WO2018210771A1

WO2018210771A1 - Method for the production of a recombinant target protein

Info

Publication number: WO2018210771A1
Application number: PCT/EP2018/062418
Authority: WO
Inventors: Stefan Winge; Jade Safta; Peter Aizawa; Tilo Schwientek
Original assignee: Octapharma Ag
Priority date: 2017-05-17
Filing date: 2018-05-14
Publication date: 2018-11-22
Also published as: EP3625329A1

Abstract

The present invention relates to a method for the production of a recombinant target protein, in particular to a method comprising culturing a human host cell in a fortified cell culture medium. Embodiments of the invention were particularly developed for increasing the cell-specific productivity of host cells transfected with a nucleic acid sequence encoding human blood proteins such as coagulation Factor VIII (FVIII) and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.

Description

Method for the production of a recombinant target protein

FIELD OF THE INVENTION The present invention relates to a method for the production of a recombinant target protein, in particular to a method comprising culturing a human host cell in a fortified cell culture medium. Embodiments of the invention have been particularly developed for increasing the cell-specific productivity of host cells transfected with a nucleic acid sequence encoding human blood proteins such as coagulation Factor VIII (FVIII) and will be described hereinafter with reference to this application. However, it will be appreciated that the invention is not limited to this particular field of use.

BACKGROUND OF THE INVENTION Any discussion of the background art throughout the specification should in no way be considered as an admission that such art is widely known or forms part of common general knowledge in the field.

Many biotechnology-derived products, whether commercially available or in development, are protein therapeutics and the recombinant production of such therapeutics is playing a key role in ensuring that the demand for safe, high-quality protein therapeutics can consistently be met.

To this end, optimisation of the host cells expressing the protein therapeutic has led to a suite of different, specifically-tailored cell lines for protein expression - often specifically tailored for the use in particular production systems, such as batch or perfusion culture, for adherent or suspension culture, for the production of particular types of proteins such as antibodies, cytokines, antigens for vaccine production, etc.

In addition, a large variety of cell culture media optimised for specific cell types or culture conditions have been developed. Generally, cell culture media comprise components of many different categories including amino acids, vitamins, fatty acids, salts, and further components.

Historically, serum has been a crucial component of cell culture media - namely, as a source of complex biological molecules beneficial to the cultured cells such as hormones, growth factors, attachment factors as well as numerous low molecular weight nutrients. Foetal bovine serum (FBS) has been routinely added to cell culture medium as it supports the successful growth of a variety of cell types and has been instrumental in the development of permanent cell lines.

Initially, serum-free media were developed to characterise and study the biological molecules present in serum itself as well as to study the effects of regulatory molecules in a defined medium context. However, the issues of reliability of supply of serum, in particular of FBS, the variability in content and, therefore, ultimately in its variability in performance and the risk for biological contaminants such as viruses or prions has since led to serious safety concerns with respect to the use of bovine serum in protein therapeutic production systems. As such, serum- and/or animal protein-free media compositions, i.e. compositions avoiding the use of serum and other supplements of animal origin, received a lot of attention and have been developed .

Notwithstanding the above-outlined achievements in developing serum-based and serum- or proteins-free cell culture media, there is a continuous need for the further development and improvement of existing, as well as for the development of new, cell culture-based methods for the production of recombinant proteins.

In particular, there is a need to increase the efficiency and/or productivity of a given cell culture-based recombinant protein production system such as to produce the desired protein therapeutic at high efficiency as well as quality, for example by enhancing growth, survival and/or productivity of the cultured cells. Media supplements known in the field to enhance growth and/or survival of the cultured cells include hormones and growth factors. However, the use of protein supplements is often costly and, depending on the source of the supplements, may still bear the risk of contaminating the culture system with animal-derived components.

Other approaches included the formulation of media compositions in which the amounts of trace metals ions such as manganese, molybdate, chromium, silicon, lithium, copper and/or zinc ions, were specifically tailored to support and enhance growth and/or survival of the cultured cells. WO 2008/008360 discloses a method for the production of a glycoprotein in a cell culture-based system wherein cells are cultured under serum-free conditions in a defined medium, wherein the defined medium is additionally supplemented with manganese, copper and ferrous ions. In particular, it is shown that a final manganese concentration between 10 and 600 nM in the cell culture medium leads to a beneficial effect with respect to the glycosylation pattern of the recombinant protein being produced, i.e. that the glycosylation pattern of the recombinantly produced protein more closely resembles the glycosylation pattern of the protein when produced by its natural host. However, the same publication indicates that the supplementation of the medium with copper ions can also lead to undesirable effects.

Similarly, the publication by Crowell et al. (Biotechnology and Bioengineering, Vol. 96, No.3, 2007) describes the addition of trace metal ions to a serum-free cell culture-based production system for recombinant human erythropoietin. It describes that the addition of manganese ions to the medium (in which the protein-producing Chinese hamster ovary (CHO) are cultured) improves the galactosylation of the recombinantly produced protein. However, Crowell et al. also describe that the effect could only be achieved in the particular system when the cell culture medium was supplemented with manganese ions late in the culturing process. In addition, the authors observed that the addition of trace metal ions has complex effects on the cultured cells (such as reduced protein yields) and discuss the unintentional consequences of selective supplementation of culture medium with trace metal ions.

The above-mentioned caveats of supplementing serum- or protein-free cell culture medium with free trace metal ions are particularly pronounced because such media compositions do not contain serum components counteracting or buffering their negative effects. For example, in a serum-based cell culture medium, albumin (one of the major protein components of serum) can serve as chelator of free copper ions and prevent them from active participation in undesirable redox cycling. In particular, and summarised at http://www.sigmaaldrich.com/life-science/cell-culture/learning- center/media-expert/copper.html, copper is essential for the growth and maintenance of healthy cells in vitro and in vivo. However, copper is a transition metal and exists, in vitro, in an equilibrium of the reduced (cuprous) Cu(l) and oxidised (cupric) Cu(ll) form. Hence, unless it is properly chelated, it can be toxic in its free form.

In particular, Cu(ll) can promote the oxidation and precipitation of cysteine and, as a result, can lead to the loss of cysteine and cystine from media compositions. Lack of cysteine and cystine in cell culture prevents the continued synthesis of cysteine- containing proteins and, importantly, of glutathione (synthesised from L-cysteine), which is an important cellular antioxidant. Glutathione can complex with Cu(l) and thereby inhibits Cu(l)'s participation in the formation of hydroxyl-free radicals. In vitro, Cu(l) spontaneously forms complexes with reduced cysteine, glutathione and presumably organic sulfhydryls, while Cu(ll) forms complexes with amino acids, for example with histidine.

As such, when considering adding copper ions to cell culture media, copper- mediated redox reactions should be considered. In particular, those that result in the formation of highly reactive and destructive hydroxyl-free radicals should be avoided. This is because, under physiological conditions, superoxide radicals or ascorbate anions can reduce cupric copper to cuprous copper, which catalyses the formation of hydroxyl-free radicals. However, in the absence of albumin, such as in serum- or protein-free medium, the copper-induced formation of hydroxyl-free radicals constitutes a real concern, as the radicals can destabilise cell membranes by attacking the membranes' polyunsaturated fatty acids leading to radical-initiated lipid peroxidation. Of course, compromised membrane stability can have detrimental effects on the cultured cells, including significantly increased cell stress and cell death. Several different approaches to managing copper delivery were developed in order to manage copper-induced oxidative stress in the culture. For example, copper chelation by way of proprietary small molecules such as synthetic polyamine tetraethylenepentamine (TEPA) was considered. Accordingly, there is a need for developing new and/or for improving existing protein production methods, including new or improved methods of copper ion management in serum- and protein-free cell culture-based recombinant protein production systems. In particular, there is a need to increase the efficiency of a given recombinant protein to be produced by such systems - both to allow lowering the overall production costs and to ensure that the demand for such therapeutics can be readily met.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

SUMMARY OF THE INVENTION

The present invention is inter alia based on the surprising finding that the efficiency of a serum- and/or protein-free mammalian cell culture-based recombinant protein production system can be improved by adding additional copper ions complexed by peptides, or fragments thereof, to an already copper containing basal medium. In particular, the beneficial addition of additional copper ions, i.e. the further increase of copper ions in the cell culture medium, can be effectively managed by adding an additional copper ion supplement, in which the copper ions are complexed by peptides, or fragments thereof, of a molecular weight of 10 kilo Dalton (kDa) or less, preferably 5 kDa or less, without invoking negative effects routinely seen when free copper ions are added to basal, chemically defined, copper containing, mammalian cell culture media.

Instead of negative effects, it was surprisingly found that the addition of copper ions complexed by peptides leads to an unexpected increase in productivity of mammalian host cells with respect to a recombinant target protein to be expressed by the cells. In particular, the cell specific productivity for the target protein is unexpectedly increased.

Accordingly, in a first aspect the present invention relates to a method for the production of a recombinant target protein, the method comprising the steps of: (a) culturing a mammalian host cell transfected with a nucleic acid encoding said recombinant target protein in a fortified cell culture medium, wherein said fortified cell culture medium is a basal, chemically defined, copper containing, cell culture medium, which: does not contain albumin, is free of non-recombinant serum proteins, and is sufficient for the recombinant production of the target protein by said host cell, to which an additional copper ion supplement is added, wherein said supplement comprises copper ions complexed by peptides, or fragments thereof, of a molecular weight of 10 kDa or less; and

(b) harvesting the recombinant target protein.

When cultured in the fortified cell culture medium, the host cell's cell-specific productivity for the recombinant target protein is increased compared to the host cell's cell-specific productivity for the recombinant target protein when cultured in the basal cell culture medium alone.

