RU2561466C2 - Expression plasmid for human recombinant coagulation factor viii with deleted b-domain in cho cells, cho cell producing human recombinant coagulation factor viii with deleted b-domain, and method for producing above factor - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and can be used for the recombinant production of human coagulation factor VIII (FVIII) with deleted B-domain. The procedure involves constructing an expression plasmid for recombinant human FVIII with deleted B-domain in dihydrofolate reductase (DHFR) gene knockout CHO cells; the above plasmid contains a region of origin of replication of the pUC plasmid, a beta-lactamase (bla) open reading frame and a bla gene prokaryotic promoter, a human Epstein-Barr virus terminal repeat (EBVTR) site, functional elements of Chinese hamster elongation factor 1 alpha gene, Kozak sequence; an open reading frame of a human FVIII gene with deleted B-domain; an internal ribosomal entry site (IRES) of EMCV virus; an open reading frame of DHFR expressed as a part of bicystronic mRNA with the gene coding human FVIII gene with deleted B-domain (IRES DHFR). The produced plasmid is used to transform a CHO cell to produce a factor producer cell.

EFFECT: invention enables substantially increasing the production effectiveness in the CHO cells and cutting the expenses for producing the human recombinant FVIII with deleted B-domain of pharmaceutical importance.

10 cl, 6 dwg, 4 tbl, 4 ex

Description

The field of technology.

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 VIII.

The prior art.

Deficiency of human coagulation factor VIII (fVIII) is the cause of hemophilia A. Congenital hemophilia A, caused by damage to the fVIII gene, is observed with a frequency of 1: 5000-7000 men. The factor VIII gene is located on the X chromosome. Its total length is about 190 Kb, the gene includes 26 exons ranging in size from 69 bp. up to 3106 p.p. (Toole, Knopf et al., Nature, 1984, Nov, 312 (5992): 342-7). Expression of the factor VIII gene is localized in liver cells, the factor VIII gene (fVIII) contains regulatory sequences of hepatocytes. The product of the fVIII gene after removal of the secretion leader peptide with a length of 19 amino acids consists of domains A1-A2-B-A3-C1-C2, the total length of 2332 amino acids.

Since fVIII is a secreted protein, it undergoes a series of post-translational modifications in the endoplasmic reticulum and Golgi apparatus. The most significant modifications of fVIII are N-glycosylation (about 25 sites, mainly in domain B), sulfation of 6 tyrosine residues, O-glycosylation and conversion to double-stranded form by specific proteolysis at the boundary of domains B and A3. The mature fVIII released from blood plasma is almost completely converted into a double-stranded form, consisting of a heavy chain (A1-A2-B) ranging in size from 90 to 200 kDa and a light chain of 80 kDa in size. The FVIII chains are connected non-covalently; their interaction is mediated by copper ions coordinated in the A1 and A3 domains. Factor VIII in the bloodstream forms a complex with a specific chaperone - von Willebrandt factor (vWF) - and leaves this complex upon activation. The heterogeneity of the heavy chain sizes is due to the decay of fVIII at many sites in the B-domain; the minimum form of the heavy chain of fVIII of 90 kDa does not contain B-domain residues at all. The normal concentration of fVIII in the bloodstream (1 international unit (MB) of fVIII in 1 ml) is about 200 ng / ml, the half-life of fVIII in humans is about 20 hours.

Since effective therapy for hemophilia A is based on regular intravenous injections of drugs fVIII at a dose of 250-2000 ME, the global consumption of fVIII is very significant. The following drugs are used in hemophilia therapy: 1) preparations of natural factor VIII, 2) preparations of recombinant full-size fVIII, 3) preparations of recombinant factor VIII with a deleted B domain, variant BDD SQ (deletion of region 746-1639, counting by mature polypeptide).

Natural fVIII drugs are usually obtained from the cryoprecipitate fraction of donor plasma using several stages of chromatographic purification. Some natural fVIII preparations are purified by immuno-affinity chromatography, and vWF is removed. Recombinant fVIII drugs also do not contain vWF and undergo multistage purification, including immuno-affinity chromatography (or the equivalent stage, purification on an immobilized peptide), and also undergo a virus inactivation procedure.

Heterologous expression of the fVIII gene is extremely ineffective. Due to the fact that fVIII mRNA is unstable and contains transcriptional silencers in the open reading frame of the protein, the translation level of the fVIII precursor protein is reduced tenfold in comparison with other proteins of similar length. About 90% of the precursor of fVIII does not leave the endoplasmic reticulum due to binding to chaperone BiP and subsequent degradation. Mature secreted fVIII is also subject to intense degradation by extracellular proteases. The deletion variant fVIII BDD SQ is less susceptible to retention in the endoplasmic reticulum and proteolytic inactivation in the culture medium, however, up to 90% of the secreted product undergoes reverse binding to the membrane of producer cells, mainly due to the specific interaction with phosphatidylserine. Due to these features of mRNA and protein fVIII, the known industrially suitable systems for producing recombinant fVIII are characterized by very low productivity - from 0.5 to 10 IU / ml, i.e. not more than 1 mg / l, while the productivity of the expression systems of the deletion variant fVIII is generally higher than the expression systems of the full-sized fVIII. The BDD SQ B domain deletion variant was used in the preparation of the registered drug fVIII “Moroccog alpha” (Lind, Larsson et al., Eur J Biochem., 1995, Aug 15, 232 (1): 19-27), other deletion variants The B-domain was not used as a medicine.

