WO2008135498A2

WO2008135498A2 - Prevention of protein degradation in mammalian cell cultures

Info

Publication number: WO2008135498A2
Application number: PCT/EP2008/055345
Authority: WO
Inventors: Henning Ralf Stennicke
Original assignee: Novo Nordisk A/S
Priority date: 2007-05-04
Filing date: 2008-04-30
Publication date: 2008-11-13
Also published as: WO2008135498A3

Abstract

A method for the production of a Factor VIII polypeptide, the method comprising a) culturing a mammalian cell transfected with an expression vector comprising a nucleic acid molecule encoding said Factor VIII polypeptide and expression. control regions operatively linked to thereto, in a cell culture medium under conditions.suitable for expression of said polypeptide; and b) isolating the expressed Factor VIII polypeptide; wherein the cell culture medium in step a) comprises a tissue inhibitor of metalloproteinase (TIMP).

Description

PREVENTION OF PROTEIN DEGRADATION IN MAMMALIAN CELL CULTURES

FIELD OF THE INVENTION

The present invention relates to a method for the production of a polypeptide of interest, e.g. Factor VIII, wherein the product (culturing of mammalian cells) takes place in the presence of a tissue inhibitor of metalloproteinases (TIMP) in order to specifically block the metalloproteinase activity.

BACKGROUND OF THE INVENTION

Classic haemophilia or haemophilia A is an inherited bleeding disorder. It results from a chromosome X-linked deficiency of blood coagulation Factor VIII and affects almost exclusively males with an incidence of between one and two individuals per 10,000. The X- chromosome defect is transmitted by female carriers who are not themselves haemophiliacs. The clinical manifestation of haemophilia A is an increased bleeding tendency. Before treatment with Factor VIII concentrates was introduced the mean life span for a person with severe haemophilia was less than 20 years. The use of concentrates of Factor VIII from plasma has considerably improved the situation for the haemophilia patients increasing the mean life span extensively, giving most of them the possibility to live a more or less normal life. However, there have been certain problems with the plasma-derived concentrates and their use, the most serious of which have been the transmission of viruses. So far, viruses causing AIDS, hepatitis B, and non-A non-B hepatitis have hit the population seriously. Since then different virus inactivation methods and new highly purified Factor VIII concentrates have recently been developed which established a very high safety standard also for plasma derived Factor VIII.

During expression of coagulation factor VIII proteolytic cleavage at Glu720 and Tyr729 has been observed (Kjalke et al., Eur. J. Biochem. 234, 773-779 (1995)). This cleavage is undesirable as it results in a heterogeneous product with, at least for cleavage at Glu720, reduced clotting activity. Recently, Sandberg et al (Biotechnology and Bioengineering) has demonstrated that CHO cells express a large contingent of proteases. Among the most prominent candidates to be linked to the degradation of Factor VIII during expression is the metalloproteinases MMP3, MMPlO and MMP12. Similar degradation problems have been identified for a number of other proteins/polypeptides.

This being, there is a need for improved production methods so as to suppress the proteolytic cleavage of the polypeptide of interest {e.g. Factor VIII polypeptides). SUMMARY OF THE INVENTION

The present invention relates to a method for the production of a polypeptide of interest in the presence of a tissue inhibitor of metalloproteinase (TIMP), the method comprising a) transfecting a mammalian cell with (i) an expression vector comprising a nucleic acid molecule encoding the polypeptide of interest and expression control regions operatively linked to thereto, b) culturing the transfected cell under conditions for expression of the polypeptide of interest, said culturing involving a cell culture medium, and c) isolating the expressed polypeptide of interest by suitable means.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Factor VIII gene sequence (cDNA) (SEQ ID NO. 1).

Figure 2. Western blot analysis of transient TIMP-I expression in freestyle HEK293 cells (Invitrogen). Mw marker: MagicMark XP Standard (Invitrogen), lane 1 : Positive Control for TIMP-I Western Blot (NeoMarkers, CA, Cat No. MS-608 - PCL), lane 2: Media from transient transfection HEK293 cells with TIMP-I in pcDNA3.2V5GW-CAT.

DETAILED DESCRIPTION OF THE INVENTION

The present invention resides in the finding that co-expression or addition of a tissue inhibitor of metalloproteinases (TIMP) in order to specifically block the metalloproteinase activity is useful in a method for the production of a polypeptide of interest, in particular a Factor VIII polypeptide. There are today four well characterized TIMPs known. Characteristic for these TIMPs are that they are fairly broad spectrum inhibitors of the MMP and adamlysins. Of particular interest to the problem associated with Factor VIII is TIMP-I which is a sub-nM inhibitor of the enzymes which may be associated with the degradation. The solution to the degradation may be to block the degradation during expression by ensuring the presence of a TIMP during fermentation.

Hence, the present invention provides a method for the production of a polypeptide of interest in the presence of a tissue inhibitor of metalloproteinase (TIMP), the method comprising a) transfecting a mammalian cell with (i) an expression vector comprising a nucleic acid molecule encoding the polypeptide of interest and expression control regions operatively linked to thereto, b) culturing the transfected cell under conditions for expression of the polypeptide of interest, said culturing involving a cell culture medium, and c) isolating the expressed polypeptide of interest by suitable means. It should be understood that in the production phase (see further below), a tissue inhibitor of metalloproteinase (TIMP) is present so as to suppress degradation, i.e. proteolytic cleavage, of the polypeptide of interest.

In the most typical embodiments, the tissue inhibitor of metalloproteinase (TIMP) is selected from the group consisting of TIMP-I, TIMP-2, TIMP-3 and TIMP-4.

For references to TIMP, see e.g. UniProtKB/Swiss-Prot database; see for example: human TIMP-I : UNIPROTKB/SWISS-PROT ENTRY P01033 (WWW.EXPASY.ORG/UNIPROT/P01033); human TIMP-2: UNIPROTKB/SWISS-PROT ENTRY P16035 (WWW. EXPASY. ORG/U N I P ROT/P 16035) human TIMP-3: UNIPROTKB/SWISS-PROT ENTRY P35625 (WWW.EXPASY.ORG/UNIPROT/P35625) ; and Human TIMP-4: UNIPROTKB/SWISS-PROT ENTRY Q99727 (WWW. EXPASY. ORG/U N I P ROT/Q99727)

In a particularly interesting variant hereof, the tissue inhibitor of metalloproteinase (TIMP) is TIMP-I.