In some embodiments of the present invention, the recombinant target protein is selected from the group consisting of recombinant copper-binding proteins, recombinant Factor IX (rFIX) and recombinant Granulocyte-Colony Stimulating Factor (rG-CSF). For example, the recombinant target protein is recombinant Factor VIII (FVIII). FIGURES

Embodiments of the invention will below be described, by way of example only, with reference to the accompanying drawings in which: Figure 1 shows a size-exclusion chromatogram of the copper ion supplement A (Batch 2) at a concentration of 100 mg/mL.

Figure 2 shows a size-exclusion chromatogram of the copper ion supplement A (Batch 3) at a concentration of 100 mg/mL.

Figure 3 shows a size-exclusion chromatogram of the copper ion supplement A (Batch 4) at a concentration of 100 mg/mL.

Figure 4 shows a size-exclusion chromatogram of Reference sample No. 14 (G-CSF; 20 kDa) at a concentration of 0.8 mg/mL. Figure 5 shows a size-exclusion chromatogram of Reference sample No. 13 (insulin; 5.8 kDa) at a concentration of 0.1 to 5 mg/mL. Figure 6 shows a size-exclusion chromatogram of Reference sample No. 12 (cyanocobalamin (B12); 1.36 kDa) at a concentration of 5 mg/mL.

Figure 7 shows a size-exclusion chromatogram of Reference sample No. 1 1 (ammonium molybdate; 1.236 kDa) at a concentration of 0.1 M.

Figure 8 shows a size-exclusion chromatogram of Reference sample No. 10 (folic acid; 0.441 kDa) at a concentration of 0.02 mg/mL.

Figure 9 shows a size-exclusion chromatogram of Reference sample No. 9 (chromium (III) chloride; 0.266 kDa) at a concentration of 50mM.

Figure 10 shows a size-exclusion chromatogram of Reference sample No. 8 (copper (II) sulfate; 0.25 kDa) at a concentration of 0.1 M. Figure 1 1 shows a size-exclusion chromatogram of Reference sample No. 7 (manganese (II) chloride; 0.198 kDa) at a concentration of 0.1 M.

Figure 12 shows a size-exclusion chromatogram of Reference sample No. 6 (L-histidine and copper (II) chloride; 0.155 kDa and 0.170 kDa) at a concentration of 0.1 M.

Figure 13 shows a size-exclusion chromatogram of Reference sample No. 5 (copper (II) chloride; 0.170 kDa) at a concentration of 0.1 M. Figure 14 shows a size-exclusion chromatogram of Reference sample No. 4 (L-histidine; 0.155 kDa) at a concentration of 0.1 M.

Figure 15 shows a size-exclusion chromatogram of Reference sample No. 3 (glycine; 0.075 kDa) at a concentration of 0.1 M. Figure 16 shows a size-exclusion chromatogram of Reference sample No. 2 (sodium chloride; 0.058 kDa) at a concentration of 0.1 M.

Figure 17 shows a size-exclusion chromatogram of Reference sample No. 1 (lithium chloride; 0.04 to kDa) at a concentration of 0.1 M.

Figure 18 shows the size-exclusion chromatogram of Figure 1 , in which the copper elution profile is indicated. Figure 19 shows a 3-dimensional cylinder chart of the average peptide size distribution in the copper ion supplement A comprising peptides of yeast origin.

Figure 20 shows a 3-dimensional cylinder chart of the average peptide size distribution in the copper ion supplement B comprising peptides of plant origin.

Figure 21 shows a bar graph of the respective copper concentrations in various batches of fortified medium.

DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear and consistent understanding of the specification and claims, and the scope to be given such terms, the following definitions are provided.

Definitions

"recombinant target protein"

In the context of this specification a recombinant target protein can be any protein of interest produced in vitro by way of culturing a suitable host cell, which has been genetically engineered to express and thereby produce the target protein. Typically, the host cell has been genetically engineered to express the target protein by introducing an expression construct for the target protein, i.e. a nucleic acid sequence encoding the target protein. For example, the nucleic acid sequence encoding the target protein can be introduced into the host cell by way of transfection such as to allow for the recombinant expression and production of the target protein when the host cell is cultured under conditions pernnissive for the production of the target protein.

"host cell"

The term "host cell" refers to genetically engineered cells into which an expression construct for the target protein, i.e. a nucleic acid sequence encoding the target protein, has been introduced, such as to allow for the recombinant expression and production of the target protein when the host cell is cultured under conditions permissive for the production of the target protein. The introduction of the nucleic acid sequence encoding the target protein can be an integration of the sequence into the genome of the host cell. Suitable host cells in the context of the present invention include mammalian cells, in particular human cells, adapted for growth in culture medium, which is free of albumin and non-recombinant serum proteins. In particular, such suitable host cells are specifically adapted to grow and produce a recombinant protein under the above-mentioned medium conditions in suspension culture.

"encodes" or "encoding"

In the context of the present invention, the terms "encodes" or "encoding" with respect to a nucleic acid sequence, mean that the sequence of the nucleic acid can be transcribed (in case of DNA) or translated (in case of mRNA) into a polypeptide such as the target protein in vitro or in i//Ve> when the sequence is placed under the control of one or more appropriate regulatory sequences and is exposed to the appropriate enzymatic context allowing for the transcription or translation of a nucleic acid sequence.

"cell culture medium"

In the context of the present invention, the term "cell culture medium" refers to liquid growth medium for mammalian cells. As such, the medium supplies the essential nutrients required to maintain and grow mammalian cells and to allow for recombinant protein production in accordance with the present invention. The essential nutrients comprised with in a cell culture medium include, but are not limited to, amino acids, carbohydrates, vitamins, minerals. In addition to such nutrients, the cell culture medium may further be supplemented with additional components to enhance, improve or boost certain cellular functions such as, for example, recombinant production of specific target proteins. Cell culture supplements known in the art include, but are not limited to, growth factors, hormones, and additional trace elements. Liquid mammalian cell culture media routinely also comprise non-ionic surface-active agents.

"basal, chemically defined, copper containing cell culture medium"

In the context of the present invention, the term "basal, chemically defined, copper containing cell culture medium" refers to a cell culture medium sufficient to ensure the growth of, as well as the recombinant production of the target protein by, the mammalian host cell. With the exception that the nasal, chemically defined, copper containing cell culture medium cannot comprise albumin or non-recombinant serum proteins but must contain copper, preferably at a concentration of between 50 and 800 nM, the composition of the basal medium is not critical to the present invention. As such, it may be based on a well-known, commercially available mammalian cell culture medium, e.g. on one of Dulbecco's Modified Eagle Medium, Eagle's Minimal Essential Medium, RPMI-1640 Medium and Ham's Medium F-12.

However, the definition of a chemically defined cell culture medium is that the content and amount of all components is known. Thus, defined/chemically defined media do not contain complex components such as serum, serum fractions or other complex components, but can contain single protein/polypeptide components like albumin, transferrin or insulin, preferably from a recombinant source. Thus, a defined/chemically defined medium can be either serum-free or protein-free or both. A chemically defined medium to which a small amount (generally less than 1 %) of a complex component is added is generally referred to as a "semi-defined medium".

'fortified cell culture medium"

In the context of the present invention, the term "fortified cell culture medium" refers to the basal, chemically defined, copper containing cell culture medium after the copper ion supplement has been added. As such, this addition "fortifies" the basal medium in the sense of the plain-English meaning of the word, namely the strengthening or improvement by addition or intensification with another ingredient. In the context of the present invention, the addition of the copper ion supplement improves, i.e. fortifies, the cell culture medium for the host cell such that cell-specific productivity of the host cell for the target protein is increased.

"copper ion supplement"

In the context of the present invention, the term "copper ion supplement" refers to a supplement to be added to a cell culture medium, preferably to a basal, chemically defined, copper containing cell culture medium to fortify said medium with additional copper ions such that the cell-specific productivity of a mammalian host cells cultured in the medium is increased. In the context of the present invention the copper ion supplement comprises between 20 and 100 nM of copper, such as between 20 and 90 nM of copper, such as between 20 and 86 nM of copper, such as between 25 and 86 nM of copper, such as between 30 and 70 nM of copper, such as between 40 and 60 nM of copper, such as 50 nM of copper. As such, the skilled person will understand that the copper ion supplement comprises between 1.27 and 6.35 parts per million (ppm) copper based on copper's molecular mass of 63.546 dalton (g/mol), such as between 1.27 and 5.72 ppm copper, such as between 1.27 and 5.47 ppm copper, such as between 1.59 and 5.47 ppm copper, such as between 1.91 and 4.45 ppm copper, such as between 2.54 and 3.81 ppm copper, such as 3.18 ppm copper. In addition, the copper ion supplement also comprises non-animal or non-human peptides and fragments thereof of a molecular weight of 10 kDa or less, preferably of a molecular weight of 5 kDa or less. In particular, more than 80% of the peptides, or fragments thereof, of the copper ion supplement have a molecular weight of less than 2 kDa; more than 70% of said peptides, or fragments thereof, have a molecular weight of less than 0.5 kDa, and/or more than 50% of said peptides, or fragments thereof, have a molecular weight of less than 0.25 kDa.