An initial method for creating cell producers of the deletion variant fVIII BDD SQ is described in US Pat. No. 4,868,112. The method involves constructing a plasmid containing the open reading frame region (OPC) of fVIII with a deletion of the B domain region under the control of the immediate early cytomegalovirus (CMV) promoter and mouse dihydrofolate reductase gene (DHFR) under the control of the SV40 promoter, subsequent transfection of this plasmid into cultured cells of the COS-1 line (monkey) or CHO DUKX-B11 (Chinese hamster, inactivation of one allele of the DHFR gene), obtaining stable colonies but transfected cells in a medium containing no hypoxanthine and thymidine, and subsequent selection of the most productive clones in a medium containing methotrexate (a DHFR inhibitor). Using this method of generating producer lines, the maximum published rate of target protein secretion into the culture medium (specific productivity) was 2 IU / million cells / day. Cell cultivation was performed in the presence of cattle serum containing bovine vWF, which binds tightly to fVIII and protects it from capture on the membrane and proteolytic degradation. A method for producing a producer without the use of animal serum has not been proposed.

Known methods for increasing the productivity of production lines of fVIII BDD SQ are based on the introduction of point amino acid substitutions or the replacement of individual sections of human fVIII with homologous fragments of fVIII mammals. In particular, in US patents US 6376463, US 7122634 described the introduction of point mutations in domains A and the creation of fusion proteins of human factor VIII and factor VIII pig. These methods do not have practical applicability in the development of fVIII drugs, although they can be the basis for solutions for hemophilia A gene therapy, since introducing mutations in the OPC of the fVIII gene can lead to an expansion of the spectrum of allo and auto antibodies in hemophilia patients, including due to the development of secondary resistance to fVIII pigs (Parker, Craddock et al., J Thromb Haemost., 2004, Apr; 2 (4): 605-11.).

Other known methods for improving producer cells that are not associated with a change in the structure of the target protein are based on the insertion into the genome of cultured cells of genetic constructs encoding bicistronic RNA, that is, RNA with two translated regions — the target gene and the selection marker. The population of cells stably transfected with such genetic constructs was subjected to prolonged selection in the presence of methotrexate (MTX), which should have led to amplification in the genome of the transfected genetic construct. This approach has been described in US Pat. No. 6,114,446 for 293 and SK-HEP1 cell lines and plasmid pCMVF / EDHPro containing ORS fVIII BDD SQ and ORF DHFR linked by a DNA site containing an internal ribosome binding site (IRES) of the encephalomyocarditis virus. When culturing colonies of stably transfected cells in the presence of increasing concentrations of MTX and subsequent production of clonal lines by the method of limiting dilutions, producer lines secreting fVIII BDD SQ were created at a rate of 0.1 to 3 IU / million cells / day. As in the cases described above, obtaining producers according to the method proposed in the patent requires the use of embryonic serum of cattle, which leads to the formation of a dense complex of factor VIII and vWF of the bovine and a significant overestimation of the measured concentration of fVIII in the culture medium (compared with the concentration of fVIII in serum free environment).

Obtaining production lines of fVIII BDD SQ using a bicistronic vector with a CMV promoter and a selection marker DHFR is described in RF patent RU 2429294, in this case a protein-free growth medium is used, but the level of production of fVIII does not exceed 0.5 IU / ml for a suspension culture with a density 1.2 million ct / ml.

The closest in technical implementation analogue of the present invention is the fVIII gene expression system described in US Pat. No. 6,358,703, which includes transfection of the bicistronic plasmid vector pCIS25DTR, encoding the fVIII gene with B domain deletion and the DHFR gene, into the NKB11 cell line. The resulting primary oligoclonal producer lines were subsequently selected in the presence of increasing concentrations of MTX, cloned by limiting dilution, and adapted to a serum-free medium containing a solution of human plasma proteins (i.e. human vWF). The use of the clonal producer line obtained by this method for cultivation in a 15-liter perfusion bioreactor made it possible to achieve a total productivity of 4 million IU / day, which corresponds to a specific productivity of 10-30 IU / million ct / day. Despite the declared very high productivity indicators, this industrial solution has a number of significant drawbacks, namely, based on the use of a hybrid cell line obtained by fusion of cultured human 293S cell lines and Burkitt’s cancer cells, actively secreting immunoglobulin and cultivated in the presence of cattle fetal serum as part of the culture medium. The cultivation medium for growth in the bioreactor did not contain serum, but was supplied with a solution of human plasma proteins, which can be contaminated with human viruses and prions. In addition, cancerous cultured human cells can also be sources of viral infections and are not optimal precursors for the production of industrial lines producing pharmaceutical proteins (US patent application US 2006/0099685 A1 p. 0058).

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 human coagulation factor VIII with a deletion of the B domain for biopharmaceutical production.

The technical result was achieved by creating a new expression plasmid DNA p1.1-F8BDD encoding an open reading frame of a human coagulation factor VIII protein with a deletion of the B domain, and creating a clonal producer cell line based on it.

The technology is based on the developed plasmid DNA p1.1-F8BDD with a length of 16282 bp. (SEQ ID NO: 1, Fig. 1), consisting of:

- a 4389 bp DNA fragment containing a 4371 bp sequence. (SEQ ID NO: 1 6460-10830), encoding the ORS of human coagulation factor VIII with a deletion of the B domain and the synthetic Kozak sequence (ribosome binding site, SEQ ID NO: 1 6451-6459), providing the initiation of translation of mRNA,

- fragment 11893 p. plasmids p1.1 (SEQ ID NO: 2, Fig. 2) containing regulatory elements that provide expression of the target protein:

- 5 'and 3' untranslated regions of the gene for 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 of 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) providing 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-F8-BDD contains unique recognition sites for the restriction endonucleases AscI (16), PvuI (1409), FseI (5213), AbsI (6446), PsiI (7737), NheI (10835).