As it will be explained in the following, the advantages of the invention may be achieved by:

1. Co-expression of the protein of interest, e.g., Factor VIII, with a tissue inhibitor of metalloproteinases (TIMP), e.g. TIMP-I.

2. Addition of purified recombinantly produced tissue inhibitor of metalloproteinases (TIMP), e.g. TIMP-I, to the culture medium when producing the protein of interest, e.g. , Factor VIII.

3. Co-culturing of mammalian cells expressing the protein of interest, e.g. , Factor VIII, with cells expressing tissue inhibitor of metalloproteinases (TIMP), e.g. TIMP-I.

In each of these variants, the tissue inhibitor of metalloproteinases (TIMP) is typically present in the cell culture medium in a concentration of 0.1-100 μg/mL, such as 0.25-10 μg/mL, in particular 0.5-5 μg/mL.

It has been found that suppression or elimination of the degradation of the protein of interest during the fermentation results in significantly increased yields and simplify processing. In the currently most interesting embodiment, the method in step a) further comprises transfecting the same cell with (ii) an expression vector comprising a nucleic acid molecule encoding the tissue inhibitor of metalloproteinase (TIMP) and expression control regions operatively linked to thereto, and the conditions in step b) also are applicable for expression of the tissue inhibitor of metalloproteinase (TIMP).

In an alternative embodiment, the tissue inhibitor of metalloproteinase (TIMP) is provided in step b).

In a particular variant hereof, the tissue inhibitor of metalloproteinase (TIMP) is expressed by another mammalian cell transfected with an expression vector comprising a nucleic acid molecule encoding the tissue inhibitor of metalloproteinase (TIMP) and expression control regions operatively linked to thereto.

Co-expression of the protein of interest, e.g.. Factor VIII, with a tissue inhibitor of metalloproteinases (TIMP), e.g. TIMP-I

The key steps of this principal embodiment involve:

1. Isolation of cDNA for the protein of interest and a suitable TIMP depending on the degradation profile based on the protease profile of the cell line used for production, e.g. TIMP-I.

2. Sub-cloning of the cDNA of the protein of interest, e.g. a Factor VIII polypeptide, and a TIMP {e.g. TIMP-I) into either a bi-cistronic construct or two separate compatible vectors with separate selection markers.

3. Cell line generation. In a bi-cistronic construct the protein of interest should precede the TIMP in order to ensure higher expression levels of the target protein than the TIMP. The major advantage of this approach would be the concomitant increase in target protein and TIMP as a function of selection pressure. Alternatively, a producer cell line may be generated via transfection and subsequent selection of the clones which efficiently produces the protein of interest. This cell line may subsequently be transfected by the TIMP vector and producer clones selected by the application of two selection agents matching the selection markers of the individual vectors used, e.g., methotrexate and neomycine. 4. Suitable producer cell lines would identified by determination of antigen levels or activity of the protein of interest and subsequently, adapted to serum free media.

Addition of purified recombinantly produced tissue inhibitor of metalloproteinases (TIMPV e.g. TIMP-I, to the culture medium when producing the protein of interest, e.g.. Factor VIII.

The key steps of this embodiment involve:

1. Isolation of cDNA for the protein of interest and sub-cloning to a suitable vector with a selection marker for selection of positive clones.

2. A producer cell line may be generated via transfection, and subsequent selection of the clones which efficiently produces the protein of interest are identified by determination of antigen levels or activity of the protein of interest and subsequently, adapted to serum free media.

3. Recombinant TIMP is produced in a suitable host and purified to homogeneity using conventional methods or suitable tags for affinity purification.

4. The purified TIMP is added to the media at a predetermined concentration suitable for eliminating degradation of the protein of interest during fermentation. This level is determined by monitoring antigen/activity levels in the media as a function of TIMP concentration.

Co-culturinq of mammalian cells expressing the protein of interest, e.g.. Factor VIII, with a cells expressing tissue inhibitor of metalloproteinases (TIMPV e.g. TIMP-I.

The key steps of this embodiment involve:

2. Sub-cloning of the cDNA of the protein of interest and TIMP-I into a vector suitable for generation of a stable cell lines for the two proteins. 3. Two separate producer cell lines may be generated via transfection and subsequent selection of the clones which efficiently produces the protein of interest, i.e., TIMP or the protein of interest. Producer clones are selected by the application a selection agents matching the marker of the vectors used, e.g., methotrexate or neomycine.

4. Efficient producers of the protein of interest or TIMP are identified by ELISA or activity assays.

5. Fermentation media are inoculated with different ratios of the two cell lines - typically the ratio will be dependent on the TIMP levels required to suppress degradation and thus, will be highly dependent on the TIMP expression levels.

The details for the expression of the polypeptide of interest and, where applicable, the tissue inhibitor of metalloproteinase (TIMP) are explained in details further below.

Polypeptides of interest

The present invention relates to a method for the production of a polypeptide of interest, whether from endogenous genes or subsequent to introduction into such cells of recombinant genes encoding the protein. Such polypeptides (proteins) include, without limitation, Factor VIII polypeptides; Factor VII polypeptides; Factor IX polypeptides; Factor X polypeptides; Protein C; tissue factor; rennin; growth hormone, including human growth hormone; bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase (uPA) or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; human macrophage inflammatory protein (MIP-1-alpha); a serum albumin such as human serum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β platelet-derived growth factor (PDGF); fibroblast growth factor such as α-FGF and β-FGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, insulin-like growth factor- I and -II (IGF-I and IGF-II); CD proteins such as CD-3, CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-I to IL-IO; T-cell receptors; surface membrane proteins; decay accelerating factor; transport proteins; homing receptors; addressin; regulatory proteins; antibodies; and fragments or genetic variants of any of the above polypeptides.

In preferred embodiments of the invention, the polypeptide is a Factor VIII polypeptide.

The invention is particularly adapted for the production of a Factor VIII polypeptide in a mammalian cell.