"complexed by peptides, or fragments thereof

In the context of the present invention, the term "complexed by peptides, or fragments thereof refers to copper ions being in a physical interaction with, i.e. being complexed by, the peptides of the copper ion supplement such as to form complexes eluting from a high-resolution size exclusion chromatography column in fractions equivalent from about 155 Da to at least 20 kDa. To mimic physiological conditions permissive for the production of the recombinant target protein in cell culture also during size exclusion chromatography, a physiological pH and a physiological sodium chloride (NaCI) concentration are maintained. As such, in the context of the present invention, copper ions are typically complexed by peptides, or fragments thereof, at a pH of about 7.2 and a NaCI concentration of about 0.15 M. "physiological conditions"

In the context of the present invention, the term "physiological condition" refers to the conditions experienced by cells in situ, but also expressly refers to the conditions, in particular to the pH and salt concentrations, considered optimal for the culture of certain cells and cell types, for example a mammalian cells, in vitro. As such, when referring to physiological conditions in the context of in vitro cell culturing systems, the conditions under which the cells are cultured mimic the cells' in situ environment, as such they mimic, for example, the normal pH and salt concentrations found in human blood plasma. While the salt concentration is largely determined by the sodium chloride concentration, other salts can also be present. Typically, under "physiological conditions" the salt concentration, predominantly the sodium chloride concentration, ranges from 0.05 to 0.20 M, such as from 0.10- 0.15 M. Most-preferably the salt concentration is about 0.15 M. Regarding the pH under "physiological conditions", the pH typically ranges from 6.5 to 8.0, such as from 6.8 to 7.6. Most-preferably the pH is between about 7.0 and 7.4.

"cell-specific productivity"

In the context of the present specification, the term "cell-specific productivity" expresses a measure of the amount of a recombinant target protein produced per host cell. In the case of FVIII- or FIX-expressing host cells, FVIII or FIX activity as a direct measure of FVIII or FIX production can be determined for a fixed number of cells and the cell-specific productivity can be calculated accordingly. For example, FVIII cell-specific productivity may be expressed as FVIII:C activity in IU per 10⁶ cells or even per single cell. Similarly, FIX activity can be determined for a fixed number of cells and the cell-specific productivity may be expressed as FIX:C activity in IU per 10⁶ cells or even per single cell. Alternatively, the de facto amount of a recombinant target protein produced by a fixed number of host cells may be determined by any suitable method known to the person skilled in the art and the cell-specific productivity for that protein may therefore also be expressed as, for example, the mask of protein determined per number of cells, such as in milligrams per 10⁶ cells or even per single cell. "copper-binding proteins"

In the context of the present invention, the term "copper-binding proteins" refers to proteins able to bind, chelate, complex copper ions, namely cupric copper ions, such as to prevent them from being reduced to cuprous copper ions. Copper- binding proteins in accordance with the present invention include, but are not limited to, superoxide dismutase, lysine oxidase, tyrosinase, ceruloplasmin, albumin as well as Factor V (FV) and Factor VIII (FVIII). "derivative"

The term "derivative" in accordance with its plain English meaning in the context of the present specification refers to something that has been derived, i.e. to a substance or compound which has been obtained from or is based on another substance or compound. As such, protein derivatives in the context of the present invention include modified proteins and protein fragments derived from an initial protein. In particular the term includes proteins and protein fragments that have been modified to have an extended half-life. Modifications for half-life prolongation include, but are not limited to, fusion proteins, proteins modified by mutagenesis and proteins linked to a conjugate by covalent or non-covalent binding. Protein derivatives generated by mutagenesis are also referred to as "muteins".

"express", "expressing" or "expression"

In the context of the present invention, the terms "express", "expressing" or "expression" refer to the transcription and translation of a nucleic acid sequence encoding a protein.

"comprise", "comprising", and the like

In addition to the above definitions, and unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

"one embodiment", "some embodiments" or "an embodiment" Further, reference throughout this specification to "one embodiment", "some embodiments" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment", "in some embodiments" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

Methods of the invention

Described herein are methods for the production of a recombinant target protein, wherein the methods of the invention comprise the steps of:

(a) culturing a mammalian host cell transfected with a nucleic acid encoding said recombinant target protein in a fortified cell culture medium, wherein said fortified cell culture medium is a basal, chemically defined, copper containing, cell culture medium, which: does not contain albumin, is free of non-recombinant serum proteins, and is sufficient for the recombinant production of the target protein by said host cell, to which an additional copper ion supplement is added, wherein said supplement comprises copper ions complexed by peptides, or fragments thereof, of a molecular weight of 10 kDa or less; and

(b) harvesting the recombinant target protein. Preferably, the copper ion supplement comprises copper ions complexed by peptides, or fragments thereof, of a molecular weight of 5 kDa or less.

In some embodiments of the present invention, the recombinant target protein is selected from the group consisting of recombinant copper-binding proteins, recombinant Factor IX (rFIX) and recombinant Granulocyte-Colony Stimulating Factor (rG-CSF). For example, the recombinant target protein is recombinant Factor VIII (FVIII). Typically, the basal, chemically defined, copper containing, cell culture medium free of non-recombinant serum proteins comprises between 50 and 800 nM of copper ions, i.e. an amount of copper ions sufficient to promote the production of the recombinant target protein by the host cells. In some embodiments the mammalian host cell is a human host cell. For example, the basal, chemically defined, copper containing, cell culture medium free of non-recombinant serum proteins comprises more than 200 nM of copper, such as 240 to 250 nM of copper. In a preferred embodiment the basal, chemically defined, copper containing, cell culture medium free of non-recombinant serum proteins has been prepared by adding between 240 and 250 nM copper chloride dihydrate pentahydrate (CuCl2°5H2O) or anhydrous copper sulphate (CuSO₄). Yet, the mass of the additional copper ion supplement is less than 1 % of the total mass of the basal, chemically defined, copper containing, cell culture medium, in particular less than 0.5%, optionally less than 0.4%, optionally less than 0.3% or, optionally, is 0.2%. Preferably the fortified medium comprises between 250 and 400 nM, preferably between about 250 and 350 nM such as about 300 nM copper. In some preferred embodiments, the fortified medium comprises 295 nM of copper.

In some embodiments of the present invention, the cell-specific productivity of the host cell for the recombinant target protein is increased to more than 1.2 μΙΙΙ/cell. For example, the cell-specific productivity of the host cell is increased to 1.21 μΐυ/cell or more, such as to 1.32 μΙΙΙ/cell, such as to 1.55 μΙΙΙ/cell, such as to 2.56 μΐυ/cell, such as to 2.88 μΙΙΙ/cell, such as to 2.95 μΙΙΙ/cell, such as to 3.17 μΐυ/cell, such as to 3.38 μΙΙΙ/cell, such as to 3.5 μΙΙΙ/cell, such as to 4.14 μΙΙΙ/cell, such as to 4.38 μΙΙΙ/cell, such as to 5.32 μΙΙΙ/cell, such as to 5.35 μΙΙΙ/cell. Generally, the cell-specific productivity of the host cell for the recombinant target protein is increased by at least 1.3-fold, such as by at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1 -fold at least 2.2-fold at least 2.3-fold at least 2.4-fold or at least 2.5-fold. For example, at or after 66 hours of culture in the fortified cell culture medium, the cell-specific productivity is increased by at least 1.4-fold compared to the cell- specific productivity of the host cell for the recombinant target protein when cultured for 66 hours in the basal cell culture medium alone. The cell-specific productivity of the host cell for the recombinant target protein is: at least 0.2 μΙΙΙ/cell/day, such as 0.22 μΙΙΙ/cell/day or 0.24 μΙΙΙ/cell/day or 0.282 μΐυ/cell/day; at least 0.3 μΙΙΙ/cell/day such as 0.32 μΙΙΙ/cell/day or 0.37 μΐυ/cell/day or 0.365 μΙΙΙ/cell/day; or at least 0.4 μΙΙΙ/cell/day such as 0.41 μΐυ/cell/day.

In the additional copper ion supplement: more than 80% of the peptides, or fragments thereof, have a molecular weight of equal to or less than 2 kDa; more than 70% of the peptides, or fragments thereof, have a molecular weight of equal to or less than 0.5 kDa; and/or more than 50% of the peptides, or fragments thereof, have a molecular weight of equal to or less than 0.25 kDa. Typically, the copper ions in the supplement are complexed by the peptides, or fragments thereof, such as to form complexes eluting from a high-resolution size exclusion chromatography column in fractions equivalent to at least about 155 Da to at least 20 kDa. For example, at least 43% of the copper ions in the supplement are complexed by peptides, or fragments thereof, of a molecular weight of 10 kDa or less such as to form complexes eluting from a high-resolution size exclusion chromatography column in fractions equivalent to at least about 1.36 kDa to at least 20 kDa.

In some preferred embodiments the mammalian host cell is: a baby hamster kidney (BHK) cell; a Chinese hamster ovary (CHO) cell, such as a CHO-S Freestyle cell (Thermo Fisher Scientific R80007); or a human cell, in particular a human embryonic kidney (HEK) cell or a derivative thereof, such as HEK293 cells (ATCC CRL-1573; DSMZ ACC 305; ECACC ref.: 85120602), HEK293T cells (DSMZ ACC 2494; ECACC: tsa201 , ref. 96121229), or FreeStyle 293 cells (HEK293F cells; Thermo Fisher Scientific R79007). In one or more preferred embodiments, the mammalian host cell is specifically adapted to growth and recombinant protein production under albumin-free and serum-free suspension cell culture conditions. In particular, the mammalian host cell is a Freestyle CHO-S cell (Thermo FisherScientific R80007) or a HEK293F cell.

In some embodiments of the present invention, the mammalian host cell is stably transfected with a nucleic acid sequence encoding the target protein. In particular, the cell is derived from a single, stably transfected clone. Most preferably, the single, stably transfected clone is a stably transfected clone of a HEK293F cell, which has the nucleic acid sequence encoding the target protein integrated into its genome. In particular, the nucleic acid sequence integrated into the host cell's genome encodes FVIII or a B-domain deleted FVIII.