The plasmid was introduced into CHO DG-44 cells by liposomal transfection (Straus SE et al, J. Virol., 1981, Jul; 39 (1): 290-4) by cultivation in a protein-free medium containing no hypoxanthine and thymidine and additionally containing 50 nM inhibitor of DHFR 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 cultivations in the presence of increasing concentrations of methotrexate. A polyclonal line of cells resistant to a high concentration of methotrexate was obtained, which produced increased amounts of the target protein, and was 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. Among them, clone DG-P1.1-F8BDD-S3 was selected, when cultured in suspension culture in a serum-free medium, the final activity of the B domain of the deleted blood coagulation factor VIII was 40 ± 9 IU / ml at a cell concentration of 1.7 million / ml . The obtained cell line was deposited in the Russian Collection of Cultures of Vertebrate Cells of the Institute of Cytology RAS, registration number No.RKKK (P) 756D.

An object of the present invention is to provide a plasmid for expression of a recombinant human coagulation factor VIII with a deleted B domain, in particular plasmid p1.1-F8BDD. Another objective of the present invention is the provision of a monoclonal line of mammalian cells, in particular Chinese hamster ovary cells, producers of a recombinant human coagulation factor VIII with a deleted B domain containing multiple copies of the expression cassette in the genome corresponding to a linearized expression plasmid, in particular plasmid p1.1 -F8BDD. An illustrative example of this monoclonal cell line are Chinese hamster ovary cells of clone DG-P1.1-F8BDD-S3.

It is also an object of the present invention to provide a method for producing a recombinant human coagulation factor VIII with a deleted B domain 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 VIII with a deletion of the B domain 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 represents a linearized expression plasmid containing a DNA fragment encoding a fact p blood clotting VIII deletion of the B domain 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 VIII with a deletion of the B domain in the expression cassette of the present invention is, but is not limited to, the promoter of the Chinese hamster elongation factor 1 alpha gene.

A DNA fragment encoding a recombinant coagulation factor VIII with a B domain deletion according to the present invention is a gene encoding the OPC of a factor VIII polypeptide with a B domain deletion, which can be obtained, for example, as described in Example 1. Also, the specified DNA fragment can be obtained, for example, using the cloning technology of Sloning BioTechnology, described in PCT application WO 2005071077.

The sequence of the gene encoding the OCR of the recombinant coagulation factor VIII with a deletion of the human B domain according to the present invention is presented in the Sequence Listing under the number SEQ ID NO: 1 (nucleotides 6460-10830). The amino acid sequence of the secreted human coagulation factor VIII with a B-domain deletion according to the present invention is presented in the Sequence Listing under the number SEQ ID NO: 3, and is shown in Figure 4, and is the translation product of nucleotides 6460-10830 of the OPC sequence SEQ ID NO: 1 without the first 19 amino acids of the secretion leader peptide. The B-domain of factor VIII is replaced by a 14-amino acid linker peptide, which is partially detached during post-translational processing of fVIII BDD (SEQ ID NO: 3, amino acids 741-754), in which the first five amino acids are the amino acid sequence of the N-terminal portion B domain fVIII, and the following nine amino acids are the amino acid sequence of the C-terminal portion of the B domain of fVIII. A part of the molecules of the mature secreted coagulation factor VIII with a deletion of the human B-domain according to the present invention may contain one or more proteolytic ruptures of the polypeptide chain, mainly after residue Arg740, and be a double-stranded form of coagulation factor VIII. The degree of conversion of the single-stranded form into a double-stranded form does not affect the biological activity of coagulation factor VIII; for the purposes of the present invention, these forms are completely equivalent.

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 6460-10830), for example, by the method of site-directed mutagenesis, so that one or more amino acid residues at a particular site will be deleted, 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.

The coagulation factor of human VIII is a non-enzymatic coagulation factor of the blood coagulation system that forms a dense non-covalent complex with activated factor IX and accelerates the proteolytic cleavage of factor X to activated factor X. The natural coagulation factor of human VIII in the bloodstream predominantly circulates in double-stranded form. a heterodimer of a heavy and light chain, while the heavy chain may partially or completely contain a region of the B domain. The main variants of the heavy chain of natural factor VIII have a mass of 90 to 150 kDa, the light chain is about 80 kDa. The recombinant human coagulation factor VIII with a deletion of the B-domain in a single-chain form has a mass of about 170 kDa, in a double-stranded one - about 90 kDa in the heavy chain and about 80 kDa in the light chain. The recombinant coagulation factor VIII with a deleted B-domain fully retains procoagulant activity, while its specific procoagulant activity, expressed in international units per milligram of pure protein, is 20-40% higher due to the lower molecular weight of the deleted variant.

Indicators of functional activity, in which it is believed that the obtained protein has the properties of a recombinant human coagulation factor VIII, are determined by its ability to reduce the coagulation time (coagulation) of human blood plasma with deleted factor VIII. For example, the activity of recombinant human coagulation factor VIII can be detected by decreasing the time of clot formation in blood plasma with removed factor VIII after mixing the test solution of factor VIII, blood plasma with deleted factor VIII, contact activator of coagulation of ellagic acid and calcium chloride solution. The clotting time of plasma mixed with several dilutions of the test solution, and the measured clot formation times are compared with the clot formation times obtained for several dilutions of the activity standard of factor VIII - normal blood plasma or a solution of isolated factor VIII. The concentration of human factor VIII is determined using an enzyme-linked immunosorbent assay in comparison with the standard - blood plasma or a solution of purified factor VIII with a known content. The specific activity of factor VIII is defined as the ratio of procoagulant activity and the concentration of factor VIII and is expressed in IU / mg. It is believed that the protein variant has the properties of recombinant human blood coagulation factor VIII, provided that the specific activity of this variant is not less than 1% of the specific activity of natural human factor VIII, i.e. at least 50 IU / mg.

