Improved titre of polypeptide of factor viii in cell cultures

FIELD: biotechnologies.

SUBSTANCE: method to produce polypeptide of factor VIII provides for cultivation of cells of mammals, which express polypeptide of factor VIII, under conditions, suitable for expression of the specified popypeptide of factor VIII, which include a cell cultural medium with O-phospho-L-serine (OPLS) and extraction of the expressed polypeptide of factor VIII from cells of mammals with the help of suitable means.

EFFECT: invention makes it possible to produce popypeptide of factor VIII with increased specific activity.

11 cl, 3 dwg, 4 tbl, 2 ex

 

The SCOPE of the INVENTION

This invention relates to a method for producing factor VIII polypeptide, including the application of ligand C2-domain, in particular O-phospho-L-serine (OPLS).

The LEVEL of TECHNOLOGY

Classic hemophilia or hemophilia a is a hereditary blood disease. It is the result of a deficiency of coagulation factor VIII associated with the X chromosome and affects almost exclusively males with a frequency of one to two cases per 10,000 people. The defect of the X chromosome is transmitted by female carriers who are not themselves hemophilia. Clinical manifestation of hemophilia a is an increased tendency to bleeding. Before treatment concentrates of factor VIII life expectancy for persons with severe hemophilia were less than 20 years. The use of concentrates of factor VIII from plasma has significantly improved the situation for patients with hemophilia, extensive increasing the average duration of their life, giving most of them the opportunity to live more or less normal life. However, there are some problems with that obtained from plasma concentrates and their use, the most serious of which is the transmission of viruses. Still the viruses that cause AIDS, hepatitis b and hepatitis no, no, seriously affect the population. Therefore, in recent years have been atrabotana different methods of inactivation of viruses and new highly purified concentrates of factor VIII, that set a very high security standard for factor VIII derived from plasma.

It is known that the factor VIII (FVIII) is expressed in mammalian cells at a very low level. It is also known that factor VIII is an unstable protein in the medium without serum or without protein. To improve the stability and titer factor VIII used the addition of various substances.

WO 9743436 discloses the addition of inhibitors of meteozavisimykh inhibitors and/or chymotripsin.

WO 88/08035 and WO 87/04187 disclose the addition of phospholipids to the culture medium of factor VIII. Also described coexpressed von Willebrand factor (vWF).

US 20050227913 A1 discloses OPLS as an inhibitor of aggregation of factor VIII by binding C2 domain (2303-2332). It is argued that less aggregated factor VIII is less immunogenic.

K.Hansen, M.Kjalke, ..Rasmussen, L.Kongerslev and M.Ezban, Cytotechnol. 24 (3), 227-234, 1997 discloses the use of bacitracin a and phosphatidylserine to prevent degradation of factor VIII in the environment.

WO 90/02175 A1 discloses processes for producing the recombinant polypeptide(s) by culturing eukaryotic cells in the presence of protease inhibitors to prevent degradation of the polypeptide(s).

EP 1707634 A1 discloses that a significant amount of factor VIII are associated with the cell surface and can be removed put the m washing buffers with high ionic strength.

At the same time, there is still a need to improve methods of production in order to increase the overall yield of factor VIII polypeptides and/or reduce manufacturing costs.

The INVENTION

The first aspect of the invention relates to a method for producing factor VIII polypeptide, comprising the steps of a) culturing mammalian cells expressing the factor VIII polypeptide, under conditions suitable for expression of the indicated factor VIII polypeptide, including cell culture medium containing ligand C2-domain, and (b) highlight the downregulation of factor VIII polypeptide from mammalian cells using the appropriate tools.

The second aspect of the invention relates to a method for producing factor VIII polypeptide, comprising the steps of a) culturing mammalian cells expressing the factor VIII polypeptide, under conditions suitable for expression of the indicated factor VIII polypeptide, including cell culture medium, and (b) highlight the downregulation of factor VIII polypeptide from mammalian cells using appropriate means, comprising adding to said cells ligand C2-domain.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

Figure 1. The sequence of a gene (cDNA) factor VIII (SEQ ID No. 1).

Figa-Century Influence of Fosfo-L-serine on the performance of FVIII and the specific activity of the FVIII protein.

Figa-C. the Influence of O-phospho-L-serine and/or hydrolyzed vegetable protein for factor VIII in the medium of cells that produce factor VIII. The peculiarity of the conditions A-D are shown in table 3.

DETAILED description of the INVENTION

As mentioned above, the first aspect of the invention relates to a method for producing factor VIII polypeptide, comprising the steps of a) culturing mammalian cells expressing the factor VIII polypeptide, under conditions suitable for expression of the indicated factor VIII polypeptide, including cell culture medium containing ligand C2-domain, and (b) highlight the downregulation of factor VIII polypeptide from mammalian cells using the appropriate tools.

The second aspect of the invention relates to a method for producing factor VIII polypeptide, comprising the steps of a) culturing mammalian cells expressing the factor VIII polypeptide, under conditions suitable for expression of the indicated factor VIII polypeptide, including cell culture medium, and (b) highlight the downregulation of factor VIII polypeptide from mammalian cells using appropriate means, comprising adding to said cells ligand C2-domain.

In both cases, the ligand C2-domain plays an important role in promoting increased levels of polypeptide titer factor VIII in cleto is Noah culture medium.

Without tied to any specific theory it is believed that the increased levels of titer factor VIII polypeptide in the cell culture medium conditioned by ligand C2-domain (in particular O-phospho-L-serine (OPLS)), which either alone or in combination with soybean trypsin inhibitor (SBTI) and/or hydrolyzed vegetable protein, or (i) increases the number of factor VIII polypeptide secreted by the cells, and/or (ii) competes with the associated with the cell a polypeptide factor VIII outside the cells, and/or (iii) reduces the degradation of the polypeptide factor VIII and thereby increases the number of functional factor VIII polypeptide present in the supernatant.

Below the invention will be described in more detail.

Ligand C2-domain is a ligand capable of binding to or associated with the C2-domain (see below) of the factor VIII polypeptide. Preferably the ligand C2-domain must be capable of displacement (competitive off) of the factor VIII polypeptide from the cell membrane.

Currently, in the preferred embodiment the ligand C2-domain is an O-phospho-L-serine (OPLS), i.e. the molecule of the formula (HO)2P(O)OCH2CH(NH2)CO2H.

Suitable alternative ligands C2 domain are those which have the formula (XO)(HO)P(O)OCH2CH(NH2)CO2H, where X is chosen among in Sogno substituted C 1-6-alkyl, possibly substituted C2-6-alkenyl, possibly substituted phenyl, possibly substituted heteroaryl, possibly substituted heterocyclyl and possibly substituted benzyl. In one of his incarnations X choose among possible substituted C1-6-alkyl, possibly substituted benzyl and possibly replaced With2-6-alkenyl.

In this context, the term "C1-6-alkyl" denotes a linear, cyclic or branched hydrocarbon group having from 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, pentyl, cyclopentyl, hexyl, cyclohexyl.

Similarly, the term "C2-6alkenyl" is used to mean a linear, cyclic or branched hydrocarbon group having from 2 to 6 carbon atoms and containing at least one unsaturated bond. Examples alkenyl groups are vinyl, allyl, butenyl, pentenyl and hexenyl. Preferred examples of alkenyl are vinyl, allyl, butenyl, especially allyl.

In this context, i.e. in connection with the terms "alkyl" and "alkenyl", the term "possibly substituted" is used to denote that the group in this case can be substituted one or several times, preferably 1-3 times, with group(s)selected from among hydroxy (which when linking with unsaturated atom at which laroda may be present in the tautomeric keto form), C1-6-alkoxy (i.e. C1-6-alkyl-oxy)2-6-alkenylamine, carboxy, oxo (forming a ketone or aldehyde functionality), C1-6-alkylsulphonyl, formyl, aryl, aryloxy, arylamino, arylcarbamoyl, heteroaryl, heteroaromatic, heteroallyl, heteroarylboronic, heterocyclyl, heterocyclic, heterocyclisation, heterocalixarenes, amino, mono - and di-(C1-6-alkyl)amino; carbarnoyl, mono - and di-(C1-6-alkyl)aminocarbonyl, amino-C1-6-alkyl-aminocarbonyl, mono - and di-(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkylcarboxylic, guanidine, urea, C1-6-alkyl-sulfonyl-amino, C1-6-alkyl-sulfonyl, C1-6-alkyl-sulfinil,1-6-alkylthio, halogen, where any aryl, heteroaryl and heterocyclyl may be substituted as specifically described below for aryl, heteroaryl and heterocyclyl.

The term "halogen" includes fluorine, chlorine, bromine and iodine.

