EP3625329A1 - Verfahren zur herstellung eines rekombinanten zielproteins - Google Patents

Verfahren zur herstellung eines rekombinanten zielproteins

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Publication number
EP3625329A1
EP3625329A1 EP18728049.0A EP18728049A EP3625329A1 EP 3625329 A1 EP3625329 A1 EP 3625329A1 EP 18728049 A EP18728049 A EP 18728049A EP 3625329 A1 EP3625329 A1 EP 3625329A1
Authority
EP
European Patent Office
Prior art keywords
cell
copper
recombinant
target protein
kda
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP18728049.0A
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English (en)
French (fr)
Inventor
Stefan Winge
Jade Safta
Peter Aizawa
Tilo Schwientek
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Octapharma AG
Original Assignee
Octapharma AG
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Filing date
Publication date
Application filed by Octapharma AG filed Critical Octapharma AG
Publication of EP3625329A1 publication Critical patent/EP3625329A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • 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.
  • FVIII coagulation Factor VIII
  • 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.
  • cell culture media optimised for specific cell types or culture conditions.
  • cell culture media comprise components of many different categories including amino acids, vitamins, fatty acids, salts, and further components.
  • FBS Foetal bovine serum
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • the same publication indicates that the supplementation of the medium with copper ions can also lead to undesirable effects.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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).
  • the recombinant target protein is recombinant Factor VIII (FVIII).
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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” 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.
  • the term "cell culture medium” refers to liquid growth medium for mammalian cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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".
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a physiological pH and a physiological sodium chloride (NaCI) concentration are maintained.
  • 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 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.
  • 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.
  • the salt concentration typically 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.
  • 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.
  • the term "cell-specific productivity" expresses a measure of the amount of a recombinant target protein produced per host cell.
  • 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.
  • FVIII cell-specific productivity may be expressed as FVIII:C activity in IU per 10 6 cells or even per single cell.
  • 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 10 6 cells or even per single cell.
  • 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 10 6 cells or even per single cell. "copper-binding proteins"
  • 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).
  • FV Factor V
  • FVIII Factor VIII
  • derivatives 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.
  • protein derivatives in the context of the present invention include modified proteins and protein fragments derived from an initial protein.
  • 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”.
  • the terms “express”, “expressing” or “expression” refer to the transcription and translation of a nucleic acid sequence encoding a protein.
  • the copper ion supplement comprises copper ions complexed by peptides, or fragments thereof, of a molecular weight of 5 kDa or less.
  • 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.
  • 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).
  • the recombinant target protein is recombinant Factor VIII (FVIII).
  • 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.
  • the mammalian host cell is a human host cell.
  • 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.
  • 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 (CuSO 4 ).
  • 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%.
  • 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.
  • the cell-specific productivity of the host cell for the recombinant target protein is increased to more than 1.2 ⁇ /cell.
  • the cell-specific productivity of the host cell is increased to 1.21 ⁇ /cell or more, such as to 1.32 ⁇ /cell, such as to 1.55 ⁇ /cell, such as to 2.56 ⁇ /cell, such as to 2.88 ⁇ /cell, such as to 2.95 ⁇ /cell, such as to 3.17 ⁇ /cell, such as to 3.38 ⁇ /cell, such as to 3.5 ⁇ /cell, such as to 4.14 ⁇ /cell, such as to 4.38 ⁇ /cell, such as to 5.32 ⁇ /cell, such as to 5.35 ⁇ /cell.
  • 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.
  • 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 ⁇ /cell/day, such as 0.22 ⁇ /cell/day or 0.24 ⁇ /cell/day or 0.282 ⁇ /cell/day; at least 0.3 ⁇ /cell/day such as 0.32 ⁇ /cell/day or 0.37 ⁇ /cell/day or 0.365 ⁇ /cell/day; or at least 0.4 ⁇ /cell/day such as 0.41 ⁇ /cell/day.
  • 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.
  • 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.
  • 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.
  • 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).
  • BHK baby hamster kidney
  • CHO Chinese hamster ovary
  • CHO-S Freestyle cell Thermo Fisher Scientific R80007
  • a human cell in particular a human embryonic kidney (HEK) cell or a derivative thereof, such as HEK293 cells (ATCC CRL
  • the mammalian host cell is specifically adapted to growth and recombinant protein production under albumin-free and serum-free suspension cell culture conditions.
  • the mammalian host cell is a Freestyle CHO-S cell (Thermo FisherScientific R80007) or a HEK293F cell.
  • the mammalian host cell is stably transfected with a nucleic acid sequence encoding the target protein.
  • the cell is derived from a single, stably transfected clone.
  • 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.
  • the nucleic acid sequence integrated into the host cell's genome encodes FVIII or a B-domain deleted FVIII.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 (CuSO 4 ) or copper chloride dihydrate (CuCl2°2H2O).
  • 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.
  • 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.
  • the basal, chemically defined, copper-containing cell culture medium does not contain albumin and is free of non- recombinant serum proteins.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • Asp Aspartic acid
  • 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.
  • the mass of the entire supplement is less than 1 % of the total mass of the basal, chemically defined, copper containing, cell culture medium.
