EA043293B1 - METHOD FOR PRODUCING RECOMBINANT ADAMTS13 IN CELL CULTURE - Google Patents

Description

Cross references to applications

The invention claims priority under U.S. Provisional Application No.

61/362635, filed July 8, 2010, the description of which is incorporated herein by reference in its entirety for any purpose.

Technical field

Recombinant expression of therapeutic proteins in cell culture (in particular, large scale cell cultures), including eukaryotic cell cultures, and more specifically mammalian cell cultures, requires the use of special culture media that provide nutrients for efficient cell growth. Cell culture media formulations often include a variety of additives, including fetal bovine serum (FBS), animal proteins and/or bovine protein hydrolysates, and protein hydrolysates derived from plants or yeasts. One of the problems associated with such cultures is that the amount of protein produced, as well as the total and specific activity of said protein, often vary between cell cultures, even when the composition of the cell culture medium is unchanged. This variability is especially evident in the case of large-scale production processes using cell culture volumes from 10 L to more than 20,000 L. Variability between cell cultures is especially often observed for cell culture media containing hydrolysates, which leads to a decrease in the production of the total amount of protein produced, as well as a decrease in total and specific activity.

One possible reason for the variability observed across cell cultures is that contaminants in additives such as hydrolysates vary from batch to batch. In general, serum or serum-derived substances, such as, for example, albumin, transferrin or insulin, may contain undesirable agents that may contaminate cell cultures and biological products derived therefrom. In addition, human serum-derived supplements must be tested for all known viruses, including hepatitis and HIV viruses, which can be transmitted through blood serum. Moreover, bovine serum and products derived from it carry the risk of TGE contamination. In addition, all whey-derived products may be contaminated with unknown substances. Numerous problems (e.g., varying quality and properties of formulations from different batches and the risk of contamination with mycoplasma, viruses, or CGE) arise when human or animal-derived sera or protein supplements are used for cell culture, in particular when these cells are used for the production of drugs. agents or vaccines for human administration. Therefore, many attempts have been made to obtain an efficient host system and culture conditions that do not require serum or other protein substances of animal origin.

Such serum-free media have been developed from protein extracts obtained from plants or yeasts. For example, soy hydrolysates are known to be suitable for fermentation processes and can enhance the growth of many nutrient complex microorganisms, yeasts and fungi. WO 96/26266 states that papain hydrolysates of soy flour are a source of carbohydrates and nitrogen, and many of its components can be used for tissue culture. Franek et al. (Biotechnology Progress (2000) 16, 688-692) describe the growth and productivity promoting effects of certain peptide fractions of soybean and wheat hydrolysates.

WO 96/15231 describes a serum-free medium, consisting of a synthetic minimal nutrient medium and a yeast extract, for the propagation of vertebrate cells and the process of reproduction of viruses. The composition of a medium consisting of a basic cell culture medium containing rice peptide, yeast extract and enzymatic hydrolysate thereof, and/or plant lipids for animal cell growth is described in WO 98/15614. A recombinant cell culture medium containing purified soy hydrolysate is described in WO 01/23527. WO 00/03000 describes a medium that contains soy hydrolyzate and yeast extract, but also requires the presence of recombinant forms of animal proteins such as growth factors.

EP-A-0 481791 describes a biochemically formulated culture medium for culturing engineered CHO cells, free of proteins, lipids and carbohydrates isolated from animal sources, also containing recombinant insulin or an insulin analog, 1% to 0.025% (w/v ) papain-cleaved soy peptone, and putrescine. WO 98/08934 describes a serum-free culture of eukaryotic cells containing hydrolyzed soy peptides (1-1000 mg/l), 0.01 to 1 mg/l putrescine, and various components of animal origin, including albumin, fetuin, various hormones and other proteins. . In this context, it should be noted that putrescine is also known to be included in standard media, for example DMEM/Ham F12 at a concentration of 0.08 mg/l.

Vegetable and/or yeast hydrolysates, however, are undefined mixtures of oligopeptides and other unknown components and contaminants. At the same time, the properties of commercially available batches of hydrolysates vary significantly. As a result, the yield of recombinant proteins or viral products varies considerably (up to a factor of three) depending on the batch of hydrolyzate used (batch-to-batch variation). This deficiency affects cell proliferation as well as protein expression in each cell. US 2007/0212770 describes various animal protein and oligopeptide-free, chemically defined culture media suitable for large-scale production of recombinant protein biopharmaceuticals.

Hemostasis involves the interaction of various pathways of hemostatic reactions, ultimately leading to the formation of a thrombus. Thrombi are deposits of blood components on the surface of vessel walls, consisting mainly of aggregated blood platelets and insoluble cross-linked fibrin. Fibrin formation occurs as a result of inhibition of fibrinogen proteolysis by the action of thrombin, a coagulation enzyme. Thrombin is the end product of a coagulation cascade, sequential zymogen activation on the surface of activated platelets and leukocytes, and various vascular cells (for reference, see K. G. Mann et al., Blood, 1990, Vol. 76, pp. 1-16).

An important function of the coagulation cascade is the activation of factor X by a complex of activated factor IX (factor IXa) and activated factor VIII (factor VIIIa). Deficiency or dysfunction of the components of this complex is associated with the blood disease known as hemophilia (J.E. Sadler & E.W. Davie: Hemophilia A, Hemophilia B, and von Willebrand's Disease), in G. Stamatoyannopoulos et al. ( Eds.): The molecular basis of blood diseases (W. B. Saunders Co., Philadelphia, 1987, pp. 576-602). Hemophilia A is associated with a deficiency in factor VIII activity, while hemophilia B is associated with a deficiency in factor IX. The current treatment is replacement therapy using pharmaceutical preparations containing a normal coagulation factor. Of these thrombopathies, hemophilia A is the most common, affecting about one in 10,000 people. Replacement therapy in patients with hemophilia A involves repeated administration of drugs containing normal factor VIII by intravenous infusion. The interval between infusions is a function of the decrease in the activity of factor VIII in the bloodstream. The half-life of factor VIII activity after infusion varies between individuals, ranging from 10 to 30 hours. Thus, prophylactic therapy requires infusion every 2-3 days. This is a heavy burden for patients with hemophilia, in particular in cases where venous access is difficult as a result of local carbonation after frequent punctures with an intravenous infusion needle.

Reducing the frequency of infusion through the use of factor VIII with an extended half-life would be very beneficial. It is well known in the art that the half-life of an unactivated factor VIII heterodimer is highly dependent on the presence of von Willebrand factor, which exhibits high affinity for factor VIII (but not factor Villa) and serves as a carrier protein (J.E. Sadler and E.W. Davie: Hemophilia A, Hemophilia B and von Willebrand's disease, in G. Stamatoynnopoulos et al (Eds.): The molecular basis of blood diseases, W. B. Saunders Co., Philadelphia, 1987, pp. 576-602). It is known that patients suffering from von Willebrand disease type 3 who do not have a detectable level of von Willebrand factor in the bloodstream also suffer from secondary deficiency of factor VIII. In addition, the half-life of intravenously administered factor VIII in these patients is 2 to 4 hours, which is significantly less than the 10 to 30 hours observed in patients with hemophilia A. From the results obtained, it follows that factor VIII tends to be rapidly eliminated from the bloodstream, and that this process is inhibited to some extent by complexation with its natural carrier, the von Willebrand factor.

Von Willebrand factor (vWF) is a glycoprotein that circulates in plasma as a series of multimers that typically range in size from about 500 to 20,000 kDa (or 2-40 vWF dimers). The dimeric and multimeric forms of vWF are composed of 250 kDa polypeptide subunits linked to each other by disulfide bonds. vWF mediates the primary adhesion of platelets to the subendothelium of the injured vessel wall; only larger multimers also exhibit haemostatic activity. The multimerized VWF binds to the platelet surface glycoprotein Gp1ba through an interaction in the A1 domain of the VWF, promoting platelet adhesion. It is assumed that endothelial cells secrete large polymeric forms of vWF, and those forms of vWF that have low molecular weights (low molecular weight vWF) arise due to proteolytic cleavage. Multimers with large molecular weights accumulate in the Weibel-Palade bodies of endothelial cells and are released upon stimulation.

Decreased FVIII binding activity due to reduced vWF protein levels or reduced FVIII binding capacity results in one of three types of von Willebrand disease. Additionally or alternatively, certain types of von Willebrand disease are characterized by an increase or decrease in the level of Gp1bα-mediated platelet binding, namely types 2A, 2B and 2M (for a summary, see Castaman et al., Disorders of Hemostasis 88(1):94-108 ( 2003)). Accordingly, modulating vWF interaction with both FVIII and Gp1ba represents a viable strategy for the treatment of both hemophilia and von Willebrand disease.

Given the biological importance of vWF, there is a continuing need to improve the techniques for obtaining vWF for therapeutic use. It is well known that vWF can be isolated from endogenous sources such as human plasma. The isolated vWF has the advantage of high specific activity with respect to its biological function and can thus be effectively used as a therapeutic protein for the treatment of related diseases such as von Willebrand's disease. Plasma vWF typically has a specific ristocetin activity of approximately 100 mU/μg, however, isolation from human plasma has disadvantages as, for example, such plasma may contain various viruses such as HIV and/or hepatitis viruses that can be transmitted patient. In addition, plasma is a limited resource and thus plasma deficiency can make it difficult to obtain enough vWF for treatment. Accordingly, recombinant methods for producing vWF are advantageous in overcoming some of the problems associated with reliance on plasma as a source of vWF. For recombinant production was cloned full-length cDNA vWF; said propolypeptide corresponds to amino acid residues 23-764 of full-length prepro-vWF (Eikenboom et al. (1995) Haemophilia 1, 77-90).

Unfortunately, vWF is a molecule subject to complex post-translational modifications. In addition, multimerization of vWF dimers into large and ultra-large multimers in the Golgi apparatus is a challenge when expressed in mammalian cells. Thus, the expression of high molecular weight vWF in cell culture, for example, human endothelial cells (primary), depends on the specific accumulation of ultra-large vWF molecules in Weibel Palade bodies. Such cell cultures are not suitable for the production of therapeutic proteins. Other cell culture methods have also been described; it is known that cell culture conditions can affect the production of vWF in various ways. For example, high concentrations of ammonium (NH4+) have been shown to disrupt post-translational modification. Mayadas et al. (J. Biol. Chem., 264(23):13498-13503, 1989) showed that ammonium levels of 25 mM led to a decrease in vWF multimerization in endothelial cells, which also negatively affected the specific ristocetin activity of recombinant vWF. The decrease in multimerization is usually associated with a decrease in the activity, in particular, the specific ristocetin activity, of recombinant vWF.

It is still difficult to predict which parameters can positively or negatively affect the production of a particular protein, especially complex glycoproteins such as factor VIII and vWF. For example, certain components of the cell culture medium have been shown to affect the production of factor VIII. According to the description in US patent No. 5804420 the addition of polyol, copper and other trace metals can positively affect the yield of factor VIII. Also, as described in WO 2009/086309, it is shown that the use of copper in the process of culturing cells increases the production of factor VIII. Mignot et al. (1989) also describes how vWF expression occurs in recombinant CHO cells. However, none of these examples contain information regarding the specific activity of vWF or its level of multimerization.

The ADAMTS (disintegrin and metalloproteinase with type I thrombospondin motifs) proteins are a family of metalloproteinases containing a number of conserved domains, including a zinc-dependent catalytic domain, a cysteine-rich domain, a disintegrin-like domain, and at least one, and in most cases many, thrombospondin-type repeats. I (for reference, see Nicholson et al., BMC Evol Biol. 2005 Feb 4;5(1):11). These proteins, evolutionarily related to the ADAM and MMP families of metalloproteinases (Jones GC, Curr Pharm Biotechnol. 2006 Feb;7(1):25-31), are secreted enzymes that have been associated with a number of diseases and conditions, including thrombotic thrombocytopenic purpura ( TTP) (Moake JL, Semin Hematol. 2004 Jan; 41(1):4-14), connective tissue disorders, cancers, inflammation (Nicholson et al.), and severe Plasmodium tropic malaria (Larkin et al., PLoS Pathog. 2009 Mar; 5(3):e1000349). Because of these links, ADAMTS enzymes have been recognized as potential therapeutic targets for a number of pathologies (Jones GC, Curr Pharm Biotechnol. 2006 Feb; 7(l):25-31). Accordingly, there is a need for methods for producing large amounts of ADAMTS proteins that have high specific activity and are free from contaminants such as viruses, CGEs, and pathogens such as Mycoplasma bacteria.

One member of the ADAMTS family, ADAMTS13, cleaves von Willebrand factor (vWF) between Tyr 1605 and Met 1606 residues; this function is responsible for the degradation of large vWF multimers in vivo. Loss of ADAMTS13 activity has been associated with several conditions such as TTP (Moake JL, Semin Hematol. 2004 Jan; 41(1):4-14), acute and chronic inflammation (Chauhan et al., J Exp Med. 2008 Sep 1; 205(9):2065-74) and, more recently, severe Plasmodium tropic malaria (Larkin et al., PLoS Pathog. 2009 Mar; 5(3):e1000349).

The ADAMTS13 protease is a 190 kDa glycosylated protein synthesized predominantly in the liver (Levy et al., Nature. 2001; 413:488-494; Fujikawa et al., Blood. 2001; 98:1662-1666; Zheng et al., J Biol Chem. 2001; 276:41059-41063; Soejima et al., J Biochem (Tokyo) 2001; 130:475-480; and Gerritsen et al., Blood. 2001; 98:1654-1661). To a large extent, and in the case of higher order rVWF multimers, recombinant expression of large ADAMTS13 in mammalian cell cultures is fraught with many difficulties.

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Thus, there is a need for cell culture conditions, in particular culture conditions for large-scale production, that provide a stable overall protein yield and/or a stable total and specific activity of the produced proteins in various cell cultures. Stability in cultures in large scale manufacturing processes is important for the production of therapeutic proteins. There is also a need for cell culture conditions for the large scale production of rVWF with levels of multimerization and specific ristocetin activity comparable to or greater than those of VWF present in normal human plasma. Similarly, since ADAMTS proteins have been implicated in a number of diseases and conditions, there is a need in the art for methods for the large scale production of high specific activity recombinant ADAMTS proteins that are suitable for pharmaceutical preparation and for pharmaceutical administration. The present invention satisfies these and other needs of the art in obtaining recombinant von Willebrand factor and recombinant ADAMTS13.

Brief description of the invention

In certain aspects, the present invention is based on the surprising discovery that the addition of additives to cell culture media used to express recombinant von Willebrand factor (rVWF) and recombinant ADAMTS13 (rA13) results in a significant increase in protein expression and enzymatic activity.

According to its first aspect, the present invention provides a method for preparing a recombinant von Willebrand factor (rVWF) composition; this method includes the steps:

(a) providing basic cell culture media;

(b) adding copper to the basic cell culture media to a final copper concentration of at least 2.4 µg/l;

(c) providing one or more cells containing a nucleic acid encoding a rVWF protein;

(d) culturing said one or more cells in a copper-supplemented cell culture medium such that rVWF is expressed and excreted from the cells into the culture supernatant; and (e) separating at least a portion of said culture supernatant, wherein said separated supernatant has a rVWF specific ristocetin cofactor activity of at least 30 IU/µg rVWF.

According to one embodiment of the methods proposed above, said method further comprises the step of adding a hydrolyzate to said basic cell culture medium prior to culturing said one or more cells.

According to one embodiment of the methods proposed above, said hydrolyzate is a vegetable hydrolyzate.

In a specific embodiment, said hydrolyzate is a soy hydrolyzate.

According to one embodiment of the methods proposed above, the basic cell culture medium is an animal protein-free culture medium.

In one embodiment of the methods proposed above, the basic cell culture media are protein-free culture media.

According to one embodiment of the methods proposed above, said basic cell culture media are chemically defined culture media.

According to one embodiment of the methods proposed above, the final concentration of copper in the basic medium for cell cultures with the addition of copper is at least 4 μg/l of copper.

According to one embodiment of the methods proposed above, the final concentration of copper in the basic medium for cell cultures with the addition of copper is from 2.4 to 20 μg/l of copper.

In one embodiment of the methods proposed above, copper is added to basic cell culture media as a copper salt, copper chelate, or a combination thereof.

In one embodiment of the above methods, said copper salt is selected from the group consisting of copper sulfate, copper acetate, copper carbonate, copper chloride, copper hydroxide, copper nitrate, and copper oxide.

According to one embodiment of the methods proposed above, said one or more cells is a mammalian cell.

In a specific embodiment, said mammalian cells are CHO cells.

According to one embodiment of the methods proposed above, culturing said one or more cells includes batch culturing said cells.

According to one embodiment of the methods proposed above, culturing said one or more cells comprises continuously culturing said cells.

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According to a specific embodiment, continuous cell culture is performed in a chemostatic mode.

According to another specific embodiment, continuous cell culture is performed in perfusion mode.

In one embodiment of the above methods, said one or more cells are cultured in at least 100 L of supplemented basic cell culture medium.

According to one embodiment of the methods proposed above, the cell density is maintained at a level of less than 2.5x10 ⁶ cells/ml during the stage of culturing said one or more cells.

According to one embodiment of the methods proposed above, the cell density is maintained at a level of less than 2.0x10 ⁶ cells/ml during the stage of culturing said one or more cells.

According to one embodiment of the methods proposed above, the cell density is maintained at a level of less than 1.5x10 ⁶ cells/ml during the stage of culturing said one or more cells.

In one embodiment of the methods proposed above, the step of separating at least a portion of said culture supernatant comprises filtration or centrifugation to remove cells from a portion of the culture supernatant.

According to one embodiment of the methods proposed above, the separated supernatant has a specific rVWF ristocetin cofactor activity of at least 40 IU/µg rVWF.

In a specific embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more specific embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 60 IU/µg rVWF.

In a more specific embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

In yet another more specific embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 80 IU/µg rVWF.

In one embodiment of the above methods, at least 10% of the rVWF in said supernatant is present as a high molecular weight VWF multimer of more than 10 dimers.

In a specific embodiment, at least 15% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 20% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 25% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 30% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

According to one embodiment of the methods proposed above, said supernatant contains high molecular weight VWF multimers of 14-22 dimers.

According to one embodiment of the methods proposed above, the content of NH ₄ ⁺ in said culture supernatant is maintained at a concentration below 10 mM.

According to one embodiment of the methods proposed above, the content of NH4+ in said culture supernatant is maintained at a concentration below 4 mM.

According to one embodiment of the methods proposed above, rVWF is co-expressed with recombinant factor VIII (rFVTII).

In a specific embodiment, said method further comprises the step of purifying the rVWF from at least 50% of the rFVTII present in the separated supernatant.

In one embodiment, the ratio of rVWF to rFVIII after the purification step is at least 10:1.

According to one embodiment of the methods proposed above, said method further comprises the step of enriching rVWF.

According to a second aspect, the present invention provides a recombinant von Willebrand factor (rVWF) composition obtained by the process described herein.

In one embodiment of the compositions described above, said composition also contains recombinant factor VIII (rFVIII).

In a specific embodiment, the ratio of rVWF to rFVIII is at least 10:1.

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In one embodiment of the compositions described above, said composition is in a form for pharmaceutical administration.

According to a specific implementation variant of the specified composition receive in the form for intravenous administration.

According to a third aspect, the present invention provides a cell culture supernatant containing recombinant von Willebrand factor (rVWF), characterized in that said supernatant is obtained by the method described in the invention.

According to a fourth aspect, the present invention provides a cell culture supernatant containing recombinant von Willebrand factor (rVWF), wherein at least 10% of the rVWF in said supernatant is present as a high molecular weight VWF multimer of more than 10 dimers.

In a specific embodiment, at least 15% of the rVWF in said supernatant is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 20% of the rVWF in said supernatant is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 25% of the rVWF in said supernatant is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 30% of the rVWF in said supernatant is present as a high molecular weight VWF multimer of more than 10 dimers.

According to another specific implementation of the above supernatants, the specified supernatant is obtained according to the method described in the invention.

According to a fifth aspect, the present invention provides a cell culture supernatant containing recombinant von Willebrand factor (rVWF), characterized in that said supernatant contains at least 0.4 IU of ristocetin cofactor activity per ml.

In a specific embodiment, said supernatant contains at least 0.5 IU of ristocetin cofactor activity per ml.

In another specific embodiment, said supernatant contains at least 0.6 IU of ristocetin cofactor activity per ml.

In another specific embodiment, said supernatant contains at least 0.7 IU of ristocetin cofactor activity per ml.

According to another specific implementation of the above supernatants, the supernatant is obtained according to the method described in the invention.

According to a sixth aspect, the present invention provides a method for preparing a recombinant ADAMTS13 (rA13) composition; this method includes the steps:

(a) providing basic cell culture media;

(b) adding copper to the basic cell culture media to a final copper concentration of at least 1.0 µg/L;

(c) providing one or more cells containing a nucleic acid encoding a rA13 protein;

(d) culturing said one or more cells in a copper-supplemented cell culture medium such that rA13 is expressed and excreted from the cells into the culture supernatant; and (e) separating at least a portion of said culture supernatant, wherein at least 1500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In one embodiment of the methods proposed above, the basic cell culture media are animal protein-free culture media.

In one embodiment of the methods proposed above, the basic cell culture media are protein-free culture media.

According to one embodiment of the methods proposed above, the basic media for cell cultures are culture media with a given chemical composition.

In one embodiment of the above methods, the final concentration of copper in the supplemented basic cell culture medium is at least 1 µg/L of copper.

According to one embodiment of the methods proposed above, the final concentration of copper in the supplemented basic cell culture medium is at least 2 μg/L of copper.

According to one embodiment of the methods proposed above, the final concentration of copper in the supplemented basic cell culture medium is at least 4 μg/l of copper.

According to one embodiment of the methods proposed above, the final concentration of copper in the supplemented basic cell culture medium is from 1 to 6 μg/l of copper.

According to one embodiment of the methods proposed above, the final concentration of copper in the supplemented basic cell culture medium is from 2 to 4 μg/l of copper.

In one embodiment of the above methods, copper is added to the basic cell culture media as a copper salt, copper chelate, or combinations thereof.

In a particular embodiment, said copper salt is selected from the group consisting of copper sulfate, copper acetate, copper carbonate, copper chloride, copper hydroxide, copper nitrate, and copper oxide.

According to one embodiment of the methods proposed above, said one or more cells is a mammalian cell.

In a specific embodiment, said mammalian cells are CHO cells.

According to one embodiment of the methods proposed above, culturing said one or more cells includes batch culturing said cells.

According to one embodiment of the methods proposed above, culturing said one or more cells comprises continuously culturing said cells.

According to a specific embodiment, said continuous cell culture is carried out in a chemostatic mode.

According to another specific embodiment, said continuous cell culture is performed in perfusion mode.

In one embodiment of the above methods, said one or more cells are cultured in at least 100 L of supplemented basic cell culture medium.

According to one embodiment of the methods proposed above, the cell density is maintained at a level of less than 4.0x10 ⁶ cells/ml during the stage of culturing said one or more cells.

According to one embodiment of the methods proposed above, the cell density is maintained at a level of less than 3.5x10 ⁶ cells/ml during the stage of culturing said one or more cells.

According to one embodiment of the methods proposed above, the cell density is maintained at a level of less than 3.0x10 ⁶ cells/ml during the stage of culturing said one or more cells.

According to one embodiment of the methods proposed above, the cell density is maintained at a level of less than 2.5x10 ⁶ cells/ml during the stage of culturing said one or more cells.

According to one embodiment of the methods proposed above, the cell density is maintained at a level of less than 2.0x10 ⁶ cells/ml during the stage of culturing said one or more cells.

According to one embodiment of the methods proposed above, the cell density is maintained at a level of less than 1.5x10 ⁶ cells/ml during the stage of culturing said one or more cells.

In one embodiment of the methods proposed above, the step of separating at least a portion of said culture supernatant comprises filtration or centrifugation to remove cells from said portion of the culture supernatant.

In one embodiment of the methods proposed above, the separated culture supernatant contains at least 2000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day.

In one embodiment of the above methods, at least 2500 FRETS-VWF73 activity units per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

According to one embodiment of the methods proposed above, the separated supernatant has a specific FRETS-VWF73 rA13 activity of at least 800 IU/μg.

In a preferred embodiment of the methods described above, the separated supernatant has a specific FRETS-VWF73 rA13 activity of at least 1200 IU/μg.

In a more preferred embodiment of the methods described above, the separated supernatant has a FRETS-VWF73 specific rA13 activity of at least 1600 IU/μg.

According to one embodiment of the methods proposed above, the content of NH ₄ ⁺ in said culture supernatant is kept below 10 mM.

According to one embodiment of the methods proposed above, the content of NH ₄ ⁺ in said culture supernatant is kept below 5 mM.

According to one embodiment of the methods proposed above, the content of NH4+ in said culture supernatant is maintained at a concentration below 4 mM.

According to one embodiment of the methods proposed above, said method further comprises the step of enriching rA13.

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According to a seventh aspect, the present invention provides a cell culture supernatant containing recombinant ADAMTS13 (rA13), characterized in that said supernatant is obtained by the method described in the invention.

According to an eighth aspect, the present invention provides a cell culture supernatant containing recombinant ADAMTS13 (rA13), characterized in that said supernatant contains at least 5 units of FRETS-VWF73 activity per ml.

In a specific embodiment, said supernatant contains at least 6 units of FRETS-VWF73 activity per ml.

In another specific embodiment, said supernatant contains at least 7 units of FRETS-VWF73 activity per ml.

In another specific embodiment, said supernatant contains at least 8 units of FRETS-VWF73 activity per ml.

In another specific embodiment, said supernatant contains at least 9 units of FRETS-VWF73 activity per ml.

In another specific embodiment, said supernatant contains at least 10 units of FRETS-VWF73 activity per ml.

According to another specific implementation of the above supernatants, the supernatant is obtained according to the method described in the invention.

According to a ninth aspect, the present invention provides a cell culture supernatant containing recombinant ADAMTS13 (rA13), said supernatant containing at least 2 μg of rA13 per ml.

In a specific embodiment, the supernatant contains at least 3 μg of rA13 per ml.

In another specific embodiment, the supernatant contains at least 4 μg of rA13 per ml.

In another specific embodiment, the supernatant contains at least 5 μg of rA13 per ml.

In another specific embodiment, the supernatant contains at least 6 μg of rA13 per ml.

According to another specific implementation of the above supernatants, the supernatant is obtained according to the method described in the invention.