In this context, the term "aryl" is used to describe fully or partially aromatic carbocyclic ring or ring system such as phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, antracol, Finntroll, pyrenyl, benzopyranyl, fluorenyl and xantinol, among which phenyl is a preferred example.

The term "heteroaryl" is used to describe fully or partially Carbo is ilichevskogo aromatic ring or ring system, where one or more carbon atom is replaced by a heteroatom such as nitrogen atom (=N - or-NH-), sulfur and/or oxygen. Examples of such heteroaryl groups are benzimidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolin, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, furyl, thienyl, hinely, triazolyl, tetrazolyl, ethanolic, indolyl, in particular benzimidazolyl, pyrrolyl, imidazolyl, pyridinyl, pyrimidinyl, furyl, thienyl, hinely, tetrazolyl and ethanolic.

The term "heterocyclyl" is used to refer to non-aromatic carbocyclic ring or ring system, where one or more carbon atom is replaced by a heteroatom such as nitrogen atom (=N - or-NH-), sulfur and/or oxygen. Examples of such heterocyclic groups (named in accordance with rings) are tetrahydrofuran, imidazolidine, piperazine, hexahydropyridine, hexahydropirimidine series, diazepan, pyrrolidine, piperidine, ASEAN, oxazine (morpholine) and diazinon.

In this context, i.e. in connection with the terms "aryl", "benzyl", "heteroaryl", "heterocyclyl" and the like (for example, "aryloxy", "heteroarylboronic" and others), the term "possibly substituted" is used to denote that the group in this case can be substituted one or several times, preferably 1-5 times, in particular 1-3 times, with group(s)you what early among the groups hydroxy, C1-6-alkyl, C1-6-alkoxy, oxo (which may be represented in the tautomeric enol form), carboxy, C1-6-alkylsulphonyl, formyl, amino, mono - and di-(C1-6-alkyl)amino; carbarnoyl, mono - and di-(C1-6-alkyl)aminocarbonyl, amino-C1-6-alkyl-aminocarbonyl,1-6-alkylcarboxylic, guanidine, urea, C1-6-alkylsulfonamides, arylsulfonamides, heteroarylboronic, C1-6-alkylsulfonyl, C1-6-alkylsulfonyl, C1-6-alkylsulfonate, sulfanyl, amino, aminosulfonyl, mono - and di-(C1-6-alkyl)aminosulfonyl or halogen, where any alkyl, alkoxy, etc. presents the substituents can be substituted by hydroxy, C1-6-alkoxy, C2-6-alkenylamine, amino, mono - and di-(C1-6- alkyl)amino, carboxy, C1-6-alkylcarboxylic, halogen, C1-6-alkylthio, C1-6-alkylsulfonyl or guanidino.

In the most interesting incarnations (relating to both the first and second aspect of the invention, the ligand C2-domain (for example OPLS) is present in the cell culture medium at a concentration of 0.1 to 100 mm, such as 5 to 30 mm, in particular 10-20 mm.

Also interesting are the embodiment (relating to both the first and the second aspect of the invention), in which the ligand C2 domain was added to the cells in step b) at a concentration of 1-200 mm, such as 50-150 mm, the company and 70-130 mm.

Detailed information about the stages of production will be disclosed in detail below.

As mentioned earlier, it was found that soybean trypsin inhibitor (SBTI) can advantageously be combined with the ligand C2-domain in cell culture medium in step a). Thus, in the present preferred embodiment of the cell culture medium further comprises soybean trypsin inhibitor.

Soybean trypsin inhibitor isolated from Glycine max. Soybean trypsin inhibitor from soybeans is a Monomeric protein containing 181 amino acid residue in the single-chain polypeptide chains linked by two disulfide bridges. Molecular weight, a specific amino acid sequence, is of 20.1 kDa. Soybean trypsin inhibitor inhibits its target protease by forming a stoichiometric complex of 1:1.

In the most typical embodiments, the concentration of soybean trypsin inhibitor in the cell culture medium is 0.01-100 mg/ml, such as 0.1-10 mg/ml, in particular from 0.3 to 3 mg/ml

In addition, it was found that hydrolyzed vegetable protein (sometimes referred to as "the product of the breakdown of plant origin", etc. can advantageously be combined with the ligand C2-domain (and possibly also with soybean trypsin inhibitor) in cell culture medium in step a). In affect, the, currently, equally preferred embodiment of the cell culture medium further comprises hydrolyzed vegetable protein.

Hydrolyzed vegetable protein can be obtained from one of various sources, for example from commercial sources. Typical types of hydrolysates are hydrolyzed soy protein, hydrolyzed wheat protein, hydrolyzed pea protein, hydrolyzed rice protein, etc. WO 01/23527 A1, which is hereby incorporated by reference, discloses receiving and total applied hydrolyzed soy protein.

In the most typical embodiments, the concentration of hydrolyzed vegetable protein in the cell culture medium is 0.1-100 mg/ml, such as 1-10 mg/ml, in particular 2-7 mg/ml

The polypeptide factor VIII

The invention is adapted for producing factor VIII polypeptide in a mammalian cell.

Mature factor VIII molecule consists of 2332 amino acids, which can be grouped into three homologous to the a-domain, two homologous to the C-domain and In-domain, which are located in the following order: A1-A2-B-A3-C1-C2. During its secretion into the plasma factor VIII is converted intracellularly in a number of heterodimers associated with metal ions, resulting single-chain factor VIII is cleaved by B-A3 border and in different parts included in In-domain. The result is that the second processing is formed by a heavy chain consisting of A1, A2 and different parts of the In-domain having a molecular size from 90 kDa to 200 kDa. Heavy chains are connected via a metal ion with a light chain, which consists of A3-, C1 - and C2-domains (Georges tarbouriech et al., 2002). In plasma, this heterodimeric factor VIII associated with high-affinity to him by von Willebrand factor, which protects it from premature catabolism. The half-life in plasma of non-activated factor VIII associated with von Willebrand factor is about 12 hours.

In the process of blood coagulation factor VIII is activated by FXa and thrombin by proteolytic cleavage of the amino acid Arg372 and Arg740 in the heavy chain and Arg1689 in the light chain, resulting in releasing the von Willebrand factor and is formed activated heterodimer factor VIII, which will form tenany complex with FIXa and FX on the phospholipid surface in the presence of CA2+. Heterodimer consists of domain A1, 50 kDa fragment, domain, A2, 43 kDa fragment and a light chain (A3-C1-C2), 73 kDa fragment. Thus, the active form of factor VIII (factor Villa consists of A1-subunit, associated through a divalent linkage metal ion with derived thrombin A3-C1-C2 light chain, and a free-A2-subunit, is relatively weakly associated with domains A1 and A3.

The factor VIII molecule comprised of a heavy chain (HC) and light chain (LC) facto is and VIII, associated with a small linker derived from In-domain (factor VIII with a remote In-domain, or BDD-FVIII) retains the biological activity of factor VIII full-length native).

Practicing the method of this invention, any polypeptide factor VIII, which is therapeutically useful, for example effective in the prevention or treatment of bleeding, can be made relevant. It includes the human factor VIII wild-type, hybrid human/porcine factor VIII and factor VIII with a remote In-domain, but not limited to.

Used in this document, the term "factor VIII polypeptide" encompasses factor VIII, as well as polypeptides associated with factor VIII, but not limited to.

The term "factor VIII" is used to denote polypeptides having the amino acid sequence described in'toole et al., Nature 1984, 312: 342-347 (human factor VIII wild-type), and factor VIII wild type obtained from other species, such as, for example, factor VIII from cattle, pig, dog, mouse and salmon, but is not limited to this. It also includes natural allelic variation of factor VIII that may exist and to move from one individual to another. In addition, the extent and location of glycosylation or other posttranslational modifications may vary depending on selected taking the affected cells (host cells) and the nature of the host cell environment. The term "factor VIII" is also used to denote polypeptides of factor VIII in their unsplit (imagenow) form, as well as those that were proteoliticeski processed to obtain their respective bioactive forms, which may be designated factor VIIIa.

The term "polypeptides associated with factor VIII"includes polypeptides of factor VIII that or chemically modified compared to human factor VIII (i.e. derived factor VIII), and/or contain one or more aminoacid sequence compared to human factor VIII (i.e. variants of factor VIII), and/or contain truncated amino acid sequences were compared to human factor VIII (i.e., the fragments of factor VIII), but is not limited to them. Such polypeptides associated with factor VIII, may have different properties compared to human factor VIII, including stability, binding to phospholipids, altered specific activity, etc. the Term "polypeptides associated with factor VIII" is used to refer to such polypeptides in their unsplit (imagenow) form, as well as those that were proteoliticeski processed to obtain their biologically active forms, which can be labeled as "polypeptides associated with factor VIII" or "aktivirovannaya, associated with factor VIII"

Used in this document, the term "polypeptides associated with factor VIII" also includes, without limitation, polypeptides, showing mostly the same or improved biological activity compared to human factor VIII wild type, as well as polypeptides, in which the biological activity of factor VIII was largely modified or reduced relative to the activity of human factor VIII of the wild type. These polypeptides include factor VIII or factor VIIIa, which has been chemically modified, and variants of factor VIII, which have made specific changes in amino acid sequences, which have altered or destroyed the biological activity of the polypeptide, but are not limited to.