In particular, the above-described methods include the culturing of the host cell in a basal, chemically defined, copper containing cell culture medium to which copper ion supplement is added. However, because the particular copper ion supplement used in the methods of the present invention provides the additional copper ions to the cells not as free copper ions but as copper ions complexed by peptides, or fragments thereof, the negative effects on recombinant protein production seen when free copper ions are added to mammalian cell culture systems are avoided. Such negative effects include a reduction in the number of viable cells in a cell culture volume and/or a reduction in recombinant protein production over time, which are particularly pronounced when free copper ions are added to perfusion mammalian cell culture systems for longer periods of time, such as for days, weeks or even months (which is routinely done when producing recombinant proteins in such systems). Instead, the addition of the copper ion supplement leads to an increase in the host cell's cell-specific productivity for said recombinant target protein compared to said host cell's cell-specific productivity for the recombinant target protein when cultured in the basal cell culture medium alone. Importantly, the present invention does not relate to the provision of copper ions essential for the production of the target protein by the host cells but relates to a further addition of copper ions to improve the recombinant production of the target protein. In particular, the copper ion supplement is added to a basal, chemically defined and already copper containing cell culture medium, which is sufficient to promote cell growth and recombinant protein production of the target protein by the host cell. In some embodiments, the basal, chemically defined, copper containing, cell culture medium comprises between 50 and 800 nM of cupric ions, either in the form of anhydrous copper sulphate (CuSO₄) or copper chloride dihydrate (CuCl2°2H2O). In some non-limiting embodiments, the basal, chemically defined, copper containing cell culture medium is a standard cultivation medium, which contains standard amounts of amino acids, vitamins, sugars, inorganic salts as well as more than 200 nM copper and is sufficient to promote cell growth and recombinant protein production of the target protein by the host cell. Suitable vitamins or sugars include, but are not limited to (a) vitamins - ascorbic acid, biotin, choline chloride, calcium-D- pantothenate, cyanocobalamin, folic acid, folinic acid, nicotine amid, pyridoxine hydrochloride, riboflavin, thiamine and (b) sugars - galactose, glucose, mannitol and mannose, whereas sodium chloride and sodium phosphate are suitable non-trace metal salts for use in the here-described basal, chemically defined, copper containing cell culture medium. As such, the addition of the copper ion supplement to the basal medium fortifies the basal medium by providing additional copper ions leading to an important improvement of the cell culture system and, in particular, to an unexpected and surprising increase of the host cells' cell-specific productivity for the target protein while, at the same time, the number of viable cells is higher than that what a corresponding cell culture system to which free copper ions have been added.

This effect is particularly unexpected because the basal, chemically defined, copper-containing cell culture medium does not contain albumin and is free of non- recombinant serum proteins. As such, the basal medium does not contain the usual serum-derived protein components able to chelate free copper ions, thereby quenching the formation of aggressive and destructive reactive oxygen species and hydroxyl radicals. Importantly, the peptides of the copper ion supplement are also not selected for their ability to chelate copper ions. Instead, they consist of a random mixture of peptides and fragments thereof of less than 10 kDa size. In particular, more than 80% of the peptides, or fragments thereof, in the copper ion supplement have a molecular weight of less than 2 kDa. In fact, more than 70% have a molecular weight of less than 0.5 kDa and more than 50% have a molecular weight of less than 0.25 kDa (see Figures 19 and 20). Based on the average molecular weight of a single amino acid of 1 10 Da, the above peptide distribution can also be described with respect to the number of amino acid residues present in the peptides of the copper ion supplement. Specifically, more than 80% of the peptides comprise less than 20 amino acids, whereby more than 70% of the peptides comprise less than 5 amino acids and more than 50% of the peptides comprise less then 3 amino acids. Even if, artificially, the molecular weight of the smallest amino acid where to be taken as the basis for the amino acid length calculation, i.e. if the molecular weight of Glycine of 75 Da were to be taken as the basis, the majority of peptides in the copper ion supplement would still be very short. Specifically, more than 80% of the peptides would comprise less than 27 amino acids, whereby more than 70% of the peptides would comprise less than 7 amino acids and more than 50% of the peptides would comprise less than 4 amino acids.

This is in stark contrast to the main serum-derived copper chelators ceruloplasmin and albumin, which have respective molecular weights of about 151 and 66.5 kDa and comprise 1065 and 585 amino acids (mature human forms). Both proteins are known to harbour multiple copper binding sites. The N-terminal copper binding site of albumin is well-known for its high affinity for cupric (i.e. oxidised) copper ions having an affinity constant Ka of 1.1 to 1.6x10¹³ M^"1 at physiological pH (designated as Site VI in Carter et al., Advances in Approach in Chemistry, Volume 45, pages 153-203, 1994). This prevents access of superoxide radicals or ascorbate ions to the cupric ions thereby preventing the reduction of the oxidised cupric ions to cuprous ions, which in turn are responsible for the undesirable generation of reactive oxygen species and hydroxyl-free radicals. The N-terminal copper binding site was initially identified to lie within a 24-residue N-terminal region of albumin, which in human and bovine serum albumin starts with an Aspartic acid (Asp) residue, which was shown to be of paramount importance to ensure the sites of high copper affinity. In particular, it was shown in US 6,787,636 B1 that a single amino acid truncation at the N-terminus of this site, i.e. removal of the N-terminal Asp residue, leads to a significantly reduced copper binding affinity. Further, the high copper affinity of the native N-terminal copper binding site for the cupric ions is extraordinary, even compared to copper binding sites in other proteins or compared to other copper binding sites within albumin, because cupric ions bound to such other copper binding sites cannot withstand superoxide radical and ascorbate ion attack leading to their reduction to cuprous ions. Notwithstanding, even in such instances, the albumin molecule itself can still quench the negative effects of generated hydroxyl radicals to some degree as, due to its size and abundance, albumin itself can serve as the primary target for such radicals.

However, the regulatory function of albumin or other serum-derived components is absent from the basal as well as from the fortified cell culture medium utilised in the methods of the present invention. In addition to the non-specific nature of the peptides present in the copper ion supplement, it is further noteworthy that the mass of the entire supplement is less than 1 % of the total mass of the basal, chemically defined, copper containing, cell culture medium. In fact, the mass of the supplement is generally less than 0.5%, optionally less than 0.4%, optionally less than 0.3%. Typically the mass of the copper ion supplement is only 0.2% of the total mass of the basal, chemically defined, copper containing cell culture medium. In contrast, typically, cell culture media sufficient for the recombinant production of target proteins by host cells are supplemented with between 2 and 20% serum of which about 50% are albumin.

In particular, the peptides in the copper ion supplement are not of animal or human origin and comprise a random collection of peptides selected by way of size limitation. As the skilled person will appreciate, such random collections of short peptides, i.e. of peptides with a maximal amino acid sequence length of between 46 and 67 amino acids can easily be synthesised, obtained by way of purified phage display libraries and/or through exposure of non-human or non-animal proteins (including plants and yeast) to proteolytic enzymes. In instances where the random peptide collections still maintain peptides of sizes above 5 kDa or even 10 kDa, separation from such larger peptides can be achieved by any means known to the skilled person - for example, by ultrafiltration using a filtration device with an appropriate pore size to ensure the desired maximum size, e.g. 5 or 10 kDa. The skilled person will further understand that the required size distribution can be achieved by successively limiting the synthesis cycle times and/or exposure times.

Accordingly, the observation that the copper ion supplement can convey the beneficial effects of not only protecting the host cells against oxidative stress caused by additional copper ions but also of making those copper ions available to the host cells to allow for the demonstrated increase in the host cells' cell-specific productivity with respect to the recombinant target protein produced in the cell culture system was surprising, even to the inventors.

Upon further analysis, it was found that at least 43% of the additional copper ions provided by the copper ion supplement are indeed delivered not as free ions, but as ions complexed by the peptides, or fragments thereof, of the supplement. The complexation of the copper ions by the peptides is such that that peptide-copper complexes are formed, which are shown to elute from a high-resolution size exclusion chromatography column under physiological conditions in fractions equivalent from about 1.36 kDa to at least 20 kDa (see Figure 18).

Typically, the copper content of the copper ion supplement and the fortified medium is determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The ICP-MS copper analysis is generally performed with a repeatability of 1.2%, a recovery of 89% and a Limit of Detection (LOD= of < 7 g/ml_). Indeed, in some embodiments at least 54% of said copper ions in said supplement are complexed by peptides, or fragments thereof, of a molecular weight of 10 kDa or less such as to form the described copper-peptide complexes eluting from a high-resolution size exclusion chromatography column in fractions equivalent to at least about 155 Da to at least 20 kDa. In other embodiments, at least 43% of the copper ions are complexed by peptides, or fragments thereof, of a molecular weight of 10 kDa or less such as to form the described copper-peptide complexes eluting from a high- resolution size exclusion chromatography column in fractions equivalent to at least about 1.36 kDa to at least 20 kDa.

In the context of the above-described degree of complexation of the copper ions in the copper ion supplement and the associated effect on the host cells' cell-specific productivity with respect to the target protein, it is again noted that almost 80% of the complexed copper ions elute from the high-resolution size exclusion chromatography column in fractions equivalent to molecular weights between 1.36 kDa and at least 20 kDa (Figures 4 to 6 and 18; Example 5). None of the copper ion-containing reference samples tested, including a mixture of L-Histidine and Copper (II) chloride dihydrate, elute in fractions equivalent of molecular weights above 1 kDa (see Example 5). However, the large majority of peptides in the supplement are also less than 1 kDa in size. As such, and without wanting to be bound by theory, the majority of copper ions in the copper ion supplement appear to be bound to/complexed by more than one peptide, possibly via multimeric complex formation between the cupric ion and nitrogen donor atoms in the peptides. For example, the cupric ions could be complexed with the peptides via nitrogen donor atoms of histidine (0.155 kDa) or arginine (0.174 kDa). Alternatively, other amino acids such as cysteine (0.121 kDa) could be involved in mediating the peptide complexation of the copper ions in the copper ion supplement.

