EA011749B1 - A process for the production of human growth hormone and use serum-free cell culture medium for mammalian cells expressing human growth hormone - Google Patents

Abstract

This invention relates to a process for the production of human growth hormone and use serum-free medium for culturing mammalian cells expressing human growth hormone.

Description

The present invention relates to the field of cultivation of mammalian cells in the absence of serum in the culture, in particular, the invention relates to the cultivation of cells producing recombinant proteins, such as, for example, human growth hormone (HOH).

The basis of the invention

The present invention relates to a serum-free medium intended for the growth and maintenance of mammalian cells in culture.

Cell culture is widely used at present to obtain various biologically active products, such as viral vaccines, monoclonal antibodies, non-antibodies, immunoregulators, growth factors of polypeptides, hormones, enzymes, tumor-specific antigens, etc. These substances are produced by normal or transformed and genetically modified cells.

In the past, cell culture media was supplemented with serum, which served as a universal nutrient for the growth and maintenance of all mammalian cell lines that produce biologically active products. Immune serum contains hormones, growth factors, transport proteins, attachment and spreading factors, nutrients, trace elements and so on. The culture medium typically contained up to about 10% animal serum, such as fetal bovine serum (EB8), also called calf fetal serum (EC8).

Despite its widespread use, serum has many limitations in use. It contains high levels of a multitude of proteins that influence the limitation of the percentage of the protein of interest produced by the cells. These proteins derived from whey must be separated from the product during the process steps, such as isolating the protein of interest, which complicates the process and increases costs.

The side effect of B8E (bovine spongiform encephalopathy), a transmissible neurodegenerative disease in cattle with a long latent or incubation period, raises the question of the regulatory involvement of the use of animal serum in the production of biologically active products.

Therefore, there is a great need to develop an alternative medium that does not contain animal serum, which supports cell growth and preserves them in the process of producing biologically active products.

Typically, the cell culture medium contains many components of various categories, such as amino acids, vitamins, salts, fatty acids and other compounds.

Amino acids.

For example, US Pat. No. 6,048,728 (Ιη1ο \ ν, etc.) describes that the following amino acids can be used in a cell culture medium: alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine , lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, threonine and valine.

Vitamins

In US patent No. 2003/0096414 (Syusatoie et al.) Or in US patent No. 5811299 (Keeg and others) it is described, for example, that the following vitamins can be used in a cell culture medium: biotin, pantothenate, choline chloride, folic acid , myoinocytol, niacinomide, pyridoxine, riboflavin, vitamin B12, thiamine, putrescine.

Salt.

In US patent No. 6399381 (B1it and others), for example, describes a medium containing CaCl ₂ , KC1, MDC1 ₂ , No. C1. monosodium phosphate, dibasic sodium phosphate, sodium selenite, Si8O _4, Ζπί'1 _2. Another example of a document which describes inorganic salts that can be used in the cell culture medium is US patent № 2003/0153042 (Lgpo1b et al.), Which discloses a medium comprising SaS1 _2, KC1 MdS1 ₂ IaS1, monosodium phosphate, dibasic sodium phosphate, CuCl ₂ -2H ₂ O, ΖηΟ ₂ .

Fatty acid.

The fatty acids known to be used in the culture medium are arachidonic acid, linoleic acid, oleic acid, lauric acid, myristic acid, as well as methyl betacyclodextrin, see, for example, US Pat. No. 5,045,468 (EagPeg). It should be noted that cyclodextrin is essentially not a lipid, but it is able to form a complex with lipids, and thus, it is used to increase the solubility of lipids in a cell culture medium.

Other components used, in particular, in an immune serum-free cell culture medium, are compounds such as glucose, glutamine, Ia-pyruvate, insulin or ethanolamine (see, for example, EP 274445), or a protective agent such like P1itoshs E68. P1itoshs® E68 (also known as Po1ochateg 188) is a blocking copolymer of ethylene oxide

- 1 011749 (EO) and propylene oxide (PO).

Standard basic media are also known to those skilled in the art. Such media already contain several components of the media mentioned above. Examples of such media that are widely used are Dulbecco’s modified environment Igla (ΌΜΕΜ), ΌΜΕΜ E12 (1: 1), the nutrient mixture Nat'k ΝιιΙποηΙ Ш1х1иге E-10, medium ΒΡΜΙ (Vokte11 Ragk Μetο ^^ a1 ΙηκΙίΙιιΚ Μеά ^ it ), Wednesday Μί, ΌΒ 131, or Wednesday ^ yyash'k Μеάшт Ε. These commercial environments are available, for example, from Clio, 1 Puigodep.

Metals, such as zinc (Ζη) and copper (Cu), are involved in metabolic interactions (Va11ee and Ea1syk, 1993 or Zhypeg, 1991).

Zinc plays a significant role in the structure and function of a large number of macromolecules and most enzymatic reactions. It plays the role of a catalyst, cocatalyst, or a structural role in the specific folding of proteins. Ζη-ATP is required for the synthesis of pyridoxine-5-phosphate and flavin-adenosine dinucleotide (ΕΑΌ), two coenzymes necessary for the biogenic synthesis of amine and metabolism of monoamine oxidase.

The activity of zinc in protecting biological structures from damage by free radicals can be due to several factors: maintaining an appropriate level of metal proteins, which are also traps from free radicals, as an essential component of superoxide dismutase, as a protective agent for thiols, and in preventing the interaction of chemical groups with iron with the formation of free radicals.

In addition, the presence of Ζη prevents lipid peroxidation. Zinc is also an effector of tubulin polymerization and acts ίη νίίτο on the formation and stabilization of actin fiber. Zinc is also a component of the zinc finger motif of DNA-related proteins, which are a common motif in transcription proteins.

Zinc ions exist mainly in the form of complexes with proteins and nucleic acids and are involved in all aspects of the intermediate metabolism, transmission and regulation of the expression of genetic information, storage, synthesis and action of peptide hormones and structural maintenance of chromatin and biological membranes.

Copper is also an important trace element for the functioning of many cellular enzymes. Copper ions can take on special redox states by oxidizing Cu (II) or reducing Cu (I), allowing the metal to play a leading role in cellular physiology as a catalytic cofactor in the redox chemistry of enzymes. Copper functions in a group of oxidases, which include cytochrome C oxidase, tyrosinase, dopamine β-monooxygenase, aminoxidase and lysyl oxidase. Copper is also involved in mitochondrial respiration, iron homeostasis as a component of ceruloplasmin, in the removal of free radicals and cross-linking of elastin.

A serum-free medium including metal ions, such as zinc or copper ions, is known to those skilled in the art, for example, from US Pat. No. 6,048,728 (1n1o and others), US Patent No. 4,767,704 (C ^ e1apy and etc.) or the International application XVO 01/16294 (Ы Т Т есес111111ощощощощ 1ps.) However, these documents do not provide data on the effect of increased productivity when these ions are added in certain concentrations to a standard production medium.

To improve and supply biologically active products, such as therapeutic proteins or vaccines, a large yield of product is required. Corresponding cells that are widely used to produce polypeptides are Chinese hamster ovary (CHO) cells.