The mature Factor VIII molecule consists of 2332 amino acids which can be grouped into three homologous A domains, two homologous C domains and a B Domain which are arranged in the order: A1-A2-B-A3-C1-C2. During its secretion into plasma Factor VIII is processed intracellular^ into a series of metal-ion linked heterodimers as single chain Factor VIII is cleaved at the B-A3 boundary and at different sites within the B-domain. This processing leads to a heavy chain consisting of the Al, the A2 and various parts of the B- domain which has a molecular size ranging from 90 kDa to 200 kDa. The heavy chains are bound via a metal ion to the light chain, which consists of the A3, the Cl and the C2 domain (Saenko et al. 2002). In plasma this heterodimeric Factor VIII binds with high affinity to von Willebrand Factor, which protects it from premature catabolism. The half-life of non-activated Factor VIII bound to vWF is about 12 hours in plasma.

During the blood coagulation process Factor VIII is activated via proteolytic cleavage by FXa and thrombin at amino acids Arg372 and Arg740 within the heavy chain and at Argl689 in the light chain resulting in the release of von Willebrand Factor and generating the activated Factor VIII heterotrimer which will form the tenase complex on phospholipid surfaces with FIXa and FX provided that Ca2+ is present. The heterotrimer consists of the Al domain, a 50 kDa fragment, the A2 domain a 43 kDa fragment and the light chain (A3-C1-C2), a 73 kDa fragment. Thus the active form of Factor VIII (Factor Villa) consists of an Al-subunit associated through the divalent metal ion linkage to a thrombin-cleaved A3-C1-C2 light chain and a free A2 subunit relatively loosely associated with the Al and the A3 domain.

A Factor VIII molecule consisting of the heavy chain (HC) and light chain (LC) of Factor VIII connected with a small linker derived from the B-domain (B-domain deleted Factor VIII or BDD-FVIII) retains the biological activity of full length (native) Factor VIII.

In practicing the method of the present invention, any Factor VIII polypeptide that is therapeutically useful, e.g. effective in preventing or treating bleeding may be relevant. This includes, without limitation, wild-type human Factor VIII, hybrid human/porcine Factor VIII and B-domain deleted human Factor VIII.

As used herein, "Factor VIII polypeptide" encompasses, without limitation, Factor VIII, as well as Factor Vlll-related polypeptides.

The term "Factor VIII" is intended to encompass, without limitation, polypeptides having the amino acid sequence as described in Toole et al., Nature 1984, 312: 342-347 (wild-type human factor VIII), as well as wild-type Factor VIII derived from other species, such as, e.g., bovine, porcine, canine, murine, and salmon Factor VIII. It further encompasses natural allelic variations of Factor VIII that may exist and occur from one individual to another. Also, degree and location of glycosylation or other post-translation modifications may vary depending on the chosen host cells and the nature of the host cellular environment. The term "Factor VIII" is also intended to encompass Factor VIII polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated Factor Villa.

"Factor Vlll-related polypeptides" include, without limitation, Factor VIII polypeptides that have either been chemically modified relative to human Factor VIII and/or contain one or more amino acid sequence alterations relative to human Factor VIII {i.e., Factor VIII variants), and/or contain truncated amino acid sequences relative to human Factor VIII {i.e., Factor VIII fragments). Such Factor Vlll-related polypeptides may exhibit different properties relative to human Factor VIII, including stability, phospholipid binding, altered specific activity, and the like. The term "Factor Vlll-related polypeptides" are intended to encompass such polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated "Factor Villa-related polypeptides" or "activated Factor Vlll-related polypeptides".

As used herein, "Factor Vlll-related polypeptides" also encompasses, without limitation, polypeptides exhibiting substantially the same or improved biological activity relative to wild- type human Factor VIII, as well as polypeptides, in which the Factor VIII biological activity has been substantially modified or reduced relative to the activity of wild-type human Factor VIII. These polypeptides include, without limitation, Factor VIII or Factor Villa that has been chemically modified and Factor VIII variants into which specific amino acid sequence alterations have been introduced that modify or disrupt the bioactivity of the polypeptide.

It further encompasses polypeptides with a slightly modified amino acid sequence, for instance, polypeptides having a modified N-terminal end including N-terminal amino acid deletions or additions, and/or polypeptides that have been chemically modified relative to human Factor VIII.

Factor Vlll-related polypeptides, including variants of factor VIII, whether exhibiting substantially the same or better bioactivity than wild-type factor VIII, or, alternatively, exhibiting substantially modified or reduced bioactivity relative to wild-type factor VIII, include, without limitation, polypeptides having an amino acid sequence that differs from the sequence of wild-type factor VIII by insertion, deletion, or substitution of one or more amino acids.

Factor Vlll-related polypeptides, including variants, encompass those that exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, and at least about 130%, of the specific activity of wild-type factor VIII that has been produced in the same cell type, when tested in the factor VIII activity assay as described in the present specification.

Factor Vlll-related polypeptides, including variants, having substantially the same or improved biological activity relative to wild-type factor VIII encompass those that exhibit at least about 25%, such as at least 50%, at least 75%, or at least 90% of the specific biological activity of wild-type human factor VIII that has been produced in the same cell type when tested in one or more of the specific factor VIII activity assay as described below in the present description ("Materials and Methods").

Factor Vlll-related polypeptides, including variants, having substantially reduced biological activity relative to wild-type factor VIII are those that exhibit less than about 25%, such as less than about 10%, or less than about 5% of the specific activity of wild-type factor VIII that has been produced in the same cell type when tested in one or more of the specific factor VIII activity assays as described below in the present description ("Materials and Methods"). Non-limiting examples of Factor VIII polypeptides include plasma-derived human Factor VIII as described, e.g., in Fulcher et al.; Proc. Acad. Nat. Sci. USA 1982; 79: 1648-1652, and Rotblat et al.; Biochemistry 1985; 24:4294-4300, and plasma-derived porcine FVIII as described, e.g., in Fass et al.; Blood 1982; 59: 594-600 and Knutson et al.; Blood 1982; 59: 615-624. Non-limiting examples of factor VIII sequence variants are described, e.g., in Lollar et al.; Blood 2000; 95(2) : 564-568 (hybrid porcine/human FVIII polypeptides) and Lollar et al.; Blood 2001; 97(1) : 169-174.

The cloning of the cDNA for Factor VIII (Wood, W. I., et al. (1984) Nature 312, 330- 336; Vehar, G.A., et al. (1984) Nature 312, 337-342) made it possible to express Factor VIII recombinantly leading to the development of several recombinant Factor VIII products, which were approved by the regulatory authorities between 1992 and 2003. The coding sequence for Factor VIII (cDNA) is shown in Figure 1. The fact that the central B domain of the Factor VIII polypeptide chain residing between amino acids Arg-740 and Glu-1649 does not seem to be necessary for full biological activity has also led to the development of a B domain deleted Factor VIII. See also Kjalke M, Heding A, Talbo G, Persson E, Thomsen J and Ezban M (1995), "Amino acid residues 721-729 are required for full factor VIII activity". Eur. J. Biochem: 234: 773-779.