According to a tenth aspect, the present invention provides a recombinant ADAMTS13 (rA13) composition prepared according to any of the methods described above.

In one embodiment of the compositions described above, said composition is in a form for pharmaceutical administration.

According to a specific implementation variant of the specified composition receive in the form for intravenous administration.

Brief description of the figures

Fig. 1-1A. Low resolution (1%) agarose gel electrophoresis of rVWF expressed in mammalian cell cultures in the presence of low (1.0 μg/L) and high (4.3 μg/L) copper concentrations, as described in Example 2. Note that culture day 3 is equivalent to batch culture day 1 of Tables 7 and 8. (1B) Relative content of VWF multimers containing 1 to 10 dimers (Zone #1) and more than 10 dimers (Zone #2) as assessed by the zones shown in fig. 1A was quantified by densitometric analysis.

Fig. 2-2A. Interval plot of mean rVWF specific activity in rVWF cell culture supernatants grown at high and low cell densities in the presence of high or low levels of copper. (FIG. 2B) Interval plot of mean NH4+ concentration found in supernatants of rVWF cell cultures grown at high and low cell densities in the presence of high or low levels of copper.

Fig. 3. Supernatants of cell cultures expressing recombinant ADAMTS13 in the presence of increasing levels of copper were examined using SDS-PAGE analysis. After SDS-PAGE, rA13 was visualized by (FIG. 3A) silver staining and (FIG. 3B) anti-A13 Western blotting.

Fig. 4. Plot of volumetric productivity (P Frets) versus copper concentration showing the extrapolated (solid line) effect of optimum copper concentration on rA13 production.

Fig. 5A-K. Step charts from continuous suspension (chemostatic) culture of cells expressing rA13 over a culture time of 8 weeks, comparing the effects of baseline copper levels (0.66 µg/L) with those in cultures supplemented to a final copper concentration of 2 µg/L. Each bar represents the weekly average of hemostatic culture. The legend refers to the specific weeks for which data is presented.

Fig. 6-6A. Low resolution agarose gel electrophoresis (1%) of rVWF expressed in

- 8 043293 mammalian cell cultures in the presence of low (1.0 µg/l) and high (4.3 µg/l) copper concentrations at high and low cell densities as described in example 3. It should be noted that cultivation days 8 and 17 (CST8 and CST17) are equivalent to day 8 and day 17 in the table. 10-13. (FIG. 6B) Relative content of VWF multimers containing 1 to 10 dimers and more than 10 dimers according to the evaluation of the zones shown in FIG. 6A was quantified by densitometric analysis.

Detailed description of the invention

I. Introduction.

Recombinant vWF (rVWF) and recombinant ADAMTS13 (rA13) can be produced by expression in large scale mammalian cell cultures. However, the activity of these proteins when obtained using standard cell culture conditions often varies from culture to cell culture, even in cases where the overall composition of the media is unchanged; the specific activity of recombinant proteins often differs from that of vWF and rA13 obtained from blood plasma. In addition, rVWF expressed in mammalian cell culture tends to form protein compounds with a low (less than 10%) percentage of higher order multimers (higher order multimers include molecules containing more than 10 VWF dimers). These shortcomings of standard methods for producing rVWF and rA13 present particular challenges when creating cultures for large scale production (ie 10 to over 20,000 liter cultures).

One potential source of variability often observed across batches of cell cultures is the presence of contaminants in components of cell culture media. These contaminants may be present in varying amounts in different batches, leading to varying results in the production of rVWF and rA13. After examining various contaminants found in various cell culture media supplements, the present inventors found that the presence of hydrolysates results in varying copper concentrations in such culture media. Further studies yielded an unexpected result: adding copper to the culture media to obtain a total copper concentration of at least about 1 µg/l to about 20 µg/l stably increased the total and specific activity of rVWF and rA13 and/or could also lead to increase in overall protein yield. Thus, in accordance with the present invention, methods and compositions are provided for the highly productive production of rVWF and rA13 proteins with high specific activity.

In one aspect, the present invention provides cell culture methods and compositions for producing large amounts of rVWF and rA13 having an activity comparable to or greater than that of plasma-derived vWF (pdVWF) or plasma-derived ADAMTS13 (pdA13).

According to further aspects, the rVWF and rA13 proteins obtained according to the present invention show consistently higher activity compared to proteins obtained using standard cell culture methods in media without the addition of copper or other additives, described in detail in this application.

Advantageously, according to certain embodiments of the methods and compositions described herein, the rVWF and rA13 proteins prepared according to the present invention exhibit consistently higher specific activity (i.e., U/mg protein) compared to proteins prepared using standard methods. culturing cells in media without the addition of copper or other additives described in detail in the invention.

Similarly, the methods of producing rVWF and rA13 according to the present invention give a greater yield of activity per culture volume (i.e., U/L/day) compared to standard cell culture methods using media without the addition of copper or other additives, described in detail in the invention.

In yet another aspect, the present invention provides methods for cell culture, wherein copper is added to the base cell culture medium to achieve a total concentration of at least about 1 μg/L.

In other embodiments, copper is added to the base cell culture medium to obtain a total concentration of at least about 2 µg/L.

In other embodiments, copper is added to the base cell culture medium to obtain a total concentration of at least about 1 µg/L to about 20 µg/L.

In some embodiments, the total copper concentration is about 1.5-4.5 µg/L.

In certain embodiments, copper is added to the cell culture medium to obtain approximately 1; 1.2; 1.4; 1.6; 1.8; 2.0; 2.2; 2.4; 2.6; 2.8; 3; 3.2; 3.4; 3.6; 3.8; 4; 4.2; 4.4; 4.6; 4.8; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10; eleven; 12; 13; 14; 15; 16; 17; 18; 19; 20 µg/l copper or more. Basic cell culture media typically contain trace copper concentrations of less than 1 µg/L.

- 9 043293

According to some embodiments of the present invention, cell culture methods are provided wherein from about 1.0 to about 20 μg/L copper is added to the basic cell culture medium to produce rVWF.

In further embodiments, approximately 1.5-15; 2.0-10; 2.5-8; 3.0-6; 4.0-5.0 µg/L copper for rVWF production.

In further embodiments, said basic cell culture medium may, in addition to the added copper, also contain one or more hydrolysates.

In other embodiments, the present invention provides methods for culturing cells in which about 1.5 to about 4 µg/L copper is added to the basic cell culture medium to produce rA13.

In further embodiments, approximately 1.6-3.8; 1.7-3.6; 1.8-3.4; 1.9-3.2; 2.0-3.0; 2.1-2.8; 2.2-2.6; 2.3-2.4 µg/l copper for rA13 production.

In further embodiments, said basic cell culture medium may, in addition to the added copper, also contain one or more hydrolysates.

In further embodiments, said basic cell culture medium comprises, in addition to copper and/or one or more hydrolyzate, from about 1.0 to about 30 μM zinc.

In further embodiments, said basic cell culture medium also contains, in addition to copper and/or one or more hydrolysates and/or zinc, about 0.5 to about 5.0 mM calcium.

In a further aspect, and in accordance with all of the foregoing, the present invention provides cell culture methods wherein ammonium levels in the cell culture solution are low (less than 10 mM).

In certain embodiments, cell culture methods of the present invention use cell culture media containing greater than 1, 2, 3, 4, or 5 μg/L copper in combination with low levels of ammonium.

One of the advantages of the methods and compositions according to the present invention is that they are suitable for large scale cell culture. The volume of said large scale cell cultures is at least 10, 50, 100, 150, 200, 250, 500, 750, 1000, 1500, 2000, 5000 L, 10,000 or 20,000 L.

In certain aspects, the methods of the present invention do not necessarily result in a greater total amount of recombinant protein, however, the resulting recombinant protein (rVWF or rA13) exhibits a higher total and specific activity than that found in proteins obtained using standard cell cultures, in particular, compared to proteins obtained in cell cultures where the cell culture medium does not contain additional copper supplements.

In further aspects, rVWF and rA13 proteins derived from cells cultured in copper supplemented media show consistently increased activity per liter of cell culture compared to cells cultured in basic cell culture media without copper supplementation.

According to further aspects, the addition of copper to the media of the present invention results in increased protein yield, increased number of cells in culture, and/or increased total activity per liter of culture compared to media without copper addition.

Further advantages of the methods and compositions of the present invention lie in the production of a protein population with a high percentage (greater than 10%) of highly multimerized rVWF.

Although much of the discussion of ADAMTS proteins in the invention relates to ADAMTS13 (A13), it should be understood that since all ADAMTS proteins share a common central domain structure and common structural-functional relationships, the methods and compositions described in the invention are applicable to the production of any protein. ADAMTS, not limited to rA13.

I. Definitions.

As used herein, the term recombinant vWF includes vWF produced using recombinant DNA technology.

In certain embodiments, the vWF proteins of the present invention may comprise a construct derived, for example, from WO 1986/06096 published 23 Oct. 1986 and US Patent Application Ser. No. 07/559509, filed July 23, 1990 on behalf of Ginsburg et al., incorporated into the invention by reference in relation to methods for obtaining recombinant vWF. vWF according to the present invention may include all potential forms, including monomeric and multimeric forms. It should also be understood that the present invention encompasses the use of combinations of different forms of vWF. For example, vWF according to the present invention may include different multimers, different derivatives; both active biological derivatives and

- 10 043293 and inactive biological derivatives.

The term recombinant, when used, for example, in relation to a cell or nucleic acid, protein or vector, means that said cell, nucleic acid, protein or vector is modified by introducing a heterologous nucleic acid or protein, or by altering the native nucleic acid or protein, or that said cell is derived from a cell thus modified. Thus, for example, recombinant cells express genes not found in native (non-recombinant) forms of said cells, or express native genes that are otherwise abnormally, underexpressed, or not expressed at all.

In the context of the present invention, recombinant vWF encompasses any members of the vWF family, for example, from mammals such as primates, humans, monkeys, rabbits, pigs, rodents, mice, rats, hamsters, gerbils, canines, felines; and their biologically active derivatives.

In a preferred embodiment, the recombinant VWF is a human VWF. Mutant and variant vWF proteins having activity are also included, as are functional fragments and hybrids of vWF proteins. In addition, the vWFs of the present invention may also contain labels to facilitate purification or detection, or both. The vWFs described in the invention may also be modified with a therapeutic agent or an agent suitable for in vitro or in vivo imaging.

The terms high multimeric vWF, high molecular weight vWF, and HMW VWF may be used interchangeably and refer to covalently linked vWF multimers containing more than 10 VWF dimers. In certain embodiments, the HMW VWF contains at least 11 VWF dimers, or at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more VWF dimers.

As used herein, the term ADAMTS protein refers to a disintegrin and metalloproteinase polypeptide from the family of metalloproteinases with type I thrombospondin motifs. Members of this family include human proteins

ADAMTS1 (NM_006988), ADAMTS2 (NM_014244; NM_021599),

ADAMTS3 (NM_014243), ADAMTS4 (NM_005099), ADAMTS5 (NM_007038), ADAMTS6 (NM_014273), ADAMTS7 (NM_0142727), ADAMTS8 (NM_007037), ADAMTS9 (NM_182920; NM_182921; NM_020 249), ADAMTS10 (NM_030957), ADAMTS12 (NM_030955), ADAMTS13 (NM_139025; NM_139026; NM_139027; NM_139028), ADAMTS14 (NM_139155; NM_080722), ADAMTS15 (NM_139055), ADAMTS16 (NM_139056), ADAMTS17 (NM_139057), ADAMTS18 (N M_199355; NM_139054), ADAMTS19 (NM_133638) and ADAMTS20 (NM_025003, NM- 175851) .

ADAMTS proteins include both full-length proteins and partial polypeptides that exhibit at least partial biological activity, e.g. a full-length protein, in particular the protease activity exhibited by a full-length protein. In certain cases, the ADAMTS protein undergoes post-translational modification in vivo or in vitro, for example, by enzymatic or chemical means. It is understood that the ADAMTS proteins of the present invention include alternative splicing isoforms, conservatively modified proteins, substantially identical proteins, homologues, and the like.

In the context of the present invention, the term ADAMTS protein encompasses any members of the ADAMTS family derived, for example, from mammals such as primates, humans, monkeys, rabbits, pigs, rodents, mice, rats, hamsters, gerbils, canines, felines, and their biologically active derivatives. Mutant and variant ADAMTS proteins having activity are also included, as are functional fragments and fusions of ADAMTS proteins. In addition, the ADAMTS proteins of the present invention may also contain labels to facilitate purification or identification, or both. The ADAMTS proteins of the invention may also be modified with a therapeutic agent or an agent suitable for in vitro or in vivo imaging.

As used herein, the term ADAMTS13 protein refers to any protein or polypeptide having ADAMTS13 activity, in particular the ability to cleave the peptide bond between Tyr-842 and Met-843 residues in VWF.

In an exemplary embodiment, an ADAMTS13 protein refers to a polypeptide containing an amino acid sequence substantially similar to that of NP_620594 (ADAMTS13 isoform 1, preproprotein) or amino acids 75-1427 of NP_620594 (ADAMTS13 isoform 1, mature polypeptide). In another embodiment, an ADAMTS13 protein refers to a polypeptide containing an amino acid sequence substantially similar to that of NP_620596 (ADAMTS13 isoform 2, preproprotein) or amino acids 75-1371 of NP_620594 (ADAMTS13 isoform 2, mature polypeptide).

In another embodiment, the ADAMTS13 proteins comprise polypeptides containing an amino acid sequence substantially similar to that of NP_620595.

- 11 043293 (ADAMTS13 isoform 3, preproprotein) or amino acids 75-1340 NP_620595 (isoform 1

ADAMTS13, mature polypeptide). As used herein, the term ADAMTS13 protein includes natural variants having vWF-cleaving activity and artificial constructs having vWF-cleaving activity.

As used herein, ADAMTS13 encompasses any natural variants, alternative sequences, isoforms, or mutant proteins that retain some degree of basic activity.

Examples of ADAMTS13 mutations found in human populations include, without limitation

R7W, V88M, H96D,

R102C, R193W, T196I, H234Q, A250V, R268P, W390C, R398H, Q448E,

Q456H, P457L, C508Y, R528G, R618A, R625H, I673F, R692C, A732V, S903L, C908Y, C951G, G982R, C1024G, A1033T, R1095W, R1123C,

C1213Y, T1226I, G1239V, R1336W, many of which have been associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 proteins also include polypeptides containing post-translational modifications. For example, ADAMTS13 has been shown to be modified by N-acetylglucosamine (GlcNAc) at residues 614, 667, and 1354, and it is predicted that residues 142, 146, 552, 579, 707, 828, and 1235 may also be modified in this way.

Proteolytically active recombinant ADAMTS13 can be obtained by performing expression in mammalian cell cultures as described by Plaimauer et al. (2002, Blood. 15; 100(10):3626-32) and US 2005/0266528, the disclosures of which are hereby incorporated by reference in their entirety for any purpose.

Methods for expressing recombinant ADAMTS13 in cell culture are described in Plaimauer B, Scheiflinger F. (Semin Hematol. 2004 Jan; 41(1):24-33 and US 2011/0086413, the disclosures of which are hereby incorporated by reference in their entirety for any purpose. .

As used herein, the term biologically active derivative, when used in the context of the ADAMTS protein, also encompasses polypeptides produced using recombinant DNA technology, which may include any methods known in the art:

(i) obtaining recombinant DNA by means of genetic engineering, for example, by reverse transcription of RNA and/or amplification of DNA, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, i.e. by electroporation or microinjection, (iii) culturing said transformed cells, e.g., continuously or batchwise, (iv) expressing said ADAMTS protein, e.g. constitutive or inducible, and (v) isolating said ADAMTS protein, e.g., from culture medium or by harvesting transformed cells, (vi) obtaining a substantially purified recombinant ADAMTS protein, for example, using ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, and the like.

The term biologically active derivative also includes hybrid molecules, such as, for example, the ADAMTS protein or a functional fragment thereof, combined with a second polypeptide, for example, an immunoglobulin Fc domain or an albumin domain, to improve biological/pharmacological parameters, such as, for example, the half-life of the protein. ADAMTS in the bloodstream of a mammal, in particular a human.

The terms isolated, purified, or biologically pure refer to a substance that is substantially or essentially free of the components normally associated with it in its natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

In one embodiment, rVWF is the predominant component in the substantially purified formulation.

In another embodiment, rA13 is the predominant component in a substantially purified formulation. The term "purified" in some embodiments means that the nucleic acid or protein produces substantially one band in an electrophoretic gel. In other embodiments, this means that the purity of said nucleic acid or protein is at least 50%, more preferably at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 , 99% or more. Purity or purification, in other embodiments, means the removal of at least one contaminant from the composition to be purified. In this sense, purification does not imply that the purified compound will be homogeneous, eg 100% pure.

The biological activity of vWF can be measured by known in vitro assay methods. For example, the ristocetin cofactor assay is based on agglutination of fresh or formalin-fixed platelets induced by the antibiotic ristocetin in the presence of vWF. The degree of platelet agglutination depends on the concentration of vWF and can be measured by a turbodimetric method, for example, using an aggregometer (Weiss et al., J. Clin. Invest. 52: 2708-2716, 1973; Macfarlane et al., Thromb. Diath. Haemorrh. 34: 306-308, 1975). In the invention, the specific ristocetin cofactor activity of vWF according to the present invention is expressed in mU/µg of vWF as assessed using in vitro assay methods.

Used in the invention, the term one unit of ADAMTS activity means the level of activity in 1 ml of mixed normal human plasma, regardless of which method of analysis is used. For example, if the ADAMTS protein is ADAMTS13, one unit of ADAMTS13 FRETS-VWF73 activity is the level of activity required to cleave the same amount of FRETS-VWF73 substrate (Kokame et al., Br J Haematol. 2005 Apr; 129( 1):93-100), which is cleaved with one ml of mixed normal human plasma. Conveniently, ADAMTS13 activity can be determined by functional assay methods such as functional assay using modified von Willebrand factor peptides as an ADAMTS13 substrate (Tripodi et al., J Thromb Haemost. 2008 Sep; 6(9): 1534-41). A preferred method for determining recombinant human ADAMTS13 activity is described in Gerritsen et al. Assay of von Willebrand factor (vWF)-cleaving protease based on decreased collagen binding affinity of degraded vWF: a tool for the diagnosis of thrombotic thrombocytopenic purpura (TTP) -binding capacity of degraded vWF: a diagnostic tool for thrombotic thrombocytopenic purpura (TTP) Thromb Haemost 1999;82:1386-1389). In one embodiment, to be considered an ADAMTS13 protein as defined above, a polypeptide or protein must have at least 1% of the vWF-cleaving activity of native ADAMTS13. In other embodiments, the ADAMTS13 protein has at least 10% of the activity of native ADAMTS13. In other embodiments, the ADAMTS13 protein has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 9o, or 100% of the activity of native ADAMTS13. The amount of ADAMTS13 protein can also be determined by measuring the ADAMTS13 antigen, for example using the ELISA method described by Rieger et al. (2006, Thromb Haemost. 2006 95(2):212-20). It is generally accepted in the art that 1 ml of mixed normal human plasma contains 1 μg of ADAMTS13. Thus, according to the rule of thumb in the art, 1 μg of plasma-derived ADAMTS13 has one unit of ADAMTS13 activity.

The terms cell culture solution, cell culture medium or media, and cell culture supernatant refer to aspects of cell culture processes generally known in the art.

In the context of the present invention, the cell culture solution may include cell culture media and cell culture supernatant. These cell culture media are added externally to the cell culture solution, optionally together with additives, to provide nutrients and other components for culturing cells expressing rVWF or rA13. The term cell culture supernatant refers to a cell culture solution containing nutrients and other components from the cell culture medium, as well as products released, metabolized and/or excreted by the cells during culture, but not the cells themselves.

Thus, in one context, cell culture supernatant can refer to the liquid phase of a cell culture solution (ie, cell culture solution, excluding cells). For example, the ammonium concentration of the culture supernatant generally refers to the ammonium concentration in the cell culture solution.

In other contexts, cell culture supernatant refers to the cell culture solution from which said cells were recovered (ie, separated cell culture supernatant).

As used herein, the terms vitamin B3, nicotinamide, niacinamide, niacin, and nicotinic acid may be used interchangeably to refer to any member of the vitamin B3 family. Accordingly, any member of this family can be used to add to the environment used in the methods according to the present invention.

As used herein, the term chemically defined medium or chemically defined medium refers to a synthetic growth medium in which all components are identified and their concentrations are known. Chemically defined media do not contain bacterial, yeast, animal, or plant extracts, although they may or may not include individual components of plant or animal origin (eg, proteins, polypeptides, etc.). Non-limiting examples of commercially available chemically formulated media include various Dulbecco's modified Eagle's media (DME) (Sigma-Aldrich Co; SAFC Biosciences, Inc), Ham's formula (Sigma-Aldrich Co; SAFC Biosciences, Inc), combinations thereof, and etc.

Methods for obtaining culture media with a given chemical composition are known in the art, for example, in US patents No. 6171825 and No. which are incorporated herein by reference in their entirety for any purpose.

As used herein, the term oligopeptide-free culture medium or oligopeptide-free culture media refers to a protein-free medium that does not contain oligopeptides, such as, for example, oligopeptides derived from a protein hydrolysate.

In one embodiment, said medium does not contain oligopeptides of twenty or more amino acids.

According to one implementation variant of the present invention, the specified environment does not contain oligopeptides containing fifteen or more amino acids.

According to another embodiment of the present invention, said medium does not contain oligopeptides containing ten or more amino acids.

According to one implementation variant of the specified environment does not contain oligopeptides containing seven or more amino acids.

According to another implementation variant of the specified environment does not contain oligopeptides containing five or more amino acids.

According to another implementation variant of the specified environment does not contain oligopeptides containing three or more amino acids.

According to a further embodiment of the present invention, said medium does not contain oligopeptides containing two or more amino acids.

Methods for preparing oligopeptide-free culture media are known in the art, for example, in US Pat. links in their entirety for any purpose.

Used in the invention, the term serum-free culture medium or serum-free culture media refers to the culture medium without the addition of animal serum. While serum-free media are often chemically defined media, individual animal or plant proteins or protein fractions can be added to serum-free media.

Methods for obtaining serum-free culture media are known in the art, for example, in US patent No. 6171825 and No. 6936441, WO 2007/077217, and published applications for US patent No. 2008/0009040 and No. 2007/0212770, the disclosures of which are incorporated into the invention by reference in its entirety for any purpose.

As used herein, the term animal protein free culture medium or animal protein free culture media refers to a culture medium without the addition of animal serum, protein or protein fraction. Although animal protein-free culture media are often chemically defined, animal protein-free culture media may contain vegetable or yeast hydrolysates.

Methods for obtaining animal protein-free culture media are known in the art, for example, in US patent No. 6171825 and No. 6936441, WO 2007/077217, and published applications for US patent No. invention by reference in its entirety for any purpose.

As used herein, the terms basic (basic) cell culture medium or basic (basic) cell culture media refer to a chemically defined culture medium, an oligopeptide-free culture medium, a serum-free culture medium, or an animal protein-free culture medium in which no a hydrolysate has been added, such as a vegetable or yeast hydrolysate. Base media are well known in the art, such as DMEM, Ham F12, DMEM/Ham F12, Medium 199, McCoy, or RPMI. Said base medium may include a number of ingredients, including amino acids, vitamins, organic and inorganic salts, and carbohydrate sources. Each ingredient may be present in an amount sufficient to culture the cell; such amounts are well known to those skilled in the art. Said medium may include excipients such as buffering agents such as sodium bicarbonate, antioxidants, stabilizers to counteract mechanical stress, or protease inhibitors. If necessary, non-ionic surfactants such as copolymers and/or mixtures of polyethylene glycols and polypropylene glycols can be added.

II. Cell culture media and cell culture supernatant.

In one aspect, the present invention relates to cell culture media for producing rVWF and/or rA13 having increased activity relative to rVWF and rA13 produced using basic cell culture media.

In one aspect, the present invention relates to cell culture media for producing rVWF and/or rA13, wherein one or more additional substances are added to the basic cell culture media.

- 14 043293

In specific embodiments and in more detail below, the cell culture conditions of the present invention include basic cell culture media supplemented with copper to a concentration of at least 1.0 µg/L.

In further embodiments, the cell culture media used and the supernatants produced by the processes of the present invention also contain low levels (less than 10 mM) of ammonium.

In a specific embodiment, the cell culture conditions used to express rVWF and/or rA13 are controlled to maintain a low ammonium level in the cell culture supernatant, i. less than 10 mm and preferably less than 5 mm.

Culture media of the present invention may be based on suitable base media commonly known in the art such as DMEM, Ham F12, DMEM/Ham F12, 199 medium, McCoy or RPMI. The base medium may include a number of ingredients including amino acids, vitamins, organic and inorganic salts, and carbohydrate sources. Each ingredient may be present in an amount to promote cell culture; such amounts are generally known to those skilled in the art. Said medium may include excipients such as buffering agents such as sodium bicarbonate, antioxidants, stabilizers to counteract mechanical stress, or protease inhibitors. If necessary, a non-ionic surfactant can be added, for example copolymers and/or mixtures of polyethylene glycols and polypropylene glycols.

Typically, basic media contain less than 1 µg/L copper - for example, DMEM/Ham F12 contains copper at a concentration of approximately 0.3 µg/L. These copper concentrations do not provide enough copper ions to support the production of the rVWF and rA13 proteins of the present invention, which exhibit high specific activity.

Copper can be introduced into the cell culture media of the present invention by a variety of methods, such as adding an additive to the media.

In some embodiments, said culture medium supplement may contain a hydrolyzate that can be used to increase the concentration of copper in said medium. Hydrolysates may include any hydrolysate commonly known in the art, such as vegetable hydrolysates, soy hydrolysates, and wheat gluten hydrolysates.

In certain embodiments, the addition of the hydrolyzate may contribute to elevated copper concentrations of about 0.2 to about 10 µg/L Cu ²⁺ .

In some embodiments, the amount of copper provided by the hydrolyzate may depend on the amount of copper in said hydrolyzate as well as the amount of hydrolyzate added. The copper content of the hydrolyzate can be determined by elemental analysis, eg adsorption atomic spectroscopy (GFAA: graphite furnace atomic absorption), or mass spectrometric methods (eg ICP-MS).