In addition, it encompasses polypeptides with a slightly modified amino acid sequence, for example, polypeptides having a modified N-end including N-terminal amino acid deletions or additions, and/or polypeptides that have been chemically modified compared to human factor VIII.

Polypeptides associated with factor VIII, including variants of factor VIII, showing mostly the same or improved biological activity compared to factor VIII wild-type, or Vice versa, demonstrirovalsya part of a modified or reduced biological activity compared to factor VIII wild-type, include polypeptides with amino acid sequence that differs from the sequence of factor VIII wild-type insert, remove or replace one or more amino acids, but are not limited to.

Polypeptides associated with factor VIII, including options encompass those that exhibit a specific activity that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120% and at least about 130% of the specific activity of factor VIII wild-type, produced by cells of the same type, and the analysis of the activity of factor VIII is carried out in accordance with the description in this manual.

Polypeptides associated with factor VIII, including variants, having mostly the same or improved biological activity compared to factor VIII wild type, encompass those that exhibit a specific activity that is at least about 25%, such as at least 50%, at least 75% or at least 90% of the specific biological activity of factor VIII wild type produced by cells that is of the same type the analysis of the activity of factor VIII is carried out in accordance with the description in this manual (the"Materials and methods").

Polypeptides associated with factor VIII, including variants, having largely reduced biological activity compared to factor VIII wild type, are those that exhibit less than about 25%, for example less than about 10%, or less than about 5% of the specific activity of factor VIII wild-type, produced by cells of the same type, and the analysis of the activity of factor VIII was conducted in accordance with the description in this manual (the"Materials and methods").

Examples of factor VIII polypeptides include obtained from plasma of human factor VIII, as described for example in Fulcher et al.; Proc. Acad. Nat. Sci. USA 1982; 79: 1648-1652 and Rotblat et al.; Biochemistry 1985; 24: 4294-4300, and FVIII derived from the plasma of pigs, as described for example in Fass et al.; Blood 1982; 59:594-600 and Knutson et al.; Blood 1982; 59: 615-624, but are not limited to. Examples of sequences of factor VIII is described for example in Lollar et al.; Blood 2000; 95(2): 564-568 (hybrid swine/human FVIII polypeptide) and Lollar et al.; Blood 2001; 97(1): 169-174, but are not limited to.

The cDNA cloning of the factor VIII (Wood, W.I., et al. (1984) Nature 312, 330-336; Vehar, G.A., et al. (1984) Nature 312, 337-342) made possible the expression of factor VIII, which allowed recombinante to get several products of recombinant factor VIII, which is and approved by regulators in the period between 1992 and 2003. The coding sequence of the factor VIII (cDNA) is shown in figure 1. The fact that the Central B-domain polypeptide chain of factor VIII, located between amino acids Arg-740 and Glu-1649, is not necessary for full biological activity, has also led to the development of factor VIII with a remote In-domain. Cm. also Kjalke M, Heding A, Talbo G, Persson E, Thomsen J and Ezban M (1995), "Amino acid residues 721-729 are required for full Factor VIII activity. Eur. J. Biochem: 234: 773-779.

Stage a) Transfection and culturing of cells

Cells

Mammalian cells expressing the polypeptide factor VIII, as a rule, selected from the group consisting of mammalian cells, which expressed endogenous polypeptide factor VIII, and mammalian cells, which was transfirieran gene polypeptide factor VIII.

In one interesting embodiment of the recent mammalian cells were transliterowany expression vector containing a nucleic acid molecule that encodes a polypeptide factor VIII, and controls the expression of functionally associated with her.

Expression of the protein in cells are well known to experts in the field of production of proteins. In the practical application of this invention the cells are mammalian cells, more preferably an established line of mammalian cells, including Cho cell line (for example ATSS CCL 61), COS-1 (for example ATSS CRL 650), kidney hamster (BHK) and HEK293 (for example ATSS CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72, 1977), but is not limited to them. The preferred cell line KSS is a cell line KSS tk-ts13 (Waechter and Baserga, Proc.Natl.Acad.Sci.USA 79:1106-1110, 1982), hereinafter referred to as cells KSS 570. Cell line KSS 570 can be obtained from the American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD 20852, under the registration number of ATSS CRL 10314. Also cell line KSS tk-ts13 can be obtained from ATSS under registration number CRL 1632. Preferred cell lines SNO are the cell line Cho K1, available in ATSS under registration number S, as well as cell lines CHO-DXB11 and CHO-DG44.

Other suitable cell lines include cells of Rat Hep I (rat hepatoma; ATS CRL 1600), Rat Hep II (rat hepatoma; ATS CRL 1548), TCMK ADS CCL 139), light person (ATSS HB 8065), NCTC 1469 (ATSS CCL 9.1); DUKX (cell line Cho (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980) (DUKX cells is also referred to as DXB11 cells) and DG44 (cell line Cho (Cell, 33:405, 1983, and Somatic Cell and Molecular Genetics 12:555, 1986), but are not limited to. Can also be used in cells C, Namalwa cells, myeloma and fusion of myeloma with other cells. In some embodiments the cells may be mutant or recombinant, such as, for example, cells that Express a spectrum of enzymes that catalyze post-translational modification of proteins (e.g. enzymes glycosyl the Finance, such as glycosyltransferases and/or glycosidase, or processing enzymes, such as propeptide), qualitatively or quantitatively different from that of the cell type from which they were obtained. Especially preferred DUKX cells (cell line Cho).

Currently, the preferred cells are cells HEK293, COS, kidney hamster (BHK) and myeloma cells, in particular cells of the Chinese hamster ovary (Cho).

Cultivation of cells

In some embodiments, the cells used in the practice of the invention, able to grow in suspension cultures. Used in this case suitable for suspension cells are cells that can grow in suspension without formation of large dense aggregates, i.e. cells that are monodisperse or grow in the form of loose aggregates with a small number of cells in the unit. Suitable for suspension cells include cells that grow in suspension without adaptation or manipulation (such as, for example, hematopoietic cells or lymphoid cells), and cells that have become suitable for the suspension by the gradual adaptation of these cells, which is necessary attachment (such as, for example, epithelial cells or fibroblasts to growth in suspension, but not limited to.

Cells used in the practice of the invention, m is may be adhesive cells (also known as cells, dependent adhesion or dependent attachment). Used in this case, the adhesive cells are cells, which for reproduction and growth need to join or be anchored to a suitable surface. In one embodiment of the invention used cells are adhesive cells. In these embodiments and phase propagation, and phase production include the use of microneedle. Used adhesive cells must be able to migrate to the media (and in the internal structure of the media, if used macroporous media) during the phase(s) of reproduction and migration to new media after their transfer to production bioreactor. If the adhesive cells do not have sufficient ability to migrate to new media on their own, they can be freed from the media by associating containing cells microsites with proteolytic enzymes or EDTA. The medium used (especially without animal ingredients) should also contain components suitable to support adhesion of cells; a suitable environment for the cultivation of adhesive cells can be obtained from commercial suppliers, such as Sigma.

Cells can also be adapted to suspension or suitable for suspension. When using such cells reproduced the tion of cells can occur in suspension, so microneedle used only in the final phase of reproduction in the culture vessel for producing and stage production. In case adapted to the cell suspension used Mironosetsky are, as a rule, macroporous media in which the cells are fixed by physical capture the internal structure of the media. However, in the case of such adapted to the cell suspension and cell proliferation, and production can occur in suspension.

In such embodiments, the mammalian cells, usually choose among Cho, BHK, HEK293, cells myeloma, etc.

Cell culture environment

In addition to the components mentioned above, namely ligand C2-domain (necessary for the invention related to the first aspect of the invention)may soybean trypsin inhibitor, and there may also be hydrolyzed vegetable protein, cell culture medium contains a number of other components that are known to the specialist, required for cell multiplication and production of factor VIII polypeptide.

The term "cell culture medium" (or simply "environment"refers to a nutrient solution used for growing mammalian cells, which typically contains at least one component from one or more of the following categories: (1) salt, for example the three is, potassium, magnesium and calcium, which contribute to the osmolality of the environment; (2) the source of energy, usually in the form of carbohydrates, such as glucose; (3) all essential amino acids, and usually the basic set of twenty amino acids; (4) vitamins and/or other organic compounds required at low concentrations; and (5) trace elements, which are defined as inorganic compounds, which are typically required at very low concentrations, usually in the micromolar. Nutrient solution may possibly be supplemented by one or more component of any of the following categories: (a) hormones and other growth factors such as insulin, transferrin, and epidermal growth factor; and (b) the hydrolysates of proteins and tissues. Preferably, the cell culture medium did not contain any components of animal origin.

