P) 755D. 15. The method according to p. 13, wherein the culture medium is serum-free.

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