However, given that the collection of very short peptides in the copper ion supplement is random, it remains surprising that the complexation of the copper ions by the peptides is sufficiently strong to avoid the negative redox-related effects of cupric ions being reduced to cuprous ions in mammalian, in particular in human, cell culture systems for the production of recombinant target proteins, while being sufficient to increase the host cells' productivity for the recombinant target protein as described above.

Mammalian host cells suitable for production of the recombinant target protein in the methods of the present invention include, but are not limited to, cell lines such as baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO) cells, human cells, in particular human embryonic kidney (HEK) cells, or derivatives thereof. The HEK cells are preferably selected from the group consisting of HEK293 cells (ATCC CRL- 1573; DSMZ ACC 305; ECACC ref.: 85120602), HEK293T cells (DSMZ ACC 2494; ECACC: tsa201 , ref. 96121229), and Freestyle 293 cells (HEK293F cells; Thermo Fisher Scientific R79007).

The use of human cells ensures an improved glycosylation pattern of the recombinant target protein produced as compared to the corresponding recombinant target protein produced in non-human cells. The improved glycosylation pattern, in turn, can lead to a reduced immunogenicity of the recombinant target protein when used as a protein therapeutic in humans. Generally, the host cell line transfected with the nucleic acid encoding the recombinant target protein is specifically adapted to grow and produce a recombinant protein in suspension culture when cultured in a medium, which does not contain albumin and is free of non-recombinant serum proteins. In particular, the host cell can be a HEK293F cell, or a derivative thereof. In some preferred embodiments, the host cell is stably transfected with the nucleic acid encoding the target protein. The recombinant target protein to be produced according to the methods of the invention is preferably selected from the group consisting of recombinant copper-binding proteins, recombinant Factor IX (rFIX) and recombinant Granulocyte-Colony Stimulating Factor (rG-CSF). Copper-binding proteins include, but are not limited to, superoxide dismutase, lysine oxidase, tyrosinase, ceruloplasnnin, albumin as well as Factor V (FV) and Factor VIII (FVIII). A particularly preferred target protein for recombinant production in the methods of the present invention is FVIII.

FVIII (NCBI GenBank entry: AAA52420.1 ; SEQ ID NO: 1) is a blood plasma glycoprotein with a molecular mass of about 280 kDa. It is involved in the cascade of coagulation reactions that lead to blood clotting. The most common bleeding disorder, haemophilia A, is caused by a deficiency of functional FVIII. Haemophilia A is treated with protein therapeutics, i.e. by substitution with human FVIII, which is either plasma-derived or produced recombinantly. FVIII protein therapeutics are used for both acute and prophylactic treatments of bleedings in haemophilia A patients. The amino acid sequence of FVIII is organised in structural domains including: a triplicated A-domain (amino acid residues 20 to 348 (A1 ), 399 to 730 (A2) and 1713 to 2040 (A3) of SEQ ID NO: 1 ), a single B-domain of 908 amino acids (amino acid residues 760 to 1667 of SEQ ID NO: 1 ), and a duplicated C-domain (amino acid residues 2040 to 2188 (C1 ) and 2193 to 2345 (C2) of SEQ ID NO: 1 ). The B-domain has no homology to other proteins and provides 18 of the 25 potential asparagine (N)-linked glycosylation sites of FVIII. The B-domain has no apparent function in coagulation and B-domain deleted FVIII (BDD-FVIII) molecules have unchanged procoagulant activity compared to full-length FVIII. Some recombinant FVIII (rFVIII) preparations are B-domain deleted. In vivo, FVIII forms a strong complex with its cofactor von Willebrand Factor (vWF). FVIII can be produced alone or with vWF or fragments of vWF (vWF is known to stabilize FVIII) and/or recombinant vWF can be produced separately and can subsequently be added to a FVIII protein therapeutic. FV and ceruloplasnnin, proteins with a molecular weight of approximately 330 kDa and 150 kDa, respectively, show structural similarity with FVIII (A- and C-domains show approximately 40% amino acid homology). Due to the structural and biochemical similarities of FV and ceruloplasnnin with FVIII, the methods of the present invention are also suitable for the recombinant production of FV and ceruloplasmin.

FIX (NCBI GenBank entry: CCA61 1 12.1 ; SEQ ID NO:2) is a so-called Vitamin K- dependent protein. Seven plasma glycoproteins are known to be dependent on vitamin K for their biosynthesis. They are: prothrombin (Factor II), FVII, FIX, Factor X, Protein C, Protein S and Protein Z. The Gla domain is a common structural feature in all these vitamin K-dependent proteins and immediately after the Gla domain, each of the proteins (except prothrombin) has one or more EGF-like domains. The vitamin K-dependent proteins require Ca²⁺ ions to exert their physiological function and the calcium binding sites involve at least the Gla domain and the EGF-like domains. Calcium binding enables these proteins to bind to phospholipids/cell membranes and thus express their full biological activities. Due to the structural and biochemical similarities between the other Vitamin K-dependent proteins and FIX, the methods of the present invention are also suitable for the recombinant production of those other Vitamin K-dependent proteins, namely prothrombin (Factor II), FVII, FIX, Factor X, Protein C, Protein S and Protein Z.

G-CSF (UniProtKB/Swiss-Prot entry: P09919.1 ; SEQ ID NO:3) is a member of the hemopoietic regulatory glycoprotein family. Proteins of this family are involved in the growth and differentiation of hemopoietic cells from stem cells. Other members of this group are granulocyte-macrophage CSF (GM-CSF), interleukin 3 (IL-3) and stem cell factor (SCF). Growth factor proteins of the family also include, for example, hepatocyte growth factor, platelet derived growth factor, epidermal growth factor, transforming growth factor alpha, transforming growth factor beta, insulin-like growth factor and fibroblast growth factor. Due to the structural and biochemical similarities between G-CSF and other growth factors/hormones, the methods of the present invention are also suitable for the recombinant production of those other growth factors/hormones, e.g. for members of the hemopoietic regulatory glycoprotein family.

In addition to the above, proteins suitable as target proteins for recombinant production in the methods of the present invention, without being limited thereto, also include: other human blood clotting factors including fibrinogen, fibrin monomer, prothrombin, thrombin, FVa, FVIIa, FIXa, FXa, FXI, FXIa, FXII, FXIIa, FXIII, FXIIIa, ADAMTS13 etc.; transport proteins such as transferrin, haptoglobin, hemoglobin, hemopexin, etc.; protease inhibitors such as β-antithrombin, a-antithrombin, a2- macroglobulin, C1 -inhibitor, tissue factor pathway inhibitor (TFPI), heparin cofactor II, protein C inhibitor (PAI-3), Protein C, Protein S, Protein Z, etc.; immunoglobulins such as polyclonal antibodies (IgG), monoclonal antibodies, lgG1 , lgG2, lgG3, lgG4, IgA, lgA1 , lgA2, IgM, IgE, IgD, Bence Jones protein etc.; cell-related plasma proteins such as fibronectin, thromboglobulin, platelet factor 4 (PF4), etc.; apolipoproteins such as apo A-l, apo A-ll, apo E; complement factors such as Factor B, Factor D, Factor H, Factor I, C3b-inactivator, properdin, C4-binding protein etc.; antiangionetic proteins such as latent-antithrombin and prelatent-antithrombin etc.; highly glycosylated proteins including alfa-1 -acid glycoprotein, antichymotrypsin, inter-a-trypsin inhibitor, a-2-HS glycoprotein, C-reactive protein, and other proteins such as histidine-rich glycoprotein, mannan binding lectin, C4- binding protein, fibronectin, GC-globulin, plasminogen, a-1 microglobulin, erythropoeitin, interferon, tumor factors, tPA, and derivatives and muteins thereof.

In one or more preferred embodiments, the present invention relates to culturing a mammalian host cell transfected with a nucleic acid encoding recombinant FVIII (SEQ ID NO:4). In particular, the rFVIII is a B-domain deleted rFVIII (BDD-rFVIII), in particular a human B-domain deleted FVIII. A human BDD-rFVIII (SEQ ID NO:5) particularly suited for production in the methods of the present invention is described in WO2001/070968 or in WO2006/103258, both of which are hereby incorporated by reference in their entirety. Briefly, an expression plasmid based on a vector of the pcDNA3.1 family (Thermo Fisher Scientific) was used for inserting a cDNA encoding the B-domain deleted rFVIII (BDD-rFVIII) with a 16 amino acid residue linker peptide consisting of the amino acid sequence of SEQ ID NO:6, i.e. of SFSQNSRHQAYRYRRG. The expression of the BDD-rFVIII from the preferred nucleic acid of SEQ ID NO: 4 is controlled by the cytomegalovirus (CMV) promoter. This promoter, in connection with the SV40 intron and the bovine growth hormone (BGH) poly (A) signal, generally provides a high level of recombinant protein expression by the stably transfected mammalian host cell. BDD-rFVIII production of stably transfected cells was typically quantified using an enzyme-linked immunosorbent assay (ELISA) and chromogenic FVIII:C assays. Preferably, the mammalian host cell is a HEK293F cell stably transfected with a nucleic acid encoding the recombinant FVIII, preferably the BDD-rFVIII described directly above. More preferably, the HEK293F cell stably transfected with a nucleic acid encoding the recombinant FVIII is derived from a single, stably transfected clone. Generation of a stably transfected FVIII-producing HEK293F cell clone is described in WO2007/003582, which is hereby incorporated by reference in its entirety. Briefly, to prepare a stably transfected human cell line for the recombinant production of the target protein, a suitable host cell is transfected under serum-free conditions with a nucleic acid sequence comprising a gene encoding the target protein, a promoter and a polyadenylation (poly (A)) signal linked to the 5' and 3' ends of the gene encoding the target protein, respectively.