CHO cells were first cultured by Risk (1. Exp. Ge. 108, 945, 1958) from biopsy material from an ovary of a female Chinese hamster. From these initial cells, several cell lines with different characteristics were obtained. One of these CHO cell lines, CHO-K1, is a proline-dependent line and is diploid in the dihydrofolate reductase gene (PNEU). Another line obtained from the indicated cell line is the SNE cell line deficient in PNEU (SNO υυκ B11) (ΡΝΑ8 77, 1980, 4216-4220), which is characterized by the loss of PNEU function due to a mutation in one PNEU gene and subsequent loss of another gene .

The following cells, often used to produce proteins intended for introduction into the human body, are human cell lines, such as the human fibrosarcoma cell line HT1080 or the human kidney embryonic cell line 293.

The C127 mouse cell line is also often used to produce recombinant proteins (Sayet et al., 1989; Oka and Virr, 1990).

One of the therapeutic proteins of interest is growth hormone. Human Growth Hormone (11CH). Also known as somatropin (ΙΝΝ) or somatotropin, it is a protein hormone produced and secreted by somatotropic cells of the anterior pituitary gland. Human growth hormone plays a key role in somatic growth in childhood and in metabolism during adulthood through its effect on the metabolism of proteins, carbohydrates and lipids.

Human growth hormone is a single polypeptide chain consisting of 191 amino acids (Be \\ '1y et al, 1972), having two disulfide bonds, one between Suk-53 and Suk-165,

- 2 011749 forming a large loop in the molecule, and another between Suk-182 and Suk-189, forming a small loop near the C-terminal portion. The DNA sequence that confirmed the amino acid sequence of the protein was described by Magya1 et al. (1979). Purified 11CH. in its lyophilized form, it is a white amorphous powder. It is readily soluble (at a concentration of> 10 mg / L) in aqueous buffers at pH in the range from 6.5 to 8.5.

In a solution, 11CH exists predominantly as a monomer, with an insignificant fraction in the form of dimers and oligomers with a high molecular weight. Under certain conditions, 11CH can be induced in the form forming a larger number of dimers, trimers and higher oligomers.

Several derivatives of JSN are known, including derivatives of natural origin, variants and metabolic products, degradation products of predominantly biosynthetic 11CH and genetically engineered derivatives of JSN obtained by genetic methods. One example of a naturally occurring 11CH derivative is CH-U, a type of growth hormone found in the placenta. Another representative of the gene locus is described by SNSP et al. (1989). Any derivative of JSN, including derivatives adapted for a long stay in the body, can be used for the purposes of the present invention, as long as they retain the biological activity of 11CH.

Methionyl-yCH was the first form of YSH obtained by recombinant DNA technology. This compound is actually a YSN derivative having an additional methionine residue at the Ν-terminal site (Сос66с1 et al., 1979).

A variety of 11CH of natural origin, found in the pituitary gland and in the bloodstream, is called 20-K-CHF (Be \\ zk et al., 1978; Betak et al., 1980). This compound, which lacks 15 amino acid residues, from C1i-32 to C1p-46, appears during alternative splicing of messenger ribonucleic acid (Ee # 1o et al., 1981). This compound has many, but not all, biological properties of 11CH.

20-K-CHF is produced in the pituitary gland and is secreted into the blood. It accounts for approximately 5% of the growth hormone produced by an adult, and approximately 20% of the growth hormone produced in childhood. It has the same growth-promoting activity as growth hormone, and, as has been revealed, has a lipolytic activity value greater than or equal to the activity of the form of 22 kDa. It binds to growth hormone receptors with an affinity equal to the growth hormone affinity of 22 kDa, and has one tenth of the lactogenic (prolactin-like) biological activity compared to the hormone of 22 kDa. Unlike the hormone of 22 kDa, 20-k-CHF has a weak anti-insulin activity.

A certain amount of 11CH derivatives is the result of proteolytic modification of the molecule. The initial pathway for the metabolism of the JS molecule involves proteolysis. The JS region around residues 130–150 is extremely prone to proteolysis, and several JS derivatives with single-strand breaks or deletions in this region have been identified (1Bog1asik-ikkdsd, 1987). This region is located in the large loop of the JSN, and cleavage of the peptide bond leads to the appearance of two chains that are connected via a disulfide bond near Suk-53 and Suk-165. It was found that many of these double-stranded formations have increased biological activity (81pd et al., 1974). Many derivatives of human growth hormone were obtained artificially using enzymes. The enzymes trypsin and subtilisin, as well as other enzymes, were used to modify 11CH in various places along the entire length of the molecule (Le ^ 1k et al., 1977; SgGG et al., 1982). One such derivative, called a double-stranded anabolic protein (2-CAP), was obtained using controlled proteolysis of JS with trypsin (Weskeg et al., 1989). It was found that 2-CAP has biological properties that are completely different from that of the intact JS molecule, in which the growth-promoting activity of the JS molecule was mainly preserved, and the effect on carbohydrate metabolism was mainly canceled.

Asparagine and glutamine residues in proteins are subject to deamidation reactions under appropriate conditions. It was shown that pituitary JSF undergoes this type of reaction, leading to the conversion of Akp-152 to aspartic acid, and also, to a lesser extent, to the conversion of Sl-137 into glutamic acid (Be \\ zk et al., 1981). It was revealed that deamidated JSN has an altered sensitivity to proteolysis under the action of the subtilisin enzyme, which indicates that deamidation may have physiological significance in the directed proteolytic cleavage of 11CH. It is known that biosynthetic JSD degrades under certain storage conditions, leading to deamidation in another asparagine (Akp-149). This represents the main site of deamidation, but deamidation in Akp-152 has also been observed (Weskeg et al., 1988). No deamidations were observed in C1-137 in biosynthetic JSF.

Methionine residues in proteins are sensitive to oxidation mainly by sulfoxide. Both the pituitary gland and the biosynthetic JSN undergo sulfoxidation in Me (-14 and Me (125 (Weskeg et al., 1988). It was found that Me (-170 oxidizes in the pituitary gland produced by JSN, but not in the biosynthetic JSN Both both deamidated YSN and Me (-14-sulfoxide-ySN were found to exhibit full biological activity (Weskeg et al., 1988).

- 3 011749

Truncated forms of BON are obtained, each through the action of enzymes or through genetic methods. In 2-CAP generated by the controlled action of trypsin, the first eight residues at the Ν-terminal portion of the BON were removed. Other truncated BON variants are obtained by modifying a gene before expressing it in a suitable host. The first 13 residues were removed, resulting in a derivative having special biological properties (Oetiet et al., 1986), in which the polypeptide chain is not disconnected.

Although human growth hormone was originally obtained from pituitary glands taken from corpses, these preparations were not electrophoretically homogeneous, and antibodies appearing in the serum of patients treated with preparations of approximately 50% purity were immunogenetically classified as inactive components. Recombinant DNA technology makes it possible to produce BON without restriction in a number of different systems. The purification of BON from the culture medium is facilitated by the presence of only a small amount of impurity proteins. In fact, it has been shown that JON can be purified on a laboratory scale through a single purification step on a reverse phase HPLC column (Nyyid e! A1. (1989).