Step a) - Transfection of cells

The mammalian cell expressing the polypeptide of interest (e.g. Factor VIII polypeptide) is typically selected from the group consisting of mammalian cells that endogenously express the Factor VIII polypeptide and mammalian cells that have been transfected with a gene for the Factor VIII polypeptide.

In one currently interesting embodiment of the latter, the mammalian cell has been transfected with an expression vector comprising a nucleic acid molecule encoding the Factor VIII polypeptide and expression control regions operatively linked to thereto.

Expression of protein in cells is well-known to the person skilled in the art of protein production. In practicing the present invention, the cells are mammalian cells, more preferably an established mammalian cell line, including, without limitation, CHO {e.g., ATCC CCL 61), COS-I (e.g., ATCC CRL 1650), baby hamster kidney (BHK), and HEK293 (e.g., ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. A preferred BHK cell line is the tk^" tsl3 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79: 1106- 1110, 1982), hereinafter referred to as BHK 570 cells. The BHK 570 cell line is available from the American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD 20852, under ATCC accession number CRL 10314. A tk^' tsl3 BHK cell line is also available from the ATCC under accession number CRL 1632. Preferred CHO cell lines are the CHO Kl cell line available from ATCC under accession number CCI61, as well as cell lines CHO-DXBIl and CHO-DG44.

Other suitable cell lines include, without limitation, Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung

(ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1); DUKX cells (CHO cell line) (Urlaub and Chasin, Proc. Natl. Acad. ScL USA 77:4216-4220, 1980) (DUKX cells also being referred to as DXBIl cells), and DG44 (CHO cell line) {Cell, 33: 405, 1983, and Somatic Cell and Molecular Genetics 12: 555, 1986). Also useful are 3T3 cells, Namalwa cells, myelomas and fusions of myelomas with other cells. In some embodiments, the cells may be mutant or recombinant cells, such as, e.g., cells that express a qualitatively or quantitatively different spectrum of enzymes that catalyze post-translational modification of proteins {e.g., glycosylation enzymes such as glycosyl transferases and/or glycosidases, or processing enzymes such as propeptides) than the cell type from which they were derived. DUKX cells (CHO cell line) are especially preferred.

Currently preferred cells are HEK293, COS, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) and myeloma cells, in particular Chinese Hamster Ovary (CHO) cells.

Step b) - Culturing the transfected cells

In some embodiments, the cells used in practicing the invention are capable of growing in suspension cultures. As used herein, suspension-competent cells are those that can grow in suspension without making large, firm aggregates, i.e., cells that are monodisperse or grow in loose aggregates with only a few cells per aggregate. Suspension-competent cells include, without limitation, cells that grow in suspension without adaptation or manipulation (such as, e.g., hematopoietic cells or lymphoid cells) and cells that have been made suspension- competent by gradual adaptation of attachment-dependent cells (such as, e.g., epithelial or fibroblast cells) to suspension growth.

The cells used in practicing the invention may be adhesion cells (also known as anchorage- dependent or attachment-dependent cells). As used herein, adhesion cells are those that need to adhere or anchor themselves to a suitable surface for propagation and growth. In one embodiment of the invention, the cells used are adhesion cells. In these embodiments, both the propagation phases and the production phase include the use of microcarriers. The used adhesion cells should be able to migrate onto the carriers (and into the interior structure of the carriers if a macroporous carrier is used) during the propagation phase(s) and to migrate to new carriers when being transferred to the production bioreactor. If the adhesion cells are not sufficiently able to migrate to new carriers by themselves, they may be liberated from the carriers by contacting the cell-containing microcarriers with proteolytic enzymes or EDTA. The medium used (particularly when free of animal-derived components) should furthermore contain components suitable for supporting adhesion cells; suitable media for cultivation of adhesion cells are available from commercial suppliers, such as, e.g., Sigma.

The cells may also be suspension-adapted or suspension-competent cells. If such cells are used, the propagation of cells may be done in suspension, thus microcarriers are only used in the final propagation phase in the production culture vessel itself and in the production phase. In case of suspension-adapted cells the microcarriers used are typically macroporous carriers wherein the cells are attached by means of physical entrapment inside the internal structure of the carriers. However, in case of such suspension-adapted cells, both propagation of cells and production may be done in suspension.

In such embodiments, the mammalian cell is typically selected from CHO, BHK, HEK293, myeloma cells, etc.

Cell culture medium

The cell culture medium typically includes a number of other constituents which - as the skilled person will know - are necessary for the propagation of the cells and production of the Factor VIII polypeptide.

The term "cell culture medium" (or simply "medium") refers to a nutrient solution used for growing eukaryote cells that typically provides at least one component from one or more of the following categories: (1) salts of e.g. sodium, potassium, magnesium, and calcium contributing to the osmolality of the medium; (2) an energy source, usually in the form of a carbohydrate such as glucose; (3) all essential amino acids, and usually the basic set of twenty amino acids; (4) vitamins and/or other organic compounds required at low concentrations; and (5) trace elements, where trace elements are defined as inorganic compounds that are typically required at very low concentrations, usually in the micromolar range. The nutrient solution may optionally be supplemented with one or more of the components from any of the following catagories: (a) hormones and other growth factors such as, for example, insulin, transferrin, and epidermal growth factor; and (b) hydrolysates of protein and tissues. Preferably, the cell culture medium does not contain any components of animal origin. The present invention encompasses cultivating eukaryote cells in medium lacking animal- derived components. As used herein, "animal-derived" components are any components that are produced in an intact animal (such as, e.g., proteins isolated and purified from serum), or produced by using components produced in an intact animal (such as, e.g., an amino acid made by using an enzyme isolated and purified from an animal to hydrolyse a plant source material). By contrast, a protein which has the sequence of an animal protein {i.e., has a genomic origin in an animal) but which is produced in vitro in cell culture (such as, e.g., in a recombinant yeast or bacterial cell or in an established continuous eukaryote cell line, recombinant or not), in media lacking components that are produced in, and isolated and purified from an intact animal is not an "animal-derived" component (such as, e.g., insulin produced in a yeast or a bacterial cell, or insulin produced in an established mammal cell line, such as, e.g., CHO, BHK or HEK cells, or interferon produced in Namalwa cells). For example, a protein which has the sequence of an animal protein {i.e., has a genomic origin in an animal) but which is produced in a recombinant cell in media lacking animal derived components (such as, e.g., insulin produced in a yeast or bacterial cell) is not an "animal- derived component". Accordingly, a cell culture medium lacking animal-derived components is one that may contain animal proteins that are recombinantly produced; such medium, however, does not contain, e.g., animal serum or proteins or other products purified from animal serum. Such medium may, for example, contain one or more components derived from plants. Any cell culture medium, in particular one lacking animal-derived components, that supports cell growth and maintenance under the conditions of the invention may be used. Typically, the medium contains water, an osmolality regulator, a buffer, an energy source, amino acids, an inorganic or recombinant iron source, one or more synthetic or recombinant growth factors, vitamins, and cofactors. In one embodiment, the medium lacks animal-derived components and lacks proteins ("protein-free"). Media lacking animal-derived components and/or proteins are available from commercial suppliers, such as, for example, Sigma, JRH Biosciences, Gibco, Hyclone and Gemini.

