WO2016198499A1 - IMPROVED METHOD FOR PRODUCTION OF γ-CARBOXYLATED POLYPEPTIDES - Google Patents

IMPROVED METHOD FOR PRODUCTION OF γ-CARBOXYLATED POLYPEPTIDES Download PDF

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Publication number
WO2016198499A1
WO2016198499A1 PCT/EP2016/063107 EP2016063107W WO2016198499A1 WO 2016198499 A1 WO2016198499 A1 WO 2016198499A1 EP 2016063107 W EP2016063107 W EP 2016063107W WO 2016198499 A1 WO2016198499 A1 WO 2016198499A1
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Prior art keywords
vitamin
cyclodextrin
culture medium
polypeptide
cell
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PCT/EP2016/063107
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English (en)
French (fr)
Inventor
Steffen Goletz
Rainer Stahn
Doreen JAHN
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Glycotope Gmbh
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Publication of WO2016198499A1 publication Critical patent/WO2016198499A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • 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 ⁇ -carboxylated polypeptide.
  • a complex of vitamin K and cyclodextrin is used to provide the host cells with a sufficient amount of vitamin K to enable y-carboxylation.
  • Vitamin K includes two natural vitamers K1 (phylloquinone) and K2 (menaquinone). Vitamin K1 can be converted into vitamin K2.
  • the function of vitamin K2 in the animal cell is to add a carboxylic acid functional group to a glutamate amino acid residue (Glu) in a protein, forming a ⁇ -carboxyglutamate (Gla) residue.
  • Glu glutamate amino acid residue
  • Gla ⁇ -carboxyglutamate
  • vitamin K undergoes electron reduction to a reduced form called vitamin K hydroquinone by the enzyme vitamin K epoxide reductase (VKOR). Another enzyme then oxidizes vitamin K hydroquinone to allow carboxylation of Glu to Gla; this enzyme is called ⁇ -glutamyl carboxylase or vitamin K-dependent carboxylase.
  • VKOR vitamin K epoxide reductase
  • Another enzyme then oxidizes vitamin K hydroquinone to allow carboxylation of Glu to Gla; this enzyme is called ⁇ -glutamyl carboxylase or vitamin K-dependent carboxylase.
  • the v- glutamyl carboxylase catalyzes the post-translational conversion of glutamic acid to v- carboxyglutamate in precursor proteins containing a ⁇ -carboxylation recognition site.
  • the following human Gla-containing proteins have been characterized to the level of primary structure: the blood coagulation factors II (prothrombin), VII, IX, and X, the anticoagulant proteins C and S, and the factor X-targeting protein Z. Further, y- carboxylation has been found in the bone Gla protein osteocalcin, the calcification- inhibiting matrix Gla protein (MGP), the cell growth regulating growth arrest specific gene 6 protein (Gas6) and the four transmembrane Gla proteins (TMGPs). The presence of the Gla residues in these proteins turned out to be essential for biological activity.
  • MGP calcification- inhibiting matrix Gla protein
  • Gas6 cell growth regulating growth arrest specific gene 6 protein
  • TMGPs transmembrane Gla proteins
  • WO 01/70968 A2 relates to a method for the recombinant synthesis of blood coagulation factors.
  • Example 4 thereof describes that 10 ⁇ g ml vitamin K is used in the culture medium for the recombinant production of Factor IX using a transiently transfected host cell. With respect to stably transfected host cells, it is mentioned that up to 100 ⁇ g ml may be used.
  • the technical problem has been solved by the provision of a method for producing a v- carboxylated polypeptide comprising culturing a host cell having ⁇ -glutamyl carboxylase activity and comprising a nucleotide sequence coding for the polypeptide in a culture medium, wherein the culture medium comprises vitamin K and cyclodextrin.
  • cyclodextrin in a method for expression of a v- carboxylated polypeptide is provided.
  • the present invention provides a cell culture medium comprising cyclodextrin and vitamin K.
  • Polypeptide means any naturally occurring or artificially designed polypeptide, including muteins thereof.
  • a “mutein” includes additions, deletions and/or substitutions with respect to the naturally occurring polypeptide.
  • the mutein does not include more than 20 additions, deletions and/or substitutions, preferably not more than 10, particularly preferred not more than 5, in particular not more than two or one amino acid addition, deletion and/or substitution compared to the parent amino acid sequence.
  • Promoter refers to a region of regulatory DNA sequences for the control of transcription of a gene to which RNA polymerases bind.
  • “Functionally linked” refers to configurations of the vector where the promoter is located within the vector in such a manner that it can stimulate transcription of the DNA sequence coding for the polypeptide.
  • Vector refers to any genetic construct, such as plasmid, phage, cosmid, etc., which is capable of replication when associated with the proper control elements.
  • the term includes cloning and expression vehicles.
  • Carrying a vector includes both, the stable and transient incorporation of a functional DNA segment into the host cell. The stable incorporation is, however, preferred.
  • Encodes or “encoding” refers to a property of the nucleic acid sequence being transcribed (in case of DNA) or translated (in case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of an appropriate regulatory sequence.
  • ⁇ -Carboxylated polypeptide refers to a polypeptide which comprises at least one v- carboxylation.