The recombinant human growth hormone, gBON, was produced by the manufacturer 8ethoio 1p1egpa1uia1 8.A. as 8ΕΚ.Ο8ΤΙΜ®, and accelerated approval ΡΌΑ has been received for the use of this product for the treatment of patients with weight loss and malnutrition among AIDS patients. 8ΑΙΖΕΝ® is a recombinant human growth hormone intended for children with OH deficiency in Turner syndrome in girls, as well as in chronic renal failure in children. ΡΚΌΤΚΌΡΙΝ®, received by Oeeee! ECB, 1f. (8oi! B 8ai Rtaiskso, CA), slightly different in structure from the natural sequence of BON, has an additional methionine residue near the Ν-terminal site. Recombinant BON is mainly supplied in vials containing BON plus an additional excipient, for example glycine and mannitol, in lyophilized form. The prescribed concomitant solvent in the vial enables the patient to obtain the product of the desired concentration before the dose is administered. Recombinant BON can also be supplied by other well-known methods, such as a pre-filled syringe, and so on.

Basically, there was no significant difference in the pharmacokinetics or biological activity of the natural recombinant sequence of BON, the recombinant Ν-methionyl-BON, or the substrate produced by the human pituitary gland (Mooge et al., 1988; 1dedei 88oi et al., 1988).

Given the various medical indications that use growth hormone, there is a need for an effective and reliable method of producing a significant amount of it in a cell culture, in particular a method in which a serum-free cell culture is used.

Serum-free medium has been described in the art.

For example, US Pat. No. 6,162,643 describes a serum-free basal medium called HECK-109, containing minor amounts of copper sulfate and zinc chloride. This medium is specifically designed for primary and secondary cultures of ordinary human cells, such as keratinocide, in order to generate tissue for human transplantation. The expression of recombinant human becks in the w νίΙΐΌ cell line is not mentioned in this US patent.

US Pat. No. 5,324,656 describes a serum-free basal medium, the so-called MSEV120 and MSEV131M, containing minor amounts of copper sulfate and zinc chloride. This medium is specifically designed for the cultivation of human muscle satellite cells in order to prevent differentiation of these cells. The production of recombinant human proteins is not provided for in this document.

Document OV 2196348 describes a synthetic medium for the cultivation of w νίΙΐΌ cells of hybridoma and myeloma. The medium contains copper, zinc and iron ions. The cultivation of hybridoma and myeloma cells in this medium is carried out exclusively with the aim of obtaining monoclonal antibodies.

US Pat. No. 6,103,529 proposes a composition for culturing w νίΙΐΌ cells of animal origin in a serum-free cell culture medium. Cells of animal origin can be used to produce viruses, monoclonal antibodies, hormones, or growth factors. However, the production of growth hormone is not provided in this document.

US Pat. No. 6,048,728 describes a protein-free cell culture medium, including Co-, Ζιτ - and Re-ions for culturing animal cells, in order to obtain, for example, natural or recombinant products, such as antibodies. The production of such products through cultured cells does not include growth hormones or growth factors, which are referred solely as constituents of serum-free medium, to increase cell growth in culture.

US Pat. No. 5,316,938 describes a biochemically characterized culture medium for Chinese Hamster Ovary (CHO) cells containing iron citrate, zinc sulfate and copper sulfate and is called \ USM5. The medium is specifically designed for the production of antibodies and! RA in CHO cells.

- 4 011749

US Pat. No. 5,222,959 describes a method for producing recombinant proteins in a serum-free culture medium containing zinc and iron ions, particularly suitable for culturing CHO cells. The production of growth hormone was not provided for in this document.

Thus, the problem underlying the present invention is related to the provision of a serum-free cell culture medium for the efficient production of growth hormone, in particular human growth hormone (BON).

SUMMARY OF THE INVENTION

The present invention is based on the improvement of a medium for culturing cells that does not contain components derived from animal serum, and at the same time highly effective for cell growth and maintenance of mammalian cells in culture, in particular enabling the production of recombinant proteins.

Thus, in a first aspect, the invention relates to a cell culture medium containing no components derived from animal serum containing small amounts of zinc and / or copper. Preferably, the medium according to the invention in addition contains iron ions in small quantities.

In a second aspect, the invention relates to a method for producing human growth hormone, comprising the step of expressing a human growth hormone cultured in a cell in a medium according to the invention.

A third aspect of the invention relates to the use of a medium according to the invention for the production of human growth hormone.

A fourth aspect of the invention relates to the use of a medium according to the invention for maintaining cells in culture during the production phase of human growth hormone.

Brief Description of the Drawings

In FIG. Figure 1 shows the productivity profile of gBON obtained in the production phase by continuously adding various elements of 10 μM to the culture medium ΌΜΕΜ (WB = roller bottle, H1-H14 = days from 1 to 14 production phases).

In FIG. Figure 2 shows the average productivity of gBON, expressed in mg gBON per roller bottle, obtained in the production phase with the continuous addition of metals (nickel, barium, cobalt, chromium) of 10 μM in culture medium ΌΜΕΜ.

In FIG. Figure 3 shows the average values of gBON productivity, increased in comparison with the control, obtained in the factorial design of the experiment for testing combinations of zinc (Ζη 0.5 μM), copper (Cu 0.02 μM), selenium (8е 0.050 μM), manganese (Μη 0.001 μM) and iron citrate (4.8 μM).

In FIG. Figure 4 shows the results of the factorial design of the experiment to determine the result of mixing metallic microelements (Ζη 0.5 μM, Cu 0.02 μM and iron citrate 4.8 μM) and amino acids: glutamine (O1n 4.8 mm), serine (8eg 0.49 mM) and cysteine (Bitch 0.29 mM).

In FIG. Figure 5 shows the effect of various concentrations of copper, in combination with zinc and iron citrate (Ζη 0.5 μM, Re citrate 4.8 μM), on the productivity of gBON in ΌΜΕΜ.

In FIG. Figure 6 shows the effect of various concentrations of zinc in combination with copper and iron citrate (Cu 0.02 μM, Re citrate 4.8 μM) on the productivity of gBON in ΌΜΕΜ.

In FIG. 7 shows the total effect in relation to the increase in the productivity of gBON obtained by adding copper, zinc and iron citrate (Ζη 1.5 and 0.5 μM, Cu 0.02 μM and iron citrate 4.8 μM), as additives to ΌΜΕΜ per based on various experiments with gBON-producing C127 cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the improvement of a cell culture medium that does not contain animal serum.

According to the present invention, the cell culture medium not containing animal serum includes zinc (Ζη) in a concentration range from 0.2 to 1.75 μM, and / or copper (Cu) in a concentration range from 10 to 75 nM, and / or iron ions (Fe) in the concentration range from 3 to 10 μM.

As shown in the examples below, the addition of Ζη and / or Cu in small amounts to the standard medium leads to an increase in the productivity of cells expressing the secreted protein of interest.

The addition of both metals, and Ζη and Cu, in the above concentrations leads to an increase in the productivity of recombinant human growth hormone (OH) in C127 cells to more than 60% of the control (the same cells cultured in standard medium ΌΜΕΜ). When OH-expressing cells were cultured in a medium containing Ζη, Cu and Fe ions at the above concentrations, productivity increased to approximately 70% compared to the control.

Preferred concentration ranges of metal ions in the medium according to the invention are as follows.

Zinc approximately 0.2, 0.25, 0.30, 0.35, 0.40, 0.45, 0.5, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75 μM.