In one embodiment, the cell culture medium is essentially serum free. In another embodiment, the medium is a medium lacking animal-derived components. In a further embodiment, the medium is lacking proteins ("protein-free") as well as lacking animal- derived components.

In one embodiment the medium is a commercially available protein-free CHO medium lacking animal-derived components, such as, e.g., EXCELL™ (SAFC Biosciences), PF-CHO, PF-CHO- LS, SFM4CH0, or CDM4CHO (all from Hyclone), and the cell line is a CHO cell.

In some embodiments, the cells used in practicing the present invention are adapted to suspension growth in medium lacking animal-derived components, such as, e.g., medium lacking serum. Such adaptation procedures are described, e.g., in Scharfenberg, et al., Animal Cell Technology Developments towards the 21^st Century, E. C. Beuvery et al. (Eds.), Kluwer Academic Publishers, pp. 619-623, 1995 (BHK and CHO cells); Cruz, Biotechnol. Tech. 11 : 117-120, 1997 (insect cells); Keen, Cytotechnol. 17: 203-211, 1995 (myeloma cells); Berg et al., Biotechniques 14:972-978, 1993 (human kidney 293 cells). In a particularly preferred embodiment, the host cells are BHK 21 or CHO cells that have been engineered to express human Factor VIII and that have been adapted to grow in the absence of serum or animal-derived components.

Cell Culture Procedures

The methods of the invention are typically performed in a stirred culture vessel and a draw- fill process type is preferably employed. In this process the cells are grown after inoculation, and when a certain density is reached, about 70% of the culture is harvested, and the remaining culture is supplied with fresh cell culture medium to its original volume. This is typically repeated about 2-10 times.

Alternatively, a microcarrier process type can be employed. In the microcarrier-based process the cells have migrated into the internal structure of the carriers (macroporous carriers) or have attached themselves to the surface of the carriers (solid carriers), or both. In a microcarrier-based process the mammalian cells, the microcarriers and the cell culture medium are supplied to a culture vessel initially. In the following days additional cell culture medium may be fed if the culture volume was not brought to the final working volume of the vessel from the start. During the following period periodic harvest of product-containing culture supernatant and replacement with new medium liquid is performed, until the culture is finally terminated. When harvesting product-containing supernatant the agitation, e.g., stirring, of the culture is stopped and the cell-containing carriers are allowed to sediment following which part of the product-containing cell culture supernatant is removed. In order to improve the overall outcome of the procedure, a cooling step may preferably be applied before harvesting of the product-containing supernatant, see, e.g., WO 03/029442. In some embodiments the cell culture medium is cooled to a temperature between about 18°C and about 32°C before allowing the carriers to sediment, or between about 20⁰C and about 30⁰C, or between about 22°C and about 28°C.

Other applicable variants of the cell culture procedure are described in WO 02/29084 (Novo Nordisk A/S). Before reaching the production phase where regular harvesting of product-containing culture supernatant for further down-stream processing is performed, the cells are propagated according to any scheme or routine that may be suitable for the particular cell in question. The propagation phase may be a single step or a multiple step procedure. In a single step propagation procedure the cells are removed from storage and inoculated directly to the culture vessel (optionally containing microcarriers) where the production is going to take place. In a multiple step propagation procedure the cells are removed from storage and propagated through a number of culture vessels of gradually increasing size until reaching the final culture vessel (optionally containing microcarriers) where production is going to take place. During the propagation steps the cells are grown under conditions that are optimized for growth. Culture conditions, such as temperature, pH, dissolved oxygen tension, concentration of dissolved CO₂, and the like, are those known to be optimal for the particular cell and will be apparent to the skilled person or artisan within this field (see, e.g., Animal Cell Culture: A Practical Approach 2^nd Ed., Rickwood, D. and Hames, B. D., eds., Oxford University Press, New York (1992)).

In one approach, the cell culture process is operated in one culture vessel : The cells are inoculated directly into the culture vessel (optionally containing microcarriers) where the production is going to take place; the cells are propagated until a suitable cell density is reached and the production phase is initiated.

In another approach, the cell culture process is operated in at least two distinct culture vessels: One or more seed culture vessel(s) (first propagation step(s)) followed by the production culture vessel (last propagation step followed by production phase). In the first propagation step the cells expressing the desired polypeptide are inoculated into a seed culture vessel containing the cell culture medium and propagated until the cells reach a minimum cross-seeding density. Subsequently, the propagated seed culture is transferred to the production culture vessel containing the cell culture medium and (optionally) microcarriers. In case of a process using microcarriers the cells are cultured in this culture vessel under conditions in which the cells migrate onto the surface of the solid carriers or the exterior and interior surfaces of the macroporous carriers, and they continue to grow in this last propagation step until the carriers are fully colonized by the cells. During this last propagation step medium exchange is performed by allowing the microcarriers to settle to the bottom of the culture vessel, after which a predetermined percentage of the tank volume is removed and a corresponding percentage tank volume of fresh medium is added to the vessel. The microcarriers are then re-suspended in the medium and this process of medium removal and replacement are repeated at a predetermined interval, for example every 24 hours. The amount of replaced medium depends on the cell density and may typically be from 10-95%, preferably from 25% to 80%, of the tank volume. In case of a suspension process, e.g. a perfusion, batch or draw-fill process, the cells are grown freely suspended without being immobilised in carriers. In a suspension cell-perfusion process the cells are inoculated into a seed culture vessel containing culture medium lacking animal-derived components and propagated until the cells reach a minimum cross-seeding density. Subsequently, the propagated seed culture is transferred to a large-scale culture vessel containing culture medium lacking animal-derived components and propagated until at least a predetermined cell density is reached. In this phase the cells are grown in suspension to allow the cell number within the culture vessel to increase to a predetermined or critical value. The medium exchange is performed by continuously perfusing the culture vessel with fresh medium.