In certain embodiments, copper can be added to the culture media, alone or together with the hydrolyzate, by introducing a media supplement including a suitable copper salt or copper chelate. Suitable copper salts may include, but are not limited to, copper sulfate, copper acetate, copper carbonate, copper chloride, copper hydroxide, copper nitrate, and copper oxide. Suitable copper chelators may include, but are not limited to, albumin, ethylenediaminetetraacetic acid (EDTA), polyamine chelating agents, ethylenediamine, diethylenetriamine, triethylenetetramine, triethylenediamine, tetraethylenepentamine, aminoethylethanolamine, aminoethylpiperazine, pentaethylenehexamine, triethylenetetramine hydrochloride, tetraethylenepentamine -hydrochloride, pentaethylenehexamine hydrochloride, tetraethylpentamine, captopril, penicillamine, N,N'-bis(3-aminopropyl)-1,3propanediamine, N,N-6uc(2-aminoethyl)-1,3-propanediamine, 1,7-dioxa-4,10-diazacyclododecane, 1,4,8,11tetraazacyclotetradecane-5,7-dione, 1,4,7-triazacyclononane trihydrochloride, 1-oxa-4,7,10triazacyclododecane, 1,4,8,12-tetraazacyclopentadecane and 1,4,7,10 -tetraazacyclododecane.

In certain embodiments, copper is added to the basic cell culture media to achieve a total copper concentration of from about 1.0 to about 20 µg/L.

In a specific embodiment, copper is added to the basic cell media to a final concentration of about 1.0 to about 10 µg/L.

In further embodiments, copper is added to the basic cell culture media to obtain a final concentration of approximately 1.0-5.0; 1.2-4.0; 1.3-3.0; 1.4-2.9; 1.5-2.8; 1.62.7; 1.7-2.6; 1.8-2.5; 1.9-2.4; 2.0-2.3; 2.1-2.2 µg/l copper.

In further embodiments, the basic cell culture media used in the methods of the present invention are supplemented to obtain copper concentrations of approximately 1.2-9.5; 1.4-9; 1.6-8.5; 1.8-8; 2.0-7.5; 2.2-7; 2.4-6.5; 2.6-6.0; 2.8-5.5; 3.0-5.0; 3.2-4.5; 3.4-4; and 2-4 µg/l.

In other embodiments, the basic cell culture media used in the methods of the present invention are supplemented to obtain copper concentrations of approximately 1-6, 2-5, 3-4 µg/L.

- 15 043293

In one embodiment, copper is added to the basic cell culture media to achieve a total copper concentration of at least 1 µg/L.

In another embodiment, copper is added to the basic cell culture media until a total copper concentration of at least 2 µg/L is obtained.

In yet another embodiment, copper is added to the basic cell culture media until a total copper concentration of at least 4 µg/L is obtained.

In certain embodiments, copper is added to the basic cell culture media to achieve a total copper concentration of at least 1 µg/L, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 µg/l copper or more.

According to certain embodiments and more detailed description below in the invention, cultures to produce rA13 may contain approximately 2-4 μg/l copper, while cultures to produce rVWF may contain at least 2 μg/l copper.

The above concentrations are relative concentrations of pure copper in the form of divalent copper ion (Cu ²⁺ ). If a copper derivative is used, such as a hydrated salt, or a copper-containing compound, such as a copper chelator, the specified amount of derivative or chelator is added such that the final concentration of copper falls within the ranges specified in the invention. For example, 2 µg/L CuSO ₄ -5H ₂ O is equivalent to a copper concentration of approximately 0.51 µg/L (without sulfate and 5H2O).

Advantageously, it has been found that the use of cell culture medium providing low concentrations of (NH4 ⁺ ) in the cell culture solution (i.e., culture supernatant) during cell culture results in the expression of recombinant VWF and/or rA13 at a higher specific activity.

Accordingly, according to certain implementation options, the concentration of NH4 ⁺ in the specified supernatant is not more than 10 mm.

In a preferred embodiment, the concentration of NH4 ⁺ in said supernatant is not more than 5 mM.

In a preferred embodiment, the concentration of NH4 ⁺ in said supernatant is not more than 4 mM.

In other embodiments, the concentration of NH4 ⁺ in said supernatant is not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mM or less.

Accordingly, in certain embodiments, the methods and compositions of the present invention rely on the use of basic cell culture media supplemented with copper (e.g., to a final concentration of at least 2 µg/L) for use in a process that results in to the concentration of NH4 ⁺ in the supernatant, which is not more than 10 mm.

In other embodiments, the base cell culture medium is supplemented to obtain final concentrations of copper and ammonium according to any of the options 1-440 shown in Table 1. 1.

Table 1 Exemplary variants of copper and ammonium concentrations in culture media and supernatant suitable for the expression of recombinant proteins according to the present description

III. IV. pmm 1 µg/l BUT. Var. 1 NBCH Yum M Var. 41 NBCh 9mm Var. 81 ai concentration MM0NI5 NBCh 5mm Var. 241 I NBC 4mM Var. 281 NBCH ZmM Var. 321 NBCh 2mm Var. 361 CBN 1mM Var. 401 CBN 8mm Var. 121 NBCh 7mm Var. 161 NBCh 6mm Var. 201 PMM Var. 2 Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 2 µg/l 42 82 122 162 202 242 282 322 362 402 PMM Var. 3 Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. No. 3 µg/l 43 83 123 163 203 243 283 323 363 403 D L PMM Var. 4 Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. H 4 µg/l 44 84 124 164 204 244 284 324 364 404 I g PMM Var. 5 Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. I 5 µg/l 45 85 125 165 205 245 285 325 365 405 And PMM Var. 6 Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 6 µg/l 46 86 126 166 206 246 286 326 366 406 PMM Var. 7 Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 7 µg/l 47 87 127 167 207 247 287 327 367 407

- 16 043293

pmm 8 µg/l Var. 8 Var. 48 Var. 88 Var. 128 Var. 168 Var. 208 Var. 248 Var. 288 Var. 328 Var. 368 Var. 408 PMM 9 µg/l Var. 9 Var. 49 Var. 89 Var. 129 Var. 169 Var. 209 Var. 249 Var. 289 Var. 329 Var. 369 Var. 409 PMM 10 µg/l Var. 10 Var. 50 Var. 90 Var. 130 Var. 170 Var. 210 Var. 250 Var. 290 Var. 330 Var. 370 Var. 410 Approximately 1 µg/l Var. eleven Var. 51 Var. 91 Var. 131 Var. 171 Var. 211 Var. 251 Var. 291 Var. 331 Var. 371 Var. 411 Approximately 1.5 µg/l Var. 12 Var. 52 Var. 92 Var. 132 Var. 172 Var. 212 Var. 252 Var. 292 Var. 332 Var. 372 Var. 412 Approximately 2 µg/l Var. 13 Var. 53 Var. 93 Var. 133 Var. 173 Var. 213 Var. 253 Var. 293 Var. 333 Var. 373 Var. 413 Approximately 2.5 µg/l Var. 14 Var. 54 Var. 94 Var. 134 Var. 174 Var. 214 Var. 254 Var. 294 Var. 334 Var. 374 Var. 414 Approximately 3 µg/l Var. 15 Var. 55 Var. 95 Var. 135 Var. 175 Var. 215 Var. 255 Var. 295 Var. 335 Var. 375 Var. 415 Approximately 3.5 µg/l Var. 16 Var. 56 Var. 96 Var. 136 Var. 176 Var. 216 Var. 256 Var. 296 Var. 336 Var. 376 Var. 416 Approximately 4 µg/l Var. 17 Var. 57 Var. 97 Var. 137 Var. 177 Var. 217 Var. 257 Var. 297 Var. 337 Var. 377 Var. 417 Approximately 4.5 µg/l Var. 18 Var. 58 Var. 98 Var. 138 Var. 178 Var. 218 Var. 258 Var. 298 Var. 338 Var. 378 Var. 418 Approximately 5 µg/l Var. 19 Var. 59 Var. 99 Var. 139 Var. 179 Var. 219 Var. 259 Var. 299 Var. 339 Var. 379 Var. 419 Approximately 5.5 µg/l Var. 20 Var. 60 Var. 100 Var. 140 Var. 180 Var. 220 Var. 260 Var. 300 Var. 340 Var. 380 Var. 420 Approximately 6 µg/l Var. 21 Var. 61 Var. 101 Var. 141 Var. 181 Var. 221 Var. 261 Var. 301 Var. 341 Var. 381 Var. 421 Approximately 7 µg/l Var. 22 Var. 62 Var. 102 Var. 142 Var. 182 Var. 222 Var. 262 Var. 302 Var. 342 Var. 382 Var. 422 Approximately 8 µg/l Var. 23 Var. 63 Var. 103 Var. 143 Var. 183 Var. 223 Var. 263 Var. 303 Var. 343 Var. 383 Var. 423 Approximately 9 µg/l Var. 24 Var. 64 Var. 104 Var. 144 Var. 184 Var. 224 Var. 264 Var. 304 Var. 344 Var. 384 Var. 424 Approximately 10 µg/l Var. 25 Var. 65 Var. 105 Var. 145 Var. 185 Var. 225 Var. 265 Var. 305 Var. 345 Var. 385 Var. 425 1-20 µg/l Var. 26 Var. 66 Var. 106 Var. 146 Var. 186 Var. 226 Var. 266 Var. 306 Var. 346 Var. 386 Var. 426 2-20 µg/l Var. 27 Var. 67 Var. 107 Var. 147 Var. 187 Var. 227 Var. 267 Var. 307 Var. 347 Var. 387 Var. 427 1-10 µg/l Var. 28 Var. 68 Var. 108 Var. 148 Var. 188 Var. 228 Var. 268 Var. 308 Var. 348 Var. 388 Var. 428 2-10 µg/l Var. 29 Var. 69 Var. 109 Var. 149 Var. 189 Var. 229 Var. 269 Var. 309 Var. 349 Var. 389 Var. 429 1-6 µg/l Var. thirty Var. 70 Var. BUT Var. 150 Var. 190 Var. 230 Var. 270 Var. 310 Var. 350 Var. 390 Var. 430 2-6 µg/l Var. 31 Var. 71 Var. 111 Var. 151 Var. 191 Var. 231 Var. 271 Var. 311 Var. 351 Var. 391 Var. 431 3-6 µg/l Var. 32 Var. 72 Var. 112 Var. 152 Var. 192 Var. 232 Var. 272 Var. 312 Var. 352 Var. 392 Var. 432 4-6 µg/l Var. 33 Var. 73 Var. 113 Var. 153 Var. 193 Var. 233 Var. 273 Var. 313 Var. 353 Var. 393 Var. 433 1-5 µg/l Var. 34 Var. 74 Var. 114 Var. 154 Var. 194 Var. 234 Var. 274 Var. 314 Var. 354 Var. 394 Var. 434 2-5 µg/l Var. 35 Var. 75 Var. 115 Var. 155 Var. 195 Var. 235 Var. 275 Var. 315 Var. 355 Var. 395 Var. 435 3-5 µg/l Var. 36 Var. 76 Var. 116 Var. 156 Var. 196 Var. 236 Var. 276 Var. 316 Var. 356 Var. 396 Var. 436 4-5 µg/l Var. 37 Var. 77 Var. 117 Var. 157 Var. 197 Var. 237 Var. 277 Var. 317 Var. 357 Var. 397 Var. 437 1-4 µg/l Var. 38 Var. 78 Var. 118 Var. 158 Var. 198 Var. 238 Var. 278 Var. 318 Var. 358 Var. 398 Var. 438 2-4 µg/l Var. 39 Var. 79 Var. 119 Var. 159 Var. 199 Var. 239 Var. 279 Var. 319 Var. 359 Var. 399 Var. 439 3-4 µg/l Var. 40 Var. 80 Var. 120 Var. 160 Var. 200 Var. 240 Var. 280 Var. 320 Var. 360 Var. 400 Var. 440

*N.O.=not defined *NBN= no more than *MMP=at least

In some embodiments, addition of copper to the media of the present invention is accomplished by supplementing basic media that do not contain animal proteins and/or media with a given chemical composition.

Methods for preparing animal protein-free and chemically defined culture media are known in the art, for example, in US Pat. , the disclosures of which are hereby incorporated by reference in their entirety for any purpose.

In one embodiment, the base culture medium used in the methods of the invention is an animal protein-free or oligopeptide-free medium.

In certain embodiments, said culture medium may be a chemically defined medium.

In certain embodiments, said culture media may contain at least one polyamine at a concentration of from about 0.5 mg/L to about 10 mg/L.

- 17 043293

In further embodiments, and in addition to any described above, the present invention provides culture media for which copper and at least one of calcium, zinc, and/or vitamin B3 are added to the base medium.

In certain embodiments, said medium may be free of animal proteins, free of oligopeptides, or be a chemically defined medium.

In certain embodiments, said animal protein-free or oligopeptide-free medium is prepared as described in US Pat. to the invention by reference in its entirety for any purpose; both sources are incorporated into the invention by reference in their entirety for any purpose; and adding additional copper and optionally one or more of calcium, zinc and vitamin B3.

In a specific embodiment, the chemically defined culture medium may be similar to a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F12 Medium (DMEM/Ham's F12) supplemented with additional copper and optionally calcium, zinc and/or vitamin B3 to increase the specific activity of rVWF or rA13 expressed in cells cultured in said medium.

In other embodiments, said culture medium does not contain animal components.

In another embodiment, said culture medium contains a protein, for example animal protein from serum, such as fetal calf serum.

In another embodiment, said culture contains added exogenous recombinant proteins.

In another embodiment, said proteins are obtained from a certified pathogen-free animal.

In certain embodiments, said culture medium contains at least one polyamine at a concentration of exactly or about 0.5 to 30 mg/L.

In another embodiment, said culture medium contains at least one polyamine at a concentration of exactly or about 0.5 to 10 mg/L.

In one embodiment, said culture medium contains at least one polyamine at a concentration of exactly or about 2 to 8 mg/L.

In certain embodiments, said polyamine belongs to the group of ornithine, putrescine, spermine or spermidine, or the like.

In a preferred embodiment, the polyamine is putrescine.

In a specific embodiment, said culture medium contains exactly or about 2 to 8 mg/l putrescine.

In one embodiment, said culture medium contains at least one polyamine at a concentration of exactly or about 0.5 mg/l to 30 mg/l, and a combination of copper and ammonium according to any one of options 1-440 shown in Table . 1.

In another embodiment, said culture medium contains at least one polyamine at a concentration of exactly or about 0.5 to 10 mg/l, and a combination of copper and ammonium according to any one of options 1-440 shown in Table 1. 1.

In one embodiment, said culture medium contains at least one polyamine at a concentration of exactly or about 2 to 8 mg/l, and a combination of copper and ammonium according to any of the options 1-440 shown in Table 1. 1.

In certain embodiments, said polyamine is in the group of ornithine, putrescine, spermine, or spermidine, or the like.

In a preferred embodiment, said polyamine is putrescine.

In a specific embodiment, said culture medium contains exactly or about 2 to about 8 mg/l putrescine and a combination of copper and ammonium according to any one of options 1-440 shown in Table 1.

In further aspects, in addition to copper, the cell culture media used according to the present invention may also contain any one or more of additional calcium, zinc, one or more vitamins, and any combinations thereof.

Generally, any calcium salt may be used to add to the media of the present invention; non-limiting examples of acceptable salts include CaCl ₂ , CaCl ₂ , CaFPO ₃ -2H2O, CaI ₂ , CaBr ₂ , (C ₂ H ₃ O ₂ ) ₂ Ca, (CHO ₂ ) ₂ Ca, (C ₆ H ₇ O ₆ ) ₂ Ca , (C ₆ H ₅ O ₇ ) ₂ Ca ₃ 2H ₂ O, etc.

In certain embodiments, a pharmaceutically acceptable calcium salt is used to add to the culture media of the present invention.

In general, any

- 18 043293 zinc salt; non-limiting examples of acceptable salts include ZnSCO ₄ 7H ₂ O, ZnSO ₃ -2H2O, (C ₆ H ₅ O ₇ )2Zn ₃ -2H2O, ZnBr ₂ , ZnBr ₂ -2H ₂ O, ZnCl ₂ , Zn(NO ₃ ) ₂ -6H ₂ O, Zn (H ₂ PO ₄ ) ₂ -H ₂ O, (C ₂ H ₃ O ₂ ) ₂ Zn-2H ₂ O, etc.

In certain embodiments, a pharmaceutically acceptable zinc salt is used to add to the culture media of the present invention.

In other embodiments, a zinc-containing peptide or protein preparation, such as insulin, may be used to add to the culture of the invention.

According to further aspects, basic cell media supplemented with copper and one or more of the additional substances described above can also be used in cultures with low levels of ammonium in the supernatant.

In certain embodiments, the supplemented cell culture media for use in accordance with the present invention results in ammonium levels in the cell culture solution of less than 10 mM.

In further embodiments, the supplemented cell culture medium of the present invention is used at cell culture ammonium levels of about 0.5-9.5; 1.0-9.0; 1.5-8.5; 2.0-8.0; 2.5-7.5; 3.0-7.0; 3.5-6.5; 4.0-6.0; 4.5-5.5 mM.

In one embodiment, the specified concentrations of copper and ammonium in the cell culture medium and cell culture supernatant are maintained for an extended period of time during the manufacturing process.

In a specific embodiment, said concentrations of copper and ammonium in cell culture are maintained throughout the manufacturing process, i.e. during the time that rVWF or rA13 is expressed and isolated from large scale cell culture.

According to certain implementation options, the indicated concentrations of copper and ammonium are maintained in the culture solution at a level according to any of the options 1-440 shown in table. 1.

In a preferred embodiment, said concentrations of copper and ammonium are maintained throughout the duration of such a manufacturing process.

In some embodiments, the culture medium of the present invention may be in liquid, dry, or powder form. Said medium may be pre-divided into aliquots containing amounts suitable for single use or larger amounts suitable for more than one cell culture. As a rule, the environment according to the present invention is presented in a sterile form.

The characteristics of cell culture media suitable for the production of rVWF or rA13 are discussed below. While they are described in relation to either rVWF or RA13, it should be understood that any descriptions below that refer to rVWF apply to RA13 and vice versa.

A. Cell culture media for obtaining recombinant VWF.

According to one aspect, the present invention relates to a cell culture solution for producing recombinant vWFs, more specifically high specific activity high molecular weight vWFs, as described below in the invention.

In one embodiment, the present invention provides a cell culture solution for producing high molecular weight recombinant vWF comprising a cell culture medium containing copper at a concentration of at least about 2.4 µg/L and a plurality of cells expressing high multimeric vWF containing from about 14 up to about 22 dimers and having a specific ristocetin cofactor activity of at least about 30 IU/μg.

In one embodiment, the cell culture solution also contains ammonium at a concentration of less than 10 mM.

In a preferred embodiment, said cell culture contains ammonium at a concentration not exceeding 5 mM.

In other embodiments, said cell culture contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less.

In other embodiments, said cell culture contains a concentration of copper and ammonium according to any of the options 1-440 shown in Table 1.

In certain embodiments, the ammonium concentration in the cell culture is maintained for an extended period at a concentration level as described above. For example, in one embodiment, the ammonium concentration is kept low for at least 3 days, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In a specific embodiment, the ammonium concentration in the cell culture is maintained at or below 5 mM for at least 7 days.

According to another specific implementation variant, the concentration of ammonium in cell culture

- 19 043293 maintain at a level not higher than 4 mm for at least 7 days.

In a specific embodiment, the ammonium concentration in the cell culture is maintained at or below 5 mM for at least 14 days.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 4 mM for at least 14 days.

According to another embodiment, the ammonium concentration in the cell culture is maintained at a low level throughout the process (ie, during the entire time that the specified culture is used to produce rVWF).

In one embodiment, the cell culture media may contain copper at a concentration of at least about 2.4 µg/L, in another embodiment, at least about 3 µg/L, in another embodiment, at least about 4 µg/L, in another embodiment at least about 8 µg/L, in another embodiment at least about 10 µg/L, in another embodiment at least about 15 µg/L , and in a further embodiment, at least about 20 µg/l.

In other embodiments, the concentration of copper in the cell culture medium of the present invention may range from about 2.4 to about 20 µg g/L, in another embodiment, from about 2.4 to about 15 µg g/L, in another embodiment. implementation - from about 2.4 to about 10 μg g / l, according to another implementation variant - from about 2.4 to about 8 μg / l, according to another implementation variant - from about 2.4 to about 6 μg / l, according to another implementation variant, from about 2.4 to about 4 µg/l, according to another implementation variant, from about 4 to about 20 µg/l, according to another implementation variant, from about 4 to about 15 µg/ l, according to another implementation variant, from about 4 to about 10 μg/l, according to another implementation variant, from about 4 to about 8 μg/l, and according to a further implementation variant, from about 4 to about 6 μg g/l .

The present invention also provides kits for expression or production of rVWF; these kits contain a culture medium suitable for the expression of rVWF, which has a high specific activity.

B. Cell culture media for the production of ADAMTS13 (A13).

According to one aspect, the present invention provides culture media suitable for the expression of high specific activity ADAMTS proteins. Successfully, it was found that the addition of copper to the culture medium significantly increases the activity of recombinant ADAMTS enzymes (for example, rADAMTS13) expressed in cells cultured in this supplemented medium, while these enzymes are expressed at levels equal to or greater than those for cells cultivated in a medium without additives.

In one aspect, the present invention provides copper-supplemented cell culture media for the expression of recombinant ADAMTS13 protein with high specific activity.

In one embodiment, these media are supplemented to obtain a total copper concentration of about 2 to about 4 µg/L.

In further embodiments, these media are supplemented to obtain a total copper concentration of approximately 1-3, 2-3, 3-4 µg/l.

In one embodiment, these media contain copper at a concentration of at least 1 µg/l.

According to another implementation variant of the specified environment contains at least 2 μg/l of copper.

According to another implementation variant of the specified environment contains at least 4 μg/l of copper.

According to other variants of implementation of the specified environment contains from 2 to 20 μg/l of copper.

According to another implementation variant of the specified environment contains from 1 to 6 μg/l of copper.

According to another implementation variant of the specified environment contains from 2 to 5 μg/l of copper.

According to another implementation variant of the specified environment contains from 3 to 4 μg/l of copper.

In other embodiments, said medium contains at least 1 µg/L copper, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 µg/l or higher concentrations of copper.

In one embodiment, the cell culture solution also contains ammonium at a concentration of less than 10 mM.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 5 mM.

In other embodiments, the cell culture solution contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less.

In other embodiments, the cell culture solution contains a concentration of copper and ammonium according to any of the options 1-440 shown in Table. 1.

- 20 043293

In certain embodiments, the ammonium concentration in the cell culture is maintained for an extended period at the concentration level as described above. For example, in one embodiment, the ammonium concentration is kept low for at least 3 days, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In a specific embodiment, the ammonium concentration in the cell culture is maintained at or below 5 mM for at least 7 days.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 4 mM for at least 7 days.

In a specific embodiment, the ammonium concentration in the cell culture is maintained at or below 5 mM for at least 14 days.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 4 mM for at least 14 days.

According to another embodiment, the ammonium concentration in the cell culture is maintained at a low level throughout the process (ie, during the entire time that the specified culture is used to produce rA13).

In one embodiment, a culture medium is prepared to express a recombinant ADAMTS protein (eg, rADAMTS13) containing at least 1 μg/L copper and at least 2 μM zinc.

In other embodiments, said medium contains at least 2 µg/l copper or at least 4 µg/l copper.

In another embodiment, when copper is added to the media, said culture media also contains at least exactly or about 5 μM zinc.

In one embodiment, said culture medium also contains exactly or about 2 to 12 μM zinc.

According to another implementation variant of the specified culture medium also contains exactly or about 5 to 12 μm zinc.

In other embodiments, said culture medium may also contain at least exactly or about 2 μM, or at least exactly or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15 , 20, 25, 30 µM or more zinc.

According to one of the options for implementing the specified culture medium contains copper and zinc in concentrations according to any of the options 441-880, are given in table. 2.