The expression plasmid according to the present invention contains a DNA fragment encoding coagulation factor VIII with a deletion of the B domain 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 implementation of the present invention is a plasmid p1.1-F8BDD with a length of 16282 p. (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 (bla) and the prokaryotic promoter of the bla gene (965-1924), allowing for preparative production of the plasmid in E. coli;

2. plot of the terminal repeat of the Epstein-Barr virus of a person (EBVTR), which is a fragment of the concatemer of terminal repeats of the Epstein-Barr virus (1960-2361), which provides 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 VIII 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 a human coagulation factor VIII with a deletion of the B domain (6460-10830);

6. the sequence of the internal binding site of the ribosome of the encephalomyocarditis virus EMCV (10855-11442), providing a cap-independent translation initiation of the second cistron of bicistronic RNA in animal cells;

7. the sequence encoding the open reading frame of dihydrofolate reductase (11456-12018) - a factor of resistance of transfected cells to methotrexate;

8. a fragment of a sequence flanking the Chinese hamster “1 downstream” elongation factor 1 alpha gene (12020-16278), including a terminator and a polyadenylation signal, as well as non-transcribed regions of this gene that provide constitutive expression of the human factor VIII gene in the CHO cell genome.

Plasmid p1.1-F8-BDD contains unique recognition sites for the restriction endonucleases AscI (16), PvuI (1409), FseI (5213), AbsI (6446), PsiI (7737), NheI (10835).

The structure of plasmid p1.1-F8-BDD is shown in Figure 1.

Plasmid p1.1-F8-BDD 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 elongation factor 1 gene of the Chinese hamster, which provides constitutive expression of the target gene in CHO cells, flanked 5 'and 5' NTO of this gene, providing euchromatization of the integration sites of the expression cassette into 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 the absence of thymidine in the medium 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-F8-BDD, 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 a population of transformed cells, clonal producer lines of recombinant human coagulation factor VIII can be obtained. Methodology and techniques for cell selection and cloning are known to those skilled in the art. Although the expression level of human coagulation factor VIII 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.

"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 is a producer of human coagulation factor VIII with a deleted B-domain” means a clonal mammalian cell line having the ability to produce and secretion of human coagulation factor VIII with a deleted B-domain when it is grown according to the present invention in said nutrient medium. Used here, the term "cell line is the producer of coagulation factor VIII person with a deleted B-domain" also means a cell line that is able to secrete the coagulation factor of human VIII with a deleted B-domain in an amount of not less than 5 IU / ml at a cell concentration of 1 -2 mln / ml, more preferably not less than 20 IU / ml, at a cell concentration of 1-2 mln / ml, when cultured in suspension culture in serum free medium. The indicated coagulation factor of human VIII with a deleted B-domain 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 VIII with a deleted B domain according to the present invention is the CHO line DG44, but the spectrum 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 ° C, CO ₂ concentration - 8%, mixing speed - 120-130 rpm.

- Cultural properties: the cultivation method is suspension, the sowing dose is 300,000 per ml, the sieving ratio is 1: 2-1: 6, the reseeding procedure is 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 × 106 cells / ml.

Transformation of CHO DG44 cell line by plasmid p1.1-F8BDD, followed by selection and cloning, leads to the production of cell lines producing recombinant human factor VIII with a deleted B domain.

The producer line DG-P1.1-F8BDD-S3 provides the synthesis and secretion of recombinant human factor VIII with a deleted B domain in the amount of 40 ± 9 ME of the final activity of the B domain of the deleted blood coagulation factor VIII per 1 ml of medium at a cell concentration of 1, 7 million / ml when cultured in suspension culture in serum-free medium. The producer line DG-P1.1-F8BDD-S3 was deposited in the Russian Collection of Vertebrate Cell Cultures of the Institute of Cytology RAS, registration number RRCK (P) 756D.

Another objective of the present invention is the provision of a method for producing recombinant human factor VIII with a deleted B-domain in mammalian cells, comprising culturing in a nutrient medium the mammalian cells described above, producers of recombinant human factor VIII with a deleted B-domain, and isolating the obtained target recombinant protein from the culture liquids.

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 VIII with a deleted B-domain, it is preferable to use a serum-free culture medium.

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 the following figures.

Short Description of Figures.

Figure 1 shows a map of the expression plasmid p1.1-F8BDD. The following notation is used: pUC origin - region of the beginning of replication of the plasmid pUC; bla - open reading frame of beta-lactamase, which provides resistance to ampicillin; bla promoter - prokaryotic promoter of the bla gene; EBV TR - plot of the terminal repeat of the Epstein-Barr virus person; CHOEEF1AUFR functional promoter of the elongation factor gene 1 of the Chinese hamster alpha, 5 'untranslated region of this gene and non-transcribed region flanking this gene; transcription start - transcription start point, TATA - TATA box, CHO EEF1A intron 1 - 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; CHOEEF1ADFR - functional terminator and polyadenylation signal of the elongation factor gene 1 of the Chinese hamster alpha, 3 'untranslated region of this gene and untranscribed region flanking this gene; Kozak is the region encoding the Kozak sequence for cap-dependent translation initiation; F8-BDD ORF is the OPC of factor VIII with a deleted B domain. 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 vector p1.1. Designations are used similarly to figure 1.

The Figure 3 shows the scheme for obtaining plasmids p1.1-F8BDD. Designations: the dashed line indicates the PCR stage, the solid line indicates the restriction-ligation stage, ring plasmids are indicated by circles, linear DNA fragments by rectangles; the names used for cloning restriction endonucleases are indicated in italics, the names of the oligonucleotide primers used in PCR are indicated in direct font.