This invention encompasses the cultivation of mammalian cells in medium without animal ingredients. Used in this case, the term "components of animal origin" means any components that are produced in intact animals (such as, for example, proteins isolated and purified from serum), or are manufactured using components produced in intact animals (such as, for example, an amino acid made by the enzyme, isolated and purified the CSOs from the animal for hydrolysis of material of plant origin). In contrast, a protein that has the sequence of animal protein (i.e. genomic origin from the animal), but which is produced in vitro in cell culture (such as, for example, recombinant yeast or bacterial cell or installed continuous line of mammalian cells, recombinant or not), in environments without components produced and isolated and purified from intact animals, is not a component of animal origin (such as, for example, insulin is produced in a yeast or bacterial cell, or the insulin produced in the prescribed lines of mammalian cells, such as, for example, cells SNO, KSS, or SOME, or interferon produced in Namalwa cells). For example, a protein that has the sequence of animal protein (i.e. genomic origin from the animal), but which is produced in a recombinant cell in environments without animal ingredients (such as, for example, insulin is produced in a yeast or bacterial cell) is not a component of animal origin". Accordingly, the cell culture medium without animal ingredients is such an environment that may contain animal proteins, which received recombinante; however, this environment does not contain, for example, animal serum or proteins or the other products, purified from animal serum. Such an environment may, for example, contain one or more component derived from plants. Can be used with any cell culture medium, in particular not containing components of animal origin, which supports the growth and existence of cells in the conditions of the invention. Typically, the environment contains water, an osmolality regulator, a buffer, an energy source, amino acids, inorganic or recombinant source of iron, one or more synthetic or recombinant growth factors, vitamins and cofactors. In one embodiment, the environment does not contain animal ingredients and proteins ("protein-free"). Environment without animal ingredients and/or proteins are available from commercial suppliers such as Sigma, JRH Biosciences, Gibco, Hyclone and Gemini.

In one embodiment the cell culture medium does not contain serum. In another embodiment, the medium contains no components of animal origin. In another embodiment, the environment does not contain proteins ("protein-free"), as well as components of animal origin.

In one embodiment, the medium is commercially available protein-free medium Cho without animal ingredients, such as, for example, EXCELL™ (SAFC Biosciences), PF-CHO, PF-CHO LS, SFM4CHO or CDM4CHO (all from Hyclone), and cell line cells are SN is.

In some embodiments, the cells used in the practice of this invention, adapted to suspension growth in the medium without animal ingredients, such as, for example, the medium without serum. Such adaptive procedures are described for example in Scharfenberg, et al., Animal Cell Technology Developments towards the 21stCentury, E. C. Beuvery et al. (Eds.), Kluwer Academic Publishers, pp.619-623, 1995 (BHK cells, Cho); Cruz, Biotechnol. Tech. 11: 117-120, 1997 (insect cells); Keen, Cytotechnol. 17: 203-211, 1995 (myeloma cells); Berg et al., Biotechniques 14: 972-978, 1993 (cells of the human kidney 293). In the private preferred embodiment the host cells are BHK cells 21 or navigational AIDS that have been developed for the expression of human factor VIII and adapted to growth in the absence of serum or animal ingredients.

Cell culture procedures

The methods of the invention are typically performed in shake culture vessel, and usually used a semi-continuous process type. During this process, after inoculation were grown cells, and when reaching a certain density collected about 70% of culture, and to the remaining culture was added to fresh cell culture medium to its original volume. This is usually repeated about 2-10 times.

In addition, there may be used a process type microsites. In the process, based on microneedle, tile and migrated into the internal structure of the media (macroporous carriers), or joined to the surface of the media (solid media), or was the one and the other. In the process, based on microneedle, initially in the culture vessel was placed mammalian cells, microsocial and cell culture medium. In the following days added an extra cell culture medium, if the culture volume did not reach the final working volume of the vessel at the beginning. In the next period performed periodically collecting the culture supernatant containing the product and replaced it with a new liquid medium, as long as the culture is finally ended. When collecting the supernatant, containing the product, mixing culture, for example the shaking was stopped and allowed to settle to the media containing the cells, and then deleted the part of the cell culture supernatant containing the product. In order to improve the overall result of the procedure may preferably be applied to the cooling phase before collecting the supernatant, containing the product, see for example WO 03/029442. In some embodiments, before allowed to set media, cell culture medium was cooled to a temperature from about 18°C to about 32°C, or from about 20°C to about 28°C, or from about 22°C to about 30°C.

Other applicable options cell culture procedures described in the WO 02/29084 (Novo Nordisk A/S).

Before advancing the phase of production, which was conducted regular collection containing the product of the culture supernatant for further processing, the cells were multiplied in the standard manner or by any scheme, suitable for a particular cell in this case. Phase propagation can include a single stage or multiple staged procedures. In a one-step procedure of reproduction cells were removed from storage vessel and inoculable directly in the culture vessel (possibly containing microneedle), where he was a production. In a multistep procedure of reproduction cells were removed from storage vessel and were propagated using a large number of culture vessels, gradually increasing in size until reaching the final of the culture vessel (possibly containing microneedle), where he was a production. During reproduction, the cells were grown under conditions optimized for growth. Culture conditions such as temperature, pH, pressure, dissolved oxygen, concentration of dissolved CO2and the like, are known that they are optimal for a particular cell, and will be apparent to the person skilled in the art (see, for example, Animal Cell Culture: A Practical Approach, 2ndEd., Rickwood, D. and Hames, B.D., eds., Oxford University Press, New York (1992)).

In one approach, the cell culture is the capacity of the process was produced in the same culture vessel: the cells were inoculable directly in the culture vessel (possibly containing microneedle), where did the products; cells multiplied to achieve an appropriate cell density, and began stage of production.

In another approach, the cell culture process has produced at least two different culture vessels: one or more vessel for sowing (first stage(s) of reproduction), and then in the culture vessel for producing (the last stage of the breeding season, followed by a production phase). At the first stage of reproduction of cells expressing the desired polypeptide, inoculable in a culture vessel for sowing containing cell culture medium, and they multiplied until, until reaching the minimum density for the next sowing. Subsequently multiplied sown culture was transferred to a culture vessel for producing containing cell culture medium and (possibly) microneedle. In the case of the process using microneedle cells cultured in the culture vessel under conditions in which cells migrated to the surface of solid media or on the outer and inner surfaces of the macroporous carrier, and they continued to grow in this last stage of breeding up until not fully occupied media. During this last stage of the multiplication carried out the change of environment, so m is canonicaly to settle to the bottom of the culture vessel, then deleted the predetermined proportion of the volume of the tank, and an appropriate proportion of the volume of the tank was filled with fresh medium. Then resuspendable microneedle in the environment and this process of removal and replacement of the medium was repeated at set intervals, for example every 24 hours. The number of substitutions environment depends on the density of cells and, as a rule, can be from 10 to 95%, preferably from 25% to 80% of the volume of the tank.

In the case of a suspension process, for example, perfusion, periodic or semi-continuous process, the cells were grown freely suspendirana them without immobilization on the media. In suspension cell-perfusion process, the cells were inoculable in a culture vessel for sowing containing culture medium without animal ingredients, and they multiplied until, until reaching the minimum density for the next sowing. Subsequently multiplied sown culture was transferred into a large culture vessel containing culture medium without animal ingredients, and the cells were multiplied until, until it reaches at least a predetermined density. In this phase, the cells were grown in suspension, allowing the number of cells in the culture vessel is increased to a predetermined or critical value. Replacement environment Khujand who have been by continuous perfusion culture vessel with fresh medium.

The number perfoirmance environment depends on the density of cells and can typically range from 10 to 95%, preferably from 25% to 80% of the volume of the tank per day (24 hours). 60-95% medium tank, for example about 80%, changed when the cell density reached values suitable for the early phase of production, usually every 24 hours. Replacement of 80% of the environment is also preferably carried out in the phase of production.

In simple periodic process cells were inoculable in a culture vessel for sowing containing nutrient medium without animal ingredients, and they multiplied until, until reaching the minimum density for the next sowing. Subsequently multiplied sown culture was transferred into a large culture vessel containing culture medium without animal ingredients.

Periodic process such as this may be extended by filing a concentrated solution of nutrients in the tank. This increases the process time and ultimately leads to an increase in the production of FVIII in the culture vessel. The collection time should be defined as a balance between the maximum possible long process in the tank and the risk of lysis of the cells.