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Preferably, the zinc is approximately 0.5 or approximately 1.5 μM. It is also preferred that the zinc is included as zinc sulfate.

Copper is approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 nM.

Preferably, the copper is approximately 25 nM. It is also preferred that the copper is included as copper sulfate.

Iron ions are approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 4.8, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 μM.

Preferably, the iron ions are approximately 5 or 6 μM. Iron ions may preferably be included in the form of iron citrate and / or iron nitrate, and it is further preferred that iron citrate is considered to contain most of the iron ions in the cell culture medium. The medium may contain, for example, about 5 μM iron citrate and about 1 μM iron nitrate.

In a preferred embodiment, the medium further comprises components of the basic medium. Preferably, the medium contains Dulbecco's modified Eagle medium (ΌΜΕΜ) as the main medium. The composition of the standard medium ΌΜΕΜ is described in the example below.

A second aspect of the present invention relates to a method for producing a protein, comprising the step of culturing a cell expressing a protein of interest in a medium according to the invention.

The cultivation step of the method according to the invention can be carried out under any suitable external conditions, such as Petri dishes, T-flasks or roller bottles, but in the same way, the cultivation step can be carried out in a vessel having a larger volume, such as, for example, a bioreactor.

The protein of the invention can be expressed using any promoter that is suitable for a particular type of cultured cell. In a particularly preferred embodiment, the protein is expressed from a metallothionein (ΜΤ) promoter. If murine cells are used to produce protein, the MT-1 murine promoter is preferred as the promoter.

Preferably, the method further includes the step of collecting the medium containing the protein of interest.

In a preferred embodiment, the method further includes the step of isolating the protein of interest.

In another preferred embodiment, the method further includes formulating the isolated protein with a pharmaceutically acceptable carrier to formulate a pharmaceutical composition.

In a third aspect, the invention relates to the use of a medium according to the invention for the production of a polypeptide of interest.

In a fourth aspect, the invention relates to the use of a medium according to the invention for maintaining cells in culture during the production phase of a polypeptide of interest.

The cells used in various aspects of the present invention are preferably mammalian cells. They can be human cells or animal cells. Examples of mammalian cells that can be cultured by the method of the present invention include, for example, C127 mouse cells, 3T3 cells, CO8 cells, human osteosarcoma cells, ΜΒί .'-5 cells. BHK cells, UEVO cells, CHO cells (Chinese hamster ovary cells), HEK 293 cells, gNEK 293 cells, normal human fibroblast cells, stromal cells, hepatocytes or RE.C6 cells. Examples of hybridomas that can be cultured by the method of the present invention include, for example, клетки4.4 cells, 123A cells, 127 A cells, ΟΑΜΜΑ cells and 67-9-B cells.

Preferably, culturing C127 murine cells according to the present invention.

Cells cultured according to the present invention can grow in suspension or, in the case of attachment-dependent cells, attached to a solid base.

The microcarriers and disks B1Ba-Ce1® are used for the cultivation of mammalian cells for the growth of attached or anchored cells, and they are used, among the generally accepted technological platforms, for the industrial production of proteins (see, for example, Voiac et al., 1987; Res and et al. 1994).

The method is preferably suitable for the production of a polypeptide of interest. The medium of the invention is thus used to produce a polypeptide of interest or protein, which may be any polypeptide whose small-scale or large-scale production is desired.

The polypeptide of interest may be, for example, a naturally secreted protein, a conventional cytoplasmic protein, a conventional transmembrane protein, or a humanized or human antibody. When the protein of interest is a natural cytoplasmic or natural transmembrane protein, the protein is preferably genetically engineered to become soluble and secreted, i.e., by size

- 6 011749 signaling peptide in front of the protein or its fragment (soluble or extracellular).

The polypeptide of interest may be of any origin. Preferably, the polypeptides of interest are derived from the human body, and more preferably the proteins of interest have a therapeutic effect.

Preferably, the protein of interest is selected from a hormone, a cytokine-bound protein, interferon, a soluble receptor, or an antibody.

Proteins with a therapeutic effect that can be produced by the method according to the invention include, for example, chorionic gonadotropin, follicle-stimulating hormone, lutropin-choriogonadotropin hormone, thyroid-stimulating hormone, growth hormone, in particular human growth hormone, interferons (e.g. interferon beta-1a , interferon beta-1b), interferon receptors (for example, the gamma interferon receptor), β-receptors p55 and p75 and their soluble variants, TAC1 receptor and its Unbound proteins, interleukins (e.g. interleukin-2, inter neukin-11), interleukin-binding proteins (e.g., interleukin-18-binding protein), anti-SP11a antibodies, erythropoietin, colony-stimulating granulocyte factor, colony-stimulating granulocyte macrophage factor, peptide hormones of the pituitary gland, menopausal gonadotropin, somatoninodin, ), keratinocyte growth factor, obtained from the glial cell line, neurotrophic factor, thrombomodulin, the main fibroblast growth factor, insulin, factor VIII, somatropin, morphogenetic protein-2 ti, platelet derived growth factor, hirudin, epoietin, recombinant fusion protein BA-3 / 1dS1, glucocerebrosidase, and muteins, fragments, soluble forms, functional derivatives, fusion proteins thereof.

In preferred embodiments, the polypeptide is selected from the group consisting of chorionic gonadotropin (CO), follicle-stimulating hormone (BBN), lutropin-choriogonadotropin hormone (BH), thyroid-stimulating hormone (TBN), human growth hormone (UN), interferon (e.g., interferon b 1a, interferon beta-1b), interferon receptors (e.g. gamma interferon receptor), ΤNB receptors p55 and p75, interleukins (e.g. interleukin-2, interleukin-11), interleukin-binding proteins (e.g., interleukin-18 binding protein), anti-s 11aantitel and muteins, fragments, soluble forms, functional derivatives, fusion proteins.

Further preferred polypeptides of interest include, for example, erythropoietin, colony stimulating factor granulocytes, colony stimulating factor granulocyte macrophages, peptide hormones of the pituitary gland, menopausal gonadotropin, insulin-like growth factors (e.g. somatomedin-C), keratinocyte growth factor cell neurotrophic factor, thrombomodulin, the main fibroblast growth factor, insulin, factor VIII, somatropin, bone morphogenetic protein-2, thrombotic growth factor itov, hirudin, epoietin, recombinant fusion protein BA-3LdS1, glucocerebrosidase, and muteins, fragments, soluble forms, functional derivatives, fusion proteins thereof.

If a formulation of the protein of interest produced by the method of the invention with a pharmaceutically acceptable carrier is developed, the result of the process will be a pharmaceutical composition.

A pharmaceutically acceptable formulation is intended to include any carrier that does not affect the effectiveness of the biological activity of the active ingredient and which is not toxic to the host organism to which the drug is administered. For parenteral administration, for example, the active protein (s) may be enclosed in a unit dosage form for injection, in combination with a carrier such as saline, dextrose solution, serum albumin and Ringer's solution.

The pharmaceutical composition according to the invention can then be administered to an individual in various ways. Methods of administration include intradermal, transdermal (e.g., slow administration of the composition), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, intracranial, epidural, local, rectal and intranasal methods. Any other therapeutically effective route of administration can be used, for example, absorption through epithelial or endothelial tissues, or using gene therapy, in which a DNA molecule encoding an active agent (for example, using a vector) is injected into the patient, resulting in the active agent expressed and secreted ίη νίνο. In addition, the protein (s) of the invention can be administered together with other components of biologically active agents, such as pharmaceutically acceptable surfactants, excipients, carriers, solvents and drug media.