  • the mass of the supplement is generally less than 0.5%, optionally less than 0.4%, optionally less than 0.3%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the copper content of the copper ion supplement and the fortified medium is determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS).
  • 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.
  • 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.
  • the large majority of peptides in the supplement are also less than 1 kDa in size.
  • 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.
  • the cupric ions could be complexed with the peptides via nitrogen donor atoms of histidine (0.155 kDa) or arginine (0.174 kDa).
  • cysteine (0.121 kDa) could be involved in mediating the peptide complexation of the copper ions in the copper ion supplement.
  • 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.
  • 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.
  • the host cell can be a HEK293F cell, or a derivative thereof.
  • 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.
  • BDD-FVIII B-domain deleted FVIII
  • rFVIII recombinant FVIII
  • FVIII forms a strong complex with its cofactor von Willebrand Factor (vWF).
  • vWF von Willebrand Factor
  • 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 Ca 2+ ions to exert their physiological function and the calcium binding sites involve at least the Gla domain and the EGF-like domains.
  • Vitamin K-dependent proteins 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.
  • prothrombin Factor II
  • FIX Factor X
  • Protein C Protein C
  • Protein S and Protein Z 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.
  • GM-CSF granulocyte-macrophage CSF
  • IL-3 interleukin 3
  • SCF stem cell factor
  • 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
  • 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.
  • proteins suitable as target proteins for recombinant production in the methods of the present invention 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 ⁇ -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,
  • the present invention relates to culturing a mammalian host cell transfected with a nucleic acid encoding recombinant FVIII (SEQ ID NO:4).
  • the rFVIII is a B-domain deleted rFVIII (BDD-rFVIII), in particular a human B-domain deleted FVIII.
  • BDD-rFVIII B-domain deleted rFVIII
  • 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.
  • an expression plasmid based on a vector of the pcDNA3.1 family 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.
  • BDD-rFVIII B-domain deleted rFVIII
  • the expression of the BDD-rFVIII from the preferred nucleic acid of SEQ ID NO: 4 is controlled by the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the transfection method comprises:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • recombinant serum proteins such as albumin, transferrin or insulin can be components of chemically defined, serum-free cell culture medium.
  • 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.
  • 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.
  • polyol detergents namely Pluronic or Tween
  • Exemplary polyol detergents routinely used in mammalian cell culture media are Pluronic F-68, Tween 20 or Tween 80.
  • 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%.
  • the cell culture medium also comprises at least 100 to 2000 nM of manganese.
  • 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.
  • the cell culture medium comprises from 10 to 1000 nM lithium.
  • 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.
  • the cell culture medium comprises from 10 to 1000 nM chromium.
  • 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.
  • the cell culture medium comprises from 5 to 500 nM molybdenum.
  • 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.
  • the silicon ion concentration of the cell culture medium is 500 to 50000 nM.
  • 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.
  • 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.
  • 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.
  • 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.
  • Bottles and up to 100 L bioreactors were used for dynamic cultivation.
  • 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/m 3 . All cell cultures were incubated at 37°C in an atmosphere containing 5% carbon dioxide and 95% relative humidity.
  • 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.
  • 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.
  • 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.
  • 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.
  • the basal cell culture medium does not contain albumin or any other non-recombinant serum protein.
  • the only protein component in the basal cell culture medium used in the below examples is recombinant insulin.
  • 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.
  • the cell numbers refer to viable cells determined in accordance with the below-described method of determining the number of viable cells.
  • 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, surochmaschine Weihenstephan, pages 44-47).
  • 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.
  • the activity assay complies with the requirements of the European Pharmacopoeia.
  • 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.
  • a cell-specific productivity of 1.49 IU per 10 6 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.
  • 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.
  • samples were taken at slightly different intervals and time points compared to Example 1.
  • 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).
  • 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 10 6 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 10 6 cells/mL (equivalent to a direct decrease of 0.91 -fold or a normalised decrease of 0.76-fold; Table 5).
  • 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.
  • 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.
  • 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.
  • 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/10 6 cells while maintaining the cultured cells at healthy viable cell numbers of 3.3 and 3.69 x 10 7 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.
  • 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 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 ⁇ _ 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.
  • 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.
  • 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.
  • 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).
  • Figure 6 for reference sample No.12, cyanocobalamin (B12), 1.36 kDa.
  • 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).
  • 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 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.
  • 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.
  • ICP-MS Inductively Coupled Plasma-Mass Spectrometry

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