- 21 043293

table 2

Exemplary embodiments of copper and zinc concentrations in culture media suitable for expression of recombinant ADAMTS 13 protein

v. VI. Zinc concentration PMM 2 µM PM M 3 µM PM M 4 µM PM M 5 µM PM M 6 µM PM M 7 µM PM M 8 µM PM M 9 µM PM M 10 µM 2- 12 microns M 5- 12 microns M Copper concentration pmm 1 µg/l Var. 441 Var. 481 Var. 521 Var. 561 Var. 601 Var. 641 Var. 681 Var. 721 Var. 761 Var 801 Var 841 PMM 2 µg/l Var. 442 Var. 482 Var. 522 Var. 562 Var. 602 Var. 642 Var. 682 Var. 722 Var. 762 Var 802 Var 842 PMM 3 µg/l Var. 443 Var. 483 Var. 523 Var. 563 Var. 603 Var. 643 Var. 683 Var. 723 Var. 763 Var 803 Var 843 PMM 4 µg/l Var. 444 Var. 484 Var. 524 Var. 564 Var. 604 Var. 644 Var. 684 Var. 724 Var. 764 Var 804 Var 844 PMM 5 µg/l Var. 445 Var. 485 Var. 525 Var. 565 Var. 605 Var. 645 Var. 685 Var. 725 Var. 765 Var 805 Var 845 PMM 6 µg/l Var. 446 Var. 486 Var. 526 Var. 566 Var. 606 Var. 646 Var. 686 Var. 726 Var. 766 Var 806 Var 846 PMM 7 µg/l Var. 447 Var. 487 Var. 527 Var. 567 Var. 607 Var. 647 Var. 687 Var. 727 Var. 767 Var 807 Var 847 PMM 8 µg/l Var. 448 Var. 488 Var. 528 Var. 568 Var. 608 Var. 648 Var. 688 Var. 728 Var. 768 Var 808 Var 848 PMM 9 µg/l Var. 449 Var. 489 Var. 529 Var. 569 Var. 609 Var. 649 Var. 689 Var. 729 Var. 769 Var 809 Var 849 PMM 10 µg/l Var. 450 Var. 490 Var. 530 Var. 570 Var. 610 Var. 650 Var. 690 Var. 730 Var. 770 Var 810 Var 850 Approximately 1 µg/l Var. 451 Var. 491 Var. 531 Var. 571 Var. 611 Var. 651 Var. 691 Var. 731 Var. 771 Var 811 Var 851 Approximately 1.5 µg/l Var. 452 Var. 492 Var. 532 Var. 572 Var. 612 Var. 652 Var. 692 Var. 732 Var. 772 Var 812 Var 852

- 22 043293

Approximately 2 µg/l Var. 453 Var. 493 Var. 533 Var. 573 Var. 613 Var. 653 Var. 693 Var. 733 Var. 773 Var 813 Var 853 Approximately 2.5 µg/l Var. 454 Var. 494 Var. 534 Var. 574 Var. 614 Var. 654 Var. 694 Var. 734 Var. 774 Var 814 Var 854 Approximately 3 µg/l Var. 455 Var. 495 Var. 535 Var. 575 Var. 615 Var. 655 Var. 695 Var. 735 Var. 775 Var 815 Var 855 Approximately 3.5 µg/l Var. 456 Var. 496 Var. 536 Var. 576 Var. 616 Var. 656 Var. 696 Var. 736 Var. 776 Var 816 Var 856 Approximately 4 µg/l Var. 457 Var. 497 Var. 537 Var. 577 Var. 617 Var. 657 Var. 697 Var. 737 Var. 777 Var 817 Var 857 Approximately 4.5 µg/l Var. 458 Var. 498 Var. 538 Var. 578 Var. 618 Var. 658 Var. 698 Var. 738 Var. 778 Var 818 Var 858 Approximately 5 µg/l Var. 459 Var. 499 Var. 539 Var. 579 Var. 619 Var. 659 Var. 699 Var. 739 Var. 779 Var 819 Var 859 Approximately 5.5 µg/l Var. 460 Var. 500 Var. 540 Var. 580 Var. 620 Var. 660 Var. 700 Var. 740 Var. 780 Var 820 Var 860 Approximately 6 µg/l Var. 461 Var. 501 Var. 541 Var. 581 Var. 621 Var. 661 Var. 701 Var. 741 Var. 781 Var 821 Var 861 Approximately 7 µg/l Var. 462 Var. 502 Var. 542 Var. 582 Var. 622 Var. 662 Var. 702 Var. 742 Var. 782 Var 822 Var 862 Approximately 8 µg/l Var. 463 Var. 503 Var. 543 Var. 583 Var. 623 Var. 663 Var. 703 Var. 743 Var. 783 Var 823 Var 863 Approximately 9 µg/l Var. 464 Var. 504 Var. 544 Var. 584 Var. 624 Var. 664 Var. 704 Var. 744 Var. 784 Var 824 Var 864 Approximately 10 µg/l Var. 465 Var. 505 Var. 545 Var. 585 Var. 625 Var. 665 Var. 705 Var. 745 Var. 785 Var 825 Var 865 1-20 µg/l Var. 466 Var. 506 Var. 546 Var. 586 Var. 626 Var. 666 Var. 706 Var. 746 Var. 786 Var 826 Var 866 2-20 µg/l Var. 467 Var. 507 Var. 547 Var. 587 Var. 627 Var. 667 Var. 707 Var. 747 Var. 787 Var 827 Var 867 1-10 µg/l Var. 468 Var. 508 Var. 548 Var. 588 Var. 628 Var. 668 Var. 708 Var. 748 Var. 788 Var 828 Var 868 2-10 µg/l Var. 469 Var. 509 Var. 549 Var. 589 Var. 629 Var. 669 Var. 709 Var. 749 Var. 789 Var 829 Var 869 1-6 µg/l Var. 470 Var. 510 Var. 550 Var. 590 Var. 630 Var. 670 Var. 710 Var. 750 Var. 790 Var 830 Var 870 2-6 µg/l Var. 471 Var. 511 Var. 551 Var. 591 Var. 631 Var. 671 Var. 711 Var. 751 Var. 791 Var 831 Var 871 3-6 µg/l Var. 472 Var. 512 Var. 552 Var. 592 Var. 632 Var. 672 Var. 712 Var. 752 Var. 792 Var 832 Var 872

- 23 043293

4-6 µg/l Var. 473 Var. 513 Var. 553 Var. 593 Var. 633 Var. 673 Var. 713 Var. 753 Var. 793 Var 833 Var 873 1-5 µg/l Var. 474 Var. 514 Var. 554 Var. 594 Var. 634 Var. 674 Var. 714 Var. 754 Var. 794 Var 834 Var 874 2-5 µg/l Var. 475 Var. 515 Var. 555 Var. 595 Var. 635 Var. 675 Var. 715 Var. 755 Var. 795 Var 835 Var 875 3-5 µg/l Var. 476 Var. 516 Var. 556 Var. 596 Var. 636 Var. 676 Var. 716 Var. 756 Var. 796 Var 836 Var 876 4-5 µg/l Var. 477 Var. 517 Var. 557 Var. 597 Var. 637 Var. 677 Var. 717 Var. 757 Var. 797 Var 837 Var 877 1-4 µg/l Var. 478 Var. 518 Var. 558 Var. 598 Var. 638 Var. 678 Var. 718 Var. 758 Var. 798 Var 838 Var 878 2-4 µg/l Var. 479 Var. 519 Var. 559 Var. 599 Var. 639 Var. 679 Var. 719 Var. 759 Var. 799 Var 839 Var 879 3-4 µg/l Var. 480 Var. 520 Var. 560 Var. 600 Var. 640 Var. 680 Var. 720 Var. 760 Var. 800 Var 840 Var 880

*PMM=at least

In one embodiment, the cell culture solution also contains a low concentration of ammonium.

In one embodiment, the cell culture solution contains ammonium at a concentration of less than 10 mM, and copper and zinc at concentrations according to any of the options 441-880 shown in Table 1. 2.

In a specific embodiment, the ammonium concentration is maintained at or below 10 mM for at least 7 days.

According to a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 6 mm, and copper and zinc at concentrations according to any of the options 441-880, are given in table. 2.

In a specific embodiment, the ammonium concentration is maintained at or below 6 mM for at least 7 days.

According to another preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 5 mm, and copper and zinc at concentrations according to any of the options 441-880 shown in table. 2.

In a specific embodiment, the ammonium concentration is maintained at or below 5 mM for at least 7 days.

According to another preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 4 mm, and copper and zinc at concentrations according to any of the options 441-880 shown in table. 2.

In a specific embodiment, the ammonium concentration is maintained at or below 4 mM for at least 7 days.

In other embodiments, the cell culture solution contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less, and copper and zinc at concentrations according to any one of options 441-880, given in table. 2.

In yet another specific embodiment, the ammonium concentration in the cell culture is maintained at a low level throughout the process (i.e., during the entire time that the culture is used to produce rA13).

In one embodiment, a culture medium for expression of a recombinant ADAMTS protein (eg, rADAMTS13) is prepared containing at least 1 μg/L copper and at least exactly or about 0.5 mM calcium.

In other embodiments, said medium contains at least 2 µg/l copper or at least 4 µg/l copper.

In another embodiment, when copper is added to the media, said culture medium also contains at least 1.5 mM calcium.

In one embodiment, said culture medium contains exactly or about 0.5 to 1.5 mM calcium.

In other embodiments, said culture medium may contain at least exactly or about 0.5 mM, or at least exactly or about 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.25; 2.5; 2.7 5; 3.0; 3.5; 4.0; 4.5; 5.0 mM or more calcium.

According to one embodiment, said culture medium contains copper and calcium at a concentration according to any one of the options 881-1320 shown in Table 1. 3.

- 24 043293

Table 3

Exemplary embodiments of copper and calcium concentrations in culture media suitable for expression of recombinant ADAMTS13 protein________

VII. VIII. Concentrate walkie-talkie calcium PM m 0.5 mM PM M 0.75 mM PM M 1.0 mM PM m 1.25 mM PM M 1.5 mM PM m 2.0 mM PM M 2.5 mM PM M 3.0 mM PM M 4 mm PM M 5 mM 0.51.5 mM Copper concentration pmm 1 µg/l Var. 881 Var. 921 Var. 961 Var. 1001 Var. 1041 Var. 1081 Var. 1121 Var. 1161 Var. 1201 Var. 1241 Var 128 1 PMM 2 µg/l Var. 882 Var. 922 Var. 962 Var. 1002 Var. 1042 Var. 1082 Var. 1122 Var. 1162 Var. 1202 Var. 1242 Var 128 2 PMM 3 µg/l Var. 883 Var. 923 Var. 963 Var. 1003 Var. 1043 Var. 1083 Var. 1123 Var. 1163 Var. 1203 Var. 1243 Var 128 3 PMM 4 µg/l Var. 884 Var. 924 Var. 964 Var. 1004 Var. 1044 Var. 1084 Var. 1124 Var. 1164 Var. 1204 Var. 1244 Var 128 4 PMM 5 µg/l Var. 885 Var. 925 Var. 965 Var. 1005 Var. 1045 Var. 1085 Var. 1125 Var. 1165 Var. 1205 Var. 1245 Var 128 5 PMM 6 µg/l Var. 886 Var. 926 Var. 966 Var. 1006 Var. 1046 Var. 1086 Var. 1126 Var. 1166 Var. 1206 Var. 1246 Var 128 6 PMM 7 µg/l Var. 887 Var. 927 Var. 967 Var. 1007 Var. 1047 Var. 1087 Var. 1127 Var. 1167 Var. 1207 Var. 1247 Var 128 7 PMM 8 µg/l Var. 888 Var. 928 Var. 968 Var. 1008 Var. 1048 Var. 1088 Var. 1128 Var. 1168 Var. 1208 Var. 1248 Var 128 8 PMM 9 µg/l Var. 889 Var. 929 Var. 969 Var. 1009 Var. 1049 Var. 1089 Var. 1129 Var. 1169 Var. 1209 Var. 1249 Var 128 9 PMM 10 µg/l Var. 890 Var. 930 Var. 970 Var. 1010 Var. 1050 Var. 1090 Var. ISO Var. 1170 Var. 1210 Var. 1250 Var 129 0 Approximately 1 µg/l Var. 891 Var. 931 Var. 971 Var. 1011 Var. 1051 Var. 1091 Var. 1131 Var. 1171 Var. 1211 Var. 1251 Var 129 1 Approximately 1.5 µg/l Var. 892 Var. 932 Var. 972 Var. 1012 Var. 1052 Var. 1092 Var. 1132 Var. 1172 Var. 1212 Var. 1252 Var 129 2 Approximately 2 µg/l Var. 893 Var. 933 Var. 973 Var. 1013 Var. 1053 Var. 1093 Var. 1133 Var. 1173 Var. 1213 Var. 1253 Var 129 3

- 25 043293

Approximately 2.5 µg/l Approximately 3 µg/l Approximately 3.5 µg/l Approximately 4 µg/l Approximately 4.5 µg/l Approximately 5 µg/l Approximately 5.5 µg/l Approximately 6 µg/l Approximately 7 µg/l Approximately 8 µg/l Approximately 9 µg/l Approximately 10 µg/l 1-20 µg/l Var. 894 Var. 895 Var. 896 Var. 897 Var. 898 Var. 899 Var. 900 Var. 901 Var. 902 Var. 903 Var. 904 Var. 905 Var. 906 Var. 934 Var. 935 Var. 936 Var. 937 Var. 938 Var. 939 Var. 940 Var. 941 Var. 942 Var. 943 Var. 944 Var. 945 Var. 946 Var. 974 Var. 975 Var. 976 Var. 977 Var. 978 Var. 979 Var. 980 Var. 981 Var. 982 Var. 983 Var. 984 Var. 985 Var. 986 Var. 1014 Var. 1015 Var. 1016 Var. 1017 Var. 1018 Var. 1019 Var. 1020 Var. 1021 Var. 1022 Var. 1023 Var. 1024 Var. 1025 Var. 1026 Var. 1054 Var. 1055 Var. 1056 Var. 1057 Var. 1058 Var. 1059 Var. 1060 Var. 1061 Var. 1062 Var. 1063 Var. 1064 Var. 1065 Var. 1066 Var. 1094 Var. 1095 Var. 1096 Var. 1097 Var. 1098 Var. 1099 Var. 1100 Var. 1101 Var. 1102 Var. 1103 Var. 1104 Var. 1105 Var. 1106 Var. 1134 Var. 1135 Var. 1136 Var. 1137 Var. 1138 Var. 1139 Var. 1140 Var. 1141 Var. 1142 Var. 1143 Var. 1144 Var. 1145 Var. 1146 Var. 1174 Var. 1175 Var. 1176 Var. 1177 Var. 1178 Var. 1179 Var. 1180 Var. 1181 Var. 1182 Var. 1183 Var. 1184 Var. 1185 Var. 1186 Var. 1214 Var. 1215 Var. 1216 Var. 1217 Var. 1218 Var. 1219 Var. 1220 Var. 1221 Var. 1222 Var. 1223 Var. 1224 Var. 1225 Var. 1226 Var. 1254 Var. 1255 Var. 1256 Var. 1257 Var. 1258 Var. 1259 Var. 1260 Var. 1261 Var. 1262 Var. 1263 Var. 1264 Var. 1265 Var. 1266 Var 129 4 Var 129 5 Var 129 6 Var 129 7 Var 129 8 Var 129 9 Var 130 0 Var 130 1 Var 130 2 Var 130 3 Var 130 4 Var 130 5 Var 130 6 2-20 µg/l Var. 907 Var. 947 Var. 987 Var. 1027 Var. 1067 Var. 1107 Var. 1147 Var. 1187 Var. 1227 Var. 1267 Var 130 7 1-10 µg/l Var. 908 Var. 948 Var. 988 Var. 1028 Var. 1068 Var. 1108 Var. 1148 Var. 1188 Var. 1228 Var. 1268 Var 130 8

- 26 043293

2-10 µg/l Var. 909 Var. 949 Var. 989 Var. 1029 Var. 1069 Var. 1109 Var. 1149 Var. 1189 Var. 1229 Var. 1269 Var 130 9 1-6 µg/l Var. 910 Var. 950 Var. 990 Var. 1030 Var. 1070 Var. 1110 Var. 1150 Var. 1190 Var. 1230 Var. 1270 Var 131 0 2-6 µg/l Var. 911 Var. 951 Var. 991 Var. 1031 Var. 1071 Var. 1111 Var. 1151 Var. 1191 Var. 1231 Var. 1271 Var 131 1 3-6 µg/l Var. 912 Var. 952 Var. 992 Var. 1032 Var. 1072 Var. 1112 Var. 1152 Var. 1192 Var. 1232 Var. 1272 Var 131 2 4-6 µg/l Var. 913 Var. 953 Var. 993 Var. 1033 Var. 1073 Var. 1113 Var. 1153 Var. 1193 Var. 1233 Var. 1273 Var 131 1-5 µg/l Var. 914 Var. 954 Var. 994 Var. 1034 Var. 1074 Var. 1114 Var. 1154 Var. 1194 Var. 1234 Var. 1274 Var 131 4 2-5 µg/l Var. 915 Var. 955 Var. 995 Var. 1035 Var. 1075 Var. 1115 Var. 1155 Var. 1195 Var. 1235 Var. 1275 Var 131 5 3-5 µg/l Var. 916 Var. 956 Var. 996 Var. 1036 Var. 1076 Var. 1116 Var. 1156 Var. 1196 Var. 1236 Var. 1276 Var 131 6 4-5 µg/l Var. 917 Var. 957 Var. 997 Var. 1037 Var. 1077 Var. 1117 Var. 1157 Var. 1197 Var. 1237 Var. 1277 Var 131 7 1-4 µg/l Var. 918 Var. 958 Var. 998 Var. 1038 Var. 1078 Var. 1118 Var. 1158 Var. 1198 Var. 1238 Var. 1278 Var 131 8 2-4 µg/l Var. 919 Var. 959 Var. 999 Var. 1039 Var. 1079 Var. 1119 Var. 1159 Var. 1199 Var. 1239 Var. 1279 Var 131 9 3-4 µg/l Var. 920 Var. 960 Var. 1000 Var. 1040 Var. 1080 Var. 1120 Var. 1160 Var. 1200 Var. 1240 Var. 1280 Var 132 0

*PMM=at least

In one embodiment, the cell culture solution also contains a low concentration of ammonium.

In one embodiment, the cell culture solution contains ammonium at a concentration of less than 10 mM, and copper and calcium at concentrations according to any of the options 881-1320 shown in Table 1. 3.

In a specific embodiment, the ammonium concentration is maintained at a level not exceeding 10 mM for at least 7 days.

In a preferred embodiment, said cell culture medium contains ammonium at a concentration not exceeding 6 mM, and copper and calcium at concentrations according to any one of the options 881-1320 shown in Table 1. 3.

In a specific embodiment, the ammonium concentration is maintained at or below 6 mM for at least 7 days.

In another preferred embodiment, said cell culture medium contains ammonium at a concentration not exceeding 5 mM, and copper and calcium at concentrations according to any one of the options 881-1320 shown in Table 1. 3.

In a specific embodiment, the ammonium concentration is maintained at or below 5 mM for at least 7 days.

In another preferred embodiment, said cell culture medium contains ammonium at a concentration not exceeding 4 mM, and copper and calcium at concentrations according to any of the options 881-1320 shown in Table 1. 3.

In a specific embodiment, the ammonium concentration is maintained at or below 4 mM for at least 7 days.

In other embodiments, said cell culture medium contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, and copper and calcium at concentrations according to any one of options 881-1320, given in table. 3.

In yet another specific embodiment, the ammonium concentration in the cell culture is maintained at a low level throughout the process (i.e., during the entire time that the culture is used to produce rA13).

- 27 043293

In one embodiment, copper, zinc, and calcium are added to the cell culture medium.

According to a specific implementation variant of the culture medium contains calcium at a concentration of at least 0.5 mm, and copper and zinc in concentrations according to any of the options 441-880, are given in table. 2.

According to another specific implementation variant of the culture medium contains calcium at a concentration of at least 1.5 mm, and copper and zinc in concentrations according to any of the options 441880 shown in table. 2.

According to another specific implementation variant of the specified culture medium contains calcium at a concentration of 0.5 and 1.5 mm, and copper and zinc at concentrations according to any of the options 441-880, are given in table. 2.

In other embodiments, the culture medium contains calcium at a concentration of at least 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1.2; 1.3; 1.4 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.25; 2.5; 2.75; 3.0; 3.5; 4.0; 4.5; 5.0 mm or more, and copper and zinc in concentrations according to any of the options 441-880, are given in table. 2.

In one embodiment, a culture medium is prepared to express a recombinant ADAMTS protein (eg, rADAMTS13) containing at least 1 μg/l copper and at least 2 mg/l nicotinamide (vitamin B3).

In other embodiments, said medium contains at least 2 µg/l copper or at least 4 µg/l copper.

In another embodiment, when copper is added to the media, said culture medium also contains at least 7 mg/L of nicotinamide (vitamin B3).

In one embodiment, said culture medium contains exactly or about 2 mg/l to 10 mg/l nicotinamide (vitamin B3).

In other embodiments, said culture medium may contain at least exactly or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 mg/l, or higher concentrations of nicotinamide (vitamin B3).

According to one of the options for implementing the specified culture medium contains copper and nicotinamide in concentrations according to any of the options 1321-1760 shown in table. 4.

Table 4 Exemplary embodiments of copper and nicotinamide concentrations in culture media suitable ____ for the expression of recombinant ADAMTS13 protein ________

IX. x. Concentrate] walkie-talkie calcium PMM 2 mg/ml pm m mg/m l pm M 4 mg/m l pm M 5 mg/m l pm M 6 mg/m l pm m 7 mg/m l PM M 8 mg/m l pm M 9 mg/m l pm M 10 mg/m l pm M 15 mg/m l 2-10 mg/m l Copper concentration pmm 1 µg/l Var. 1321 Var. 1361 Var. 1401 Var. 1441 Var. 1481 Var. 1521 Var. 1561 Var. 1601 Var. 1641 Var. 1681 Var. 1721 pmm 2 µg/l Var. 1322 Var. 1362 Var. 1402 Var. 1442 Var. 1482 Var. 1522 Var. 1562 Var. 1602 Var. 1642 Var. 1682 Var. 1722 pmm 3 µg/l Var. 1323 Var. 1363 Var. 1403 Var. 1443 Var. 1483 Var. 1523 Var. 1563 Var. 1603 Var. 1643 Var. 1683 Var. 1723 pmm 4 µg/l Var. 1324 Var. 1364 Var. 1404 Var. 1444 Var. 1484 Var. 1524 Var. 1564 Var. 1604 Var. 1644 Var. 1684 Var. 1724 pmm 5 µg/l Var. 1325 Var. 1365 Var. 1405 Var. 1445 Var. 1485 Var. 1525 Var. 1565 Var. 1605 Var. 1645 Var. 1685 Var. 1725 pmm 6 µg/l Var. 1326 Var. 1366 Var. 1406 Var. 1446 Var. 1486 Var. 1526 Var. 1566 Var. 1606 Var. 1646 Var. 1686 Var. 1726 pmm 7 µg/l Var. 1327 Var. 1367 Var. 1407 Var. 1447 Var. 1487 Var. 1527 Var. 1567 Var. 1607 Var. 1647 Var. 1687 Var. 1727

- 28 043293

pmm 8 µg/l Var. 1328 Var. 1368 Var. 1408 Var. 1448 Var. 1488 Var. 1528 Var. 1568 Var. 1608 Var. 1648 Var. 1688 Var. 1728 PMM 9 µg/l Var. 1329 Var. 1369 Var. 1409 Var. 1449 Var. 1489 Var. 1529 Var. 1569 Var. 1609 Var. 1649 Var. 1689 Var. 1729 PMM 10 µg/l Var. 1330 Var. 1370 Var. 1410 Var. 1450 Var. 1490 Var. 1530 Var. 1570 Var. 1610 Var. 1650 Var. 1690 Var. 1730 Approximately 1 µg/l Var. 1331 Var. 1371 Var. 1411 Var. 1451 Var. 1491 Var. 1531 Var. 1571 Var. 1611 Var. 1651 Var. 1691 Var. 1731 Approximately 1.5 µg/l Var. 1332 Var. 1372 Var. 1412 Var. 1452 Var. 1492 Var. 1532 Var. 1572 Var. 1612 Var. 1652 Var. 1692 Var. 1732 Approximately 2 µg/l Var. 1333 Var. 1373 Var. 1413 Var. 1453 Var. 1493 Var. 1533 Var. 1573 Var. 1613 Var. 1653 Var. 1693 Var. 1733 Approximately 2.5 µg/l Var. 1334 Var. 1374 Var. 1414 Var. 1454 Var. 1494 Var. 1534 Var. 1574 Var. 1614 Var. 1654 Var. 1694 Var. 1734 Approximately 3 µg/l Var. 1335 Var. 1375 Var. 1415 Var. 1455 Var. 1495 Var. 1535 Var. 1575 Var. 1615 Var. 1655 Var. 1695 Var. 1735 Approximately 3.5 µg/l Var. 1336 Var. 1376 Var. 1416 Var. 1456 Var. 1496 Var. 1536 Var. 1576 Var. 1616 Var. 1656 Var. 1696 Var. 1736 Approximately 4 µg/l Var. 1337 Var. 1377 Var. 1417 Var. 1457 Var. 1497 Var. 1537 Var. 1577 Var. 1617 Var. 1657 Var. 1697 Var. 1737 Approximately 4.5 µg/l Var. 1338 Var. 1378 Var. 1418 Var. 1458 Var. 1498 Var. 1538 Var. 1578 Var. 1618 Var. 1658 Var. 1698 Var. 1738 Approximately 5 µg/l Var. 1339 Var. 1379 Var. 1419 Var. 1459 Var. 1499 Var. 1539 Var. 1579 Var. 1619 Var. 1659 Var. 1699 Var. 1739 Approximately 5.5 µg/l Var. 1340 Var. 1380 Var. 1420 Var. 1460 Var. 1500 Var. 1540 Var. 1580 Var. 1620 Var. 1660 Var. 1700 Var. 1740 Approximately 6 µg/l Var. 1341 Var. 1381 Var. 1421 Var. 1461 Var. 1501 Var. 1541 Var. 1581 Var. 1621 Var. 1661 Var. 1701 Var. 1741 Approximately 7 µg/l Var. 1342 Var. 1382 Var. 1422 Var. 1462 Var. 1502 Var. 1542 Var. 1582 Var. 1622 Var. 1662 Var. 1702 Var. 1742 Approximately 8 µg/l Var. 1343 Var. 1383 Var. 1423 Var. 1463 Var. 1503 Var. 1543 Var. 1583 Var. 1623 Var. 1663 Var. 1703 Var. 1743 Approximately 9 µg/l Var. 1344 Var. 1384 Var. 1424 Var. 1464 Var. 1504 Var. 1544 Var. 1584 Var. 1624 Var. 1664 Var. 1704 Var. 1744 Approximately 10 µg/l Var. 1345 Var. 1385 Var. 1425 Var. 1465 Var. 1505 Var. 1545 Var. 1585 Var. 1625 Var. 1665 Var. 1705 Var. 1745 1-20 µg/l Var. 1346 Var. 1386 Var. 1426 Var. 1466 Var. 1506 Var. 1546 Var. 1586 Var. 1626 Var. 1666 Var. 1706 Var. 1746 2-20 µg/l Var. 1347 Var. 1387 Var. 1427 Var. 1467 Var. 1507 Var. 1547 Var. 1587 Var. 1627 Var. 1667 Var. 1707 Var. 1747 1-10 µg/l Var. 1348 Var. 1388 Var. 1428 Var. 1468 Var. 1508 Var. 1548 Var. 1588 Var. 1628 Var. 1668 Var. 1708 Var. 1748 2-10 µg/l Var. 1349 Var. 1389 Var. 1429 Var. 1469 Var. 1509 Var. 1549 Var. 1589 Var. 1629 Var. 1669 Var. 1709 Var. 1749 1-6 µg/l Var. 1350 Var. 1390 Var. 1430 Var. 1470 Var. 1510 Var. 1550 Var. 1590 Var. 1630 Var. 1670 Var. 1710 Var. 1750 2-6 µg/l Var. 1351 Var. 1391 Var. 1431 Var. 1471 Var. 1511 Var. 1551 Var. 1591 Var. 1631 Var. 1671 Var. 1711 Var. 1751 3-6 µg/l Var. 1352 Var. 1392 Var. 1432 Var. 1472 Var. 1512 Var. 1552 Var. 1592 Var. 1632 Var. 1672 Var. 1712 Var. 1752 4-6 µg/l Var. 1353 Var. 1393 Var. 1433 Var. 1473 Var. 1513 Var. 1553 Var. 1593 Var. 1633 Var. 1673 Var. 1713 Var. 1753 1-5 µg/l Var. 1354 Var. 1394 Var. 1434 Var. 1474 Var. 1514 Var. 1554 Var. 1594 Var. 1634 Var. 1674 Var. 1714 Var. 1754 2-5 µg/l Var. 1355 Var. 1395 Var. 1435 Var. 1475 Var. 1515 Var. 1555 Var. 1595 Var. 1635 Var. 1675 Var. 1715 Var. 1755 3-5 µg/l Var. 1356 Var. 1396 Var. 1436 Var. 1476 Var. 1516 Var. 1556 Var. 1596 Var. 1636 Var. 1676 Var. 1716 Var. 1756 4-5 µg/l Var. 1357 Var. 1397 Var. 1437 Var. 1477 Var. 1517 Var. 1557 Var. 1597 Var. 1637 Var. 1677 Var. 1717 Var. 1757 1-4 µg/l Var. 1358 Var. 1398 Var. 1438 Var. 1478 Var. 1518 Var. 1558 Var. 1598 Var. 1638 Var. 1678 Var. 1718 Var. 1758 2-4 µg/l Var. 1359 Var. 1399 Var. 1439 Var. 1479 Var. 1519 Var. 1559 Var. 1599 Var. 1639 Var. 1679 Var. 1719 Var. 1759 3-4 µg/l Var. 1360 Var. 1400 Var. 1440 Var. 1480 Var. 1520 Var. 1560 Var. 1600 Var. 1640 Var. 1680 Var. 1720 Var. 1760

*PMM=at least

In one embodiment, the cell culture solution also contains a low concentration of ammonium.