In particular, the transfection method comprises:

- culturing host cells under serum-free cell culture conditions;

- transfecting the host cells under serum-free cell culture conditions with a transfection vector comprising said nucleic acid sequence, an origin of replication and at least one gene encoding a selectable marker by using, for example, a cationic lipid transfection agent such as Fugene (Roche), ViaFect (Promega); Lipofectamine (Thermo Fisher), SuperFect (Qiagen), 293-free (Merck Millipore), or the like, or by using the calcium phosphate method according to Chen eta/. 1987 (Mol. Cell Biol. 7(8), 2745-2752); - exposing the cells to a suitable selection agent, preferably hygromycin, for example to 200 ng/mL of hygromycin, 72 hours after transfection, to apply selective pressure under serum-free, adherent cell culture conditions, for example for 10 days; and

- selecting one or more stably transfected cells under serum-free, adherent cell culture conditions, wherein the nucleic acid sequence, the origin of replication and the at least one gene encoding a selectable marker are integrated into the genome of the one or more stably transfected cells, wherein each of the one or more stably transfected adherent cells can be individually picked and expanded such as to establish the cell line for the recombinant production of the target protein under serum-free suspension cell culture conditions. In some preferred embodiments, the individually-picked hygromycin-resistant clones are isolated, expanded and subcloned through two consecutive rounds of single cell cloning such as to establish the cell line for recombinant production of the target protein under serum-free suspension cell culture conditions.

The above described transfection method comprises culturing and transfecting host cells which are specifically adapted for growth and protein production under serum- free suspension cell culture conditions under exactly those conditions, and subsequently exposing the selected cells to selection pressure under serum-free cell culture conditions. The switch from suspension to adherent cell culture conditions allows for the identification of individual, stably transfected host cell clones, which can be individually picked and expanded such as to establish the immortalized human cell line of the present invention. This unique method using host cells specifically adapted for growth in suspension cultures, transferring those cells for selection to an adherent cell culture to allow for the successful isolation of individual, stably transfected cell clones and subsequently again establishing an cell line from single clones for protein production in suspension culture, produces cells particularly suitable for use in the methods of the present invention.

A large number of different mammalian cell culture media are known to the person skilled in the art. One way of distinguishing different cell culture media is by way of their components. If the chemical composition of a cell culture medium is entirely known, the medium is called chemically defined, whereas cell culture media, which comprise complex components with unknown composition, are referred to as undefined media. For example, cell culture media to which large quantities of serum or other complex components such as hydrolysates have been added are undefined media, because the exact composition of the additives is not known. While chemically defined media may still contain serum-derived proteins, they are specifically selected, purified and the amounts added are specified. If a chemically defined medium free of animal- or serum-derived components is to be prepared, recombinant proteins such as recombinant growth factors or hormones can be added, while corresponding proteins purified from animal sources or from serum fractions cannot be included. For example, recombinant serum proteins such as albumin, transferrin or insulin can be components of chemically defined, serum-free cell culture medium.

In the context of the present invention, the basal, chemically defined, copper containing cell culture medium does not comprise albumin of any source and is otherwise also free of non-recombinant serum proteins. Notwithstanding, in some embodiments of the present invention, the basal, chemically defined, copper containing cell culture medium comprises recombinant serum proteins. In particular embodiments, the medium comprises recombinant transferrin and/or insulin.

In addition, mammalian cell culture media suitable for use in the methods of the present invention may comprise other useful components such as, for example, non-ionic detergents. In particular, polyol detergents, namely Pluronic or Tween, can be included. Exemplary polyol detergents routinely used in mammalian cell culture media are Pluronic F-68, Tween 20 or Tween 80. Typically, the concentration of such non-ionic detergents in the mammalian cell culture medium ranges from 0.00001 wt% to 1 wt%, in particular 0.0001 wt% to 0.5 wt%, most suitably 0.001 wt% to 0.1 wt%. In one or more embodiments of the present invention, the cell culture medium also comprises at least 100 to 2000 nM of manganese. In particular, the cell culture medium comprises from 200 to 2000 nM manganese, such as 250 to 1500 nM, such as 250 to 1000 nM, such as 250 to 750 nM, such as 250 to 500 nM, such as about 300, 350, 400 or 500 nM manganese.

In some embodiments, the cell culture medium comprises from 10 to 1000 nM lithium. In particular, the cell culture medium comprises between 10 and 750 nM, such as between 10 and 500 nM, such as between 25 and 500 nM, such as between 50 and 500 nM, such as between 75 and 500 nM, such as between 100 and 500 nM, such as between 100 and 400 nM, such as between 100 and 350 nM, such as between 150 and 350 nM, such as between 200 and 350 nM, such as between 250 and 350 nM, such as about 260, 270, 280, 290, 300, 310, 320, 330, 340 or 350 nM lithium. In further preferred embodiments, the cell culture medium comprises from 10 to 1000 nM chromium. In particular, the cell culture medium comprises between 10 and 750 nM, such as between 10 and 500 nM, such as between 25 and 500 nM, such as between 50 and 500 nM, such as between 75 and 500 nM, such as between 100 and 500 nM, such as between 100 and 400 nM, such as between 100 and 350 nM, such as between 150 and 350 nM, such as between 200 and 350 nM, such as between 250 and 350 nM, such as about 260, 270, 280, 290, 300, 310, 320, 330, 340 or 350 nM chromium. In further preferred embodiments, the cell culture medium comprises from 5 to 500 nM molybdenum. In particular, the cell culture medium comprises between 20 and 500 nM, such as between 50 and 500 nM, such as between 75 and 500 nM, such as between 100 and 500 nM, such as between 150 and 500 nM, such as between 200 and 500 nM, such as between 200 and 450 nM, such as between 200 and 400 nM, such as between 200 and 350 nM, such as about 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 or 350 nM molybdenum.

In a further preferred embodiment the silicon ion concentration of the cell culture medium is 500 to 50000 nM. In particular, the silicon ion concentration 500 to 25000 nM, or 500 to 20000 nM, or 500 to 18000 nM, or 1000 to 18000 nM, or 5000 to 18000 nM, or 10000 to 18000 nM, or 10000 to 15000 nM, or 12000 to 15000 nM, or 1000 to 18000 nM, 500 to 10000 nM, or 500 to 5000 nM, or 500 to 4000 nM, or 500 to 3000 nM, or 500 to 2000 nM, or 500 to 1000 nM. In accordance with the methods of the present invention, the host cells were cultured in suspension culture in shaker flasks. Once a sufficient number of viable cells of the required quality had been achieved, a series of sequential batches consisting of the major part of the cell suspension were transferred to a baffled stainless steel bioreactor or to a single use wave bioreactor. In some embodiments, the production bioreactor was operated as a perfusion process using a membrane- based cell retention system. In such instances, the perfusion rate was adjusted daily after each cell density measurement to maintain the cell specific perfusion rate until the product was collected in a batch harvest. In another embodiment, the production bioreactor was operated as a perfusion process using a membrane-based cell retention system, whereas the product was continuously captured/harvested during perfusion. All stainless steel bioreactors were baffled, continuously controlled and maintained in regard of pressure, temperature, pH, oxygen and carbon dioxide according to cultivation standard procedures and the cells were agitated during the process.

In preferred embodiments of the present invention where a stably transfected HEK293F-derived FVIII producing cell line was used, a batch harvest process relying on the addition of sodium chloride and a final concentration of 0.3 M was employed. This step was used in order to ensure detachment of FVIII from the cells as previously described in WO 2006/103258, the disclosure of which is herewith incorporated by reference in its entirety. Cells were subsequently removed by way of centrifugation and/or filtration before harvesting the recombinantly produced target protein. EXAMPLES

Cell cultivation

Bottles and up to 100 L bioreactors were used for dynamic cultivation. For agitation purpose the bottles where placed on a horizontal shaker situated within the incubator. Agitation introduced a power density into the culture of between 3 and 30 W/m³. All cell cultures were incubated at 37°C in an atmosphere containing 5% carbon dioxide and 95% relative humidity. To determine the FVIII production during cultivation, a small, representative sample of cell suspension was collected and a salt solution containing NaCI was added to obtain a final NaCI concentration of 0.3 M. After addition of the salt solution, the sample was incubated for 10 minutes. Subsequently, cells were removed and the remaining cell-free solution/supernatant was used for FVIII analysis. For FIX and G-CSF, no salt addition was performed but the cell-free solutions/supernatants remaining after cell removal were used directly to analyse FIX and G-CSF, respectively.

Basal cell culture medium

As described above, the basal cell culture medium used as reference/control medium in many of the below examples and as the base medium for the fortified cell culture medium, is a standard chemically defined basal cell culture medium, containing standard amounts of amino acids, vitamins, sugars and inorganic salts. In particular, the basal cell culture medium used in the below examples is prepared to contain 2.5 nM manganese (i.e. 2.5 nM manganese chloride tetra hydrate), 3 nM molybdenum (i.e. 3 nM ammonium molybdate tetrahydrate) and 240 to 250 nM copper (i.e. 240 to 250 nM copper chloride dihydrate). No lithium or silica has been added. Further, the basal cell culture medium does not contain albumin or any other non-recombinant serum protein. In fact, the only protein component in the basal cell culture medium used in the below examples is recombinant insulin. Further, the medium comprises a non-ionic detergent (Pluronic F-68). Suitable vitamins or sugars include, but are not limited to (a) vitamins - ascorbic acid, biotin, choline chloride, calcium-D-pantothenate, cyanocobalamin, folic acid, folinic acid, nicotine amid, pyridoxine hydrochloride, riboflavin, thiamine and (b) sugars - galactose, glucose, mannitol and mannose, whereas sodium chloride and sodium phosphate are suitable non-trace metal salts for use in the here-described basal, chemically defined, copper containing cell culture medium.