For parenteral administration (e.g., intravenous, subcutaneous, intramuscular), the active protein (s) must be formulated as a solution, suspension, emulsion or lyophilized powder, together with a pharmaceutically acceptable parenteral excipient (e.g. water, saline, dextrose solution) and additives that maintain isotonicity (e.g., mannitol) or chemical stability (e.g., preservatives and buffers). The composition is sterilized using commonly used techniques.

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According to the present invention, considerable preference is given to using the medium according to the invention for the production of growth hormone.

CH can be native, i.e. CH of natural origin. Preferably, the HF to be produced is a human hormone. Since CH is a soluble secreted protein, it is released into the cell culture supernatant either through its natural signal peptide or through a heterologous signal peptide, i.e., a signal peptide derived from another secreted protein, which may be more effective in the particular expression system used .

The term growth hormone is used herein as a synonym for the term CH. This term includes natural or native CH, recombinantly produced CH, as well as various CH, described in detail in the section `` Fundamentals of the invention. The term `` CH ', as used herein, further includes muteins, functional derivatives, active fractions, fusion proteins, circularly permuted proteins and CH salts. CH is preferably a human growth hormone, but may also be of a different variety, in particular mammalian growth hormone.

As used herein, the term muteins refers to analogues of CH in which one or more amino acid residues of natural CH are replaced or removed, or one or more amino acid residues are added to the natural sequence of CH, without significantly reducing the activity of the resulting products compared to wild CH type. These muteins are obtained by known synthesis and / or by the site-specific mutagenesis technique, or by any other suitable technique.

Muteins in accordance with the present invention include proteins encoded by a nucleic acid, such as DNA or RNA, which under stringent conditions hybridize with DNA or RNA encoding CH. DNAs encoding HF are known in the art. The term stringent conditions refers to hybridization and sequential flushing conditions, which the specialist in this field traditionally refers to stringent. See Li8iLe1 et al., Stews! ArgoCoil ίη Mo1ci1ag Βίo1odu, above, 1n1eg5C1epse. Ν.Υ., § 6.3 and 6.4 (1987, 1992). Not limited to these examples, stringent conditions include washing conditions at 12-20 ° C below the calculated TT of the hybrid when studied, for example, in 2x88C and 0.5% 8I8 for 5 min, 2x88C and 0.1% 8I8 for 15 min; 0.1x88C and 0.5% 8I8 at 37 ° C for 30-60 minutes and then 0.1x88C and 0.5% 8I8 at 68 ° C for 30-60 minutes. One skilled in the art will recognize that stringent conditions also depend on the length of the DNA sequences, oligonucleotide probes (for example, 10-40 bases) or a mixture of oligonucleotide probes. If you use a mixture of probes, it is preferable to use tetramethylchloridammonium (TMAC) instead of 88C. See Ai8iBe1, above.

Identity determined by sequence comparison reflects the relationship between two or more polypeptide sequences or two or more polynucleotide sequences. In general, identity is based on the exact correspondence of the nucleotide to the nucleotide or amino acid for the amino acid of two polynucleotides or two polypeptide sequences, respectively, along the entire length of the compared sequences.

For sequences between which there is no exact match,% identity can be determined. Typically, the two sequences to be compared are aligned to maximize the correlation between the sequences. This method may include inserting gaps in any, one or both sequences, increasing the degree of alignment. % identity can be determined along the entire length of each of the sequences that were compared (the so-called global alignment), which, in particular, is convenient for sequences of the same or very similar length, or shorter than the determined length (the so-called local alignment), which is more suitable for sequences of unequal length.

Methods for comparing the identity and homology of two or more sequences are well known in the art. So, for example, the programs at the disposal of \ U15sop5sh 8ec. | Iepse Apa1u§18 Raskade, version 9.1 (Jeuegeih et al., 1984), for example, the ΒΕ8ΤΕΙΤ and САР programs, can be used to determine% identity between two polynucleotides and% identity and% homology between two polypeptide sequences. The ΒΕ8 программе program uses the local homology algorithm 8π11 and \ Wa1eppap (1981), and it best detects a single region of similarity between two sequences. Other programs for determining the identity and / or similarity between sequences are also known in the art, for example, the BAB8T family of programs (AYUSYY 8.E. et al., 1990, A11xNi1 8.E. et al., 1997, available on the NCI homepage on \\ l \ lu.psy.p1t.sh11.dow) and EA8TA (Reagkop HUL., 1990).

Any such mutein preferably has an amino acid sequence that sufficiently repeats the sequence of CH so that it has mainly activity similar to CH. One type of CH activity is its ability to bind to the CH receptor. As long as mutein has significant binding activity to the CH receptor (START), it can consider

- 01 01749 as having substantially similar OH activity. Thus, it is possible to determine whether any given mutein has substantially similar OH activity through standard experiments, including, for example, a simple competitive sandwich assay of such a mutein, to determine whether it binds to appropriately labeled START or cells expressing START, an analysis such as radioimmunological analysis or EHBA.

In a preferred embodiment, any such mutein has at least 40% identity or homology with the amino acid or DNA sequence of CH. Such sequences are known in the art, for example, ΩοΝοΙο et al., 1981 or Magya 1 et al., 1979.

More preferably, it has at least 50%, at least 60%, at least 70%, at least 80%, or most preferably at least 90 or 95% identity or homology with the amino acid or DNA sequence of CH.

Preferred substitutions for the muteins of the present invention are those that are known as conservative substitutions. Conservative amino acid substitutions of CH polypeptides can include synonymous amino acids within a group that has fairly similar physicochemical properties, so that the biological function of the molecule is preserved during substitutions between representatives within the group (Cgapyash, 1974). It is clear that insertions and deletions of amino acids can also occur in the sequences defined above without changing their function, in particular if the insertions or deletions consist of only a few amino acids, for example less than thirty, and preferably less than ten, and amino acids that are not removed or not replaced necessary for functional conformation, for example, cysteine residues. Proteins and muteins produced by such deletions and / or insertions are included in the scope of the present invention.

The term fusion protein refers to a polypeptide containing CH, or a mutein, or a fragment thereof, fused to another protein, which, for example, has a long residence time in the body’s liquid medium. CH can thus be fused to another protein, polypeptide or the like, for example, an immunoglobulin or a fragment thereof. Its GdC sites are suitable for the production of fusion proteins with immunoglobulin. Proteins fused with HD are described, for example, in the patents EP 314 317 A1 (Sieie1esy) or EP 0325224 A2 (2 archives! 1C8 1ps.).

Under the active fractions of CH or muteins and fusion proteins, the present invention provides for any fragment or precursors of the polypeptide chain of a protein molecule, alone or together with associated molecules or associated residues, such as sugar or phosphate residues, or protein molecule aggregates or sugar residues per se determining the specified fraction have essentially similar activity with CH.