The process is semi-continuous cultivation (Draw-Fill) is similar to a recurring periodic fermentation. If periodic is eskay fermentation cells were grown in the culture vessel and the medium was collected at the end of the cycle. In the process of semi-continuous cultivation of a culture vessel used to collect before any of the nutrients were exhausted. Instead of deleting the entire contents of the vessel were removed only a portion of the volume of the tank (usually 80% of the volume of the tank). After collecting in the vessel was added the same volume of fresh medium. Then the cells were allowed to grow in the vessel again, and after a certain number of days again took 80%. When re-periodic processes of cells remaining in the vessel after harvest, can be used as inoculum for the next batch.

The process is semi-continuous cultivation was carried out in two stages. The first phase of the process carried out is identical to the simple periodic process. After the first harvest of the culture vessel again acted as if simple periodic process; however, the duration of the party was shorter than the first because of the high initial density of the cells. These short "re-periodic phase can continue indefinitely.

Recharging process is semi-continuous cultivation is a fermentation of the semi-continuous cultivation with a concentrated nutrient similar to the type proposed in the recharging process. Case when simple process semi-continuous cultivation concludes what I is that added fresh environment may not be sufficient to maintain the cells to re-periodic fermentati. Power solves this problem. The power also allows the use of a culture vessel with a long periodic intervals during semi-continuous cultivation.

The culture vessel can be used in a wide range of temporal cycles and a wide range of volumes of semi-continuous cultivation. The ranges and preferred values can be seen in table 1 below.

Table 1
Set valueRangeThe preferred rangeThe most preferred value
Initial periodic phase
pH6-8of 6.6 and 7.67,0 for SNO and 6.6 to 7.4 for BHK
Temperature28-40°C30-37°C37°C for SNO and 36°C for BHK
Delta t is Imperator (optional)
the temperature drop to26-39°C30-36°C32°C
the temperature difference atof 0.5 to 12.0×106cells·ml-1of 0.5 to 12.0×106cells·ml-1the 2.0-10×106cells·ml-1

The pressure of the dissolved oxygen10-100%20-60%30%
Collection
- tank10-99%10-90%80%
the time collection2-10 days5-10 days9 days after start
Entered nutrient6-0 g·l-13-0 g·l-1When glucose <2 g·l-1
Repeated periodic stages
pH6-8of 6.6 and 7.67,0 for SNO and 6.6 to 7.4 for BHK
Temperature28-40°C30-37°C37°C for SNO and 36°C for BHK
Temperature (optional)
The temperature drop to26-39°C30-36°C32°C
The temperature difference atof 0.5 to 12.0×106cells·ml-1of 0.5 to 12.0×106cells·ml-1the 2.0-10×106cells·ml-1
The pressure of the dissolved oxygen10-100%20-60%30%
Collection
- tank10-99% 10-90%80%
the time collection1-7 days1-7 days5 days after collection
Entered nutrient3-0 g·l-13-0 g·l-1When glucose <2 g·l-1

It should be understood that in the process, where the phase of reproduction is a multi-step process, reproduction can take place in the culture vessels gradually increasing in size until it receives a sufficient number of cells for introduction in recent culture vessel. For example, you may consistently use one or more culture vessel for planting a volume of 5 l, 50 l, 100 l or 500 l Culture vessel for planting, as a rule, has a capacity of 5 l to 1000 L. Normally, cells inoculant into the culture vessel with an initial seeding density of about 0.2 to 0.4×106cells/ml, and they breed up until the culture reaches the density of cells of about 1.0×106cells/ml As a rule, the minimum density for subsequent sowing is from about 0.8×106up to about 1.5×106cells/ml

Some of the preset values, which are suitable for producera the project for factor VIII, not necessarily suitable for initial cell growth or planted in the culture, or at microsites. For example, temperature, pressure, dissolved oxygen and/or pH may be different for the two phases. Replacement of the medium during reproduction carried out in order to keep cells alive and growing, not to collect the culture supernatant for further processing.

Perhaps, given the magnitude of the temperature differential cultivation can be used when making and during the phase of production. In addition, when making temperature phase of production, the current pH and the frequency of replacement of the medium, as a rule, changes to values that are optimal for production.

Microneedle

Used in this case, microsocial represent particles that are small enough to be used in suspension cultures (with the same rate of shaking, which does not cause significant cell damage). They are solid, porous or have a solid core with a porous coating on the surface. Microneedle can be, for example based on cellulose or dextran, and their surfaces (external and internal surfaces in the case of porous media can be positively charged, but they are not limited to these examples. Other details which can be found in WO 02/29083 and "Microcarrier cell culture, principles and methods. Amersham Pharmacia Biotech. 18-1140-62. Edition AA".

Used solid microneedle include Cytodex 1™ and Cytodex 2™ (Amersham Pharmacia Biotech, Piscataway NJ), but are not limited to. Solid carriers are, in particular, carriers suitable for adhesion of cells (subtractability cells). Used macroporous carriers include Cytopore 1™ and Cytopore 2™ (Amersham Pharmacia Biotech, Piscataway NJ), but are not limited to. In particular, preferred are the holders of Cytopore 1™ with an average particle diameter of 230 μm, an average pore size of 30 μm and density of the positive charge of 1.1 mEq/g

Culture conditions for large-scale cultivation

The invention, in particular, true for large-scale production. The term "large scale production" refers to production with the participation of the culture vessel with a volume of at least 100 L. However, in preferred embodiments the scale, as a rule, not less than 250 l, such as at least 500 l, for example at least 1000 litres or even 5000 litres or more. The term "large-scale" can be used interchangeably with the terms "industrial scale" and "industrial scale".

Method for large-scale production of the polypeptide generally spend at least 120 hours, for example 1-26 weeks.

If the cell culture process is carried out at me is e in two different culture vessels, for example, one or more of the culture vessel(s) for sowing (first stage(s) of reproduction), then in the culture vessel for producing (last breeding stage and the subsequent stage of production), this process usually includes the transfer of approximately 50 l multiplied seed culture (about 1.0×106cells/ml) in the culture vessel with a volume of 500 l, containing 150 l of cell culture medium. For large-scale culture support appropriate conditions, such as temperature, pH, pressure, dissolved oxygen (DOT) and the rate of mixing, and the volume is gradually increased by adding medium in the culture vessel. In the case of a process with microneedles culture vessel also contains a number of microsetella corresponding to a final concentration of microsites in the range from 1 to 10 g/L. After migrating cells usually move on the surface of the media or in the inner part of the media within the first 24 hours.

Culture vessel

Culture vessels suitable for this invention may be placed, for example, on a standard reactor with stirrer (CSTR), where the mixing is carried out using conventional impellers types or using air reactor by introducing air into the lower part of the vessel. Among other parameters, which are usually controlling is carried out within the specified limits, there are pH, pressure, dissolved oxygen (DOT), the concentration of dissolved CO2and the temperature. Pressure of dissolved oxygen can be maintained, for example, by bubbling pure oxygen. The concentration of dissolved CO2can be maintained by sparging with air. Environment for temperature control is usually water, optionally heated or cooled. Water can be filtered through a jacket around the vessel or through a coiled tube immersed in the culture.

The term "culture vessel" may be used interchangeably with the terms "reservoir", "reactor", "fermenter and bioreactor".

Stage b) - allocation of the expressed polypeptide

At this stage, b) the factor VIII polypeptide was isolated from mammalian cells using the appropriate tools. In a typical embodiment, the cells can be removed from the environment, and the environment can be cleaned by sequential filtration of the collected material through the filter of 1.0 μm and 0.2 μm.

You can then increase the concentration of factor VIII in the medium (cell culture supernatant) using cation-exchange chromatography, where they are combined fractions rich in factor VIII. The factor VIII polypeptide then can be cleaned on a column by binding with antibodies anti-factor VIII (for example on a column with antibodies F25, see for example WO 95/13301 and/or Nordfanget al. 1995 (Thromb. Haemostas. 54:586-590)), and then eluted under conditions that preserve the activity of the factor VIII polypeptide. Other impurities can be removed by buffer exchange by gel filtration.

According to the second aspect of the invention, but is also applicable to the first aspect of the invention, the ligand C2 domain was added to facilitate the selection of the factor VIII polypeptide from the cells, i.e. the ligand C2-domain (for example OPLS) was added to the release associated with the cell of the factor VIII polypeptide.

A feature of this invention is that the factor VIII polypeptide can be isolated from cells without inactivation or destruction of mammalian cells. Thus, in the particular embodiment of the expressed polypeptide factor VIII is extracted from the cell culture medium with almost no decrease in cell viability. In addition, it is advantageous if the production can continue using the same batch of cells.