Figure 4 shows the amino acid sequence of a polypeptide secreted by DG-P1.1-F8BDD-S3 cells (expression product of a plasmid with p1.1-F8BDD without a secretion leader peptide).

The Figure 5 shows the results of immunoblotting of secreted and intracellular fVIII BDD for line 3-B8. The following notation is used: “heavy chain” and “light chain” - hybridization with monoclonal antibodies to the heavy and light chains of fVIII, respectively; "CMV" and "CHEF1" are samples corresponding to cells of the DG-BDDFVIII-18 line (CMV promoter described in RF patent RU 2429294) and 3-B8 (CHEF1 promoter), respectively. The paths for each antibody correspond to one membrane, separation in 7.5% SDS-PAGE under reducing conditions, molecular weights are indicated in kDa.

The Figure 6 shows the electrophoregram of the protein fractions of the washing columns and eluates during purification fVIII. Restorative conditions, Coomassie blue. The studied fractions are concentrated and desalted by protein reprecipitation with trichloroacetic acid. The following designations are used: light chain bands — LC, heavy chain band — HC, single chain precursor band — HC + LC. MMS wash - fractions of the washing column Capto MMS, SP el - fraction of elution of the column Sepharose SP, VIII ff - fraction of the slip for the column VIII Select, VIII el - fraction of elution from the column VIII Select, M - molecular weight marker. Molecular weights are indicated in kDa.

The practical applicability of the present invention is illustrated by the following examples.

Example 1. Obtaining a genetic construct p1.1-F8BDD encoding a protein of human blood coagulation factor VIII with a deletion of the B domain, for transfection of the cell line CHO-DG-44.

The expression construct was prepared according to the general scheme shown in FIG. 3. To obtain the ORS of factor VIII with a B-domain delegated by PCR using specific primers O1KpnIfor and O1HindIIIrev, 02HmdIIIfor and 02B1pIrev (SEQ ID NO: 4-7, respectively), two PCR products F1 and F2 flanking the deletion region were obtained. A commercially available clone pCMV6-XL4 / NM_000132 was used as a matrix. Oligonucleotides were synthesized by ZAO Evrogen, RF. PCR was performed using a Tersus polymerase mix (ZAO Evrogen, RF) using a RTS-100 Thermal Cycler (MJ Reseach, United States).

PCR products were purified using a reagent kit ″ Wizard SV Gel and PCR Clean-Up System ″ (″ Promega ″, USA) according to the manufacturer's protocol, and cloned into the pAL-TA (Eurogen) vector by T4 DNA ligase (Fermentas, Lithuania) according to the method enzyme producer. The obtained ligase mixture was used to transform E. coli cells of strain DH5α with the genotype F-φ80lacZΔM15 Δ (lacZYA-argF) U169 recAl endAl hsdR17 (rk-, mk +) phoA supE44 λ-thi-1 gyrA96 relAl. For this, 5 μl of the ligase mixture was added to 200 μl of a frozen suspension of E. coli cells, incubated on ice for 30 minutes to sorb plasmid DNA, heated to 42 ° C for 60 seconds, and incubated on ice for 5 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 MgCl2) 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-agar (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 at 37 ° C for 18 hours. Individual transformant clones were inoculated in 5 ml of LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, grown 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 from T7 and SP6 primers were completely sequenced 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).

Fragment F3 fVIII was obtained without PCR, with preparative restriction of plasmid pCMV6-XL4 / NM_000132, NotI and KpnI endonucleases. Restriction products were mixed 6: 1 with 6x Loading dye application buffer (Fermentas, Lithuania) and separated in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, the UV-visualized strip was excised from an agarose gel, placed in a 1.5 ml tube, and then DNA was isolated using a set of Wizard SV Gel and PCR Clean-Up System reagents (Promega, USA) according to the protocol of the manufacturer.

The F1-F3 fragments were assembled in the pAL-TA vector to form the plasmid pALTA / F123. Clones of E. coli were selected by PCR from the clones using the Odelf primer and standard primers for the T7prom vector sequence (SEQ ID NO: 9) and SP6 (SEQ ID NO: 10). PCR from E. coli colonies was performed using the ScreenMix-HS reagent kit (CJSC Evrogen, Russia), according to the manufacturer's method, by placing bacterial clone samples in tubes with 10 μl of the PCR ready mix. PCR products were analyzed by electrophoresis on a 1% agarose gel, positive clones were grown in 5 ml of LB medium with the addition of ampicillin at a concentration of 50 μg / ml for 18 hours at 37 ° C and plasmid DNA was isolated using a set of Wizard Plus SV Minipreps reagents (Promega, USA) according to the manufacturer's protocol. The insertion regions of the obtained plasmids from the T7prom primers (SEQ ID NO: 9) and SP6 (SEQ ID NO: 10) and the specific Odelf primer (SEQ ID NO: 8) were completely sequenced as described above.

The obtained artificial fragment of the B-domain of the deleted factor VIII gene was ligated with the site-restricted BlpI and dephosphorylated plasmid pOptivec / F8.