According to one embodiment, said culture medium contains ammonium at a concentration of less than 10 mM, and copper and nicotinamide at concentrations according to any of the options 1321-1760 shown in Table 1. 4.

In a specific embodiment, the ammonium concentration is maintained at or below 10 mM for at least 7 days.

According to a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 6 mm, and copper and nicotinamide at concentrations according to any of the options 1321-1760 shown in table. 4.

In a specific embodiment, the ammonium concentration is maintained at or below 6 mM for at least 7 days.

According to another preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 5 mm, and copper and nicotinamide at concentrations according to any of the options 1321-1760 shown in table. 4.

According to a specific implementation variant, the ammonium concentration is maintained at a level not

- 29 043293 above 5 mm for at least 7 days.

According to another preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 4 mm, and copper and nicotinamide at concentrations according to any of the options 1321-1760 shown in table. 4.

In a specific embodiment, the ammonium concentration is maintained at or below 4 mM for at least 7 days.

In other embodiments, the cell culture solution contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less, and copper and nicotinamide at concentrations according to any one of options 1321-1760, given in table. 4.

In yet another specific embodiment, the ammonium concentration in the cell culture is maintained at a low level throughout the process (i.e., during the entire time that the culture is used to produce rA13).

In one embodiment, copper, zinc, and nicotinamide are added to the cell culture medium.

According to a specific implementation variant of the specified culture medium contains nicotinamide at a concentration of at least 2 mg/ml, and copper and zinc at concentrations according to any of the options 441-880, are given in table. 2.

According to another specific implementation variant of the specified culture medium contains nicotinamide at a concentration of at least 7 mg/ml mm, and copper and zinc at concentrations according to any of the options 441-880, are given in table. 2.

According to another specific implementation variant of the specified culture medium contains nicotinamide at a concentration of 2 and 10 mg/ml, and copper and zinc at concentrations according to any of the options 441-880, are given in table. 2.

In other embodiments, the culture medium contains nicotinamide at a concentration of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mg/ml or more, and copper and zinc in concentrations according to any of the options 441-880 shown in table. 2.

In one embodiment, copper, calcium, and nicotinamide are added to the cell culture medium. According to a specific implementation variant of the culture medium contains nicotinamide at a concentration of at least 2 mg/ml, and copper and calcium at concentrations according to any of the options 881-1320 shown in table. 3. According to another specific implementation variant of the culture medium contains nicotinamide at a concentration of at least 7 mg/ml mm, and copper and calcium at concentrations according to any of the options 881-1320 shown in table. 3. According to another specific implementation variant of the culture medium contains nicotinamide at a concentration of 2 mg/ml to 10 mg/ml, and copper and calcium at concentrations according to any of the options 881-1320 shown in table. 3. In other embodiments, the culture medium contains nicotinamide at a concentration of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mg/ml or more, and copper and calcium in concentrations according to any of the options 881-1320, are given in table. 3.

XI. Methods for obtaining blood factors with high specific activity.

A. Methods for culturing cells.

The present invention provides methods for the large scale production of recombinant proteins (such as rVWF and rA13).

In certain embodiments, such large-scale production methods use agitated/stirred reactors to produce said therapeutic recombinant proteins.

In certain embodiments, the methods of the present invention may include the use of cell culture systems in a batch or continuous mode. For example, if batch cell cultures are used, the cells may be subjected to a single batch, fed-batch, or repeated batch mode. Similarly, for continuous cell cultures, for example, a perfusion, turbidostatic, or chemostatic regimen may be applied. Batch and continuous cell culture can be carried out under suspension or adhesive culture conditions. Under suspension culture conditions, the cells are freely suspended and distributed in the culture medium. Alternatively, under adhesion culture conditions, the cells are bound to a solid phase such as a microcarrier, a porous microcarrier, a disc carrier, a ceramic cartridge, a hollow fiber, a flat plate, a gel matrix, and the like.

Batch culture is generally a large scale cell culture in which the cell inoculum is cultured to maximum density in a reactor or fermenter and harvested and processed as in single batch culture. The fed culture is typically a batch culture receiving either fresh nutrients (eg, growth-limiting substrates) or supplements (eg, product precursors). The nutrient solution is usually highly concentrated to avoid dilution in the bioreactor. In repeated batch culture, cells are placed in a culture medium and

- 30 043293 grown to the desired cell density. The culture is then diluted with complete growth medium before the cells reach maximum concentration to avoid the onset of a decay phase and cell death. The number and frequency of dilutions varies widely and depends on the growth characteristics of the cell line and the convenience of the culture process. This process can be repeated as many times as needed and, if the cells and medium are not discarded at subculture, the culture volume will be increased stepwise with each successive dilution. The issue of increasing volume can be resolved by using a reactor having sufficient size for dilutions within said vessel, or by spreading the diluted culture over several vessels. The principle of this type of cultivation is to maintain cells in an exponential growth phase. Serially subcultured culture is characterized by the fact that the volume of said culture always increases gradually, there may be several fees, the cells continue to grow and this process can continue for as long as necessary.

In certain embodiments, the recombinant ADAMTS protein (eg, rADAMTS13) can be isolated after collection of the batch culture supernatant.

In other embodiments, the recombinant VWF may be isolated after collection of the batch culture supernatant.

The continuous culture may be a suspension culture continuously fed with fresh medium influx, where the culture volume is generally maintained at a constant volume by concomitant removal of the spent medium.

According to chemostatic and turbidostatic methods, the extracted medium contains cells. Thus, the cells remaining in the cell culture vessel must grow to maintain a steady state.

According to the chemostatic method, the growth rate is generally controlled by controlling the degree of dilution, i.e. rate of addition of fresh medium. The growth rate of cells in culture can be maintained, for example, at a submaximal level, by changing the degree of dilution. In contrast, in the turbidostatic method, the dilution rate is set to provide the maximum growth rate that cells can achieve under given process conditions such as pH and temperature.

In certain embodiments, rVWF or rA13 is isolated after collection of the continuous culture supernatant. An exemplary continuous cell culture method is described in WO/2011/012725 (Grillberger et al.), the contents of which are hereby incorporated by reference in their entirety for any purpose.

In perfusion culture, the extracted medium is poor in cells, which remain in the culture vessel, for example, due to the use of filtration or centrifugation methods, resulting in the re-introduction of cells into culture. However, as a rule, those used for filtration do not retain 100% of the cells, and thus some of them are removed when the medium is removed. Very high growth rates for cultivation in perfusion mode are not critical, since most of the cells remain in the culture vessel.

In certain embodiments, rVWF or rA13 is isolated after collection of the perfusion culture supernatant.

The stirred tank reaction system can be used for batch and continuous cell culture in suspension or adhesive modes. In general, said stirred tank reaction system can be operated as any standard stirred tank reactor with any type of agitation, such as Rushton, hydraulic, pitched blade, or propeller type.

In certain embodiments, cell culture methods of the present invention may include the use of a microcarrier.

In some embodiments, cell culture according to embodiments of the invention may be carried out in large bioreactors under conditions suitable for obtaining high specific surface areas relative to culture volume to achieve high cell densities and protein expression.

One way to achieve such growth conditions is to use cell culture microcarriers in agitated bioreactors. The principle of growing cells on microcarriers was first described by van Wezel (van Wezel, A.L., Nature 216:64-5 (1967)); it provides attachment of cells to the surface of small solid particles suspended in the growth medium.

These methods provide a high surface-to-volume ratio and thus ensure efficient utilization of nutrients. In addition, when secreted proteins are expressed in eukaryotic cell lines, increasing the surface-volume ratio results in higher levels of secretion and thus higher protein yield in the culture supernatant.

Finally, these methods make it easy to scale up eukaryotic expression cultures.

Cells expressing vWF and/or rA13 may be associated with spherical or porous

- 31 043293 microcarrier during cell culture growth. Said microcarrier may be a microcarrier selected from the group of microcarriers based on dextran, collagen, plastic, gelatin, cellulose and others as described by Butler (1988. In: Spier & Griffiths, Animal Cell Biotechnology 3:283-303). It is also possible to grow cells to a certain biomass on spherical microcarriers, and subculture these cells, when they reach the final biomass for the fermenter, before obtaining the expressed protein, onto a porous microcarrier, or vice versa. Suitable spherical microcarriers may include smooth surface microcarriers such as Cytodex™ 1, Cytodex™ 2, and Cytodex™ 3 (GE Healthcare) and large pore microcarriers such as Cytopore™ 1, Cytopore™ 2, Cytoline™ 1 and Cytoline™ 2 (GE healthcare).

As described above, the present invention includes cell culture media containing an elevated concentration of copper. It is understood that all embodiments of the invention and the concentrations described above in the Cell Culture Media section apply to the methods of the present invention described in the invention.

In certain embodiments, said culture may be maintained for at least about 7 days, or at least about 14, 21, 28 days, or at least about 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or at least about 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months, or longer. The cell density that is maintained in a cell culture to produce a recombinant vWF protein or a recombinant rA13 protein depends on the culture conditions and the medium used to express the protein.

One skilled in the art will readily be able to determine the optimal cell density for culturing cells to produce rVWF or rA13.

In one embodiment, the cell density in said culture is maintained at about 0.5x106 to 4x10 ⁷ cells/mL for an extended period of time.

In other embodiments, the cell density is maintained at a concentration of about 1.0x106 to about 1.0x107 cells/mL for an extended period of time.

In other embodiments, the cell density is maintained at a concentration of about 1.0x106 to about 4.0x106 cells/mL for an extended period of time.

In other embodiments, the cell density is maintained at a concentration of about 1.0x106 to about 4.0x106 cells/mL for an extended period of time.

In other embodiments, cell density may be maintained at a concentration of about 2.0x106 to about 4.0x106 cells/ml, or about 1.0x106 to about 2.5x106 cells/ml, or about 1.5x106 to about 3. 5x106 cells/ml, or in any similar range, for an extended period of time.

In one embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 4.0 x 106 cells/mL for at least 7 days.

In a specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 4.0 x 106 cells/mL for at least 14 days.

In a more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 4.0 x 106 cells/mL for at least 21 days.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 4.0 x 106 cells/mL for at least 28 days.

In another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 4.0 x 106 cells/mL for at least 5 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 4.0 x 106 cells/mL for at least 6 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 4.0 x 106 cells/mL for at least 7 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 4.0 x 106 cells/mL for at least 8 weeks.

- 32 043293

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 4.0 x 10 ⁶ cells/mL for at least 9 weeks.

In one embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.5 x 10 ⁶ cells/mL for at least 7 days.

In a specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.5 x 10 ⁶ cells/mL for at least 14 days.

In a more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.5 x 10 ⁶ cells/ml for at least 21 days.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.5 x 10 ⁶ cells/mL for at least 28 days.

In another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.5 x 10 ⁶ cells/mL for at least 5 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.5 x 10 ⁶ cells/mL for at least 6 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.5 x 10 ⁶ cells/mL for at least 7 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.5 x 10 ⁶ cells/mL for at least 8 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.5 x 10 ⁶ cells/mL for at least 9 weeks.

In one embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.0 x 10 ⁶ cells/mL for at least 7 days.

In a specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.0 x 10 ⁶ cells/mL for at least 14 days.

In a more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.0 x 10 ⁶ cells/mL for at least 21 days.

In another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.0 x 10 ⁶ cells/mL for at least 28 days.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.0 x 10 ⁶ cells/mL for at least 5 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.0 x 10 ⁶ cells/mL for at least 6 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.0 x 10 ⁶ cells/mL for at least 7 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.0 x 10 ⁶ cells/mL for at least 8 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 3.0 x 10 ⁶ cells/mL for at least 9 weeks.

In one embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.5 x 10 ⁶ cells/mL for at least 7 days.

- 33 043293

In a specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.5 x 10 ⁶ cells/mL for at least 14 days.

In a more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.5 x 10 ⁶ cells/ml for at least 21 days.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.5 x 10 ⁶ cells/mL for at least 28 days.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.5 x 10 ⁶ cells/mL for at least 5 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.5 x 10 ⁶ cells/mL for at least 6 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.5 x 10 ⁶ cells/mL for at least 7 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.5 x 10 ⁶ cells/mL for at least 8 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.5 x 10 ⁶ cells/mL for at least 9 weeks.

In another embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.0 x 10 ⁶ cells/mL for at least 7 days.

In a specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.0 x 10 ⁶ cells/mL for at least 14 days.

In a more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.0 x 10 ⁶ cells/mL for at least 21 days.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.0 x 10 ⁶ cells/mL for at least 28 days.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.0 x 10 ⁶ cells/mL for at least 5 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.0 x 10 ⁶ cells/mL for at least 6 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.0 x 10 ⁶ cells/mL for at least 7 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.0 x 10 ⁶ cells/mL for at least 8 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 2.0 x 10 ⁶ cells/mL for at least 9 weeks.

In one embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 1.5 x 10 ⁶ cells/mL for at least 7 days.

In a specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 1.5 x 10 ⁶ cells/mL for at least 14 days.

In a more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 1.5 x 10 ⁶ cells/mL for at least 21 days.

- 34 043293

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 1.5 x 10 ⁶ cells/mL for at least 28 days.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 1.5 x 10 ⁶ cells/mL for at least 5 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 1.5 x 10 ⁶ cells/mL for at least 6 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 1.5 x 10 ⁶ cells/mL for at least 7 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 1.5 x 10 ⁶ cells/mL for at least 8 weeks.

In yet another more specific embodiment, the cell density of the continuous cell culture of the present invention is maintained at a concentration not exceeding 1.5 x 10 ⁶ cells/mL for at least 9 weeks.

Below are the specific details of the methods for obtaining rVWF and rA13.

It should be understood that although the conditions are given specifically for rVWF or rA13, the specified conditions for rVWF can be used to obtain rA13, and vice versa.

B. Methods for producing high molecular weight recombinant vWF.

According to another aspect, the present invention also relates to methods for producing vWF under cell culture conditions, including a cell culture medium containing an increased concentration of copper.

In certain embodiments, said culture also contains a low concentration of ammonium. As used herein, the terms cell culture (cell culture) and cell culture solution are used interchangeably.

In one embodiment, the present invention provides a method for producing high molecular weight recombinant vWF, comprising:

a) providing cell culture;

b) introducing a nucleic acid sequence encoding vWF; c) selecting cells carrying said nucleic acid sequence; And

d) effecting expression of vWF in said cells under cell culture conditions comprising a cell culture medium containing copper at a concentration of at least about 2.4 μg/L and a cell culture supernatant containing ammonium at a concentration of less than about 1 mM, wherein said vWF is a high multimeric vWF containing from about 14 to about 22 dimers and having a specific ristocetin activity of at least about 30 IU/µg.

In further embodiments, the multimeric rVWF produced using the methods of the present invention contains approximately 10-30, 12-28, 14-26, 16-24, 18-22, 20-21 dimers. According to further implementation options rVWF obtained according to the present invention, has a specific activity of at least about 20; 22.5; 25; 27.5; thirty; 32.5; 35; 37.5; 40; 42.5; 45; 47.5; 50; 52.5; 55; 57.5; 60; 62.5; 65; 67.5; 70; 72.5; 75; 77.5; 80 or more IU / mcg.

In a specific embodiment, the cell density in the continuous cell culture to produce rVWF is maintained at a concentration not exceeding 2.5 x 10 ⁶ cells/mL for an extended period.

In other specific embodiments, the cell density is maintained at or below 2.0x10 ⁶ , 1.5x10 ⁶ , 1.0x10 ⁶ , 0.5x10 ⁶ cells/mL or less.

In one embodiment, cell density is maintained between 1.5 x 10 ⁶ and 2.5 x 10 ⁶ cells/mL.

In one embodiment, the present invention provides a method for preparing a recombinant von Willebrand factor (rVWF) composition; this method includes the steps:

(a) providing basic cell culture media;

(b) adding copper to the basic cell culture media to a final copper concentration of at least 2.0 µg/l;

(c) providing one or more cells containing a nucleic acid encoding a rVWF protein;

(d) culturing said one or more cells in copper-supplemented cell culture medium such that rVWF is expressed and excreted from the cells into the culture medium.

- 35 043293 pernatant; and (e) separating at least a portion of said culture supernatant, wherein said separated supernatant has a rVWF specific ristocetin cofactor activity of at least 30 IU/µg rVWF.

In one embodiment, the cell culture solution also contains ammonium at a concentration of less than 10 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration not exceeding 10 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 5 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration not exceeding 5 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 4 mM.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at an ammonium concentration not exceeding 4 mM for at least 7 days.

In other embodiments, the cell culture solution contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less.

According to another embodiment, the ammonium concentration in the cell culture is maintained at a low level throughout the process (ie, during the entire time that the specified culture is used to produce rVWF).

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, copper is added to the basic cell culture media to a final copper concentration of at least 2.4 µg/l.

In one embodiment, the cell culture solution also contains ammonium at a concentration of less than 10 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 10 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 5 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 5 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 4 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 4 mM for at least 7 days.

In other embodiments, the cell culture solution contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less.

According to another embodiment, the concentration of ammonium in the cell culture solution is maintained at a low level throughout the process (ie, during the entire time during which the specified culture is used to obtain rVWF).

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, copper is added to the basic cell culture media to a final copper concentration of at least 3 µg/L.

In one embodiment, the cell culture solution also contains ammonium at a concentration of less than 10 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 10 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium in

- 36 043293 concentration not higher than 5 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 5 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 4 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 4 mM for at least 7 days.

In other embodiments, the cell culture solution contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less.

According to another embodiment, the concentration of ammonium in the cell culture solution is maintained at a low level throughout the process (ie, during the entire time during which the specified culture is used to obtain rVWF).

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, copper is added to the basic cell culture media to a final copper concentration of at least 4 µg/l.

In one embodiment, the cell culture solution also contains ammonium at a concentration of less than 10 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 10 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 5 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 5 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 4 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 4 mM for at least 7 days.

In other embodiments, the cell culture media contain ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less.

According to another embodiment, the concentration of ammonium in the cell culture solution is maintained at a low level throughout the process (ie, during the entire time during which the specified culture is used to obtain rVWF).

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

In one embodiment of the method described above, copper is added to the basic cell culture media to a final copper concentration of approximately 4.3 µg/L.

In one embodiment, the cell culture solution also contains ammonium at a concentration of less than 10 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 10 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 5 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 5 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 4 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 4 mM for at least 7 days.

In other embodiments, the cell culture solution contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less.

According to another embodiment, the concentration of ammonium in the cell culture solution is maintained at a low level throughout the process (ie, during the entire time during which the specified culture is used to obtain rVWF).

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, copper is added to the basic cell culture media to a final copper concentration of 2 to 20 µg/l.

In one embodiment, the cell culture solution also contains ammonium at a concentration of less than 10 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 10 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 5 mM.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 5 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 4 mM. In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 4 mM for at least 7 days.

In other embodiments, the cell culture solution contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less.

According to another embodiment, the ammonium concentration in the cell culture is maintained at a low level throughout the process (ie, during the entire time that the specified culture is used to produce rVWF).

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, copper is added to the basic cell culture media to a final copper concentration of 3 to 10 µg/l.

In one embodiment, the cell culture solution also contains ammonium at a concentration of less than 10 mM.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 10 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 5 mM.

In another specific embodiment, the ammonium concentration in the cell culture solution is maintained at an ammonium concentration of no more than 5 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 4 mM.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 4 mM for at least 7 days.

In other embodiments, the cell culture solution contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less.

According to another embodiment, the concentration of ammonium in the cell culture solution is maintained at a low level throughout the process (ie, during the entire time during which the specified culture is used to obtain rVWF).

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, copper is added to the basic cell culture media to a final copper concentration of 4 to 7.5 µg/L.

In one embodiment, the cell culture solution also contains ammonium at a concentration of less than 10 mM.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 10 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium in

- 38 043293 concentration not higher than 5 mM.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 5 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 4 mM.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 4 mM for at least 7 days.

In other embodiments, the cell culture solution contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less.

According to another embodiment, the ammonium concentration in the cell culture is maintained at a low level throughout the process (ie, during the entire time that the specified culture is used to produce rVWF).

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

In one embodiment, the present invention provides a method for preparing a recombinant von Willebrand factor (rVWF) composition; this method includes the steps:

(a) providing basic cell culture media;

(b) adding copper to the basic cell culture media;

(c) providing one or more cells containing a nucleic acid encoding a rVWF protein;

(d) culturing said one or more cells in a copper-supplemented cell culture medium such that rVWF is expressed and excreted from the cells into the culture supernatant; and (e) separating at least a portion of said culture supernatant, wherein the NH ₄ ⁺ concentration in said supernatant is maintained at a low level for at least 7 days, wherein said separated supernatant also has a rVWF specific ristocetin cofactor activity of at least 30 IU/µg rVWF.

According to a specific embodiment, the concentration of copper and the concentration of NH ₄ ⁺ in the cell culture solution is maintained at the concentration level according to any of the options 1-440 shown in table. 1.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one of the embodiments of the method described above, the concentration of copper and the concentration of NH4+ in the cell culture is maintained at the concentration level according to any of the options 1-440 shown in table. 1, for at least 14 days.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one of the embodiments of the method described above, the concentration of copper and the concentration of NH4+ in the cell culture is maintained at the concentration level according to any of the options 1-440 shown in table. 1 for at least 21 days.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one of the embodiments of the method described above, the concentration of copper and the concentration of NH4+ in the cell culture is maintained at the concentration level according to any of the options 1-440 shown in table. 1 for at least 28 days.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one of the embodiments of the method described above, the concentration of copper and the concentration of NH4+ in the cell culture is maintained at the concentration level according to any of the options 1-440 shown in table. 1 for at least 5 weeks.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

- 39 043293

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, the concentration of copper and the concentration of NH ₄ ⁺ in cell culture is maintained at the concentration level according to any of the options 1-440 shown in table. 1 for at least 6 weeks.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, the concentration of copper and the concentration of NH ₄ ⁺ in cell culture is maintained at the concentration level according to any of the options 1-440 shown in table. 1 for at least 7 weeks.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, the concentration of copper and the concentration of NH ₄ ⁺ in cell culture is maintained at the concentration level according to any of the options 1-440 shown in table. 1 for at least 8 weeks.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, the concentration of copper and the concentration of NH ₄ ⁺ in cell culture is maintained at the level of concentrations according to any of the options 1-440 shown in table. 1 for at least 9 weeks.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

In one embodiment, the present invention provides a method for preparing a recombinant von Willebrand factor (rVWF) composition; this method includes the steps:

(a) providing basic cell culture media;

(b) adding copper to the basic cell culture media to a final copper concentration of at least 2.0 µg/L;

(c) providing one or more cells containing a nucleic acid encoding a rVWF protein;

(d) culturing said one or more cells in a copper-supplemented cell culture medium such that rVWF is expressed and excreted from the cells into the culture supernatant;

(e) monitoring the concentration of ammonium in said culture supernatant; and (f) separating at least a portion of the culture supernatant, wherein said culture supernatant containing ammonium at a concentration of more than 10 mM is not used to prepare the rVWF composition, and wherein said separated supernatant has a specific rVWF ristocetin cofactor activity of at least 30 mU/µg rVWF.

In certain embodiments, the final concentration of copper in the supplemented basic culture media is at least 2.4, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 µg/l or more.

In other embodiments, the final concentration of copper in the supplemented basic culture media is 2-20, 2-10, 3-8, or 4-6 µg/L.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, the culture supernatant containing ammonium at a concentration greater than 6 mM is not used to prepare the rVWF composition.

In certain embodiments, the final concentration of copper in the supplemented basic culture media is at least 2.4, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 µg/l or more.

In other embodiments, the final concentration of copper in the supplemented basic culture media is 2-20, 2-10, 3-8, or 4-6 µg/L.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a specific rVWF ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, the culture supernatant containing ammonium at a concentration greater than 5 mM is not used to prepare the rVWF composition.

In certain embodiments, the final concentration of copper in the supplemented basic culture media is at least 2.4, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 µg/l or more.

In other embodiments, the final concentration of copper in the supplemented basic culture media is 2-20, 2-10, 3-8, or 4-6 µg/L.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

According to one embodiment of the method described above, the culture supernatant containing more than 4 mM ammonium is not used to prepare the rVWF composition.

In certain embodiments, the final concentration of copper in the supplemented basic culture media is at least 2.4, 3, 4, 5, 6, 7, 8, 9, 10, 15 L, 20 µg/L or more.

In other embodiments, the final concentration of copper in the supplemented basic culture media is 2-20, 2-10, 3-8, or 4-6 µg/L.

In a preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 50 IU/µg rVWF.

In a more preferred embodiment, the separated supernatant has a rVWF specific ristocetin cofactor activity of at least 70 IU/µg rVWF.

Recombinant vWF can be obtained by carrying out the expression in a suitable eukaryotic host system. Examples of eukaryotic cells include, without limitation, mammalian cells such as CHO, COS, HEK293, BHK, SK-Hep, and HepG2; insect cells, eg SF9 cells, SF21 cells, S2 cells and High Five cells; and yeast cells, for example Saccharomyces or Schizosaccharomyces cells. In one embodiment, said vWF can be expressed in yeast cells, insect cells, avian cells, mammalian cells, and the like. For example, in a human cell line, a hamster cell line, or a mouse cell line. In one particular embodiment, said cell line is a CHO, BHK, or HEK cell line. Generally, mammalian cells, such as CHO cells from a continuous cell line, are used to express vWF according to the present invention.