After the medium containing the polypeptide factor VIII was isolated from the cells, it can be subjected to one or more processing steps to purify the desired protein, including affinity chromatography, chromatography with hydrophobic interactions; ion-exchange chromatography, exclusion chromatography; electrophoretic procedures (e.g., preparative isoelectric focusing), differential dissolve the cost (for example, precipitation with ammonium sulfate), or a selection, etc., but not limited to. See, generally, Scopes, Protein Purification, Springer-Verlag, New York, 1982; and Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989.

Purification of factor VIII polypeptides, in particular, may include affinity chromatography on a column with antibodies against factor VIII and activation of proteolytic cleavage.

The following examples are used to illustrate this invention without limiting it.

EXAMPLES

Materials and methods

Cell line: cell line used for transfection, dhfr-Cho cells, DUKX-B11 cells (Urlaub, G. & Chasin, L.A. (1980) Proc. Natl. Acad. Sci. USA, 77, 4216-4220), was adapted to growth in suspension culture in medium without serum with dobavleniem of ribonucleosides and deoxyribonucleosides.

The vector expression: transcription factor VIII was performed using promoter of SV40 adenovirus, and selection using selective marker digidrofolatreduktazy. Downregulation of the factor VIII molecule comprises the heavy chain (HC) and light chain (LC) of factor VIII, connected with a short linker, obtained from In-domain. B-domain deleted, as it allows you to save the high expression of factor VIII and biological activity of factor VIII.

Transfection: β-lactamase gene was removed by cleavage with enzymes from plasmids No. 815 F8-500B-pTSV7, and the resulting fragment containing the gene for factor VIII, was purified in the gel and using the and for transfection DUKX-B11, Cho cells using the reagent FuGENE 6 (Roche). The transfection was performed in 6-hole boards in the medium α-MEM (Gibco)supplemented with ribonucleoside and deoxyribonucleoside and 10% detalizirovannoi FBS (fetal bovine serum). Two days after transfection cells transferred into vials TS on Wednesday α-MEM (Gibco) without ribonucleosides and deoxyribonucleosides, but with 10% detalizirovannoi FBS. After selection of the survivors of transfectants within 15 days started step amplification with methotrexate. Cells amplified with methotrexate to 1000 nm with several sublimirovanny performed during this process.

SF-adaptation and cultivation of cells: cells adapted for growth in medium without serum by step reduce the concentration of FBS in the medium SF. Cells adapted and maintained in vials with a volume of 125 ml shaker.

Cultivation of the cells during the experiments with supplemented medium without serum: For experiments with augmented environments, cells were cultured under perfusion model a high density of cells in test tubes of 50 ml volume with a ventilated lid on the shaker at 35°C in medium without serum, as described below. Cells were cultured in large flasks on a shaker at 37°C. cell Viability was assessed by collection of cells, and it was always more than 95%. The collected cells resuspendable in fresh cf is de. 2.5 ml were collected and resuspending cells were added to 2.5 ml of fresh medium with addition, so as to obtain a total volume of 5 ml with a concentration of 1×107cells/ml. Tubes shaker was placed in a shaker with temperature of 35°C and a speed of 250 rpm After 24 hours the samples were analyzed by cell density, viability, COA, ELISA and integrity of the protein of factor VIII using Western blotting.

Cell viability: the viability of the cell culture can be measured, for example, in accordance with the description of the Mammalian Cell Culture; essential techniques, 1997 (Wiley) Editors: A. Doyle and J. Bryan Griffiths (see, for example, protocols 13 and 14).

Analysis COA analysis of the activity of factor VIII)In the presence of calcium and phospholipids factor X becomes active factor XA with factor IXa. This formation is largely driven by factor VIII, which can be viewed as a cofactor for this reaction. When using the optimal number of CA2+and phospholipids and an excess of factor IXa and X the rate of activation of factor X depends only on the number of factor VIII. Factor XA hydrolyzes the chromogenic substrate S-2765 so that the released chromophore group, pNA. The color is then assessed photometrically at a wavelength of 405 nm. The resulting factor XA and, consequently, the intensity of color is proportional to the activity of factor VIII in the sample. Hydro is of the S-2765 educated thrombin prevent by adding a synthetic thrombin inhibitor, I-2581, together with the substrate (Chromogenix Coatest SP Factor VIII, diaPharma).

Other tests on the activity of factor VIII: other suitable assays to detect the activity of factor VIII can be carried out as a simple in vitro tests described in, for example, Kirkwood TBL, Rizza CR, Snape TJ, Rhymes IL, Austen DEG. Identification of sources of interlaboratory variation in factor VIII assay. In J Haematol 1981; 37; 559-68.; or Kessels et al., British Journal of Haematology, Vol.76 (Suppl.1) pp.16 (1990). The biological activity of factor VIII can also be quantified by measuring the ability of the drug to normalize clotting time of plasma deficient in factor VIII, for example as described in Nilsson et al., 1959.(Nilsson IM, Blombaeck M, Thilen A, von Francken I., Carriers of haemophilia A - A laboratory study, Acta Med Scan 1959; 165:357). In this assay, the biological activity is expressed in units/ml plasma (1 unit corresponds to the amount of FVIII in normal polerowanie plasma).

ELISA: Stripemania wells pre-coated sheep polyclonal antibody to human factor VIII. The samples were diluted and added into the wells. Available antigen factor VIII was associated with attached antibody. After washing unbound material was made peroxidase labeled sheep detectable antibodies and gave them contact with a fixed factor VIII. The wells are again washed, made the TMB solution (peroxidase substrate tetramethylbenzidine) and allowed to react for about the certain period of time. Developed blue color, which changes to yellow when the stop reaction with acid. The colour was measured spectrophotometrically in a microplate photometer at 450 nm. The absorbance at 450 nm is directly proportional to the amount of antigen factor VIII, fixed in a hole (VisuLize, FVIII antigen kit, Affinity biologicals). The analysis was calibrated using purified factor VIII with a remote In-domain.

F25 ELISA: ELISA: Stripemania wells pre-coated sheep polyclonal antibody to human factor VIII. The samples were diluted and added into the wells. Available antigen factor VIII was associated with attached antibody. After washing unbound material was made of diluted mouse monoclonal antibodies F25 against factor VIII that can recognize With the end of the heavy chain of factor VIII, and gave them contact with a fixed factor VIII. The wells are again washed, made of diluted peroxidase labeled goat protivomaskitnye IgG (DAKO) and gave them contact with the fixed antibodies F25. The wells are again washed, made the TMB solution (peroxidase substrate tetramethylbenzidine) and allowed to react for a certain period of time. Developed blue color, which changes to yellow when the stop reaction with acid. The colour was measured spectrophotometrically in a microplate photometer at 450 nm. The absorbance of CR is 450 nm is directly proportional to the amount of antigen factor VIII, fixed in the hole. The analysis was calibrated using its own standard heavy chains of factor VIII, affinity purified antibodies F25.

(Antibody F25: Cm. for example WO 95/13301 and/or Nordfang et al. 1995 (Thromb. Haemostas. 54:586-590).

Example 1: Add OPLS in serum-free culture medium of cells that produce factor VIII

OPLS were added in serum-free cell culture medium at the indicated concentration in accordance with the details of the experiment described in materials and methods. The results can be seen below in table 2 and in figa and 2B.

Table 2
Add OPLS in serum-free cell culture medium with cells producing factor VIII
The concentration of OPLS (mm)Performance factor VIII (PCG/cell / day)The specific activity of the protein factor VIII (U/g))
00,06111,0
0,030,06811,8
0,10,06212,8
0,3 0,05212,2
10,05812,6
3of 0.06615,8
100,1015,4
300,1815,0

Conclusion:

Add OPLS increase the specific productivity of the cells that produce factor VIII (see figa), and add OPLS increases the specific activity of factor VIII (see figv).

Example 2: Adding O-phospho-L-serine and/or vegetable hydrolyzed in serum-free culture medium of cells that produce factor VIII

Cells producing BDD factor VIII (cell line 1C5-SF), was cultured in 50 ml test tubes with lids with filter tubes with a filter 50 for bioreactor, RTR). a 2.5×106cells in 5 ml medium CDM4CHO, augmented O-phospho-L-serine concentrations up to 20 mm and/or vegetable hydrolysate to a concentration of 5 mg/ml, as shown in Table 3. Each condition was tested in four cultures in 5 ml of Culture were incubated in a shaker-incubator (37°C, 8% CO2and 250 rpm). Four days after seeding 1.2 ml of each culture was centrifuged at 2000g out within 5 minutes and remove cellular precipitate. The supernatant stabilized by the addition of imidazole pH 7.2 to a final concentration of 20 mm and Tween 80 to a final concentration of 0.02%, and froze in the aliquot of 0.2 ml at a temperature of -80°C.