The amount of perfused medium depends on the cell density and may typically be from 10- 95%, preferably from 25% to 80%, of the tank volume per day (24 hours). When the cell density reaches the value suitable for initiation of production phase, 60-95% of the tank medium in the tank is typically changed every 24 hours, such as e.g. about 80%. An 80% medium exchange is also preferably used in the production phase.

In a simple batch process the cells are inoculated into a seed culture vessel containing culture medium lacking animal-derived components and propagated until the cells reach a minimum cross-seeding density. Subsequently, the propagated seed culture is transferred to a large-scale culture vessel containing culture medium lacking animal-derived components.

A batch process such as this can be extended by feeding a concentrated solution of nutrients to the tank. This extends the process time and ultimately leads to an increase in FVII production within the culture vessel. The time of harvest has to be determined as a balance between the longest possible operation of the tank and the risk of cell lysis.

A simple Draw-Fill process closely resembles a repeated batch fermentation. In batch fermentation the cells grow in the culture vessel and the medium is harvested at the end of the run. In a Draw-Fill process the culture vessel is harvested before any of the nutrients become exhausted. Instead of removing all of the contents from the vessel, only a proportion of the tank volume is removed (typically 80% of the tank volume). After the harvest, the same volume of fresh medium is added back to the vessel. The cells are then allowed to grow in the vessel once more and another 80% harvest is taken a set number of days later. In repeated batch processes the cells left in the vessel after a harvest may be used as the inoculum for the next batch.

A Draw-Fill process is operated in two phases. The first phase of the process is operated identically to a simple batch process. After the first harvest, the culture vessel is again operated as a simple batch process; however, the length of the batch is shorter than the first batch because of the higher initial cell density. Theses short 'repeated batch phases' are continued indefinitely.

A fed-batch Draw-Fill is a draw-fill fermentation with a concentrated feed similar to the type proposed in the fed-batch process. A concern with a simple draw-fill process is that the fresh medium added may not be sufficient to sustain the cells over repeated batch fermentations. The inclusion of a feed would remove this worry. A feed would also allow operating the culture vessel with long batch times in a draw-fill process.

The culture vessel may be operated within a broad range of cycle times and a broad range of draw-fill volumes. Ranges and preferred values can be seen from Table 1, below.

Table 1

It will be understood that in a process where the propagation phase is a multiple step procedure the propagation may take place in culture vessels of progressively increasing size until a sufficient number of cells is obtained for entering the final culture vessel. For example, one or more seed culture vessels of 5 L, 50 L, 100 L or 500 L may be used sequentially. A seed culture vessel typically has a capacity of between 5 L and 1000 L. Typically, cells are inoculated into a seed culture vessel at an initial density of about 0.2 to 0.4 x 10⁶ cells/mL and propagated until the culture reaches a cell density of about 1.0 x 10⁶ cells/ml_. Typically, a minimum cross-seeding density is between about 0.8 and about 1.5 x 10⁶ cells/ml_.

Some of the set-points that are suitable for the production of Factor VIII are not necessarily suitable for the initial growth of the cells, either in seed culture or on the microcarriers. For example, temperature, dissolved oxygen tension, and/or pH may be different for the two phases. The medium exchanges during propagation is done to keep the cells alive and growing, not to harvest culture supernatant for down-stream processing. Optionally, a drop in temperature set point of the cultivation may be employed when entering, and during, the production phase. Furthermore, when entering the production phase temperature, operating pH and medium exchange frequency are typically changed to values that are optimal for production.

Microcarriers

As used herein, microcarriers are particles which are small enough to allow them to be used in suspension cultures (with a stirring rate that does not cause significant shear damage to cells). They are solid, porous, or have a solid core with a porous coating on the surface. Microcarriers may, for example, without limitation, be cellulose- or dextran-based, and their surfaces (exterior and interior surface in case of porous carriers) may be positively charged. Further details can be found in WO 02/29083 and in "Microcarrier cell culture, principles and methods. Amersham Pharmacia Biotech. 18-1140-62. Edition AA".

Useful solid microcarriers include, without limitation, Cytodex 1™ and Cytodex 2™ (Amersham Pharmacia Biotech, Piscataway NJ). Solid carriers are particularly suitable for adhesion cells (anchorage-dependent cells). Useful macroporous carriers include, without limitation, Cytopore 1™ and Cytopore 2™ (Amersham Pharmacia Biotech, Piscataway NJ). Particularly preferred are Cytopore 1™ carriers, which have a mean particle diameter of 230 um, an average pore size of 30 um, and a positive charge density of 1.1 meq/g.

Large-Scale Culture Conditions

The invention is particularly relevant for large-scale production. By the term "large-scale production" is meant production involving a culture vessel of at least 100 L. In preferred embodiments, however, the scale is typically at least 250 L, such as at least 500 L, e.g. at least 1000 L or even 5000 L or more. The term "large-scale" may be used interchangeably with the terms "industrial-scale" and "production-scale".

The method for large-scale production of the polypeptide is typically conducted over a period of at least 120 hours, e.g. 1-26 weeks.

As used herein, a large-scale culture vessel has a capacity of at least about 100 L, preferably at least about 500 L, more preferably at least about 1000 L and most preferably at least about 5000 L. In case that the cell culture process is operated in at least two distinct culture vessels, such as one or more seed culture vessel(s) (first propagation step(s)) followed by the production culture vessel (last propagation step followed by production phase), then the process typically involves transferring about 50 L of the propagated seed culture (having about 1.0 x 10⁶ cells/mL) into a 500 L culture vessel containing 150 L of cell culture medium. The large-scale culture is maintained under appropriate conditions of, e.g., temperature, pH, dissolved oxygen tension (DOT), and agitation rate, and the volume is gradually increased by adding medium to the culture vessel. In case of a microcarrier process the culture vessel also comprises an amount of microcarriers corresponding to a final microcarrier concentration in the range of 1 to 10 g/L. After the transfer, the cells typically migrate onto the surface of the carriers or into the interior of the carriers within the first 24 hours.