Viable cell count In all examples where reference is made to particular cell numbers, i.e. where particular biological activities are expressed in relation to cell numbers, the cell numbers refer to viable cells determined in accordance with the below-described method of determining the number of viable cells. In particular, the applied method is known to the skilled person and has previously been published in Freshney 2000 (Culture of animal cells (a manual of basic techniques). Wiley-Liss, 4th edition, pages 309-31 1 ) and in Kesper 2003 (Implementation of a 5 L bioreactor system for the process development of the production of a B-domain deleted recombinant factor VIII in human 293T cells. Diploma Thesis, Fachhochschule Weihenstephan, pages 44-47). Briefly, cell suspension is diluted with a 0.4% trypan blue staining solution and the cells are thereafter counted by way of visual inspection through a transmission microscope, thus making it possible to determine the total cell number per volume. Due to the appearance of the cells, it is also possible to visually distinguish between viable and dead/dying cells. The number of viable cells in a given volume is therefore calculated by dividing the number of dead/dying cells with the number of viable cells.

FVIII activity assay

In all of the examples relating to FVIII, the biologic activity of the recombinantly produced FVIII was measured with a chromogenic FVIII:C assay (COATEST SP FVIII kit, 82 4086 63, Chromogenix/lnstrumentation Laboratory, US). This essay is based on a 2-stage photometric method that measures the biological activity of FVIII as a cofactor.

An activity of 1 International Unit (IU) FVIII as determined by the above assay corresponds to the activity of about 0.1

BDD-rFVIII (=170 kDa; SEQ ID NO: 5) or to about 0.25

of wild type FVIII (=300 kDa; SEQ ID NO: 1 ). The activity assay complies with the requirements of the European Pharmacopoeia.

Example 1

Example 1 relates to a small-scale bioreactor culturing system and compares the effects of culturing a stably transfected HEK293F host cell, which has integrated the nucleic acid sequence encoding for BDD-rFVIII in its genome, in the basal, chemically defined, copper containing cell culture medium as well as in the fortified cell culture medium of the present invention, i.e. in the basal medium to which the copper ion supplement of the present invention has been added.

As shown in Tables 1 and 2, using the fortified cell culture medium of the present invention (i.e. basal cell culture medium fortified with 0.2% of copper ion supplement B), cell cultures can be produced, which show an increased FVIII:C activity at t = 92 h as well as an increased cell-specific productivity with respect to BDD-rFVIII at t = 92 h compared to corresponding cultures grown in the basal, chemically defined, copper containing cell culture medium.

At the end of the culturing period, i.e. at t = 92 h, a cell-specific productivity of 1.49 IU per 10⁶ cells could be determined. In comparison to the cell-specific productivity seen in the basal medium alone this is equivalent to a direct increase of 2.5-fold or a normalised increase of 1.49-fold.

As such, it can be concluded that the addition of the copper ion supplement leads to a significant increase in the host cells' production of BDD-rFVIII, in particular with respect to the host cells' cell-specific productivity.

Table 1, Accumulated FVIII:C activity

Example 2

Similar to Example 1 , Example 2 relates to a small-scale bioreactor culturing system and compares the effects of culturing a stably transfected HEK293F host cell, which has integrated the nucleic acid sequence encoding for BDD-rFVIII in its genome, in the basal, chemically defined, copper containing cell culture medium as well as in the fortified cell culture medium of the present invention, i.e. in the basal medium fortified with 0.2% copper ion supplement B. In Example 2 samples were taken at slightly different intervals and time points compared to Example 1.

Notwithstanding, Tables 3 and 4 again show that using the fortified cell culture medium of the present invention, cell cultures can be produced having an increased FVIII:C activity (Table 3) as well as an increased cell-specific productivity with respect to BDD-rFVIII compared to corresponding cultures grown in the basal, chemically defined, copper containing cell culture medium (Table 4) while no increase in overall call numbers could be observed during the same culturing period (Table 5).

In particular, at t = 76 h the accumulated FVIII:C activity was determined as 8.0 lU/mL (equivalent to a direct increase of 1.45-fold or a normalised increase of 1.56 fold; Table 3) and the cell-specific productivity was 3.5 IU per 10⁶ cells (equivalent to a direct increase of 1.56-fold or a normalised increase of 2.02 fold; Table 4), while the viable cell density at the same time point had decreased to 2.44 x 10⁶ cells/mL (equivalent to a direct decrease of 0.91 -fold or a normalised decrease of 0.76-fold; Table 5).

As such, Example 2 also supports the conclusion that the addition of the copper ion supplement to the basal medium leads to a significant increase in the host cells' production of BDD-rFVIII, in particular with respect to the host cells' cell-specific productivity.

The specific productivity, i.e. the productivity of a single cell per day, achieved after 76 h of culture in the fortified cell culture medium of the invention was 1.1 1 μΐυ FVI I l/cell/day (calculated based on the cell-specific productivity values of Table 4). As such, the specific productivity was also increased by 2.02-fold when compared to the corresponding measurement in the basal medium and normalized against the differences already present at t = 0. Table 3, Accumulated FVIII:C activity

Table 5, Specific productivity

FVIIhC FVIIhC FVIIhC FVIIhC (μΐυ/cell/day), (μΐυ/cell/day), (μΐυ/cell/day), (μΐυ/cell/day), t = O h t = 21 h t = 45 h t = 76 h

Basal medium - 3.00 2.50 2.24

Fortified medium 2.95 2.56 3.50

(i.e. Basal medium + 0.2% copper

ion supplement)

Fold change (direct) - 0.98 1.02 1.56

Fold change (normalised against - 1.27 1.44 2.02 t = 0) Table 6, Viable cell density

Example 3 In Example 3, the effects of culturing a stably transfected HEK293F host cell, which has integrated the nucleic acid sequence encoding for BDD-rFVIII in its genome, in the fortified cell culture medium of the present invention (comprising 0.4% of copper ion supplement A) were compared to those seen in basal medium to which additional free cupric copper ions in the form of CuCl2 dihydrate were added to a final concentration of 1000 nM as well as to the basal medium containing 0.05% Albumin.

As can be seen from the data presented in Table 7 below, while the addition of free cupric ions to a final concentration of 1000 nM may have boosted cell-specific productivity (3.77 IU/10⁶ cells), it also led to a reduced number of viable cells (1.64 x 10⁷ viable cells per ml_) compared to the other culture conditions tested. In contrast, the addition of 0.05% albumin seemed to have a very positive effect on overall growth of the cells (3.3 and 3.69 x 10⁷ viable cells per ml_) but not on the cell-specific productivity of the cell for the recombinantly expressed target protein (1.01 and 1.73 IU/10⁶ cells). The surprisingly low cell-specific productivity seen in basal medium comprising 0.05% albumin was confirmed for two separate culture volumes, i.e. in a 0.6 L and a 2 L bioreactor. In comparison, cells cultured in the fortified medium of the present invention displayed a much higher, i.e. a significantly increased cell-specific productivity of 2.45 and 2.3 IU/10⁶ cells while maintaining the cultured cells at healthy viable cell numbers of 3.3 and 3.69 x 10⁷ viable cells per mLover a 12 day long culture period. The surprising benefits of culturing the cells in the fortified medium of the present invention is also illustrated by the increased accumulated FVIII:C activity (i.e. by the FVIII titer expressed as IU per ml_) determined seen in comparison to cells cultured either in the presence of 1000nM copper or 0.05% albumin (in the 2 L reactor: 100.69 lU/mL in fortified medium versus 63.66 lU/mL in basal medium comprising 0.05% albumin and 61.65 lU/mL in basal medium comprising 1000nM copper).

Table 7, Effects seen after 12 days of culture in different cell culture media

Example 4

In Example 4, five cultures of stably transfected HEK293F host cells, all of which have the nucleic acid sequence encoding for BDD-rFVIII integrated in their genome, were cultured under otherwise identical conditions in five separately-produced batches of the fortified cell culture medium of the invention (i.e. in five batches of basal cell culture medium fortified with 0.4% of copper ion supplement A). After 13 days, the cultures were tested for the active BDD-rFVIII produced. Table 8, Comparison of active BDD-rFVIII produced after 13 days of culture in fortified cell culture media of the invention

Example 4 shows significantly increased values for FVIII titre, cell specific productivity and specific productivity in a perfused batch bioreactor harvest after 13 days compared to corresponding values previously reported in the field.

Example 5

In Example 5, the copper ion supplement according to embodiments of the present invention was analysed using an Akta Pure 150 system with 0.75 mm i.d. tubing, software Unicorn 6.4.1 and a Superdex Peptide 10/300 GL size exclusion chromatography column at a physiological pH and a physiological salt concentration, i.e. at concentrations mimicking the pH and salt conditions of the preceding cell culture.

The copper ion supplement was analysed as a 10% solution in 10 mM Phosphate pH 7.2/20°C with 0.15 M NaCI. Sample volume was 500 μΙ_ and the flow rate 1 mL/min. All fractions were stored at -70°C until being further analysed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Figures 1 to 3 show chromatograms of the analysis for different batches (namely batches 2, 3 and 4) of copper ion supplement A. The results of the ICP-MS analysis of the fractions of Figure 1 are outlined in Table 1 1 below and the copper elution profile determined is indicated in Figure 18. The reference samples listed in Table 9 below were similarly prepared in 10 mM Phosphate pH 7.2/20°C with 0.15 M NaCI and applied to the same column in separate runs. The sample volume was again 500 μΙ_ and the flow rate 1 mL/min in all reference experiments.