If the HF according to the invention is used as a pharmaceutical composition, such a pharmaceutical composition can be used to treat and / or prevent a number of diseases or disorders. Such diseases or disorders are preferably associated with insufficient endogenous production of HF. Purified HF can be used, for example, to treat and / or prevent HF deficiency, AIDS depletion, lipodystrophy (also called NAVB-NSU syndrome associated dystrophy / dysmetabolism), or a shortened small bowel syndrome, in particular in pediatrics. In addition, diseases in which the administration of growth hormone may be indicated include liver cirrhosis, adult growth failure, atherosclerosis, Crohn's disease and ulcerative colitis, osteoarthritis, cardiac cachexia, congestive heart failure, chronic renal failure, recovery or mobilization blood cells, male infertility, hematopoietic stem cell mobilization, multiple sclerosis, stroke, multiple systemic atrophy or cancer.

Having now a complete description of the invention, one skilled in the art will appreciate that such a thing can be reproduced within a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without undue experimentation.

Although this invention is described in connection with its specific embodiments, it should be understood that other modifications are possible. The present patent is intended to include any variations, applications or adaptations of the invention, generally following the principles of the present invention and including such derogations from the present description that fall under normal practice within the scope of the invention, and which can be used in relation to the main features formulated above in this document, to the extent covered by the attached claims.

All references cited herein, including journal articles or synopsis published or unpublished in the United States, or a foreign patent application published in the United States, or foreign patents, or any other references are fully incorporated into this description by reference, including all data , tables, figures and text presented in these links. In addition, the full contents of the references referred to in the documents cited in the context of the references cited in this document are also fully incorporated into the present description by reference.

- 9 011749

Reference to known method steps, traditionally used process steps, known methods or conventionally used methods does not in any way mean that any aspect, description or embodiment of the present invention has already been disclosed, described or recommended in the relevant field.

The above description of specific embodiments should thoroughly disclose the general nature of the invention so that others can, using knowledge within this field (including the contents of the references cited in this document), easily modify and / or adapt these specific embodiments for various applications without undue experimentation without deviating from the basic concept of the present invention. Therefore, such adaptations and modifications are contemplated within the scope of equivalent embodiments disclosed based on the concepts and methods presented herein. It should be clear that the phraseology or terminology of this document is used to describe, and not limit, the invention, so that the terminology or phraseology of the present description should be interpreted by a specialist in this field only as a guide that, combined with the skills of an ordinary specialist in this field will allow to realize the invention.

Example. Improvement of a new serum-free production medium for C127 cells expressing 11CH.

1. Introduction.

This study describes the experimental work related to the improvement of the producing culture medium (ΌΜΕΜ, Dulbecco's modified Eagle medium) through the addition of trace amounts of metal elements. As a result of this improvement, a striking increase in the productivity of g-CHF in a C127 cell culture was obtained.

In the following table. Figure 1 shows the cumulative effect of composition ΌΜΕΜ and the enhancement demonstrated as part of this example.

Table 1

The composition of the growth medium, ΌΜΕΜ and production medium

Components Growth medium mg / L EMEM, mg / l Production medium mg / L amino acids B-alanine 49.45 B-arginine 227.5 84.0 84, 0 L-ASPARAGIN N ₂ O 27,50 B-aspartate 21.65 Kommersant НС1 Н ₂ О 17.56 B-cystine 2ns1 31.29 62.0 62.0 GLUTAMATE 40.35 B-glutamine 693.5 584.0 584.0 GLYCINE 43.75 30,0 30, 0 B-Histidine HC1, H ₂ O 41.48 42.0 42.0 B-hydroxyproline 6, 50 B-isoleucine 114.5 105.0 105.0 B leucine 119.1 105.0 105.0 D-LYSIN-NS1 151.3 146.0 146.0 B-methionine 52.24 30,0 30,0 B-phenylalanine 70.48 66, 0 66.0 B-proline 37.25 B-serine 39.25 42.0 42.0 B-threonine 93, 45 95.0 95.0 B-tryptophan 15.02 16,0 16,0 L-TYR03IN 2Ha 2H ₂ O 75.79 104.0 104.0 B-balin 92, 85 93, 0 93.0 SALTS CALCIUM CHLORIDE 121.0 200 200 Ge (IOz) s-9H ₂ O 0,050 0.1 0.1 IRON SULPHATE 7Н ₂ О 1,251 KC1 334.2 400 400 MAGNESIUM CHLORIDE 40.94 MDZOA ANHYDRID 48.84 97 97

- 10 011749

Ma ₂ NRA " 71.02 SODIUM SELENIUM 5Н ₂ О 0,030 ΝθΌΙ 7500 6400 6400 Jan ₂ RO ₄ H ₂ O 62.5 125 125 ΝβΗΟΟ ₃ 1200 3700 3700 Lithium CHLORIDE 1.00 Ζη3Ο ₄ · 7Η ₂ Ο 0.432 0.144 CARBOHYDRATES Ό-Glucose 3650 4500 4500 VITAMINS VITAMIN C 0,500 BIOTIN 0.1035 CHOLINE CHLORIDE 11.48 4.00 4.00 VITAMIN B12 1, 68 CA Pantothenate 2,34 4:00 4.00 FOLIC ACID 3, 65 4:00 4.00 1-INOZIT 17.1 7.00 7.00 NICOTINAMID 2.27 4.00 4:00 PARA AMINO BENBENIC ACID one hundred PIRIDOXAL-NS1 0.25 4.00 4.00 Pyridoxine HC1 4,531 RIBOFLAVIN 0.419 0, 40 0.40 Thiamine HC1 2.92 4:00 4.00 LIPIDS LINOIC ACID 0,042 LITTLE ACID 0.205 METILLINOLEATE 0.1 OTHER IRON CITRATE 122.5 1.22 HEPES 3600 Hypoxanthine 2, 900 IRON (II) ά-Gluconate Dihydrate 4,820 B-glutathione 0,500 ME RKAPTOETHANOL 0.234 Pyruvate Sodium 57.00 110, 0 110.0 PUTRESCIN, 2CH1 0, 241 Thymidine 0, 505 DETERGENTS Ethanolamine (as base) 6.25 Micronutrients SILVER CHLORIDE 0.0000044 BARIUM CHLORIDE 2H ₂ O 0, 002 COBALT CHLORIDE 6H ₂ O 0, 002 CHROMIUM SULPHATE POTASSIUM 0, 001 POTASSIUM BROMIDE 0.0001 POTENTIUM IODIDE 0.0001 MOLYBDENIC ACID (Ammonium molybdate 4Ngo) 0.0001 SODIUM FLUORIDE 0, 004 METAVANDATE AMMONIA 0,0006 NICKEL NITRATE 6H ₂ O 0,0002 RUBIDIUM CHLORIDE 0.00001 TIN Dichloride 2Nzo 0.0001 COPPER SULFATE 5H ₂ O 0.0064 0.0064 MAGNESIUM CHLORIDE 4H ₂ O 0.0001 TITANIUM OXIDE 0.001 ADDITIONS RECOMBINANT HUMAN INSULIN 10 mg / l PHYSICAL INSTALLATIONS pH 7.1 + 0.1 7.0 + 0.2 7.0 ± 0.2 OSMOTIC CONCENTRATION (tozt / kg) 325 + 25 330 + 30 330 ± 30

- 11 011749

2. Materials and methods.

Reagents and solutions.