To create the pOptivec / F8 plasmid, the pOptivec vector was used, obtained by recirculating the linear vector pOptivec-TOPO (Invitrogen, USA) after incubation in deionized water for 30 minutes, followed by ligation of T4 phage DNA ligase (Fermentas, Lithuania) according to the method of the enzyme manufacturer. The plasmid pOptivec was restricted with NotI endonuclease and ligated with the NotI-NotI fragment of the plasmid pCMV6-XL4 / NM_000132 (Origene, USA) to form the plasmid pOptivec / F8. The BlpI-BlpI pOptivec / FS fragment was replaced with the BlpI-BlpI pALTA / F123 fragment to form the plasmid pOptivec / F8BDD. Colony PCR analysis was performed using two pairs of specific primers 8sq4f (SEQ ID NO: 11) and 8sq5r (SEQ ID NO: 12) or CMV for (SEQ ID NO: 13) and 8sq15r (SEQ ID NO: 14). Using primers AD-8-AbsF (SEQ ID NO: 15), and AD-ONheI-R (SEQ ID NO: 16), and pOptivec / F8BDD, a PCR product containing an open reading frame of factor VIII blood coagulation was obtained as a matrix a person with a deleted B domain. The resulting PCR product was cloned into the PAL-TA vector and analyzed by sequencing the insertion region using primers for the T7prom vector sequences (SEQ ID NO: 9) and SP6 (SEQ ID NO: 10), as well as specific primers for the factor VIII gene sequence deleted by B- domain - 8sq1f, 8sq2f, 8sq3f (SEQ ID NO: 17-19, respectively), 8sq4f (SEQ ID NO: 11), 8sq5f, 8sq12f, 8sq13f, 8sq14f, 8sq15f (SEQ ID NO: 20-24, respectively), 8sq5r (SEQ IDr: ID NO: 12), 8sq15r (SEQ ID NO: 14), 8sq2r, 8sq3r, 8sq4r, 8sq12r, 8sq13r, 8sq14r (SEQ ID NO: 25-30, respectively). After the detection of clones with the correct nucleotide sequence of the insertion region, the PAL-F8BDD plasmid was restricted with AbsI and NheI and the fragment containing OPC F8BDD with a Kozak consensus sequence was transferred into vector p1.1 (SEQ ID NO: 2), which was restricted with AbsI and NheI endonucleases, to form an expression plasmid p1.1-F8BDD (SEQ ID NO: 1). The open reading frame of the deletion variant of BDD-FVHI, as well as the region of the main functional elements of the expression construct, were sequenced as described above. For sequencing of the OPC region and functional elements of the expression plasmid, specific primers for the factor VIII gene sequence with a deleted B domain were used: 8sq1f, 8sq2f, 8sq3f (SEQ ID NO: 17-19, respectively), 8sq4f (SEQ ID NO: 11), 8sq5f, 8sq12f , 8sq13f, 8sq14f, 8sq15f (SEQ ID NO: 20-24, respectively), 8sq5r (SEQ ID NO: 12), 8sq15r (SEQ ID NO: 14), 8sq2r, 8sq3r, 8sq4r, 8sq12r, 8sq13r, 8sq14r (SEQ ID NO: 12 : 25-30, respectively), as well as primers for the sequences of the vector SQ-3CH1-R, SQ-5CH6-F, IRESArev (SEQ ID NO: 31-33, respectively). The results were compared with theoretically predicted sequences of the human coagulability factor FVIII gene with a deleted B domain and the corresponding regulatory elements; no nucleotide substitutions were found.

The resulting expression construct of human coagulation factor VIII with a deleted B domain in the p1.1-F8BDD vector has a size of 16282 bp. and contains unique recognition sites for restriction endonucleases AscI (16), PvuI (1409), FseI (5213), AbsI (6446), PsiI (7737), NheI (10835). This design is intended for transfection of eukaryotic cells with the human coagulation factor VIII genome with a deleted B domain, followed by selection and amplification based on the linked DHFR ⁺ trait. It informs the recipient cells of the following phenotypic signs: during the transformation of prokaryotes, resistance to ampicillin, carbenicillin; with transfection of eukaryotes, limited resistance to methotrexate, increasing with selective selection, expression of recombinant human coagulation factor FVIII with a deleted B-domain.

The created genetic construct was used to obtain a producer line.

Example 2. Obtaining primary polyclonal cell lines of CHO DG-44 stably transfected with plasmid p1.1-F8BDD.

The expression plasmid p1.1-F8BDD encoding the recombinant human coagulation factor VIII with a deletion of the B domain according to the SQ variant was constructed according to the method described in Example 1, grown in preparative quantities in E. coli strain TOP 10 (Invitrogen, Ltd. , USA), purified using the EndoFree Plasmid MaxiKit kit, (Qiagen, USA), linearized with Pvul restriction endonuclease (Fermentas, Lithuania) with destruction of the beta-lactamase gene and sterilized by filtration.

Cells of the CHO-DG-44 line (Invitrogen, Ltd., USA) were cultured in protein-free CD DG-44 medium (Gibco, USA) with 8 tM L-glutamine and 0.018% Pluronic F-68 surfactant (BASF Inc., USA). This culture medium contains sufficient amounts of hypoxanthine and thymidine to support the growth of cells defective in the dhfr gene. Cells were grown in sterile disposable Erlenmeyer flasks (30 ml of suspension in 125 ml flasks) on an orbital shaker at 130 rpm in an atmosphere containing 8% carbon dioxide at 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 ct / ml, inoculum concentration of 0.3 million ct / ml. 48 hours and 24 hours before transfection, the cells were additionally passaged to an inoculum concentration of 0.3 million ct / ml.

Transfection of the linearized plasmid p1.1-F8BDD was performed using Unfectin-56 liposome reagent (Unifect Group, Russia) in serum-free CD DG-44 medium (Invitrogen) using 1.5 × 10 ⁷ cells in 30 ml of medium in an Erlenmeyer flask, 18 μg plasmid DNA and 15 μl transfection agent. The mixing of DNA and transfection reagent was carried out according to the method of the manufacturer of the agent.