Table 9

The main retention volumes for the reference samples on the above-described chromatography system/size exclusion column used are indicated in Table 10.

Table 10

Particularly noteworthy are the following observations:

Reference sannples Nos. 5, 6 and 8 contain cupric copper ions. The first of the copper containing reference sannples is eluting from the column is reference sample No. 8, eluting already at about 18.3 mL, while sample No. 6 comprising both L- histidine and cupric copper ions elutes in later, i.e. "smaller", fractions at about 19.3 mL. As described above, cupric copper is able to form complexes with histidine but, given the size elution profile in the present system, complex formation between the cupric ions and the histidine does not appear to be significant as the copper of the mixture of L-histidine and copper (II) chloride dihydrate (reference sample No. 6) shows substantially the same elution profile as the copper of copper (II) chloride dehydrate alone (reference sample No. 5). Notwithstanding, and as outlined in Table 1 1 below, deternnination of the copper elution profile of copper ion supplement A from the above-described chromatography system/size exclusion column revealed that, at physiological pH and physiological salt concentrations, a significant proportion of copper eluted in fractions equivalent to from about 155 Da to at least 20 kDa.

Table 11

n.d. = not determined, LOD = limit of detection, 7 pg/L/ n.a. = not applicable From the above results, it is apparent that about 43.9% of the copper in the copper ion supplement elute in fractions A2 to B5.

Fraction B5 consists of 1 mL of eluate eluting after about 16.4 mL have already passed through the column. The reference samples run on the same column under the same conditions indicate that Fraction B5 corresponds to a molecular weight of about 1.36 kDa (see Figure 6 for reference sample No.12, cyanocobalamin (B12), 1.36 kDa). As such, it can be concluded that more than 43% of the copper in the copper ion supplement elutes in fractions comprising molecules and/or complexes larger than 1.36 kDa.

Further, it is apparent that about 54.7%, i.e. the majority of the copper in the copper ion supplement elutes in fractions A2 to C1. Fraction C1 consists of 1 millilitre of eluate eluting after about 18.4 ml_ have already passed through the column. The reference samples run on the same column under the same conditions indicate that Fraction C1 includes molecules of a molecular weight as low as about 0.155 kDa (see Figure 14 for reference sample No. 4, L-histidine, 0.155 kDa, and Table 10 above) and also includes the major peaks determined for the copper containing reference samples No. 5 and No. 6 (see Figures 13 and 12 and Table 10 above). However, as is also apparent, and despite the fact that it has been established here that Fractions B6 to C1 include molecules of weights including that of the copper - containing reference samples 5, 6 and 8, namely from about 0.155 to up to 1.36 kDa, these Fractions only contain about 1 1 % of the copper ions of the copper ion supplement. The majority of the copper ions detected in measurable quantities (43.9% of the total copper ions as explained above), however, elute in fractions containing molecules of molecular weights greater than 1.36 kDa. Surprisingly, about 29.3% of the copper ions were detected in Fractions A2 to B3, i.e. in fractions eluting after only about 7.4 ml_ have passed through the column. The reference samples run on the same column under the same conditions indicate that molecules of molecular weight greater than 1.36 kDa and up to at least 20 kDa elute in these fractions. Accordingly, it can be concluded that a significant portion of the copper ions in the copper ion supplement elute in complex with, i.e. are complexed by, the peptides of the copper ion supplement.

Further, and as copper ion supplement A was used in the present example, it is noted that Figure 19 shows that copper ion supplement A comprises very few peptides exceeding 2 kDa in size (less than 10% of 2 to 5 kDa in size and substantially none exceeding 5 kDa). As such, and without wanting to be bound by theory, it would appear that copper ions eluting in Fractions A2 to B3 are complexed by more than one peptide. Example 6

As shown in Figure 21 , copper analysis was performed on selected samples from 20 separate batches of fortified medium according to the present invention. The batches analysed were prepared by adding 0.4% copper ion supplement A to the basal medium comprising Analysis was performed using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Different samples from the same batch of fortified medium were also tested after storage at either 4°C or -20°C. No significant difference was observed in samples stored at either 4°C or -20°C.

The range of copper ions in the fortified medium samples was determined to lie between 200 and 400 nM and on average at about 295 nM.

Example 7

Copper analysis was also performed on selected samples from 4 separate batches of 0.4% copper ion supplement A in buffer (batches 2 to 4 and 6). Analysis was performed using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). The range of copper ions in the analysed batches of copper ion supplement A was determined to lie between about 47 and 90 nM, with an average of about 66 nM and a standard deviation of about 16.1.

Many modifications and other embodiments of the invention set forth herein will come to mind to the one skilled in the art to which the invention pertains having the benefit of the teachings presented in the foregoing description and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

C l a i m s

1. A method for the production of a recombinant target protein, said method comprising the steps of:

(a) culturing a mammalian host cell transfected with a nucleic acid encoding said recombinant target protein in a fortified cell culture medium, wherein said fortified cell culture medium is a basal, chemically defined, copper containing, cell culture medium, which does not contain albumin, is free of non-recombinant serum proteins, and is sufficient for the recombinant production of the target protein by said host cell, to which an additional copper ion supplement is added, wherein said supplement comprises copper ions complexed by peptides, or fragments thereof, of a molecular weight of 10 kDa or less; and

(b) harvesting the recombinant target protein.

2. The method of claim 1 , wherein, when cultured in said fortified cell culture medium, said host cell's cell-specific productivity for said recombinant target protein is increased compared to said host cell's cell-specific productivity for said recombinant target protein when cultured in the basal cell culture medium alone.

3. The method of claim 1 or claim 2, wherein said recombinant target protein is selected from the group consisting of recombinant copper-binding proteins, recombinant FIX (rFIX) and recombinant Granulocyte-Colony Stimulating Factor (rG-CSF).

4. The method of any one of the preceding claims, wherein said recombinant target protein is rFVIII.

5. The method of any one of the preceding claims, wherein said basal, chemically defined, copper containing, cell culture medium free of non-recombinant serum proteins comprises between 50 and 800 nM of copper.

6. The method of any one of the preceding claims, wherein said mammalian host cell is a human host cell.

7. The method of any one of the preceding claims, wherein said cell-specific productivity of said host cell for the recombinant target protein is increased to more than 1.2 μΐυ/cell.

8. The method of any one of the preceding claims, wherein said cell-specific productivity of said host cell for the recombinant target protein is increased by at least 1.3-fold.

9. The method of any one of the preceding claims, wherein at or after 66 hours of culture in said fortified cell culture medium, said cell-specific productivity is increased by at least 1.4-fold.

10. The method of any one of the preceding claims, wherein said cell-specific productivity of said host cell for the recombinant target protein is at least 0.2 μΐυ/cell/day.

1 1. The method of any one of the preceding claims, wherein said basal, chemically defined, copper containing, cell culture medium comprises more than

200 nM of copper.

12. The method of any one of the preceding claims, wherein the mass of said supplement is less than 1 % of the total mass of the basal, chemically defined, copper containing, cell culture medium, in particular less than 0.5%, optionally less than 0.4%, optionally less than 0.3%, or optionally 0.2%.

13. The method of any one of the preceding claims, wherein more than 80% of said peptides, or fragments thereof, have a molecular weight of equal to or less than 2 kDa; more than 70% of said peptides, or fragments thereof, have a molecular weight of equal to or less than 0.5 kDa, and/or more than 50% of said peptides, or fragments thereof, have a molecular weight of equal to or less than 0.25 kDa.

14. The method of any one of the preceding claims, wherein copper ions in said supplement are complexed by said peptides, or fragments thereof, such as to form complexes eluting from a high-resolution size exclusion chromatography column in fractions equivalent from about 155 Da to at least 20 kDa.

15. The method of claim 14, wherein at least 54% of said copper ions are complexed by peptides, or fragments thereof, of a molecular weight of 10 kDa or less such as to form complexes eluting from a high-resolution size exclusion chromatography column in fractions equivalent to at least about 155 Da to at least 20 kDa, preferably at least 43% of said copper ions are complexed by peptides, or fragments thereof, of a molecular weight of 10 kDa or less such as to form complexes eluting from a high-resolution size exclusion chromatography column in fractions equivalent to at least about 1.36 kDa to at least 20 kDa.

16. The method of any one of the preceding claims, wherein said mammalian host cell is: a baby hamster kidney (BHK) cell; a Chinese hamster ovary (CHO) cell, such as a CHO-S FreeStyle cell (Thermo Fisher Scientific R80007); or a human cell, in particular a human embryonic kidney (HEK) cell or a derivative thereof, such as HEK293 cells (ATCC CRL-1573; DSMZ ACC 305; ECACC ref.: 85120602), HEK293T cells (DSMZ ACC 2494; ECACC: tsa201 , ref. 96121229), or FreeStyle 293 cells (HEK293F cells; Thermo Fisher Scientific R79007).

17. The method of any one of the preceding claims, wherein said mammalian host cell is stably transfected with a nucleic acid sequence encoding the target protein, optionally said cell is derived from a single, stably transfected clone, in particular from a stably transfected clone of a HEK293F cell, which has the nucleic acid sequence encoding the target protein integrated into its genome.

18. The method of claim 17, wherein said nucleic acid sequence integrated into the host cell's genome encodes FVIII or a B-domain deleted FVIII.