The total amount of antigen factor VIII each culture was determined using ELISA "sandwich"-type. Aliquots stable and frozen medium was thawed and analyzed in accordance with the description in Materials and methods. It was determined the content of factor VIII recognized by the antibodies F25, which selectively bind factor VIII inaktivirovannye With the end of the heavy chain. Aliquots stable and frozen medium was thawed and analyzed using F25 ELISA in accordance with the description in Materials and methods.

To test the activity of aliquots stable and frozen medium was thawed and analyzed using analysis of COA in accordance with the description in Materials and methods.

The quality of factor VIII in the environment of each culture was assessed by the specific activity calculated by the activity and the total content of antigen factor VIII. The share of factor VIII with inaktivirovannye With the end of the heavy chain was assessed by the ratio between the amount of antigen factor VIII, as defined by F25 ELISA, and the total number of antigen factor VIII.

The results obtained with the two additives shown in figas. These data suggest that the positive effect of added O-phospho-L-serine or vegetable hydrolysate to a cell culture producing factor VIII. Both additives improved the yield and quality of recombinant factor VIII from cell cultures, and both additives increased the share of factor VIII with inaktivirovannye With the end of the heavy chain in the environment. Furthermore, an additional positive impact on the share of factor VIII with inaktivirovannye With the end of the heavy chain was observed when O-phospho-L-serine and vegetable hydrolysate used in combination.

Table 3
The additives tested on the cells that produce factor VIII
ProductProviderNo. in catalogue
AndNo additives--
InHydrolyzed wheat glutenKerry BioscienceNorer 4605
O-phospho-L-serineSigmaR
DHydrolyzed wheat gluten and O-phospho-L-serine--

1. Method of producing factor VIII polypeptide, comprising stages of: a) culturing mammalian cells expressing the factor VIII polypeptide, under conditions suitable for expression of the indicated polypeptide factor VIII, which include cell culture medium with O-phospho-L-serine (OPLS), and (b) highlight the downregulation of factor VIII polypeptide from mammalian cells using the appropriate tools.

2. Method of producing factor VIII polypeptide, comprising stages of: a) culturing mammalian cells expressing the factor VIII polypeptide under conditions suitable for expression of the indicated polypeptide factor VIII, which include cell culture medium, and (b) highlight the downregulation of factor VIII polypeptide from mammalian cells using appropriate means, comprising adding to said cells O-phospho-L-serine (OPLS).

3. The method according to claim 1 or 2, where O-phospho-L-serine (OPLS) is found in cell culture medium at a concentration of 0.1-100 mm.

4. The method according to claim 1 or 2, where O-phospho-L-serine (OPLS) are added to cells in step b) at a concentration of 1-200 mm.

5. The method according to claim 1 or 2, where the cell culture medium further comprises soybean inhibitor three is sin.

6. The method according to claim 1 or 2, where the cell culture medium further comprises hydrolyzed vegetable protein.

7. The method according to claim 1 or 2, where mammalian cells selected from the group consisting of mammalian cells, which Express endogenous polypeptide factor VIII, and mammalian cells, which was transfirieran gene polypeptide factor VIII.

8. The method according to claim 1 or 2, where in mammalian cells was transfirieran the expression vector containing the nucleic acid molecule encoding the factor VIII polypeptide, and controls the expression of functionally associated with her.

9. The method according to claim 1 or 2, where the cell culture medium does not contain serum.

10. The method according to claim 1 or 2, where the expressed polypeptide factor VIII obtained from the cell culture medium with almost no decrease in cell viability.

11. The method according to claim 10, where production continues using the same batch of cells.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing and purifying interferon β-1b. The present method of producing and purifying interferon β-1b involves washing inclusion bodies with 0.3% solution of trifluoroacetic acid in water, followed by centrifuging, extracting protein from the washed inclusion bodies with a solution containing 1-propanol and water in ratio of 1:1, with addition of 0.3% trifluoroacetic acid, purification by reversed-phase chromatography on a C4 sorbent, refolding in a buffer solution containing 1% sodium laurate, 50 mM Na2HPO4 at pH=9.0 with addition of 10 mcM copper sulphate dissolved in ammonia, extracting interferon β-1b from the aqueous solution using 75% phenol solution in water and purification by reversed-phase chromatography on a C18 sorbent. Use of 0.3% trifluoroacetic acid solution for washing inclusion bodies containing β-1b is also disclosed.

EFFECT: invention enables to obtain purified interferon β-1b on industrial scales in an effective amount.

3 cl, 2 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: producing protein is ensured by bacterial host cell culture containing a recombinant nucleic acid in a medium containing crude glycerol as a carbon source, and protein recovery expressed by the cell. Crude glycerol represents a side product of bio fuel or soap which is found in the medium in the concentration of 0.1% to 75% (vol./vol.). The culture process is performed in an enclosed volume, injection or continuous fermentation. The bacterial cell is specified in Streptomyces Hvidans, Bacillus subtilis and Streptomyces rubiginosis.

EFFECT: higher accumulation of cell biomass and protein production in the culture.

11 cl, 14 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically to obtaining factor VII (FVII) and factor Vila (FVIIa) albumin linked polypeptides, and can be used in medicine. A polypeptide, which is a FVII or FVIIa polypeptide is obtained in a recombinant manner, said peptide being linked with albumins through a glycerine-serine peptide linker of a special structure, which separates part associated with FVII or FVIIa from the albumin part, wherein the FVII or FVIIa polypeptide lies on the N-end of the fused protein. The linked polypeptide or vector structure, which contains its coding nucleic acid, is used as a medicinal agent for treating or preventing blood-clotting disorders.

EFFECT: invention enables to obtain a protein with FVII or FVIIa biological activity and longer functional half-time in plasma in vitro compared to non-linked FVII or FVIIa.

12 cl, 4 dwg, 6 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: hybrid polypeptide is a polypeptide 1 to which a polypeptide 2 is covalently bonded, where polypeptide 1 is a human endostatin sequence with 135-184 amino acid residues, wherein at position 173, Cis is replaced with Ala with a relatively native endostatin sequence, and polypeptide 2 is a human plasminogen sequence with 82-341 or 463-511 amino acid residues. Also disclosed is a method of obtaining the hybrid polypeptide using an E.coli producer, which involves methods for extraction and purification thereof.

EFFECT: polypeptide is capable of inhibiting human endothelial cell proliferation in vitro and can be used when producing nontoxic preparations for inhibiting angiogenesis.

4 cl, 4 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: method involves culturing mammal cells containing a gene which codes the blood-coagulation factor IX, in a cell culturing medium containing approximately from 10 nM to 600 nM manganese under conditions and during a time sufficient for expression of the blood-coagulation factor IX.

EFFECT: invention enables to obtain an expressed blood-coagulation factor IX with high degree of glycosylation compared to the degree of glycosylation observed using the same conditions and the same medium but without manganese.

25 cl, 8 dwg, 4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: what is produced is the yeast strain Saccharomyces cerevisiae able to produce secreted human somatotropin. Said strain contains a promoter contolled DNA sequence coding mature human somatotropin fused with a leader peptide in the same reading frame. The leader peptide includes a double pro-site of α-factor of yeast Saccharomyces cerevisiae. It also can contain a triple pro-site of α-factor of yeast Saccharomyces cerevisiae, or a double pro-site of HSP150 protein of yeast Saccharomyces cerevisiae, or a combination of said pro-sites. A method for producing human somatotropin provides cultivation of the human somatotropin producer strain.

EFFECT: use of the invention provides higher end product yield.

2 cl, 1 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: method involves providing a cell culture containing mammal cells which contain a gene coding rTNF-lg expression of which occurs in the cell culture medium. The medium containing glutamine and possessing the essential properties is used. Said culture is maintained in an initial growth phase at a first set of cultivation conditions during a first period of time. At least one of the cultivation conditions are modified to produce a second set of cultivation conditions with said cultivation condition at the specified stage of modification of at least one of cultivation conditions specified from the group consisting of: (i) temperature; (ii) pH; (iii) osmolality; (iv) a level of the chemical activator and their combinations. Said culture is maintained at the specified second set of conditions during a second period of time so that rTNF-lg is accumulated in the specified culture.

EFFECT: invention enables the scale rTNF-lg production in the cell culture.

48 cl, 76 dwg, 27 tbl, 17 ex

FIELD: medicine.

SUBSTANCE: method for producing an immortalised human cell involves transfection of an immortalised human host cell in a serum-free medium with using a transfection vector containing a gene coding a target human protein, a promoter and a bovine growth hormone polyadenilation (polyA) signal wherein said promoter and the polyA signal are binded with 5'- and 3'-terminus of the gene coding a target human protein respectively, and an origin of replication. Said transfection vector additionally bears at least one genetic selection marker. Stable transfected cells are selected.

EFFECT: invention allows producing the stable immortalised transfected human cells for preparing recombinant human proteins.