The cell culture was grown for 48 h without changing the medium, after which an aliquot of the culture fluid was taken to determine the concentration of fVIII (0.048 IU / ml by ELISA) and cells were sown in 96-well culture plates of 50 thousand cells per well in CHO A medium (Invitrogen ) with 8 mM alanyl-glutamine (Invitrogen) and 50 nM methotrexate, 200 μl of medium per well and grown for 14 days at 37 ° C, 5% CO ₂ . After selection of stably transfected cells dividing in the absence of hypoxanthine and thymidine and in the presence of a DHFR inhibitor methotrexate, culture medium was selected in the wells with large fissile cell colonies to determine the concentration of secreted fVIII by enzyme-linked immunosorbent assay. In 96 wells containing fVIII at maximum concentration (from 0.1 to 1 IU / ml), the medium was changed and retested after 4 days. Cells from 48 wells with the highest concentration of fVIII were transplanted into the wells of 24-well plates, grown to cover with a monolayer of at least 50% of the surface in 80% of the wells, and re-tested by ELISA. Cells from 18 wells with the highest concentration of fVIII were transferred to wells of 6-well non-adhesive plates and 5 ml of Opti CHO medium (Invitrogen) with 8 mM alanyl-glutamine (Invitrogen) and 50 nM methotrexate were added to the wells. Since the Opti CHO medium contains the Pluronic F-68 surfactant, the cells lose their ability to flatten at the bottom of the wells and form an unattached layer. Cultures were grown to reach a cell concentration of about 1 million ct / ml, the concentration of secreted fVIII was measured, and the three most productive oligoclonal lines were transplanted into Erlenmeyer flasks and further grown with constant stirring. For further work, the 3-B8 oligoclonal line was chosen (Table 1), which secreted predominantly correctly processed variants of the FVIII chains (Figure 5).

Table 1 The concentration of secreted fVIII for primary oligoclonal producer lines. Measurement by ELISA with monoclonal antibodies against the heavy chain fVIII and polyclonal antibodies against the complete molecule fVIII Sample Name The concentration of fVIII, IU / ml Standard deviation, ME / ml 2-f6 2.1 0.35 3-B8 5,4 0.37 4-H6 2.2 0.09

Example 3. Amplification of the target gene under the action of methotrexate and obtaining clonal lines of producer fVIII.

The target gene was amplified in the cell genome of the selected 3-B8 line in suspension culture, adding methotrexate in increasing concentrations to the culture medium until the culture viability decreased. When a drop in viability was detected, the culture was cultured at a constant concentration of methotrexate until viability> 85% was restored, after which the concentration of methotrexate was doubled. The amplification process was considered complete when cell viability ceased to recover after 20 days of cultivation. The amplification progress and culture productivity indicators are shown in Table 2.

table 2 The dynamics of secretion fVIII during amplification of the target gene Name of cell line / population Methotrexate concentration at time of measurement The level of secretion fVIII, IU / ml 3-B8 50 nM 5.4 ± 0.4 * 3-B8 1K 1 μM 20.1 * 3-B8 1K 4K 4 μm 18.6 ** 13B4F 50 nM → 500 nM → 1 μM → 4 μM → cloning 39.0 ± 5.5 *** 11A4H 50 nM → 500 nM → 1 μM → 4 μM → cloning 39.4 ± 3.4 *** 21A7B 50 nM → 500 nM → 1 μM → 4 μM → cloning 34.4 ± 0.8 *** Notes: * - measurement of the concentration of fVIII by ELISA, standard - full-size recombinant fVIII. ** - measurement of fVIII activity by a single-stage coagulometric method.

The obtained polyclonal population of 3-B8 1K 4K cells was used to obtain clonal producer lines by the limiting dilution method. Cells were cloned under adhesion cultivation in CD CHO-A medium with 8 mM alanyl-glutamine ("Invitrogen"), 200 μl of medium in each well of the plate at 37 ° C, 5% CO ₂ for 21 days. Methotrexate was not included in the medium during cloning and was not used for further cultivation. The growth of single colonies was observed and documented on the 14th day of cultivation. In 10 tablets, 361 single colonies were obtained. The grown colonies were transferred to 24-well plates and, using ELISA, the conditioned medium was screened from wells containing actively growing colonies. The clonal lines with the best levels of secretion were continued to grow in an adhesion culture and then adapted to the suspension culture conditions for three consecutive passages in the wells of 6-well plates using Pro CHO 4 medium (Lonza, USA) with 8 mM L-glutamine. Screening of the conditioned medium from the wells of 6-well plates was performed using ELISA and coagulometric measurement of activity vVIII. Three selected lines with high productivity and a dividing time of less than 40 hours were grown under suspension cultivation in stirred Erlenmeyer flasks. The main parameters of the three selected lines are shown in table 3.

Table 3 The main properties of clonal producer lines fVIII Line name The final level of secretion fVIII, IU / ml * Specific productivity, ME / million ct / day * Division time, h 13B4F 39.0 ± 5.5 6.45 26 DG-P1.1-F8 BDD-S3 (11A4H) 39.4 ± 3.4 7.74 29th 21A7B 34.4 ± 0.8 6.21 43 Notes: * - measurement of fVIII activity by a chromogenic test.

Clone 11A4H was selected as the main producer line, which had a maximum specific productivity and cell division time of less than 30 hours. The producer line was designated as DG-P1.1-F8 BDD-S3, saved in the form of a research cell bank in cryo-ampoules, deposited to the Specialized Collection of Vertebrate Cell Cultures of the Russian Collection of Cell Cultures 05.02.2013, under the number RKKK (P) 756D. The DG-P1.1-F8 BDD-S3 line was tested for consistent secretion during continuous cultivation. It was found that the final activity of fVIII in the conditioned medium, determined at the moments of passage of the culture, does not change significantly during 60 days of sequential cultivation (data not shown). Thus, the resulting cell line is potentially suitable for industrial use, i.e. maintains constant productivity for a period of time sufficient for the preparation of seed and 10 cycles of periodic cultivation in a large volume bioreactor.

Example 4. The selection of the target protein from the supernatant of the culture line DG-P1.1-F8BDD.