Culture Vessel

Culture vessels applicable within the present invention may, e.g., be based on conventional stirred tank reactors (CSTR) where agitation is obtained by means of conventional impeller types or airlift reactors where agitation is obtained by means of introducing air from the bottom of the vessel. Among the further parameters that are typically controlled within specified limits are pH, dissolved oxygen tension (DOT), concentration of dissolced CO₂ and temperature. Dissolved oxygen tension may be maintained by, e.g., sparging with pure oxygen. The concentration of dissolved CO₂ may be maintained by sparging with air. The temperature-control medium is typically water, heated or cooled as necessary. The water may be passed through a jacket surrounding the vessel or through a piping coil immersed in the culture.

The term "culture vessel" may be used interchangeably with "tank", "reactor", "fermentor" and "bioreactor".

Step c) - Isolation of expressed polypeptide

In this step c), the polypeptide of interest (e.g. Factor VIII polypeptide) is to be isolated from the mammalian cells by suitable means. In a typical embodiment, the cells can be removed from the medium and the medium can be clarified by means of sequential filtration of harvest through 1.0 μm and 0.2 μm filters.

Once the medium containing the polypeptide of interest (e.g. Factor VIII polypeptide) has been isolated from the cells, it may be subjected to one or more processing steps to purify the desired protein, including, without limitation, affinity chromatography, hydrophobic interaction chromatography; ion-exchange chromatography; size exclusion chromatography; electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction and the like. See, generally, Scopes, Protein Purification, Springer-Verlag, New York, 1982; and Protein Purification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989.

Purification of polypeptides may in particular involve affinity chromatography, e.g., on an anti-polypeptide antibody column or an immobilized ligand or peptide specific for the polypeptide, and activation by proteolytic cleavage. For example, the Factor VIII polypeptide in the medium (cell culture supernatant) may then advantageously be up-concentrated cation-exchange chromatography where Factor VIII rich fractions are pooled. The Factor VIII polypeptide may then be purified by binding to an anti-Factor VIII antibody column {e.g. an F25 antibody column, see, e.g., WO 95/13301 and /or Nordfang et al. 1995 (Thromb. Haemostas. 54: 586-590)) followed by elution under conditions that preserve the Factor VIII polypeptide activity. Further impurities may be removed by buffer exchange by gel-filtration.

A list of embodiments of the present invention is given below:

Embodiment 1. A method for the production of a polypeptide of interest in the presence of a tissue inhibitor of metalloproteinase (TIMP), the method comprising a) transfecting a mammalian cell with (i) an expression vector comprising a nucleic acid molecule encoding the polypeptide of interest and expression control regions operatively linked to thereto, b) culturing the transfected cell under conditions for expression of the polypeptide of interest, said culturing involving a cell culture medium, and c) isolating the expressed polypeptide of interest by suitable means.

Embodiment 2. The method according to embodiment 1, wherein the method in step a) further comprises transfecting the same cell with (ii) an expression vector comprising a nucleic acid molecule encoding the tissue inhibitor of metalloproteinase (TIMP) and expression control regions operatively linked to thereto, and wherein the conditions in step b) also are applicable for expression of the tissue inhibitor of metalloproteinase (TIMP).

Embodiment 3. The method according to embodiment 1, wherein the tissue inhibitor of metalloproteinase (TIMP) is provided in step b).

Embodiment 4. The method according to embodiment 3, wherein the tissue inhibitor of metalloproteinase (TIMP) is expressed by another mammalian cell transfected with an expression vector comprising a nucleic acid molecule encoding the tissue inhibitor of metalloproteinase (TIMP) and expression control regions operatively linked to thereto. Embodiment 5. The method according to any one of the preceding embodiments, wherein the polypeptide of interest is a Factor VIII polypeptide.

Embodiment 6. The method according to any one of the preceding embodiments, wherein the tissue inhibitor of metalloproteinase (TIMP) is selected from the group consisting of TIMP-I, TIMP-2, TIMP-3 and TIMP-4.

Embodiment 7. The method according to embodiment 6, wherein the tissue inhibitor of metalloproteinase (TIMP) is TIMP-I.

Embodiment 8. The method according to any one of the preceding embodiments, wherein the tissue inhibitor of metalloproteinases (TIMP) is present in the cell culture medium in a concentration of 0.1-100 μg/mL.

The following examples are intended as non-limiting illustrations of the present invention.

EXAMPLES

Materials and Methods

CoA assay (Factor VIII activity assay) : In the presence of calcium and phospholipids, Factor X is activated to Factor Xa by Factor IXa. This generation is greatly stimulated by Factor VIII, which may be considered as a cofactor in this reaction. By using optimal amounts of Ca²⁺ and phospholipids and an excess of Factors IXa and X, the rate of activation of Factor X is solely dependent on the amount of Factor VIII. Factor Xa hydrolyses the chromogenic substrate S- 2765 thus liberating the chromophoric group, pNA. The colour is then read photometrically at 405 nm. The generated Factor Xa and thus the intensity of colour is proportional to the

Factor VIII activity in the sample. Hydrolysis of S-2765 by thrombin formed is prevented by the addition of the synthetic thrombin inhibitor, 1-2581, together with the substrate (Chromogenix Coatest SP Factor VIII, diaPharma)

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

Example 1: Effect of TIMP-I on FVIII productivity

CHO cells expressing recombinant human B-domain deleted FVIII (BDD-FVIII) were generated by transfecting cells with a plasmid containing BDD-FVIII cDNA and the dihydrofolate reductase selection marker using procedures known to persons skilled in the art. Clones stably producing BDD-FVIII were isolated and adapted to serum-free medium. One clone, labeled 1C5, was selected for further studies.

A stock culture of TIMP-I protein (Lab Vision Corporation, CA, USA) was generated by dissolving 10 microgram TIMP-I in 1 ml_ buffer (50 mM Tris; pH 7.5, 10 mM CaCI2, 150 mM NaCI, 0.01% Tween-80).