16 cl, 16 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, particularly genetic and protein engineering, as well as a method of producing recombinant human C-peptide. The disclosed method involves culturing an Escherichia coli producing strain, breaking down bacterial cells by disintegration, separating "inclusion bodies" containing a hybrid protein, dissolution thereof in a buffer containing urea and dithiothreitol, renaturating and purifying the renatured hybrid protein, disintegration thereof with trypsin and carboxypeptidase B, separation via chromatography on SP-sepharose, followed by purification and obtaining the end product.

EFFECT: method enables to obtain C-peptide with high output and purity of not less than 95% from wastes formed when producing recombinant human insulin.

4 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: method involves preparing a cell culture containing mammal cells which contain a gene which encodes a polypeptide whose expression takes place in cell culture conditions. A medium containing glutamine and having the required properties is used. Said culture is kept at the initial growth phase under a first set of culturing conditions for a first period of time. At least one of the culturing conditions is changed to obtain a second set of culturing conditions, wherein said culturing condition at said step for changing at least one culturing condition is selected from a group consisting of: (i) temperature; (ii) pH; (iii) osmolality; (iv) level of chemical activator and combinations thereof. Said culture is kept at said second set of conditions for a second period of time so that polypeptide accumulates in said culture.

EFFECT: invention enables large-scale production of polypeptides in a cell culture.

46 cl, 76 dwg, 27 tbl, 17 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns area of molecular biology and biochemistry, and can be used in medicine. There is offered mutein conjugate of the blood coagulation factor VIII (FVIII) wherein a residue not being cysteine in position 41, 129, 377, 388, 468, 491, 556, 1804, 1808, 1810, 1812, 1813, 1815 and/or 2118 is substituted by a cysteine residue with polyethylene glycol (PEG) where a PEG molecule is bound with a polypeptide in a mutant cysteine residue.

EFFECT: improved pharmacokinetic properties of the FVIII as an ingredient of the conjugate under the invention with preserved a procoagulant factor activity allows presenting new FVIII PEG-muteins for producing of a pharmaceutical compositions for treating hemophilia.

12 cl, 38 dwg, 8 tbl, 1 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to obtaining versions of glycoprotein IV alpha polypeptide of human thrombocytes (GPIbalpha) and can be used in medicine to treat vascular disorders. Using a recombinant technique, a polypeptide is obtained, which contains substitutes in SEQ ID NO:2 selected from: Y276F K237V C65S; K237V C65S; Y276F C65S; or Y276F Y278F Y279F K237V C65S. The obtained polypeptide is used to inhibit bonding of leucocytes to biological tissue or for treating disorders associated with activation of thrombocytes.

EFFECT: invention enables to obtain GPIbalpha polypeptide which bonds with von Willebrand factor with affinity which is at least 10 times higher than in natural GPIbα polypeptide, and also has low affinity for bonding with alpha-thrombin, lower aggregation and/or high resistance to proteolysis relative the polypeptide with SEQ ID NO:2.

41 cl, 3 dwg, 8 ex

FIELD: biotechnology, in particular production of modified swine factor VIII (POL1212).

SUBSTANCE: DNA molecule encoding of modified swine factor VIII is cloned in expression vector, having functionality in mammalian cells. Modified swine factor VIII protein is obtained by cultivation of mammalian cell line BHK CRL-1632 (ATCC), BHK 1632, or CHO-K1, transfected with vector. Therapeutic composition for treatment of subjects suffering from deficit of factor VIII, such as haemophilia, contains effective amount of swine factor VIII protein.

EFFECT: effective agent for treatment of factor VIII deficit.

13 cl, 8 dwg, 7 ex

The invention relates to medicine, in particular to Hematology

FIELD: biotechnology, in particular production of modified swine factor VIII (POL1212).

SUBSTANCE: DNA molecule encoding of modified swine factor VIII is cloned in expression vector, having functionality in mammalian cells. Modified swine factor VIII protein is obtained by cultivation of mammalian cell line BHK CRL-1632 (ATCC), BHK 1632, or CHO-K1, transfected with vector. Therapeutic composition for treatment of subjects suffering from deficit of factor VIII, such as haemophilia, contains effective amount of swine factor VIII protein.

EFFECT: effective agent for treatment of factor VIII deficit.

13 cl, 8 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to obtaining versions of glycoprotein IV alpha polypeptide of human thrombocytes (GPIbalpha) and can be used in medicine to treat vascular disorders. Using a recombinant technique, a polypeptide is obtained, which contains substitutes in SEQ ID NO:2 selected from: Y276F K237V C65S; K237V C65S; Y276F C65S; or Y276F Y278F Y279F K237V C65S. The obtained polypeptide is used to inhibit bonding of leucocytes to biological tissue or for treating disorders associated with activation of thrombocytes.

EFFECT: invention enables to obtain GPIbalpha polypeptide which bonds with von Willebrand factor with affinity which is at least 10 times higher than in natural GPIbα polypeptide, and also has low affinity for bonding with alpha-thrombin, lower aggregation and/or high resistance to proteolysis relative the polypeptide with SEQ ID NO:2.

41 cl, 3 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns area of molecular biology and biochemistry, and can be used in medicine. There is offered mutein conjugate of the blood coagulation factor VIII (FVIII) wherein a residue not being cysteine in position 41, 129, 377, 388, 468, 491, 556, 1804, 1808, 1810, 1812, 1813, 1815 and/or 2118 is substituted by a cysteine residue with polyethylene glycol (PEG) where a PEG molecule is bound with a polypeptide in a mutant cysteine residue.

EFFECT: improved pharmacokinetic properties of the FVIII as an ingredient of the conjugate under the invention with preserved a procoagulant factor activity allows presenting new FVIII PEG-muteins for producing of a pharmaceutical compositions for treating hemophilia.

12 cl, 38 dwg, 8 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: method to produce polypeptide of factor VIII provides for cultivation of cells of mammals, which express polypeptide of factor VIII, under conditions, suitable for expression of the specified popypeptide of factor VIII, which include a cell cultural medium with O-phospho-L-serine (OPLS) and extraction of the expressed polypeptide of factor VIII from cells of mammals with the help of suitable means.

EFFECT: invention makes it possible to produce popypeptide of factor VIII with increased specific activity.

11 cl, 3 dwg, 4 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: method of purifying coagulation factor VIII protein from a solution involves: a) contacting the protein with a multimodal resin or a mixed action-type resin containing ligands having a hydrophobic portion and a negatively charged portion; b) elution of the protein with an elution buffer containing at least 1.5M salt and at least 40% (wt/vol) ethylene glycol, propylene glycol or mixture thereof, and calcium ions. The method is a single-step chromatographic process where the coagulation factor VIII protein is captured and does not require adjusting pH or electroconductivity at the feeding step. The method of stabilising the coagulation factor VIII protein, where the coagulation factor VIII protein is captured and stabilised when the protein passes once through a column with a multimodal resin containing ligands having a hydrophobic portion and a negatively charged portion, and elution of the protein with an elution buffer containing at least 1.5M salt and at least 40% (wt/vol) ethylene glycol, propylene glycol or mixture thereof, and calcium ions.

EFFECT: invention reduces the volume of the column about 250-fold and a coefficient of purification equal to 30, high stability of the protein product.

2 cl, 4 dwg, 4 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to recombinant factor VIII, which contains one or more mutations, resulting in an increased stability of both the factor VIII and factor VIIIa, as well as to a pharmaceutical composition for treating haemophilia containing it. Also described is a molecule of nucleic acid, coding the said recombinant factor VIII, and an expression vector and host-cells, containing the said molecule of nucleic acid. The invention also relates to a method of obtaining the said factor VIII, as well as to its application in the method of treating haemophilia A in an animal.

EFFECT: invention makes it possible to obtain a biologically active factor VIII with an increased stability.

50 cl, 12 dwg, 5 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biotechnologies, namely to a method of purification or enrichment of blood-coagulation factor FVIII with the application of chromatography. The method includes provision of a fraction, which contains recombinant FVIII, in a water solution with high ionic power, where the water solution contains recombinant FVIII in a solution with the high concentration of salt, corresponding to conductivity in the range from approximately 25 to approximately 200 mC/cm at 25°C, bringing the fraction, which contains recombinant FVIII, in contact with multimodal resin, which represents Capto Adhere or Capto MMC. The fractions which contain recombinant FVIII are eluted with a water eluting buffer, which contains, at least, one amino acid, which is positively charged at a pH value of 6-8, where the amino acid, which is positively charged at the pH value of 6-8, is selected from the group of amino groups, including amino acids, such as lysine, arginine, histidine and their combinations, in the concentration over 0.4 M.

EFFECT: invention makes it possible to obtain the purified or enriched factor of blood-coagulation FVIII with high output.

14 cl, 8 dwg, 22 tbl, 13 ex

Up!