All chemicals were obtained from Megsk®: zinc sulfate (Ζη8Ο ₄ · 7Η ₂ Ο), copper sulfate (Cu8О ₄ -5Н ₂ О), barium chloride (ВаС1у2Н ₂ О), cobalt chloride (СС1у6Н ₂ О), potassium chromosulfate ( K [Cr (8ObH ₄ ) ₂ (H ₂ O) ₂ ph6H ₂ O), nickel nitrate (No. (IO ₃ ) y6 H ₂ O), sodium selenite (Ia ₂ 8eO ₃ ^ 5H ₂ O).

Iron citrate (FeC ₆ H ₅ O ₇ 81 dta®, catalog number P3388) was used as a source of iron.

A mixture of microelements for the growth medium (100000X) was provided by DYAN® Vyukaepsek.

All solutions of trace elements for the experiments were prepared in the form of concentrated solutions in distilled water and sterilized by filtration through a 0.2 μm filter.

The addition of various analyzed additives (metal solutions, etc.) in different experiments was carried out directly in a fresh culture medium.

Cell culture.

Genetically modified murine C127 cells (ATCC CAB 1616) were used to express recombinant human growth hormone (gHCH). The vector is based on BPU69T containing the plurally cloned pBYA322 site and containing the 1.6 kb mini-gene gSN. under the control of the mouse promoter metallothionein-1 (MT-1). The cultures used in the experiments were obtained in Khuogishche Ce11 Vapk from different series producing hkSN.

The cell culture was incubated at 36 ± 0.5 ° C and 0.4 rpm in 2125 cm roller bottles containing 375 ± 15 ml of culture medium, or in 1700 cm ² roller bottles containing 300 ml of culture medium.

The cultural environment.

The culture medium used for the gFH production phase was EMEM with 4.5 g / L glucose buffered with sodium bicarbonate (3.7 g / L).

Titration

Measurement of hBS in the culture medium was performed daily by reverse phase titration of HBS.

Materials

8ups11gorak YaR4, 100x4.6 mm inner diameter, 30A Ca !. No. С4Я103-10 (Ekkgot).

Yaekoigs YaRS 1 ml, 30x6.4 mm inner diameter, 15 microns, art. 17-1181-01, LtegSat Vyukaepsek.

8utte! Gu 300, 50x4.6 mm inner diameter, C4 5 μm, P / Ν 186000287 (\ Wa! Eg5).

Reagents

Trifluoroacetic acid (TFU) (P1egse, Ca !. No. 28904 or equivalent).

Acetonitrile (ASI) (Megsk 1,00030 or equivalent).

Purified water, P \ ¥ (for example, water M1Shr, or equivalent).

Helium.

Solutions.

Mobile phase A: TFA 0.08% in H ₂ O (v / v).

To the required amount of water volume P \ U in a graduated flask was added TFA according to the following table. Shook and affixed the label.

VOLUME OF PHASE A VOLUME OF WATER MGYO VOLUME OF TFU 1 liter 1 liter 0.8 ml 0.5 l 0.5 l 0.4 ml 0.25 L 0.25 L 0.2 ml

Mobile phase B: TFA 0.08% in acetonitrile (v / v).

To the required amount of volume P \ ¥ water in a volumetric flask was added TFA, according to the following table. Shook and affixed the label.

VOLUME OF PHASE IN VOLUME ΆΟΝ VOLUME OF TFU 1 liter 1 liter 0.8 ml 0.5 l 0,5l 0.4 ml 0.25 L 0.25 L 0.2 ml

Chromatography parameters.

Gradient elution: first with a mixture of phase A / phase B 60/40, at the end phase A / phase B 20/80. The gradient is completed in 5 minutes (small variations depend on the equipment used).

The injection volume is 50 μl.

Registration: UV absorption at 215 nm.

Calibration curve: g-LCH standard sample at 10, 50, 100, 120 and 150 μg / ml.

The concentration of the g-LCH sample was determined by comparison with the concentration of the standard g-LCH sample.

Productivity CH.

Productivity is expressed as mg hSN per roller bottle. The approximate data are the concentration of hBS during cell collection (as measured by HPLC) and the total amount

- 12 011749 roller bottles when collecting cells. Typically, the roller bottle during the collection phase contains approximately 10 ⁹ cells.

3. Results.

In the first series of experiments, the effect of a high concentration of cobalt (20 μM CoC1 ₂ -6H ₂ O) was analyzed with periodic addition of it to the culture medium. The addition of cobalt was carried out during two medium changes at the initial stage (washing and ΡΜ = producing medium, i.e., 0 point of the collection phase) and in the intermediate stage (collections 6 and 7) of the production phase in one series. Results (not shown) indicate that the promoter is active and can be modulated.

The increase in productivity was further confirmed using other metal elements. The analysis was carried out, respectively, with barium (BaCl ₂ -2H ₂ O), cobalt (CoCl ₂ -6H ₂ O), chromium (K [Cr (8O ₆ H ₄ ) ₂ (H ₂ O) ₂ ] -6H ₂ O) and nickel (N1 (NO ₃ ) ₂ -6H ₂ O), at concentrations of 10 μM, continuously added continuously to the medium. The results of this experiment are depicted in FIG. 1 and 2.

In FIG. Figure 1 shows the amount of BON secreted by C127-expressing BON cells when cultured in a medium containing analyzed elements during the experiment (14 days). In FIG. 2 shows the average values of productivity achieved for each analyzed element. The obtained average productivity continues to increase the percentage compared with the control, i.e. without trace amounts of elements, between 1.1% for barium and 32.4% for cobalt. It was also noted that the productivity of gBON increases from 9 to 32.5% compared with the control value with a continuous method of introducing cobalt compared to periodic. Neither barium nor chromium caused an increase in productivity at the studied concentrations in ΌΜΕΜ. Measurements in the presence of trace amounts of individual elements ΌΜΕΜ were supplemented with trace elements Ζη, Cu, 8e, and Co at the concentrations presented in the table below. 2. These concentrations correspond to the concentrations of metal elements in the growth medium. The increase in GBON productivity achieved by adding ΌΜΕΜ trace amounts of elements is shown in Table. 2.

table 2

The increase in GBON productivity in percent when studying the effect of metal ions in ΌΜΕΜ at the indicated concentrations in μM

And he CONCENTRATION (μM) % INCREASED PRODUCTIVITY Ζη 1,50300 12.10% Si 0,02560 37.50% 5th 0.11400 -3.10% With 0,00840 -3.20%

It should be noted that in the case of zinc, an estimated concentration of 1.5 μM is the cause of the exfoliation phenomenon, i.e. separation of the cell monolayer from the plastic substrate of the roller bottles. In some cases, this peeling leads to a loss of culture in the last stages of cell collection (especially between cell harvests 12-14). The exfoliation phenomenon can be obtained by using appropriate cell culture vessels, such as Sotn®® roller bottles under the trademark Ce11btb®.

Measurement of the interaction between metals.

Based on the effect of copper and zinc on the level of production of BON, we conducted a plan of factor experiments to test the possible combined effect of metals.

Statistical analysis of the results is shown in table. 3. A persistent synergistic effect was observed with respect to productivity (p <0.0001) when using Cu and Ζη together.