In certain embodiments, the nucleic acid sequence containing the vWF encoding sequence may be a vector. Said vector may be delivered by a virus or may be a plasmid. The nucleic acid sequence encoding said protein may be a specific gene or a biologically functional part thereof.

In one embodiment, said protein is at least the biologically active portion of the vWF.

A variety of vectors can be used to express said vWF; they may be selected from eukaryotic expression vectors. Examples of vectors for eukaryotic expression include:

(i) yeast expression vectors such as pAO, pPIC, pYES, pMET using promoters such as AOX1, GAP, GAL1, AUG1, etc.;

(ii) vectors for expression in insect cells such as pMT, pAc5, pIB, pMIB, pBAC and the like using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh and the like, and (iii) vectors for expression in mammalian cells such as pSVL, pCMV, pRc/RSV, rcDNA3, pBPV and the like, and vectors derived from viral systems such as vaccinia virus, adeno-associated viruses, herpesviruses, retroviruses, etc. .p using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV and β-actin.

An exemplary vector for rVWF expression is described in Kaufman et al. (Mol Cell Biol. 1989 Mar; 9(3):123342); the contents of said source are incorporated into the invention by reference in their entirety for any purpose.

According to some embodiments of the present invention, said nucleic acid sequence also contains other sequences suitable for controlled protein expression, such as promoter sequences, enhancers, TATA boxes, transcription initiation sites, polylinkers, restriction sites, poly A sequences, processing sequences proteins, selection markers, and the like, which are well known to those skilled in the art.

In addition to cell culture media containing elevated concentrations of copper, cell culture conditions of the present invention may include an ammonium concentration of less than about 25 mM throughout the upstream process in culture systems.

- 41 043293

In one embodiment, cell culture conditions include an ammonium concentration of less than about 25 mM, in another embodiment less than about 20 mM, in another embodiment less than about 15 mM, in another embodiment less than about 10 mM, and in a further embodiment, less than about 5 mM.

In some embodiments, the ammonium concentrations of the present invention are maintained at a constant level throughout the preparatory process in the cell culture system. The cells used in accordance with the present invention may be cultured, for example, by methods modified from standard batch culture and continuous culture well known in the art. However, such standard techniques can lead to the formation of high concentrations of ammonium at the end of cultivation.

The methods of the present invention overcome this problem by employing production systems that can provide a constant supply of culture medium using techniques such as, for example, perfusion or chemostatic culture. After culturing host cells, vWF can be isolated from spent media using standard techniques such as ultrafiltration or centrifugation. If necessary, the vWF can be purified, for example, by ion exchange and/or size exclusion chromatography and the like.

A continuous culture (eg, perfusion or chemostatic culture) may be a suspension culture that continuously receives nutrients from the influx of fresh medium, while the culture volume is usually unchanged. Similarly, continuous fermentation may refer to a process in which the cells or microorganisms in culture are maintained in an exponential growth phase by the constant addition of fresh medium, exactly compensated by the removal of the cell suspension from the bioreactor. In addition, the stirred tank reaction system can be used for suspension, perfusion, chemostatic and/or microcarrier cultures. Generally, any standard stirred tank reactor with any type of agitator, such as Rushton type, hydraulic, inclined blade, or propeller type, can operate as said stirred tank reaction system. WITH.

Methods for obtaining recombinant ADAMTS13 (A13).

According to another aspect, the present invention also relates to methods for producing rA13 under cell culture conditions, comprising a cell culture medium containing an elevated concentration of copper.

In certain embodiments, said culture also contains a low concentration of ammonium.

In one embodiment, the present invention provides a method for preparing a recombinant ADAMTS13 (rA13) composition; this method includes the steps:

(a) providing basic cell culture media;

(b) adding copper to the basic cell culture media to a final copper concentration of at least 1.0 µg/L;

(c) providing one or more cells containing a nucleic acid encoding a rA13 protein;

(d) culturing said one or more cells in a copper-supplemented cell culture medium such that rA13 is expressed and excreted from the cells into the culture supernatant; and (e) separating at least a portion of said culture supernatant, wherein at least 1500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant (i.e., 1500 units of FRETS-VWF73 activity per day per liter of cell culture; P FRETS).

In certain embodiments, the final concentration of copper in the supplemented basic culture media is at least 2, 3, 4, 5, 6 µg/l or more.

In other embodiments, the final concentration of copper in the supplemented basic culture media is 1-6, 2-5, 2-4, or 3-4 µg/L.

In a preferred embodiment, at least 2000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In a more preferred embodiment, at least 2500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In the most preferred embodiment, at least 3,000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

- 42 043293

In certain embodiments, these methods provide a sustained increase in FRETS-VWF73 (P FRETS) volumetric productivity. For example, in certain embodiments, at least 1500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media are isolated daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56 , 63, 70 or more days.

In a preferred embodiment, at least 2000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media are isolated daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63 , 70 or more days.

In a more preferred embodiment, at least 2500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media are isolated daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In the most preferred embodiment, at least 3000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media are isolated daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In a specific embodiment, the cell density in the continuous cell culture for rA13 production is maintained at a concentration not exceeding 4.0 x 106 cells/mL for an extended period.

In other specific embodiments, the cell density is maintained at or below 3.5x106, 3.0x106, 2.5x106, 2.0x106, 1.5x106, 1.0x106 cells/ml.

In one embodiment, the cell density is maintained at 3.0x106 to 4.0x106 cells/mL.

In one embodiment of the methods described above, the separated supernatant contains at least 4 units of FRETS-VWF73 activity per ml of supernatant (FRETS).

In a preferred embodiment, the separated supernatant contains at least 6 units of FRETS-VWF73 activity per ml of supernatant.

In a more preferred embodiment, the separated supernatant contains at least 8 units of FRETS-VWF73 activity per ml of supernatant.

In the most preferred embodiment, the separated supernatant contains at least 10 units of FRETS-VWF73 activity per ml of supernatant.

In certain embodiments, these methods provide a sustained increase in FRETS productivity. For example, in certain embodiments, a supernatant having at least 4 units of FRETS-VWF73 activity per ml is isolated daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63 , 70 or more days.

In a preferred embodiment, a supernatant having at least 6 units of FRETS-VWF73 activity per ml is isolated daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In a more preferred embodiment, a supernatant having at least 8 units of FRETS-VWF73 activity per ml is isolated daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In the most preferred embodiment, a supernatant having at least 10 units of FRETS-VWF73 activity per ml is isolated daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In one embodiment of the methods described above, cell culture produces at least 800 IU FRETS-VWF73 activity per 10 ⁶ culture cells per day (ie, q Frets).

In a preferred embodiment, cell culture produces at least 1 U FRETS-VWF73 activity per 10 ⁶ culture cells per day.

In a more preferred embodiment, cell culture produces at least 1.2 U FRETS-VWF73 activity per 10 ⁶ culture cells per day.

In a most preferred embodiment, cell culture produces at least 1.4 U FRETS-VWF73 activity per 10 ⁶ culture cells per day.

In certain embodiments, these methods provide a sustained increase in FRETS-VWF73 specific productivity per cell (q Frets). For example, in certain embodiments, cell culture produces at least 800 IU FRETSVWF73 activity per 10 ⁶ cell culture daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63 , 70 or more days.

In a preferred embodiment, cell culture produces at least 1 U FRETS-VWF73 activity per 10 ⁶ cell culture daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63 , 70 or more days.

In a more preferred embodiment, cell culture produces at least 1.2 U FRETS-VWF73 activity per 10 ⁶ culture cells daily for up to

- 43 043293 at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In the most preferred embodiment, cell culture yields at least 1.4 U FRETS-VWF73 activity per 106 culture cells daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56 , 63, 70 or more days.

In one embodiment of the methods described above, cell culture produces at least 1 mg of rA13 as measured by ELISA per liter of culture per day (P ELISA).

In a preferred embodiment, cell culture produces at least 1.5 mg of rA13 as measured by ELISA per liter of culture per day.

In a more preferred embodiment, cell culture yields at least 2 mg of rA13 as measured by ELISA per liter of culture per day.

In certain embodiments, these methods provide a sustained increase in rA13 production. For example, in certain embodiments, cell culture produces at least 1 mg of rA13, as measured by ELISA, per liter of culture daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In a preferred embodiment, cell culture yields at least 1.5 mg of rA13 as measured by ELISA per liter of culture daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In a more preferred embodiment, cell culture yields at least 2 mg rA13, as measured by ELISA, per liter of culture daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56 , 63, 70 or more days.

In one embodiment of the methods described above, cell culture produces at least 0.5 μg of rA13 as measured by ELISA per 10 ⁶ culture cells per day (ie, q ELISA).

In a preferred embodiment, cell culture produces at least 0.7 μg of rA13, as measured by ELISA, per 10 ⁶ culture cells per day.

In a more preferred embodiment, cell culture yields at least 0.9 μg of rA13 as measured by ELISA per 10 ⁶ culture cells per day.

According to certain implementation options, these methods provide a stable increase in production according to q ELISA. For example, in certain embodiments, cell culture produces at least 0.5 μg of rA13, as measured by ELISA, per 10 ⁶ cell culture daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In a preferred embodiment, cell culture produces at least 0.7 μg of rA13, as measured by ELISA, per 10 ⁶ cell culture daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In a more preferred embodiment, cell culture produces at least 0.9 μg of rA13, as measured by ELISA, per 10 ⁶ cell culture daily for at least 7 days, or at least 14, 21, 28, 35, 42 , 49, 56, 63, 70 or more days.

In one embodiment of the methods described above, the separated supernatant contains at least 3 μg of rA13, as determined by ELISA analysis, per ml of supernatant.

In a preferred embodiment, the separated supernatant contains at least 4 μg of rA13, as determined by ELISA analysis, per ml of supernatant.

In a more preferred embodiment, the separated supernatant contains at least 5 μg of rA13, as determined by ELISA, per ml of supernatant.

According to the most preferred implementation variant of the separated supernatant contains at least 6 μg of rA13, according to ELISA analysis, per ml of supernatant.

In certain embodiments, these methods provide a sustained increase in rA13 production. For example, in certain embodiments, a supernatant having at least 3 μg of rA13 per ml is isolated daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In a preferred embodiment, a supernatant having at least 4 μg of rA13 per ml is isolated daily for at least 7 days, or for at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days .

In a more preferred embodiment, a supernatant having at least 5 μg of rA13 per ml is isolated daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In the most preferred embodiment, a supernatant having at least 6 μg of rA13 per ml is isolated daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70 or more days.

In one embodiment of the methods described above, the cell culture solution also contains ammonium at a concentration of less than 10 mM.

- 44 043293

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 10 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 5 mM.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 5 mM for at least 7 days.

In a preferred embodiment, the cell culture solution contains ammonium at a concentration not exceeding 4 mM.

In another specific embodiment, the ammonium concentration in the cell culture is maintained at or below 4 mM for at least 7 days.

In other embodiments, the cell culture solution contains ammonium at a concentration of not more than 10 mM, or not more than 9, 8, 7, 6, 5, 4, 3, 2, 1 mM, or less.

According to another embodiment, the ammonium concentration in the cell culture is maintained at a low level throughout the process (ie, during the entire time that the specified culture is used to produce rA13).

According to a specific implementation variant of the culture solution contains copper and ammonium in a concentration according to any of the options 1-440 shown in table. 1.

In one embodiment, the present invention provides a method for preparing a recombinant ADAMTS13 (rA13) composition; this method includes the steps:

(a) providing basic cell culture media;

(b) adding copper and zinc to basic cell culture media;

(c) providing one or more cells containing a nucleic acid encoding a rA13 protein;

(d) culturing said one or more cells in a copper-supplemented cell culture medium such that rA13 is expressed and excreted from the cells into the culture supernatant; and (e) separating at least a portion of said culture supernatant, wherein at least 1500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

According to one embodiment, said culture medium contains at least 1 μg/L copper and at least 2 μM zinc.

In other embodiments, said medium contains at least 2 µg/l copper or at least 4 µg/l copper.

In one embodiment, when copper is added to the media, said culture media also contains at least exactly or about 5 μM zinc.

In one embodiment, said culture medium also contains exactly or about 2 to 12 μM zinc.

According to another implementation variant of the specified culture medium also contains exactly or about 5 to 12 μm zinc.

In other embodiments, said culture medium may also contain at least exactly or about 2 μM, or at least exactly or about 3, 4, 5,

6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 µM or more zinc.

According to one of the options for implementing the specified culture medium contains copper and zinc in concentrations according to any of the options 441-880, are given in table. 2.

In a preferred embodiment, at least 2000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In a more preferred embodiment, at least 2500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In the most preferred embodiment, at least 3,000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In one embodiment, the present invention provides a method for preparing a recombinant ADAMTS13 (rA13) composition; this method includes the steps:

(a) providing basic cell culture media;

(b) adding copper and calcium to the basic cell culture media;

(c) providing one or more cells containing a nucleic acid encoding a rA13 protein;

(d) culturing said one or more cells in a copper-supplemented cell culture medium such that rA13 is expressed and excreted from the cells into the culture supernatant; And

(e) separating at least a portion of said culture supernatant, wherein at least 1500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

According to one embodiment, said culture medium contains at least 1 μg/L copper and at least 0.5 mM calcium.

In other embodiments, said medium contains at least 2 µg/l copper or at least 4 µg/l copper.

In another embodiment, when copper is added to the media, said culture medium also contains at least 1.5 mM calcium.

In one embodiment, said culture medium contains exactly or about 0.5 to 1.5 mM calcium.

In other embodiments, said culture medium may contain at least exactly or about 0.5 mM, or at least exactly or about 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.25; 2.5; 2.75; 3.0; 3.5; 4.0; 4.5; 5.0 mM or more, calcium. According to one embodiment, said culture medium contains copper and calcium at a concentration according to any one of the options 881-1320 shown in Table 1. 3.

In a preferred embodiment, at least 2000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In a more preferred embodiment, at least 2500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In the most preferred embodiment, at least 3,000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In one embodiment, the present invention provides a method for preparing a recombinant ADAMTS13 (rA13) composition; this method includes the steps:

(a) providing basic cell culture media;

(b) adding copper, zinc and calcium to basic cell culture media;

(c) providing one or more cells containing a nucleic acid encoding a rA13 protein;

(d) culturing said one or more cells in a copper-supplemented cell culture medium such that rA13 is expressed and excreted from the cells into the culture supernatant; and (e) separating at least a portion of said culture supernatant, wherein at least 1500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

According to one embodiment, the culture medium contains calcium at a concentration of at least 0.5 mm, and copper and zinc at concentrations according to any of the options 441-880, are given in table. 2.

According to another specific implementation variant of the culture medium contains calcium at a concentration of at least 1.5 mm, and copper and zinc in concentrations according to any of the options 441880 shown in table. 2.

According to another specific implementation variant of the specified culture medium contains calcium at a concentration of 0.5 and 1.5 mm, and copper and zinc at concentrations according to any of the options 441-880, are given in table. 2.

In other embodiments, the culture medium contains calcium at a concentration of at least 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.25; 2.5; 2.7 5; 3.0; 3.5; 4.0; 4.5; 5.0 mm or more, and copper and zinc in concentrations according to any of the options 441-880, are given in table. 2.

In a preferred embodiment, at least 2000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In a more preferred embodiment, at least 2500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In the most preferred embodiment, at least 3,000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In one embodiment, the present invention provides a method for preparing a recombinant ADAMTS13 (rA13) composition; this method includes the steps:

(a) providing basic cell culture media;

- 46 043293 (b) adding to the basic media for cell cultures of copper and nicotinamide;

(c) providing one or more cells containing a nucleic acid encoding a rA13 protein;

(d) culturing said one or more cells in a copper-supplemented cell culture medium such that rA13 is expressed and excreted from the cells into the culture supernatant; and (e) separating at least a portion of said culture supernatant, wherein at least 1500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

According to one embodiment, said culture medium contains at least 1 μg/l copper and at least 2 mg/l nicotinamide (vitamin B3).

In other embodiments, said medium contains at least 2 µg/l copper or at least 4 µg/l copper.

In another embodiment, when copper is added to the media, said culture medium also contains at least 7 mg/L nicotinamide (vitamin B3).

In one embodiment, said culture medium contains exactly or about 2 to 10 mg/l of nicotinamide (vitamin B3).

In other embodiments, said culture medium may contain at least exactly or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 mg/l, or higher concentrations of nicotinamide (vitamin B3).

According to one of the options for implementing the specified culture medium contains copper and nicotinamide in concentrations according to any of the options 1321-1760 shown in table. 4

In a preferred embodiment, at least 2000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In a more preferred embodiment, at least 2500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In the most preferred embodiment, at least 3,000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In one embodiment, the present invention provides a method for preparing a recombinant ADAMTS13 (rA13) composition; this method includes the steps:

(a) providing basic cell culture media;

(b) adding copper, zinc, and nicotinamide to basic cell culture media;

(c) providing one or more cells containing a nucleic acid encoding a rA13 protein;

(d) culturing said one or more cells in a copper-supplemented cell culture medium such that rA13 is expressed and excreted from the cells into the culture supernatant; and (e) separating at least a portion of said culture supernatant, wherein at least 1500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In one embodiment, said cell culture medium contains nicotinamide at a concentration of at least 2 mg/mL, and copper and zinc at concentrations according to any of the options 441-880 shown in Table 1. 2.

According to another specific implementation variant of the specified culture medium contains nicotinamide at a concentration of at least 7 mg/ml mm, and copper and zinc at concentrations according to any of the options 441-880, are given in table. 2.

According to another specific implementation variant of the specified culture medium contains nicotinamide at a concentration of 2 and 10 mg/ml, and copper and zinc at concentrations according to any of the options 441-880, are given in table. 2.

In other embodiments, said culture medium contains nicotinamide at a concentration of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more, and copper and zinc in concentrations according to any of the options 441-880 shown in table. 2.

In a preferred embodiment, at least 2000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In a more preferred embodiment, at least 2500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In the most preferred embodiment, the separated culture supernatant contains at least 3000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day.

In one embodiment, the present invention provides a method for preparing a recombinant ADAMTS13 (rA13) composition; this method includes the steps:

(a) providing basic cell culture media;

(b) adding copper, calcium, and nicotinamide to basic cell culture media;

(c) providing one or more cells containing a nucleic acid encoding a rA13 protein;

(d) culturing said one or more cells in a copper-supplemented cell culture medium such that rA13 is expressed and excreted from the cells into the culture supernatant; and (e) separating at least a portion of said culture supernatant, wherein at least 1500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In one embodiment, the cell culture medium contains nicotinamide at a concentration of at least 2 mg/ml, and copper and calcium at concentrations according to any of the options 881-1320 shown in Table 1. 3.

According to another specific implementation variant of the culture medium contains nicotinamide at a concentration of at least 7 mg/ml mm, and copper and calcium at concentrations according to any of the options 881-1320 shown in table. 3.

According to another specific implementation variant of the specified culture medium contains nicotinamide at a concentration of 2 mg/ml and 10 mg/ml, and copper and calcium at concentrations according to any of the options 881-1320 shown in table. 3.

In other embodiments, said culture medium contains nicotinamide at a concentration of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mg/ml or more, and copper and calcium in concentrations according to any of the options 881-1320, are given in table. 3.

In a preferred embodiment, at least 2000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In a more preferred embodiment, at least 2500 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

In the most preferred embodiment, at least 3,000 units of FRETS-VWF73 activity per liter of supplemented basic cell culture media per day are present in the separated culture supernatant.

Recombinant ADAMTS proteins can be generated by expression in any suitable prokaryotic or eukaryotic host system. Examples of eukaryotic cells include, without limitation, mammalian cells such as CHO, COS, HEK293, BHK, SKHep, and HepG2; insect cells, such as SF9 cells, SF21 cells, S2 cells, and High Five cells; and yeast cells, for example Saccharomyces or Schizosaccharomyces cells.

In one embodiment, said ADAMTS proteins can be expressed in bacterial cells, yeast cells, insect cells, avian cells, mammalian cells, and the like. For example, in a human cell line, a hamster cell line, or a mouse cell line.

In one particular embodiment, said cell line is a CHO, BHK, or HEK cell line.

In a preferred embodiment, the cell line is a CHO cell line.

In a specific embodiment, CHO clones capable of stable expression of rA13 are generated by co-transfection of CHO cells with coding sequences for rA13 and dihydrofolate reductase (eg, the mouse dhfr gene) and growth selection in the presence of increasing levels of methotrexate.

In one embodiment, said cells may be any mammalian cell suitable for culture, preferably during the manufacturing process (i.e. at least 10 L, preferably at least 100 L), to obtain the desired ADAMTS protein, such as ADAMTS13. Examples include the CV1 monkey kidney cell line transformed with SV40 (COS-7, ATCC CRL 1651); human kidney embryonic cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); newborn hamster kidney cells (BHK, ATCC CCL 10); X-DHFR Chinese Hamster Ovary cells such as subclone DUKX-B11 (CHO, Uriaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod, 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); dog kidney cells (MDCK,

- 48 043293

ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci., 383:44-68 (1982)); cells

MRC 5; FS4 cells; and a human hepatoma line (Hepp G2). Preferably the cell line is a rodent cell line, in particular a hamster cell line such as CHO or BHK.

A significant number of vectors can be used to express the ADAMTS protein (eg, ADAMTS13) and can be selected from eukaryotic and prokaryotic expression vectors. In certain embodiments, the plasmid vector is for use in the expression of an ADAMTS protein (eg, ADAMTS13). Typically, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in conjunction with said hosts. Said vector may carry a replication site as well as marker sequences capable of providing phenotypic selection in transformed cells. The plasmid contains a nucleotide sequence encoding an ADAMTS protein (eg, ADAMTS13) operably linked to one or more control sequences, such as a promoter.

A preferred method for obtaining stable clones of CHO cells expressing recombinant ADAMTS protein is as follows. The DHFR-deficient CHO cell line DUKX-B11 was transfected with a DHFR expression vector to allow expression of the corresponding recombinant protein. An exemplary method is described in Plaimauer et al. (Blood. 2002 Nov 15; 100(10):3626-32. Epub 2002 Jul 12), the contents of which are incorporated into the invention by reference in their entirety for any purpose. The selection is carried out by cultivation in not containing hypoxanthine/thymidine (HT) environments; amplification of the corresponding region encoding the expression of the recombinant ADAMTS protein and the DHFR gene is achieved by cell expansion in increasing concentrations of methotrexate. If necessary, CHO cell lines can be adapted to grow in a serum-free and/or protein-free medium essentially as described in US 6,100,061 (Reiter et al., lmmuno Aktiengesellschaft).

In another preferred embodiment, stable HEK293 cells are generated by transfecting them with a construct containing a selectable hygromycin resistance marker and selecting transformants for antibiotic resistance.

The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, integrate into the host cell genome, and stably and efficiently express viral genes makes them attractive candidates for the transfer of foreign nucleic acids into cells (eg, mammalian cells).

Accordingly, in certain embodiments, a viral vector is used to introduce a nucleotide sequence encoding an ADAMTS protein (eg, ADAMTS13) into a host cell for expression. Said viral vector must contain a nucleotide sequence encoding an ADAMTS protein (eg ADAMTS13) operably linked to one or more control sequences, eg a promoter. Alternatively, said viral vector may not contain a control sequence, instead using the control sequence of the host cell to direct expression of the ADAMTS protein. Non-limiting examples of viral vectors that can be used for nucleic acid delivery include adenoviral vectors, AAV vectors, and retroviral vectors.

In one embodiment, the adenoviral expression vector includes constructs containing adenoviral sequences sufficient to package said construct and ensure expression of an ADAMTS construct cloned therein. Adenovirus vectors allow the introduction of foreign sequences up to 7 kb. (Grunhaus et al., Seminar in Virology, 200(2):535-546, 1992)).

In another embodiment, an adeno-associated virus (AAV) can be used to introduce a nucleotide sequence encoding an ADAMTS protein (eg, ADAMTS13) into a host cell for expression. AAV systems have been described previously and are generally well known in the art (Kelleher and Vos, Biotechniques, 17(6):1110-7, 1994; Cotten et al., Proc Natl Acad Sci USA, 89(13):6094 -6098, 1992; Curiel, Nat Immun, 13(2-3):141-64, 1994; Muzyczka, Curr Top Microbiol Immunol, 158:97-129, 1992). Details of the preparation and use of rAAV vectors are described, for example, in US Pat.

In one embodiment, a retroviral expression vector can be used to introduce a nucleotide sequence encoding an ADAMTS protein (eg, ADAMTS13) into a host cell for expression. These systems have been described previously and are generally well known in the art (Mann et al., Cell, 33:153-159, 1983; Nicolas and Rubinstein, in: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt , eds., Stoneham: Bufferworth, pp. 494-513, 1988; Temin, in: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986). In a specific embodiment, the retroviral vector is a lentiviral vector (see, e.g., Naldini et al., Science, 272 (5259) :263-267, 1996; Zufferey et al., Nat Biotechnol, 15(9) :871-875, 1997; Blomer et al., J Virol., 71(9):6641-6649, 1997; U.S. Patent Nos. 6,013,516 and

5994136).

Non-limiting examples of prokaryotic expression vectors include plasmids such as pRSET, pET, pBAD, and the like, while promoters useful in prokaryotic expression vectors include lac, trc, trp, recA, araBAD, and the like. Examples of vectors for eukaryotic expression include:

(i) yeast expression vectors such as pAO, pPIC, pYES, pMET using promoters such as AOX1, GAP, GAL1, AUG1, etc.;

(ii) vectors for expression in insect cells such as pMT, pAc5, pIB, pMIB, pBAC and the like using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh and the like, and (iii) vectors for expression in mammalian cells such as pSVL, pCMV, pRc/RSV, rcDNA3, pBPV and the like, and vectors derived from viral systems such as vaccinia virus, adeno-associated viruses, herpesviruses, retroviruses, etc. .p/, using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV and β-actin.

An exemplary vector for the expression of rA13 is described in Plaimauer et al. (Blood. 2002 Nov. 15; 100(10):3626-32. Epub 2002 Jul 12); the contents of said source are incorporated into the invention by reference in their entirety for any purpose.

In certain embodiments, cell culture methods of the present invention may include the use of a microcarrier. The present invention provides, among other aspects, methods for large scale expression of the ADAMTS protein.