1C5 cells were cultured in commercial serum-free medium (Hyclone, UT, USA) in 5 ml_ shaker cultures with a starting density of 2.5 million cells/mL BDD-FVIII productivity was compared between cultures with or without addition of TIMP-I protein (Table 1). Results demonstrated that the relative production was increased by addition of TIMP-I protein.

Table I

Example 2: Generation of a TIMP-I expression plasmid.

According to GeneCards the mRNA for TIMP-I (Genbank accession # NM_003254) can be detected in all human tissues. As a consequence of this the A5 Human liver cDNA library from ProQuest (pPC86 cDNA) was used as template for cloning TIMP-I. PCR was performed using the Phusion High-Fidelity PCR (New England Biolabs, cat # F-553) according to the manufactures recommendations using the primers listed in Table II below. PCR was performed for 35 cycles of 1' 94°C, 2' 55°C, 2' 72°C, yielding a single distinct band of the desired size. Name Length Sequence

Table II

The PCR products were gel purified using conventional techniques and cloned directly into the pcDNA 3.2 /V5 Gateway Directional TOPO Expression kit (Invitrogen) utilizing the CACC sequence in the primer which precedes the coding sequence and ensures directional control of the insert. Cloning and transformation into TOPlO cells were performed according to manufactures recommendation and positive clones were verified by Dyedeoxy sequencing at MWG Biotech AG (Bersberg, Germany). The resulting plasmid was termed TIMP-I in pcDNA3.2V5GW-CAT.

In order to validate the functionality of the construct it was tested in a transient mammalian expression system. Briefly, 85 μl of plasmid DNA (15 μg) was diluted in 0.5 ml_ of Opti-MEM I Reduced Serum Medium and 20 μl_ of 293fectin was diluted in 0.5 ml_ of Opti-MEM I Reduced Serum Media. After 5 minutes incubation at room temperature the diluted DNA and 293fectin were mixed and incubated for 30 minutes. The DNA-293fectin complexes were added to a 125 ml_ Erlenmeyer shaker flask containing 14 ml_ of 1.1 ^■ 10⁶ cells/mL cell suspension grown in Freestyle 293 Expression medium (serum-free media containing Glutamax-I). The expression continued for 4 days at 8% CO₂, 37°C and 120 rpm. A media sample was drawn and analyzed for expression by SDS-PAGE and western blot analysis using the TIMPl antibody [2A5] from Abeam (www.abcam.com) and a One step western blot kit (Genscript cat # L00205) according to the manufactures recommendations. The analysis revealed a single band with a size corresponding to TIMP-I.

Example 3: Co-expression of TIMP-I and BDD-FVIII

Since there is a clear effect on BDD-FVIII levels of addition of exogenous TIMP-I to the expression medium, the effect of TIMP-I co-expression on BDD-FVIII productivity in serum- free cultures can be assessed in two ways: 1) by generating two stable cell lines which habour either the TIMP-I or the BDD-FVIII cDNA using a single or two separate selection markers. Briefly, CHO cell line expressing BDD-FVIII or TIMP-I can be generated by transfecting cells with a plasmid containing the corresponding cDNA and the dihydrofolate reductase selection marker using procedures known to persons skilled in the art. Clones stably producing BDD-FVIII or TIMP-I are isolated and adapted to serum-free medium. The two cell lines may subsequently be co-cultured to produce predictable levels of BDD-FVIII and TIMP-I in the expression medium depending on the mixing ratio. 2) A single stable cell line transfected with both TIMP-I and BDD-FVIII plasmid can be generated by using two separate selection markers, e.g. metotrexate and neomycine resistance. The first step in this process would be to generate a stable cell line for BDD-FVIII as described in example 1 and subsequently transfect this cell line with TIMP-I cDNA and select for the second resistance marker. Clones stably producing both BDD-FVIII and TIMP-I are isolated and adapted to serum-free medium. Positive clones are cultured in commercial serum-free medium (Hyclone, UT, USA) in 5 ml_ shaker cultures with a starting density of 2.5 million cells/mL BDD-FVIII productivity is compared as a function of TIMP-I protein levels and FVIII activity to identify an optimal producer cell line.

Claims

1. A method for the production of a Factor VIII polypeptide, the method comprising: a) culturing a mammalian cell transfected with an expression vector comprising a nucleic acid molecule encoding said Factor VIII polypeptide and expression control regions operatively linked to thereto, in a cell culture medium under conditions suitable for expression of said polypeptide; and b) isolating the expressed Factor VIII polypeptide; wherein the cell culture medium in step a) comprises a tissue inhibitor of metalloproteinase (TIMP).

2. The method according to claim 1, wherein the mammalian cell cultured in step a) is further transfected with an expression vector comprising a nucleic acid molecule encoding said tissue inhibitor of metalloproteinase (TIMP) and expression control regions operatively linked to thereto, and wherein the conditions in step a) are also suitable for expression of the tissue inhibitor of metalloproteinase (TIMP).

3. The method of claim 1, wherein step a) further comprises culturing a second mammalian cell transfected with an expression vector comprising a nucleic acid molecule encoding said tissue inhibitor of metalloproteinase (TIMP) and expression control regions operatively linked to thereto, and wherein the conditions in step a) are also suitable for expression of the tissue inhibitor of metalloproteinase (TIMP).

5. The method according to claim 1, wherein the tissue inhibitor of metalloproteinase (TIMP) is added to the cell culture medium in step a).

6. The method according to claim 5, wherein said tissue inhibitor of metalloproteinase (TIMP) is purified recombinantly produced tissue inhibitor of metalloproteinase (TIMP).

7. The method according to any one of the preceding claims, wherein the Factor VIII polypeptide is selected from the group of: wild-type human Factor VIII, hybrid human/porcine Factor VIII, and B-domain deleted human Factor VIII.

8. The method according to any one of the preceding claims, wherein the tissue inhibitor of metalloproteinase (TIMP) is selected from the group consisting of TIMP-I, TIMP-2, TIMP-3 and TIMP-4.

9. The method according to claim 8, wherein the tissue inhibitor of metalloproteinase (TIMP) is TIMP-I.

10. The method according to any one of the preceding embodiments, wherein the tissue inhibitor of metalloproteinases (TIMP) is present in the cell culture medium in a concentration of 0.1-100 μg/mL.