Table 3

Statistical analyzes of a 3-factor plan of factor research

Productivity Change Analysis A source Sumy Squares About £ Average square G-coefficient P-magnitude A: copper 406, 143 one 406,143  350.74 0.0000 B: zinc 84,6308 one 84,6308 73.09 0.0000 C: c £ r1 water 0.626472 one 0.626472 0.54 0.4807 AB 47,7896 one 47,7896 41.27 0.0001 AC 2,49008 one 2,49008 2, 15 0.1766 sun 0.438906 one 0.438906 0.38 0.5534 Total error 10,4215 nine 1,15795 Total (corr.) 552,541 fifteen

B squared = 98.1139%.

B-square (adjusted for bg) = 96.8565%.

Exfoliation was observed in all cultures treated with zinc at a concentration of 1.5 μM, whether copper was present or not.

- 13 011749

Subsequent plans of factor experiments were carried out to study the combined effect of zinc and copper, when the zinc concentration was reduced (Ζη 0.5 μM) and other trace elements were added to the growth medium, such as magnesium, selenium or iron citrate, which can increase the productivity and adhesion of the cultures to the substrate .

Continuous growth continued from the overall starting point of productivity, approximately 20 mg tySN per bottle, with final values of about 70 mg tySN / KV, compared with 30 mg tySN in a control culture with standard ΌΜΕΜ (not shown). Average values of increasing CH productivity (i.e., productivity measured in mg / KB on day 14) compared to the control are shown in FIG. 3.

In these series of experiments, the phenomenon of exfoliation was reduced.

The addition of amino acids.

A study of the action of amino acids was carried out in order to determine whether the increase in 11CH productivity is limited by the presence of any amino acid. In the table. Figure 4 shows the percentage of cell harvest after two days of culture in a medium with and without zinc and copper.

Table 4 The percentage of amino acids in the untreated medium in the collection of cells after two days of cultivation, created from cultures supported by standard ΌΜΕΜ or ΌΜΕΜ, supplemented with Ζη 0.5 μm, Cu 0.02 μm and iron citrate 4.8 μm

AMINO ACID STANDARD βΜΕΜ ADDED ϋΜΕΜ B-glutamine 31% thirteen% B-serine 28% eleven% B-cystine twenty% thirteen% residual amino acids 50% -100% 39% -86%

A mixture of copper, zinc and iron citrate (Ζη 0.5 μM, Cu 0.02 μM and iron citrate 4.8 μM) was analyzed in combination with those amino acids that are consumed in the greatest amount: L-glutamine (C1.8 4.8 mmol) , B-serine (§ 0.49 mM) and b-cysteine (Bitch 0.29 mM). The results are shown in FIG. 4.

A positive effect on the productivity of any of the tested amino acids was not observed, either separately or in a mixture.

When metals were added together with amino acids, only a slight effect on productivity was observed: a slight positive effect from glutamine (from 78.2 to 81.1%) and a negative effect from cysteine (from 78.2 to 69.9%). Such differences were not considered significant.

Productivity undoubtedly differs in cultures treated with a mixture of metals in ΌΜΕΜ (average value of approximately 50 mg tySH / KV per cell collection) from cultures not containing metals (approximately 30 mg tySCH / KV per cell collection). Given these results, it was not considered necessary to modify the original amino acid composition of the producing medium.

The effect of titration of copper and zinc.

In order to measure the optimal concentration of copper, the experiment was carried out by keeping the concentration of iron citrate (4.8 μM) and the concentration of zinc (0.5 μM) constant and varying the concentration of copper (Fig. 5).

As shown in FIG. 5, the dose-response experiment confirmed that the optimal concentration of copper added in ΌΜΕΜ is 25.6 nM at the indicated concentrations of zinc and iron citrate.

In another experiment, the concentration of iron citrate (4.8 μM) and copper (0.025 μM) was kept constant and the zinc concentration was varied (Fig. 6): the maximum productivity level was observed (plateau) at zinc concentrations above 200 nM, productivity stabilization was observed at approximately 60% above the average control value.

Summary of Results

In FIG. 7 summarizes the results obtained with Ζη and Cu ions in insignificant concentrations together with or without iron ions.

The following values were obtained on the basis of data obtained by increasing productivity by adding trace amounts of metal elements to the medium ΌΜΕΜ:

1.5 μM zinc: 10.3 ± 5.0%, 0.5 μM zinc: 16.8 ± 1.6%, 0.02 μM copper: 32.1 ± 9.4%, 1.5 μM zinc + copper 0.02 μM: 66.3 ± 16%, zinc 0.5 μM + copper 0.02 μM: 61.2% (± 10%), zinc 0.5 μM + copper 0.02 μM + iron citrate 4.8 μM: 69.4 ± 19%.

Considering these data, the following mixture is optimal as an additive to the medium ΌΜΕΜ.

- 14 011749

Copper as copper sulfate (Cu8O ₄ -5H ₂ O) at 25 nM.

Zinc as zinc sulfate (Ζη8Ο ₄ · 7Η ₂ Ο), between 50 and 1500 nM with preferred concentrations of 200 to 500 nM.

Iron citrate at an ion concentration of 4.8 μM to a final concentration of approximately 6 μM, given that iron nitrate is already present in ΌΜΕΜ.

Conclusion

The use of insignificant amounts of copper, zinc, and iron ions as additives to the medium ΌΜΕΜ increases the efficiency of SRS by more than 50% compared to the standard ΌΜΕΜ.

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Claims (15)

CLAIM 1. A method of producing human growth hormone, comprising the step of culturing a cell line expressing growth hormone in a culture medium containing no components derived from animal serum, the medium containing zinc in a concentration in the range from 0.2 to 1.75 μM, copper in a concentration in the range of 10 to 75 nM and iron ions in a concentration in the range of 1 to 10 μM, where the cells are C127 mouse cells and the medium additionally contains components of the basic medium. 2. The method according to claim 1, where the medium contains zinc in a concentration of 0.2 μm. 3. The method according to any one of the preceding paragraphs, where the medium contains zinc in a concentration of 0.5 μm. 4. The method according to any one of the preceding paragraphs, where the medium contains zinc in the form of zinc sulfate. 5. The method according to any one of the preceding paragraphs, where the medium contains copper at a concentration of 25 nm. 6. The method according to any one of the preceding paragraphs, where the medium contains copper in the form of copper sulfate. 7. The method according to any one of the preceding paragraphs, where the medium contains iron ions in a concentration of 5 or 6 μm. 8. The method according to any one of the preceding paragraphs, where the medium contains iron ions in the form of iron citrate and / or iron nitrate. 9. The method according to any one of the preceding paragraphs, where the main environment is a modified Dulbecco environment Needle (ΌΜΕΜ). 10. The method according to any one of the preceding paragraphs, where the human growth hormone is expressed under the control of the metallothionein (MT) promoter. 11. The method of claim 10, where the metallothionein promoter is a murine MT-1 promoter. 12. The method according to any one of the preceding paragraphs, further comprising the step of isolating human growth hormone from the cell culture. 13. The method of claim 12, further comprising the step of purifying human growth hormone. 14. The use of the medium, as defined in any one of claims 1 to 9, for the production of human growth hormone. 15. The use of the medium, as defined in any one of claims 1 to 9, for maintaining cells in culture during the phase of production of human growth hormone.