Preparative cultivation of the DG-P1.1-F8BDD line was carried out in stirred Erlenmeyer flasks using serum-free Pro CHO 4 medium supplemented with 8 mM L-glutamine. The culture was inoculated at a density of 0.3 million ct / ml and 7/8 of the volume of the culture suspension was replaced every 96 hours with fresh culture medium.

The resulting portions of the cell suspension were immediately clarified by centrifugation and applied to a Capto MMS sorbent column (GE Healthcare, USA). The column was washed with a high salt solution and eluted with fVIII solution containing 50% ethylene glycol and 0.8 M arginine hydrochloride. The eluate was diluted 12-fold with water and applied to an SP Sepharose FF column (GE Healthcare), the column was washed with a solution containing 0.15 M NaCl, and eluted with vVIII solution containing 0.4 M NaCl. Mannitol was added to the solution of the semi-purified target protein to a final concentration of 0.2 M, and the resulting stabilized solution was frozen and stored for subsequent purification.

The combined portions of the frozen solution of semi-purified fVIII were subjected to final purification using the VIII Select affinity sorbent (GE Healthcare) according to the instructions of the sorbent manufacturer. The target protein solution eluted from the affinity column was diluted 12 times with water, applied to a Capto Q sorbent column (GE Healthcare), the proteolytic fragments vIII were removed by washing with a solution containing 0.3 M NaCl, after which a gel filtration column was attached to the column outlet Superdex 200 sorbent (GE Healthcare), previously balanced with an isotonic solution containing all the auxiliary substances for the preparation of dosage form fVIII (sodium chloride, sucrose, calcium chloride, histidine hydrochloride, polysorbate-80). PhVIII was eluted from the Capto Q column using a solution containing 0.4 M NaCl, and the eluting solution was continued to be fed to the tandem connected columns until the fraction of the FVIII monomer fraction exited from the gel filtration column. The resulting solution of purified monomeric fVIII was aliquoted and frozen. In eluate samples from all stages of purification, the activity of fVIII, the total protein content by the Bradford method, the protein content of the producer strain and the DNA of the producer strain were determined. The main parameters of purification of fVIII are shown in Table 4, the results of gel electrophoresis of eluate fractions and column washing are shown in Figure 6.

Table 4 The fractions of the eluate fVIII by stages of chromatographic purification Type of chromatography Sorbent Volume ml Protein, mg / ml Activity, IU / ml * The specific activity, IU / mg Exit stage Common output Culture medium - 320 - 36.3 - - - Multimodal Anion Exchange Capto MMS 17 0.233 496.9 2133 73% 73% Cation exchange SP Sepharose 10 0.271 538.1 1986 64% 46% Affinity VIII Select eleven 0,046 382.0 8304 78% 36% Anion exchange + gel filtration Capto Q + Superdex 200 10 0.026 ** 284.1 10925 68% 24% Note: * - the activity of fVIII was determined by the one-stage coagulometric method, 1 IU when this method determined the equivalent of 1.4 IU, determined by the chromogenic method for deletion variants of fVIII. ** - the concentration of total protein is determined by the optical density of the solution at 280 nm.

The obtained prototype of the formulated solution of the active pharmaceutical substance fVIII had a specific activity of about 11 thousand IU / mg of total protein when measured by the one-step method, which corresponds to the pharmacopoeial requirements for drugs of the deletion variant fVIII.

The techniques described in these examples are applicable to create producer cell lines of a recombinant coagulation factor VIII with a deletion of the B domain based on any eukaryotic cell lines with a genetic knockout of the dhfr gene, either attached or suspension, as well as transfected with both the described method and and using other liposome agents, as well as calcium phosphate transfection, nucleofection, or microinjection methods.

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.

Claims (10)

1. Plasmid for expression of recombinant human coagulation factor VIII with a deleted B domain in Chinese hamster ovary (CHO) cells knocked out by the dihydrofolate reductase gene, in the following sequence essentially consisting of the pUC plasmid replication origin, an 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; Kozak Consensus Sequence; open reading frame of a gene encoding a coagulation factor of human blood VIII with a deleted B-domain; encephalomyocarditis virus (EMCV) internal ribosome binding site (IRES); an open reading frame of dihydrofolate reductase (DHFR) expressed as part of a bicistronic mRNA together with a gene encoding human coagulation factor VIII with a deleted B domain (IRES DHFR); 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 coagulation factor VIII gene encodes a polypeptide of human coagulation factor VIII with a deleted B domain, the amino acid sequence of which is shown in SEQ ID NO: 3. 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 the plasmid is the vector p1.1-F8BDD, presented in the List of sequences under the number SEQ ID NO: 1. 6. Chinese hamster ovary cell (CHO) - producer of recombinant human coagulation factor VIII with a deleted B-domain, transformed with the plasmid according to paragraphs. 1-5. 7. The cell according to claim 6, characterized in that the cell is an ovary cell of a Chinese hamster of the monoclonal line DG-P1.1-F8BDD-S3 deposited in the Russian Culture Collection of Vertebrate Cells of the Institute of Cytology RAS under registration number RRCK (P) 756D. 8. A method of obtaining a recombinant coagulation factor VIII with a deleted B-domain, comprising the following stages:
- cultivation in a nutrient medium of cells according to claim 6; and
- isolation of the resulting recombinant protein from the culture fluid. 9. The method according to p. 8, characterized in that the ovary cells of the Chinese hamster of the Chinese hamster of the monoclonal line DG-P1.1-F8BDD-S3, deposited in the Russian Culture Collection of Vertebrate Cells of the Institute of Cytology RAS, registration number No.RKKK (P) 756D. 10. The method according to p. 8, wherein the culture medium is serum-free.

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