In some embodiments, cell culture according to embodiments of the invention may be carried out in large bioreactors under conditions suitable to obtain a high surface to volume ratio in culture to achieve high cell density and protein expression. One way to provide such growth conditions is the use of microcarriers for culturing cells in bioreactors with a stirrer.

According to another implementation variant of these growth requirements are met through the use of suspension cell culture.

XII. specific implementations.

A. Recombinant background factor.

Willebrand (rVWF) Recombinant vWF can be expressed in mammalian cells, but the specific activity of vWF can vary widely depending on cell culture conditions; it has not been shown to be comparable or equal to that of vWF isolated from plasma. The present invention is based in part on the unexpected discovery that cell culture media containing at least 2.4 µg/l of copper provide the beneficial effect of promoting the expression of high molecular weight vWF having a high specific activity. In particular, the high molecular weight recombinant vWF of the present invention may include a high multimeric form containing from about 14 to about 22 dimers and having a specific ristocetin activity of at least about 30 mU/μg. The cell culture processes of the present invention also allow low levels of NH ₄ ⁺ (eg, less than 10 mM) to be maintained during the preparatory process in cell culture systems, thus reducing detrimental effects on post-translational modifications. It is believed that the present invention provides for the first time cell culture conditions comprising a medium with a suitable concentration of copper in combination with suitable levels of ammonium in the supernatant for the expression of high multimeric vWFs with high specific activity.

According to one aspect, the present invention relates to cell culture conditions for producing recombinant high molecular weight vWF with high specific activity. Cell culture conditions according to the present invention may include, for example, a cell culture medium with a high concentration of copper and/or a cell culture supernatant with a low concentration of ammonium (NH ₄ ⁺ ).

The present invention also provides methods for culturing cells under cell culture conditions to express high molecular weight vWF with high specific activity.

In one aspect, the present invention provides a cell culture solution for producing a high molecular weight recombinant vWF protein; said cell culture solution contains: cell culture medium containing copper at a concentration of at least about 2.4 μg/L; cell culture supernatant containing ammonium at a concentration of less than 10 mM; and a plurality of cells expressing a highly multimeric vWF protein, said vWF protein having a specific ristocetin activity of at least about 30 mU/μg.

In one specific embodiment of the cell cultures described above, the cell culture solution contains a copper-containing media additive.

According to one of the specific embodiments of the cell cultures described above, the specified

- 50 043293 the environmental additive contains a hydrolyzate, optionally a soy hydrolyzate.

According to one specific embodiment of the cell cultures described above, said medium supplement contains a copper salt, a copper chelate, or combinations thereof.

In one specific embodiment of the cell cultures described above, said copper salt is selected from the group consisting of copper sulfate, copper acetate, copper carbonate, copper chloride, copper hydroxide, copper nitrate, and copper oxide.

In one specific embodiment of the cell cultures described above, the copper concentration is at least about 4 µg/L.

In one specific embodiment of the cell cultures described above, the copper concentration is from about 2.4 to about 20 µg/L.

In one specific embodiment of the cell cultures described above, the vWF protein contains from about 14 to about 22 dimers.

According to one aspect, the present invention provides a method for producing a high molecular weight recombinant vWF protein; this method includes the steps:

a) providing a cell culture containing a nucleic acid encoding a recombinant vWF protein;

b) expressing the vWF protein in said cells under cell culture conditions comprising a cell culture medium containing copper at a concentration of at least about 2.4 μg/L and a cell culture supernatant containing ammonium at a concentration of less than about 10 mM, wherein said vWF protein is a highly multimeric vWF protein and has a specific ristocetin activity of at least about 30 IU/µg.

According to one specific embodiment of the methods described above, said cells are mammalian cells.

According to one specific embodiment of the methods described above, said cells are taken from a continuous cell line.

In one specific embodiment of the methods described above, said cells are CHO cells.

In one specific embodiment of the methods described above, the copper concentration is at least about 4 µg/L.

In one specific embodiment of the methods described above, the copper concentration is from about 2.4 to about 20 µg/L.

In one specific embodiment of the methods described above, the recombinant vWF protein has a specific ristocetin cofactor activity of at least about 50 IU/µg.

In one specific embodiment of the methods described above, the recombinant vWF protein has a specific ristocetin cofactor activity of about 30 IU/µg to about 100 IU/µg.

In one specific embodiment of the methods described above, the recombinant vWF protein contains about 14 to about 22 dimers.

In one aspect, the present invention provides a high molecular weight recombinant vWF protein produced by a process comprising the steps of:

a) providing a cell culture containing a nucleic acid encoding a recombinant vWF protein; And

b) expression of the vWF protein in said cells under cell culture conditions comprising a cell culture medium containing copper at a concentration of at least 2.4 μg/L and a cell culture supernatant containing ammonium at a concentration of less than 10 mM, wherein said protein vWF is a highly multimeric vWF protein and has a specific ristocetin activity of at least about 30 IU/μg.

In one specific embodiment of the rVWF compositions described above, the recombinant vWF protein has a specific ristocetin cofactor activity of at least about 50 IU/µg.

In one specific embodiment of the rVWF compositions described above, the recombinant vWF protein has a specific ristocetin cofactor activity of about 30 IU/µg to about 100 IU/µg.

In one specific embodiment of the rVWF compositions described above, the recombinant vWF protein contains from about 14 to about 22 dimers.

In one aspect, the present invention provides a cell culture solution for producing a high molecular weight recombinant vWF protein, said cell culture solution comprising: a cell culture medium containing copper at a concentration of at least about 2.4 µg/L; cell culture supernatant containing ammonium at a concentration of less than 10 mM; and a plurality of cells expressing a highly multimeric vWF protein, wherein said vWF protein contains from about 14 to about 22 dimers and has a specific ristocetin activity of at least about 30 mU/µg.

In one aspect, the present invention relates to cell culture conditions for producing recombinant high molecular weight vWF in a high multimeric form with high specific activity. Cell culture conditions according to the present invention may include, for example, a cell culture medium with a high concentration of copper and a cell culture supernatant with a low concentration of ammonium (NH ₄ ⁺ ).

The present invention also provides methods for culturing cells under cell culture conditions to express high molecular weight vWF with high specific activity.

According to one aspect, the present invention includes a cell culture solution for producing high molecular weight recombinant vWF, comprising a cell culture medium containing copper at a concentration of at least about 2.4 μg/L; cell culture supernatant containing ammonium at a concentration of less than 10 mM; and a plurality of cells expressing high multimeric vWFs containing from about 14 to about 22 dimers and having a specific ristocetin activity of at least about 30 IU/μg.

In one embodiment of the cell culture solutions described above, at least 10% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In a specific embodiment, at least 15% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 20% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 25% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 30% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In certain embodiments, the copper concentration may be at least about 4 μg/l, or the copper concentration may vary from about 2.4 μg/l to about 20 μg/l.

In some embodiments, the cell culture media comprise a media supplement containing copper.

In certain embodiments, said media additive may contain a hydrolyzate or a copper salt, a copper chelate, or combinations thereof.

In some embodiments, said copper salt may include copper sulfate, copper acetate, copper carbonate, copper chloride, copper hydroxide, copper nitrate, or copper oxide.

In certain embodiments, the cells may be from a continuous cell line and may include mammalian cells such as CHO cells.

In some embodiments, said recombinant vWF has a specific ristocetin cofactor activity of at least about 50 mU/µg, or the specific ristocetin cofactor activity can range from about 30 to about 100 mU/µg.

According to another aspect, the present invention includes a method for producing high molecular weight recombinant vWF, comprising:

a) providing cell culture;

b) introducing a nucleic acid sequence encoding vWF;

c) selecting cells carrying said nucleic acid sequence; And

d) performing vWF expression in said cells under cell culture conditions comprising a cell culture medium containing copper at a concentration of at least about 2.4 μg/L and a cell culture supernatant containing ammonium at a concentration of less than about 10 mM, wherein said vWF is a high multimeric vWF containing from about to about 22 dimers and having a specific ristocetin activity of at least about 30 IU/μg.

In one embodiment of the cell culture supernatant described above, at least 10% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In a specific embodiment, at least 15% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 20% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 25% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 30% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In some embodiments, cells that may be from a continuous cell line may include mammalian cells such as CHO cells.

In certain embodiments, the copper concentration may be at least about 4 µg/L, or the copper concentration may range from about 2.4 to about 20 µg/L.

In some embodiments, the cell culture media comprise a media supplement containing copper.

In certain embodiments, said media additive may contain a copper hydrolyzate or salt, a copper chelate, or combinations thereof.

In some embodiments, said copper salt may include copper sulfate, copper acetate, copper carbonate, copper chloride, copper hydroxide, copper nitrate, or copper oxide.

In some embodiments, said recombinant vWF has a specific ristocetin cofactor activity of at least about 50 mU/µg, or the specific ristocetin cofactor activity can range from about 30 to about 100 mU/µg.

According to another aspect, the present invention encompasses a high molecular weight recombinant vWF obtained by a process comprising the steps of:

a) providing cell culture;

b) introducing a nucleic acid sequence encoding vWF;

c) selecting cells carrying said nucleic acid sequence; And

d) expressing vWF in said cells under cell culture conditions comprising a cell culture medium containing copper at a concentration of at least 2.4 μg/L and a cell culture supernatant containing ammonium at a concentration of less than 10 mM, wherein said vWF is is a highly multimeric vWF containing from about 14 to about 22 dimers and having a specific ristocetin activity of at least about 30 IU/µg.

In one embodiment of the cell culture supernatant described above, at least 10% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In a specific embodiment, at least 15% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 20% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 25% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 30% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In some embodiments, the cells may be from a continuous cell line and may include mammalian cells such as CHO cells.

In certain embodiments, the copper concentration may be at least about 4 µg/L, or the copper concentration may range from about 2.4 to about 20 µg/L.

In some embodiments, the cell culture media comprise a media supplement containing copper.

In certain embodiments, said media additive may contain a copper hydrolyzate or salt, a copper chelate, or combinations thereof.

In some embodiments, said copper salt may include copper sulfate, copper acetate, copper carbonate, copper chloride, copper hydroxide, copper nitrate, or copper oxide.

In some embodiments, said recombinant vWF has a specific ristocetin cofactor activity of at least about 50 mU/µg, or said specific ristocetin cofactor activity can range from about 30 to about 100 mU/µg.

In one aspect, the present invention provides a composition comprising a recombinant von Willebrand factor (rVWF) having a specific ristocetin cofactor activity of at least 30 IU/µg.

In a preferred embodiment, said composition has a specific ristocetin cofactor activity of at least 40 IU/μg.

In a more preferred embodiment, said composition has a specific ristocetin cofactor activity of at least 50 IU/μg.

In a more preferred embodiment, said composition has a specific ristocetin cofactor activity of at least 60 IU/μg.

In a more preferred embodiment, said composition has a specific ristocetin cofactor activity of at least 70 IU/μg.

In an even more preferred embodiment, said composition has

- 53 043293 specific ristocetin-cofactor activity of at least 80 IU/μg.

In one embodiment of the above compositions, at least 10% of the rVWF in said composition is present as a high molecular weight VWF multimer of more than 10 dimers.

In a specific embodiment, at least 15% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 20% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 25% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

In another specific embodiment, at least 30% of the rVWF is present as a high molecular weight VWF multimer of more than 10 dimers.

According to one embodiment of the above compositions, said composition contains a culture supernatant.

In one particular embodiment, said culture supernatant is a mammalian cell culture supernatant.

In a more specific embodiment, said mammalian cell culture supernatant is a CHO cell supernatant.

According to one embodiment of the above compositions, rVWF is expressed in cell culture containing at least 2.4 μg/l of copper.

According to a specific implementation variant of the specified cell culture contains at least 4 μg/l of copper.

According to a more specific implementation variant of the specified culture contains from 2.4 to 20 μg/l of copper.

In one embodiment, the copper is in the form of a copper salt, a copper chelate, or a combination thereof.

In a particular embodiment, said copper salt is selected from the group consisting of copper sulfate, copper acetate, copper carbonate, copper chloride, copper hydroxide, copper nitrate, and copper oxide.

According to one embodiment of the above compositions, said cell culture is a batch culture.

According to one embodiment of the above compositions, said cell culture is a continuous culture.

According to a specific implementation variant of the specified continuous cultivation is performed in chemostatic mode.

According to another specific implementation variant of the specified continuous cultivation is performed in perfusion mode.

According to one embodiment of the above compositions, the level of NH ₄ ⁺ in said culture is maintained at a concentration below 4 mM.

According to one embodiment of the above compositions, the cell density in culture is maintained at a level of less than 2.5x10 ⁶ cells/ml.

According to one embodiment of the above compositions, the cell density in culture is maintained at a level of less than 2.0 x 10 ⁶ cells/ml.

According to one embodiment of the above compositions, said culture is maintained at a level of less than 1.5 x 10 ⁶ cells/ml.

In one embodiment of the compositions described above, rVWF is co-expressed with recombinant factor VIII (rFVTII).

In a specific embodiment, most of the co-expressed rFVTII is removed.

In a more specific embodiment, the ratio of rVWF to rFVIII in said composition is at least 10:1.

According to one embodiment of the above compositions, said composition is in a form for pharmaceutical administration.

According to a specific implementation variant of the specified composition receive in the form for intravenous, subcutaneous or intramuscular administration.

According to one embodiment of the above compositions, said composition is lyophilized.

According to another aspect, the present invention includes a high molecular weight recombinant vWF obtained by a process comprising the steps of:

a) providing cell culture;

b) introducing a nucleic acid sequence encoding vWF;

c) selecting cells carrying said nucleic acid sequence; And

- 54 043293

d) carrying out the expression of vWF in said cells under cell culture conditions comprising a cell culture medium containing copper at a concentration of at least 2.4 μg/l and a cell culture supernatant containing ammonium at a concentration of less than 10 mM, said vWF is a highly multimeric vWF containing from about 14 to about 22 dimers and having a specific ristocetin activity of at least about 30 IU/µg.

Obviously, all embodiments and concentrations described in the Cell Culture Media and Methods for Producing Recombinant vWF sections above can be used here.

The recombinant vWF of the present invention may include a high molecular weight recombinant vWF protein having a high specific activity.

In one embodiment, the vWF of the present invention is a high multimeric form of vWF.

In some embodiments, said high multimeric form of vWF contains at least up to about 14 dimers, and in other embodiments, at least up to about 22 dimers.

In other embodiments, the high multimeric form of vWF may range from about 10 to about 20 dimers, or from about 15 to about 25 dimers, or from about 20 to about 40 dimers.

In certain embodiments, recombinant vWF is comparable to plasma vWF.

According to the description given in the invention, in accordance with the present invention, an unexpected result is obtained, namely, that an increased concentration of copper in the cell culture medium allows the production of high molecular weight vWF with high specific activity. Cell culture media containing copper at a concentration, for example, greater than about 2.4 µg/L, can increase the yield of recombinant multimeric vWF compared to media that does not contain copper.

In certain embodiments, the percentage of multimeric vWF (ie, rVWF containing at least 2 dimers) may be higher than about 50%, or higher than about 75%, or higher than about 90%. The level of vWF multimerization can be analyzed using standard techniques such as, for example, in agarose electrophoresis under non-reducing conditions.

According to the invention, recombinant vWF obtained using the methods of the present invention may have a high specific activity, for example, a high specific ristocetin-cofactor activity.

In one embodiment, the recombinant vWF produced using the methods of the present invention may have a specific ristocetin cofactor activity of at least 30 mU/µg, and in another embodiment, at least 50 mU/µg.

In other embodiments, the specific ristocetin cofactor activity may range from about 30 to about 100 IU/µg, or from about 50 to about 100 IU/µg.

B. Recombinant ADAMTS13 (rA13).

The ADAMTS proteins (i.e., ADAMTS-1-ADAMTS-20) are a family of secreted zinc metalloproteinases that share a common modular domain organization (for reference, see Flannery C.R., Front Biosci. 2006 Jan 1; 11:544-69). All ADAMTS proteins share a common structure of a central domain consisting of a signal peptide followed by a prodomain, a zinc-dependent metalloproteinase catalytic domain, a disintegrin-like domain, a type I thrombospondin repeat, a cysteine-rich domain, and a spacer domain (Apte S.S., J Biol Chem 2009 Nov 13;284(46):314937). In addition, all except ADAMTS-4 contain at least one more type I thrombospondin repeat domain, and many of the ADAMTS proteins contain one or more additional accessory domains. In particular, it has been reported that all ADAMTS proteins appear to contain at least one calcium binding site and at least one zinc binding site located within the metalloproteinase catalytic domain (Andreini et al., J. Proteome Res., 2005, 4(3), pp. 881-888).

The biological role of ADAMTS proteins in various diseases and conditions has been described, including antiangiogenesis, renal interstitial fibrosis, bone remodeling, ovarian folliculogenesis, atherosclerosis, genitourinary development, and tumor growth/remodeling (ADAMTS-1); Ehlers-Danlos syndrome type 7C and bovine dermatosparaxis (ADAMTS-2); arthritis, atherosclerosis and tendinopathy (ADAMTS-4); arthritis and glioblastoma (ADAMTS-5); arthritis (ADAMTS-7); antiangiogenesis, malignant neoplasms of the brain, arthritis and atherosclerosis (ADAMTS-8); arthritis (ADAMTS-9, -12); thrombotic thrombocytopenic purpura (ADAMTS-13); and antithrombosis/stroke (ADAMTS18) (for review, see Lin and Liu, Open Access Rheumatology Research and Reviews 2009:1 121-131).

Recombinant ADAMTS13 (A13) has been expressed in mammalian cells before, however

- 55 043293 specific activity varies widely depending on the cell culture conditions. Many commercially available culture media have been found to be unsuitable for expressing rA13 with high specific activity expressed as the ratio of activity measured by the FRETS-VWF73 assay to antigen content as assessed using ELISA. In one aspect, the methods of the present invention are based on several useful discoveries that allow expression in cell culture of the protein rA13, which has increased levels of total and specific activity.

Accordingly, due to the common structural-functional relationships of the ADAMTS family of secreted metalloproteinases, the methods according to the present invention allow all ADAMTS proteins to be expressed in cell culture and isolated from the cellular environment.

In one aspect, the present invention provides a composition comprising recombinant ADAMTS13 (rA13) having a specific FRETS-VWF activity of at least 1600 IU/μg.

According to one embodiment of the above compositions, said rA13 has a specific FRETS-VWF activity of at least 800 IU/μg.

According to one embodiment of the above compositions, said composition contains a culture supernatant.

In a particular embodiment, said culture supernatant is a mammalian cell culture supernatant.

In a more specific embodiment, said mammalian cell culture supernatant is a CHO cell supernatant.

In one embodiment of the compositions described above, said rA13 is expressed in a cell culture containing at least 1 μg/L of copper.

According to a specific implementation variant of the specified cell culture contains at least 2 μg/l of copper.

According to a more specific implementation variant of the specified culture contains from 2 to 20 μg/l of copper.

In one embodiment of the compositions described above, the copper is present as a copper salt, a copper chelate, or a combination thereof.

In a particular embodiment, said copper salt is selected from the group consisting of copper sulfate, copper acetate, copper carbonate, copper chloride, copper hydroxide, copper nitrate, and copper oxide.

According to one embodiment of the above compositions, said cell culture is a batch culture.

According to one embodiment of the above compositions, said cell culture is a continuous culture.

According to a specific implementation variant, continuous cultivation is performed in a chemostatic mode.

According to another specific implementation variant, continuous cultivation is performed in perfusion mode.

According to one embodiment of the above compositions, the level of NH ₄ ⁺ in said culture is maintained at a concentration below 5 mM.

According to one embodiment of the above compositions, the level of NH ₄ ⁺ in said culture is maintained at a concentration below 4 mM.

In one embodiment of the compositions described above, the cell density in culture is maintained at less than 4.0 x 106 cells/mL.

In a specific embodiment, the cell density in the culture is maintained at less than 3.0 x 106 cells/mL.

In a specific embodiment, the cell density in the culture is maintained at less than 2.0 x 106 cells/mL.

In a more specific embodiment, cell density in culture is maintained at less than 1.5 x 106 cells/mL.

According to one embodiment of the above compositions, said composition is in a form for pharmaceutical administration.

According to a specific implementation variant of the specified composition receive in the form for intravenous, subcutaneous or intramuscular administration.

According to one embodiment of the above compositions, said composition is lyophilized.

XIII. Compositions.

In one aspect, formulations containing the recombinant rVWF or rA13 therapeutic proteins of the present invention are lyophilized prior to administration.

- 56 043293

Lyophilization is carried out using techniques well known in the art and must be optimized for the formulation being developed [Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res. 13:243-9 (1996)].

Methods for preparing pharmaceutical compositions may include one or more of the following steps: adding a stabilizing agent as described in the invention to said mixture prior to lyophilization, adding at least one agent selected from a bulking agent, an osmolarity regulating agent, and a surfactant, each of which corresponds to that given in the invention description, to the specified mixture before lyophilization. The lyophilized formulation consists, in one aspect, of at least one or more of a buffer, a bulking agent, and a stabilizer.

According to this aspect, the usefulness of a surfactant is evaluated and used in cases where sticking during the lyophilization or reconstitution process step is a problem. A suitable buffering agent is added to maintain the pH stability of the formulation during lyophilization.

The standard way to reconstitute lyophilized material is to re-add a volume of pure water or sterile water for injection (WFI) (generally equivalent to the volume removed during lyophilization), although dilute solutions of antibacterial agents are sometimes used in the preparation of parenteral pharmaceuticals [Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)].

Accordingly, methods are provided for preparing reconstituted recombinant VWF compositions comprising the step of adding a diluent to a lyophilized recombinant VWF composition of the present invention.

The lyophilized material may be reconstituted as an aqueous solution. Various aqueous vehicles, for example, sterile water for injection, water with preservatives for multi-dose use, or water with a suitable amount of surfactants (for example, an aqueous suspension that contains the active compound in admixture with excipients suitable for the preparation of aqueous suspensions).

In various aspects, such excipients are suspending agents, for example, and without limitation, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic; dispersing or wetting agents are naturally occurring phosphatides, for example, and without limitation, lecithin, or alkylene oxide condensates with fatty acids, for example, and without limitation, polyoxyethylene stearate, or ethylene oxide condensates with long chain aliphatic alcohols, for example, and without limitation, heptadecaethyleneoxycetanol , or condensates of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitan monooleate, or condensates of ethylene oxide with partial esters derived from fatty acids and hexitan anhydrides, for example, and without limitation, such as polyethylene sorbitan monooleate.

In various aspects, said aqueous suspensions also contain one or more preservatives, for example, and without limitation, ethyl or n-propyl p-hydroxybenzoate.

For administration of the compositions to humans or experimental animals, in one aspect, said compositions comprise one or more pharmaceutically acceptable carriers. The terms pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that are stable, inhibit protein degradation such as aggregation and cleavage products, and furthermore do not cause allergic or other undesirable reactions when administered using routes well known in the art, according to the description below. Pharmaceutically acceptable carriers include any and all of the clinically useful diluents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, including those described above.

These pharmaceutical compositions are administered intravenously, orally, topically, transdermally, parenterally, by inhalation, vaginally, rectally or by intracranial injection. As used herein, the term parenteral includes subcutaneous injections, intravenous, intramuscular, intracisternal injections or infusion techniques. Also included is administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a specific site. As a rule, the compositions are essentially free of pyrogens, as well as other impurities that may be harmful to the recipient.

Single or multiple administration of these compositions is performed using dosages and regimens selected by the attending physician. The appropriate dosage for preventing or treating a disease depends on the type of disease being treated, the severity and course of the disease, whether the drug is being administered prophylactically or therapeutically, on prior therapy, the patient's medical history and drug response, and based on choice of the attending physician.

In one aspect, the formulations of the present invention are administered first as a bolus followed by a continuous infusion to maintain therapeutic circulating drug product levels. In another example, a compound of the present invention is administered as a single dose. Those skilled in the art will readily be able to optimize effective dosages and administration schedules in accordance with good medical practice and the clinical condition of the patient. The frequency of dosing depends on the pharmacokinetic parameters of the agents and the route of administration. The optimal pharmaceutical composition is determined by a person skilled in the art depending on the route of administration and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, ^18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042), pp. 1435-1712, incorporated herein by reference. Such formulations affect the physical condition, stability, in vivo release rate, and in vivo clearance rate of the administered agents. Depending on the route of administration, a suitable dose is calculated according to body weight, body surface area or organ size. Appropriate dosages can be ascertained by standard methods of determining blood dose levels in conjunction with appropriate dose-response data. The final dosing regimen is determined by the attending physician, taking into account various factors that modify the effect of drugs, for example, the specific activity of the drug, the severity of the lesion and the response of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, the time of administration, and others. clinical factors. For example, a typical dose of recombinant vWF according to the present invention is approximately 50 U/kg, which is equal to 500 μg/kg. As research progresses, additional information is emerging regarding appropriate dosage levels and duration of treatment for various diseases and conditions.

XIV. Methods of treatment.

The present invention also encompasses methods of treating a patient in need of rVWF or rA13 obtained according to the methods described in the invention. Such treatments may include administering pharmaceutical formulations containing recombinant rA13 or high molecular weight recombinant vWF of the present invention.

According to another aspect, the present invention provides methods of therapeutic or prophylactic treatment, comprising the introduction of the composition of rVWF or rA13 proposed in the present invention. Typically, for therapeutic use, the formulations are administered to a patient with a disease or condition associated with dysfunction of ADAMTS13 or VWF, or otherwise in need thereof, at a therapeutically effective dose. Formulations and amounts effective for said use will depend upon the severity of said disease or condition, and upon the general health of the patient. Depending on the dosage and frequency required and tolerated by the patient, a single or multiple administration of these formulations may be carried out.

In one embodiment, the present invention provides methods for treating or preventing diseases or conditions associated with ADAMTS13 or VWF dysfunction.

In a further embodiment, pharmaceutical formulations containing recombinant vWF may be administered to treat diseases associated with vWF, such as von Willebrand's disease or hemophilia.

In another embodiment, the present invention provides methods for treating or preventing diseases or conditions associated with the formation and/or presence of one or more thrombi, comprising administering an rA13 composition of the invention.

In another embodiment, the present invention provides methods for disrupting one or more thrombi in a patient in need thereof.

In other embodiments, the present invention provides methods for treating or preventing a heart attack in a patient in need thereof. Generally, the methods of the present invention comprise administering a rADAMTS13 composition of the present invention to a patient in need thereof.

Non-limiting examples of disorders associated with the formation and/or presence of one or more thrombi are hereditary thrombotic thrombocytopenic purpura (TTP), acquired TTP, arterial thrombosis, acute myocardial infarction (AMI), stroke, sepsis, and disseminated intravascular coagulation (DIC).

Non-limiting examples of disorders associated with a heart attack include, but are not limited to, myocardial infarction (heart attack), pulmonary embolism, cerebrovascular disorders such as stroke, peripheral arterial occlusive disease (eg, gangrene), antiphospholipid syndrome, sepsis, giant cell arteritis (GCA), hernia and volvulus.

XV. Examples.

The present invention will also be illustrated by the following examples, without being limited to them.

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Example 1

Continuous cell culture experiments were performed using a culture of a recombinant CHO cell line expressing vWF. The main medium was DMEM/F12 containing approximately 0.3 μg/l Cu ²⁺ . Soy hydrolyzate and copper sulfate (CuSO ₄ -5H ₂ O) were added to this medium to produce a final copper concentration in the medium greater than at least 2.4 µg/l.

Recombinant CHO cells expressing vWF were cultured in continuous cell culture such that ammonium (NH ₄ ⁺ ) levels were at a concentration level of less than about 10 mM. It has been found that continuous media production systems (eg, perfusion or chemostatic cultures) are preferred because standard batch or fed-batch culture techniques result in high NH ₄ ⁺ concentrations at the end of culture. At the end of cultivation, the highly multimeric vWF was isolated and the specific ristocetin-cofactor activity of vWF was measured.

Example 2

Recombinant factor VIII (rFVIII) and von Willebrand factor (rVWF) were co-expressed in batch GD8/6 cell cultures to determine the effect of culture medium composition on VWF expression and activity. Briefly, GD8/6 cells were cultured continuously in BAV-SP medium, consisting of a powder of modified DMEM/F12 basic medium (Table 5) and additional supplements also containing 4 g/l soy hydrolysate, see Table. 6, with or without the addition of additional copper. Basic BAV-SP media were used to study the effect of low copper concentrations on rVWF expression and activity. These basic media contained 0.3 µg/l copper and added soy hydrolyzate, which added an additional 0.7 µg/l copper, which was experimentally determined to give a final copper concentration of 1.0 µg/l. For comparison, BAV-SP media used for batch cultures were also supplemented with an additional 3.3 μg/l Cu ²⁺ , resulting in a final copper concentration of 4.3 μg/l, to determine the effect of high copper concentrations on VWF expression and activity.

Table 5

Composition of culture media BAV-SP

XVI. Medium BAV-SP Components mg/l Amino acids L-Alanine 4.45 L-Arginine HC1 147.50

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Iron nitrate (Fe(NO3) Z-ENGO) 0.050 Ferrous sulfate (Ee3O4-7HgO) 0.417 Magnesium chloride (MgC12) 28.640 Magnesium sulfate (MgSO4) 48.840 Potassium chloride (KC1) 311,800 Sodium chloride (NaCl) 6995.50 0 Sodium Phosphate (Na2HPO4) 71.020 Sodium Phosphate (NaH2PO4) 62,500 Zinc sulfate heptahydrate (ZnSO47H2O) 0.432 sodium selenite 0.0131 Other D-Glucose 3151 Linoleic acid 0.042 Lipoic acid 0.105 Putrescine 2HC1 0.081 thymidine 0.365 Hypoxanthin Na 2,390 sodium pyruvate 55,000

Table 6

Additive composition for BAV-SP

Components of the final composition L-Glutamine 600.00 ethanolamine 1,530 Synperonic F68 250,000 Sodium bicarbonate (NaHCO3) 2000,000 soy peptone 4000.00 General composition 18596.8

GD8/6 cells expressing rVWF were cultured in either low copper medium (Table 7) or high copper medium (Table 8) for 7 days. After 2 days, the cultures were subcultured to carry out periodic cultivation for 5 days. Samples of this culture supernatant were tested daily for rVWF content (vWF ELISA), total (ristocetin) and specific (specific activity) using ristocetin cofactor analysis. Various culture parameters including cell count, cell viability and ammonium concentration were also monitored daily (except for batch culture days 3 and 4).

Surprisingly, cell cultures grown at high copper concentrations were found to produce supernatants having significantly higher total and specific rVWF activity (cf. results in Tables 7 and 8). For example, on day 4 of batch culture, a cell culture grown at high concentrations of copper had 1.52 IU rVWF activity/mL compared to 0.2 IU rVWF activity/mL for a low copper culture, despite the fact that the low copper cell culture yielded almost twice as much rVWF as the high copper cell culture. In addition, the specific activity of the supernatant derived from the high copper culture was more than 13 times higher than that of the low copper culture supernatant (831 IU/10 µg rVWF to 62 IU/10 µg rVWF).

As can be seen from Table. 7 and 8, the total and specific ristocetin-cofactor activity of the high copper culture was twice that of the low copper culture on day 1 of batch culture. In addition, in contrast to the subsequent increase in activity observed in high copper culture, no increase in ristocetin cofactor activity was observed in low copper culture after 1 day of batch culture. Consistent with this result, analysis of the multimeric status of rVWF by agarose gel electrophoresis showed that the low copper cell culture supernatant on day 1 of batch culture contained a low concentration of high molecular weight rVWF relative to the concentration of low molecular weight rVWF species, and also that the indicated relative concentration of high molecular weight rVWF

- 61 043293 Traction decreased over time (FIGS. 1A and 1B). On the contrary, the addition of Cu ²⁺ to the culture medium stably led to the formation of an antigen with ristocetin-cofactor (RiCoF) activity throughout the fourth day. Consistently, there was no decrease in stable high molecular weight rVWF multimers during day 4 of batch culture (FIGS. 1A and 1B). The results of agarose electrophoretic gel densitometry shown in FIG. 1B showed that under conditions of low copper levels, the culture was only able to produce a population of rVWF with more than 10% of the rVWF (i.e., 16.3%) represented by more than 10 dimer molecules during one day of batch culture (i.e., e. sample day 3 in lane 2 of the agarose gel in Fig. 1A); notably, this population dropped to as little as 4% on day 5 of batch culture (day 7 sample in lane 6). In contrast, under conditions of high copper content, the relative amount of VWF multimers consisting of more than 10 dimers is stable at about 30% throughout day 4 in batch culture (day 3-day 6 in lanes 7-10; 28-31.4%) .

Notably, starting on day 5 of the high copper batch culture, when NH4 ⁺ levels exceeded 100 mg/l (higher than about 5.0 mM), additional antigen expression (18.3-35.4 µg/ l; cf. batch culture days 4 and 5 in Table 8) did not result in a concomitant increase in RiCoF activity in the supernatant. Consistent with this result, the level of high molecular weight rVWF multimers on day 5 of batch culture (day 7 of culture) decreased relative to the concentration of low molecular weight rVWF multimers. Also, only on day 5 of batch culture (sample of day 7 culture in lane 11) the relative amount of vWF consisting of more than 10 dimers is reduced to 21.4%.

Taken together, the above data show that the additional concentration of copper in cultures of cells expressing rVWF significantly increases the total and specific rVWF ristocetin cofactor activity, as well as stable production of high molecular weight rVWF multimers. In addition, these data reveal a correlation between high concentrations of NH4 ⁺ in cell culture and a decrease in the ristocetin-cofactor activity of rVWF and the production of high molecular weight multimeric rVWF.

Table 7

Expression of rVWF in mammalian cell cultures under batch culture with

P application day periodical cultivation BAV-SP media with low copper concentration (1.0 µg/l) vWF count Cell specific NH ₄ ⁺ Viability Ristocetin ELISA activity [10E6 [mg/l] [%] [IU/ml] [[µg/ml] mU/ 10µg cells/mL] 0 0.42 21 98.8 But. But. But. 1 0.78 43 But. 6.3 0.23 365 2 1.24 64 99.1 12.8 0.23 180 3 1.86 But. But. 22.7 0.21 93 4 2.49 But. But. 32.2 0.20 62 5 h,i 106 98.3 44.4 0.20 45

Table 8

Expression of rVWF in mammalian cell cultures cultured in a batch mode using BAV-SP media with a high concentration of copper (4.3 μg/l)

day periodic cultivation Number of cells [10E6 cells/ml] NHL [mg/l] Viability [%] vWF ELISA [µg/ml] Ristocetin [IU/ml] Specific activity mU/10mkg 0 0.40 21 99.3 But. But. But. 1 0.71 42 But. 4.8 0.40 833 2 1.23 63 99.5 8.7 0.62 713 3 1.85 But. But. 15.0 1.07 713 4 2.54 But. But. 18.3 1.52 831 5 3.32 109 98.9 35.4 1.55 438

Example 3

Recombinant factor VIII (rFVTII) and von Willebrand factor (rVWF) were co-expressed in continuous cultures of GD8/6 cells operated under chemostatic conditions to determine the effect of culture medium composition on VWF expression and activity. Briefly, GD8/6 cells were cultured in BAV-SP medium containing 4 g/L soy hydrolysate with and without copper addition as described in Example 2. To investigate the effect of low copper concentrations on expression and

- 62 043293 rVWF activity BAV-SP basic media were used. These basic media contained 0.3 µg/l copper and added soy hydrolyzate, which gave an additional 0.7 µg/l copper to give a final copper concentration of 1.0 µg/l. For comparison, BAV-SP media, also supplemented with an additional 3.3 µg/L Cu ²⁺ , was used in batch cultures to give a final copper concentration of 4.3 µg/L, to determine the effect of high copper concentrations on VWF expression and activity. Cultures growing in the presence of high and low concentrations of copper were cultured at both high (2.8 x ¹⁰⁶ cells/ml) and low (approx. 1.4 x 10E06 cells/ml) cell densities.

As before, samples of said culture supernatant were tested for rVWF content (vWF ELISA), total (ristocetin) and specific (specific activity) activity by ristocetin cofactor analysis. Various culture parameters were also monitored, including cell count, cell viability, and ammonium concentration. Data were obtained based on the steady state phase of weeks 2 and 3 of the chemostatic culture (Tables 9-13).

Table 9

Average data on rVWF expression in chemostatic ____________ cell culture during weeks 2 and 3____________

XVII. Number of cells XVIII. Copper concentration XIX. Number of cells [10E6 cells/ml] XX. NH4+ XXI. [mm] XXII. Viability XXIII. [%] XXIV. vWF ELISA [µg/ml] XXV. Ristocetin [IU/ml] XXVI. specific activity [mU/ 10mkg] XXVII. big XXVIII. low XXIX. 2.88 XXX. 3.88 XXXI. 97.74 XXXII. 44.56 XXXIII. 0.10 XXXIV. 21.62 XXXV. big XXXVI. high XXXVII. 2.79 XXXVI II. 4.04 XXXIX. 98.25 XL. 38.38 XLI. 0.19 XLII. 53.11 XLIII. small XLIV. low XLV. 1.55 XLVI. 3.33 XLVII. 98.63 XLVIII. 18.96 XLIX. 0.10 L.50.16 L.I. small LII. high III. 1.43 LIV. 3.17 Lv. 98.59 LVI. 11.76 LVII. 0.70 LVIII. 598.76

Table 10

Expression of rVWF in chemostatic cell culture under conditions of high cell count and low _________ copper levels for weeks. 2 and 3 _________

LIX. Day LX. Number of cells [10E6 cells/ml] LXI. NH4+ LXII. [mm] LXIII. Viability LXIV. [%] LXV. vWF ELISA [µg/ml] LXVI. Ristocetin [IU/ml] LXVII. specific activity [μΕ/ΙΟμκγ] LXVIII. 8 LXIX. 2.54 LXX. 3.7 LXXI. 98.20 LXXII. 39.8 LXXIII. 0.095 LXXIV. 23.86934673 LXXV. 9 LXXVI. 3.02 LXXVII. 4.2 LXXVIII. 97.40 LXXIX. LXXX. LXXXI. LXXXII. 10 LXXXIII. 2.97 LXXXIV. 3.9 LXXXV. 97.90 LXXXVI. 41.3 LXXXVII. 0.095 LXXX VIII. 23.00242131 LXXXIX. eleven XC. 2.78 XCI. XXIII. hash. xciv. XCV. XCVI. 13 XCVII. 2.91 XCVIII. 3.8 XXIX. 97.60 C. 41.7 C.I. 0.095 II. 22.78177458 cm. 14 CIV 2.90 CV 3.8 VI. 97.40 evil. CVIII. CIX. CX. 15 CXI. 3.05 CXII. 3.9 CXIII. 97.70 CXIV. 44.7 CXV. 0.095 CXVI. 21.25279642 CXVII. 16 CXVIII. 2.99 CXIX. 3.8 CXX. 98.30 CXXI. CXXII. CXXIII. CXXIV 17 CXXV. 2.76 CXXVI. 3.9 CXXVII. 97.40 CXXVIII. 55.3 CXXIX. 0.095 exxx. 17.17902351 CXXXI. CXXXII. 2.88 CXXXIII. 3.88 CXXXIV. 97.74 CXXXV. 44.56 CXXXVI. 0.10 CXXXVII. 21.62

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Table 11

Expression of rVWF in chemostatic cell culture under conditions of high cell numbers and high ________ copper levels during weeks 2 and 3 _________

CXXXVIII. Day CXXXIX. Number of cells [10E6 cells/ml] CXL. NH4+ CXLI. [mm] CXLII. Viability CXL III. [%] CXLIV. vWF ELISA [µg/ml] cxlv. Ristocetin [IU/ml] CXLVI. specific activity [mU/10mkg] CXLVII. 8 CXLVIII. 2.52 CXLIX. 3.9 CL. 98.30 CLI. 31.8 CLIL 0.35 CLIII. 110.0628931 CLIV. 9 CLV. 2.92 CLVI. 4.3 CLVII. 98.50 CL VIII. CLIX. CLX. 10 2.80 4.2 98.60 37.4 0.32 85.56149733 eleven 2.57 13 2.81 3.9 98.50 37.6 0.095 25.26595745 14 2.92 3.9 97.80 15 2.77 3.9 97.80 43.0 0.095 22.09302326 16 2.72 4.0 98.30 17 3.04 4.1 98.20 42.1 0.095 22.56532067 2.79 4.04 98.25 38.38 0.19 53.11

Table 12 Expression of rVWF in chemostatic cell culture under low cell count, low ______________ copper levels for weeks. 2 and 3 _________

Day Number of cells [10E6 cells/ml] ML [mm] Viability [%] vWF ELISA [µg/ml] Ristocetin [IU/ml] specific activity [mU/Jmkg] 8 1.61 3.3 99.10 19.3 0.095 49.22279793 9 1.65 3.3 98.00 10 1.49 3.3 98.90 18.6 0.095 51.07526882 eleven 1.58 13 1.58 3.4 98.10 18.1 0.095 52.48618785 14 1.56 3.3 99.30 15 1.52 3.3 98.50 18.9 0.095 50.26455026 16 1.50 3.3 98.40 17 1.50 3.4 98.70 19.9 0.095 47.73869347 1.55 3.33 98.63 18.96 0.10 50.16

Table 13 Expression of rVWF in chemostatic cell culture under low cell count, ________ high copper levels during weeks 2 and 3 _________

Day Number of cells [10E6 cells/ml] NH4+ [mM] Viability [%] vWF ELISA [µg/ml] Ristocetin [IU/ml] specific activity [mU/10mkg] 8 1.46 3.2 98.80 11.6 0.73 629.3103448 9 1.45 3.2 98.20 10 1.37 3.1 98.90 11.0 0.7 636.3636364 eleven 1.43 13 1.33 3.2 98.10 H.1 0.68 612.6126126 14 1.39 3.2 97.20 15 1.51 3.2 99.70 12.4 0.69 556.4516129 16 1.49 3.2 98.60 17 1.43 3.2 99.20 12.7 0.71 559.0551181 1.43 3.17 98.59 11.76 0.70 598.76

As shown in FIG. 2A, the supernatant harvested from a continuous rVWF cell culture grown at low cell density and high copper concentrations had a high specific activity (average 600 mU/10 μg), while supernatants harvested from rVWF cell cultures grown at at high cell density in the presence of high or low copper concentrations, and rVWF cell cultures grown at low cell density in the presence of low copper concentration had low specific activities (less than 100 mU/10 μg). In accordance with the results obtained for batch cultures, as seen from FIG. 2B, continuous cultures of mammalian cells expressing high specific activity rVWF contain lower NH4' concentrations than cultures producing low specific activity rVWF. These data also confirm the correlation between the concentration of NH4+ in the cell culture and the specific activity of rVWF produced by this culture. It is noteworthy that the combination of high copper concentration and low ammonium concentration in cell culture made it possible to achieve a significant increase in rVWF activity.

Consistent with this result, analysis of the multimeric status of rVWF in chemostatic cultures by agarose gel electrophoresis (Fig. 6) showed that only the supernatants of cultures operated at high copper and low cell density and thus low ammonium provided sustained expression of high multimeric vWF (approximately 23-27% on CST days 8, 17, and 24, lanes 4, 8, and 12, respectively). All other conditions did not provide a sustainable achievement of a large amount - more than 10% of vWF containing more than 10 dimers, over a long period of cultivation.

Example 4

To determine the effect of copper concentration in the culture medium on the expression and specific activity of rA13, mammalian cell cultures for rA13 expression were cultured for 4 weeks under chemostatic continuous culture conditions in ADAMTS13 medium, including modified DMEM/F12 BESP845 basic media and additional additives (Table 14) containing copper in the concentration range from 0.66 µg/l (without additional addition of copper) and with additional addition of copper up to 4 µg/l. As can be seen from Table. 15, increasing concentrations of copper in the cell culture medium resulted in a significant increase in volumetric (P) and specific (q) productivity, expressed as total rA13 activity produced per liter of culture per day and total rA13 produced activity per cell per day, respectively. .

Table 14

The composition of the environment for obtaining ADAMTS13

L-Alanine 13.3500 L-Arginine HC1 147.5000 L-Asparagine-HgO 45.1600 L-Aspartic Acid 19.9500 L-Cysteine HC1-H ₂ O 32.5500 L-Cystine 2HC1 102.3500 L-Glutamic acid 22.0500 Glycine 26.2500 L-Histidine-HgO HC1 51.4800 L-Isoleucine 74.4700 L-Leucine 119.0500 L-Lysine HC1 146.2500 L-Methionine 100.0000 L-Phenylalanine 60.4800 L-Proline 63.7400 L-Serine 36.7500 L-Threonine 53.4500 L-Tryptophan 29.0100 L-Tyrosine 2Na 2H2O 75.7900 L-Valine 82.8500 salt mg/l Calcium chloride (CaC12) 116.6000

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Copper sulfate (CuSC>4-5H2O) 0.0026 Iron nitrate (Fe (NOz) 3-9H ₂ O) 0.0500 Ferrous sulfate (FeSO4-7H2O) 1.0170 Magnesium chloride (MgC12) 28.6400 Magnesium sulfate (Mg3O4) 48.8400 Potassium chloride (KC1) 311.8000 Sodium chloride (NaCl) 5495.5000 Na2HPO4 Anhydrous 213.0200 NaH2PO4 Anhydrous 54.3500 Zinc sulfate heptahydrate (ZnSO427H2O) 0.4320 Sodium selenite, anhydrous 0.0087 Vitamin mg/l Ascorbic acid 3.4990 Biotin 0.2035 Choline chloride 26.9800 D-Ca-Pantothenate 6, 2400 Folic acid 6.6500 1-Inositol 36.6000 Nicotinamide 7.0200 Pyridoxine HC1 6.0310 Riboflavin 0.6590 Thiamine HC1 6.5100 Vitamin B12 2.6800 other mg/l D-Glucose 5000.0000 Linoleic acid 0.0420 Lipoic acid 1.0050 Putrescine 2HC1 3.6810 thymidine 0.3650 Hypoxanthin Na 2.3900 sodium pyruvate 55.0000 DMEM/F12 BESP845: total 12738.3 ADAMTS-13 Media Additives mg/l L-Glutamine 1300.0000 Pluronic F68 1000.0000 ethanolamine 1.5300 ZnSo4*7 H2O 1.0000 Na-Hydrocarbonate 1500.0000 Total Additives 3802.5 Total Ingredients 16540.8

To determine whether elevated copper concentrations affect retention of rA13 expressed, supernatant harvested from tL13 cell cultures grown in media containing 0.66 µg/L, 1 µg/L, and 4 µg/L copper was examined by SDS assay. -PAAG. As can be seen from FIG. FOR (silver staining) and FIG. SR (anti-A13 Western blotting), no obvious changes in the quality of the product during gel electrophoresis are observed. Thus, the expression of tL13 in the presence of elevated concentrations of copper does not lead to an increase in the level of truncated 170 kDa or other low molecular weight variants of tL13, and to the appearance of additional or increased bands corresponding to HCPs (host cell proteins).

When calculating the optimal concentration of copper for the activity of tL13, extrapolation of data from Table. 15 of P Frets versus copper concentration (FIG. 4) shows that the optimal effect appears to be at about 2 µg/L, with a conservative negative effect at more than about 4 µg/L.

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Table 15

Expression of rA13 in mammalian cell cultures under chemostatic mode of continuous cultivation using a medium containing various concentrations of copper

Volume 10 l R&D (R&D) experiment Average over 4 weeks CST ZZ-NC P Frets q Frets Oud. activity [10E6 cells/ml] [E/(l*d)] [E/ (10E9 cells * e)] U/mg 0.66 μg/l Cu ²⁺ 1.35 1322 1028 1759 1 μg/l Cu ²⁺ 1, 64 1962 1247 1690 4 μg/l Cu ²⁺ 2.26 2960 1338 1768

Based on the results described above, an additional experiment was performed to compare the expression of rA13 in cell culture medium containing 0.66 μg/l copper and containing 2.0 μg/l copper. As can be seen from Table. 16, the addition of copper to basic cell media to a final concentration of 2.0 μg/l copper resulted in a significant increase in volumetric (P) and specific (q) productivity, expressed as total rA13 activity obtained per liter of culture per day and total rA13 activity obtained per cell per day, respectively, for 8 weeks. Specific data for each week of rA13 production in these two cultures is shown in FIG. 5. It is clear from these data that copper supplementation has a measurable beneficial effect on cell metabolism, specific growth rate, and rA13 production.

Table 16

Expression of rA13 in mammalian cell cultures under chemostatic mode of continuous cultivation using a medium containing various concentrations of copper

Volume 10 l Average over 8 weeks CST ZZ-NC [10E6 class. /ml] P Frets [E/(l*d)] q Frets [E/ (10E9 cells*d)] Oud. activity U/mg 0.66 μg/l Cu ²⁺ 1.29 1049 821 1862 2 μg/l Cu ²⁺ 2.17 2470 1146 1749

It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations therefrom will be understood by those skilled in the art, are to be included within the spirit and scope of the invention, and fall within the scope of the appended claims. All publications, patents and patent applications cited in the invention are hereby incorporated by reference in their entirety for any purpose.

Claims (21)

1. A method for obtaining a supernatant of recombinant ADAMTS13 (disintegrin and metalloproteinase with thrombospondin motif type I, representative 13), including the steps: culturing one or more mammalian cells containing a nucleic acid encoding a recombinant ADAMTS13 protein in a cell culture medium containing 1 to 4 μg/L copper and 2 to 12 μM zinc such that the recombinant ADAMTS13 is expressed and isolated from mammalian cells in culture supernatant, where the content of NH ₄ ⁺ in cell culture is maintained at a concentration below 10 mm, where mammalian cells are grown under continuous cultivation conditions; and separating at least a portion of the culture supernatant, wherein at least 1500 units of FRETS-VWF73 activity per liter of cell culture medium per day are present in said separated culture supernatant. 2. The method of claim 1, wherein the cell culture medium contains 2 to 4 µg/l copper. 3. The method of claim 1, wherein the cell culture medium contains 1.3 to 3 µg/l copper. 4. The method of claim 1, wherein the cell culture medium contains 4 µg/l copper. 5. The method according to any one of claims 1 to 4, wherein the cell culture medium contains 5 to 12 μM zinc. 6. The method according to any one of claims 1 to 5, wherein the cell culture medium further comprises at least 7 mg/ml of nicotinamide. 7. The method according to any one of claims 1 to 6, wherein the cell density is maintained at a level of less than 4.0 x 10 ⁶ - 67 043293 cells per ml, less than 3.5 x 10 ⁶ cells per ml or less than 3.0 x 10 ⁶ cells per ml during the stage of culturing one or more mammalian cells. 8. The method according to any one of claims 1 to 7, wherein culturing one or more mammalian cells comprises continuous culturing of mammalian cells. 9. The method according to claim 8, where the continuous cultivation of mammalian cells is carried out in a chemostatic mode. 10. The method according to claim 8, where the continuous cultivation of mammalian cells is carried out in perfusion mode. 11. The method according to any one of claims 1 to 10, wherein the mammalian cells are CHO cells. 12. The method of any one of claims 1-11, wherein the recombinant ADAMTS13 protein is a recombinant human ADAMTS13 protein. 13. The method according to any one of claims 1 to 12, wherein at least 2000 units of FRETS-VWF73 activity per liter of cell culture medium per day are present in said separated culture supernatant. 14. The method according to any one of claims 1 to 12, wherein at least 2500 units of FRETS-VWF73 activity per liter of cell culture medium per day are present in said separated culture supernatant. 15. Cell culture supernatant for obtaining a composition of recombinant ADAMTS13 for the treatment or prevention of a disease or condition associated with ADAMTS13 deficiency and/or dysfunction, where the specified supernatant is obtained by the method according to any one of claims 1-14, where at least 1500 units of activity FRETS- VWF73 per liter of cell culture medium per day are present in said supernatant. 16. The composition of recombinant ADAMTS13 for the treatment or prevention of a disease or condition associated with deficiency and/or dysfunction of ADAMTS13, where the specified composition contains an effective amount of recombinant ADAMTS13 protein purified from the supernatant according to claim 15, and a pharmaceutically acceptable carrier, where the recombinant ADAMTS 13 has a specific FRETSVWF activity of at least 1600 IU/μg. 17. The composition of claim 16, wherein the disease or condition associated with ADAMTS13 deficiency and/or dysfunction is a disorder associated with the formation and/or presence of blood clots. 18. The composition of claim 17 wherein the disorder associated with the formation and/or presence of thrombi is thrombotic thrombocytopenic purpura. 19. The composition of claim 17, wherein the disorder associated with the formation and/or presence of thrombi is an infarction. 20. The composition of claim 17 wherein the disorder associated with the formation and/or presence of blood clots is stroke. 21. A composition according to any one of claims 17-20, wherein the composition is formulated for intravenous administration.