  • a ⁇ -carboxylation in particular is a modification of a glutamic acid residue within the polypeptide resulting in a ⁇ -carboxyglutamate. This modification may be catalyzed by the enzyme ⁇ -glutamyl carboxylase.
  • the inventors of the present invention have found that when cyclodextrin is added to the culture medium the above technical problems can be overcome.
  • using cyclodextrin a homogeneous culture medium containing vitamin K is obtained.
  • the process conditions can be better controlled and reproducible production conditions and results are obtained.
  • a significant achievement is that the concentration of vitamin K in the culture medium can be lowered to 1 ⁇ g ml or less, in particular to about 0.2 ⁇ g ml or even as low as 0.05 ⁇ g/ml. It is readily apparent that the process according to the invention is more efficient and less expensive than the process of the prior art.
  • the present invention provides a method for producing a v- carboxylated polypeptide comprising culturing a host cell having ⁇ -glutamyl carboxylase activity and comprising a nucleotide sequence coding for the polypeptide in a culture medium, wherein the culture medium comprises vitamin K and cyclodextrin.
  • the vitamin K used in the method may be any vitamin K available, including vitamin K derivatives.
  • vitamin K1 or vitamin K2 or a mixture of vitamin K1 and vitamin K2 are used.
  • vitamin K1 is used.
  • the culture medium comprises the vitamin K in a concentration of 10 ⁇ g ml or less.
  • the concentration of vitamin K in the culture medium is 5 ⁇ g ml or less, especially 4 ⁇ g ml or less, 3 ⁇ g ml or less or 2 ⁇ g ml or less.
  • the concentration of vitamin K in the culture medium is 1.5 ⁇ g/ml or less, in particular 1 ⁇ g ml or less or 0.8 ⁇ g ml or less.
  • the concentration of vitamin K in the culture medium is 0.5 ⁇ g ml or less.
  • the concentration of vitamin K in the culture medium may in particular be at least 0.001 ⁇ g/ml, especially 0.005 ⁇ g/ml, 0.01 ⁇ g/ml, 0.02 ⁇ g/ml, 0.03 ⁇ g/ml, 0.04 ⁇ g/ml or 0.05 ⁇ g/ml.
  • the concentration of vitamin K in the culture medium is in the range from 0.04 to 1.5 ⁇ g/ml.
  • the concentration of vitamin K in the culture medium is in the range from 0.05 to 1 ⁇ g/ml.
  • the concentration of vitamin K in the culture medium is in the range from 0.05 to 0.5 ⁇ g/ml.
  • Cyclodextrins are cyclic oligosaccharides which may be produced from starch.
  • the cyclodextrin used in the method may be any cyclodextrin, in particular a cyclodextrin capable of forming a complex with vitamin K.
  • the vitamin K may be present in the culture medium in complex with the cyclodextrin.
  • vitamin K and cyclodextrin are mixed prior to adding them to the culture medium.
  • the method may comprise the step of adding a composition comprising vitamin K and cyclodextrin to the culture medium.
  • a cyclodextrin composed of 6 to 10 glucose monomers, in particular 6 to 8 glucose monomers is used, such as a-cyclodextrin having 6 glucose monomers, ⁇ -cyclodextrin having 7 glucose monomers and ⁇ -cyclodextrin having 8 glucose monomers.
  • the glucose monomers are in particular 1 -4 linked a-D-glucopyranoside units.
  • the glucose units of the cyclodextrin are further modified, for example by methyl groups.
  • a specific example of the cyclodextrin is randomly methylated ⁇ -cyclodextrin.
  • the cyclodextrin is used in the culture medium in a concentration which results in a ratio by weight of vitamin K to cyclodextrin in the range of from 1 :50 to 1 :250 (weight of vitamin K : weight of cyclodextrin).
  • the ratio by weight of vitamin K to cyclodextrin is in the range of from 1 :80 to 1 :150, especially in the range of from 1 :100 to 1 :130 or from 1 :110 to 1 :120, such as about 1 :114.
  • At least 100 parts per weight cyclodextrin are used for each 1 part per weight vitamin K, in particular at least 1 10 or even at least 114 parts per weight cyclodextrin for each 1 part per weight vitamin K. That is, the concentration of cyclodextrin in the culture medium is at least 100 times higher, in particular at least 110 times higher or at least 1 14 times higher, than the concentration of vitamin K.
  • the ⁇ -carboxylated polypeptide produced by the method comprises at least one ⁇ - carboxylation. It may comprise more than one ⁇ -carboxylation, such as at least 2, at least 3, at least 4 or at least 5.
  • the ⁇ -carboxylated polypeptide in particular comprises at least one recognition motif for a ⁇ -glutamyl carboxylase in its amino acid sequence. Suitable recognition sequences for a ⁇ -glutamyl carboxylase have the amino acid sequence FVTXXXXHXXXH (SEQ ID NO: 1 ) or a similar sequence as described in Presnell and Strafford (2002) Thromb Haemost 87:937-46.
  • the polypeptide is a blood coagulation factor or a mutein thereof.
  • the polypeptide may for example be selected from the group consisting of blood coagulation factors II (prothrombin), VII, IX and X, including the activated forms thereof, the anticoagulant proteins C and S, the factor X-targeting protein Z, osteocalcin, the calcification-inhibiting matrix Gla protein (MGP), the cell growth regulating growth arrest specific gene 6 protein (Gas6), the four transmembrane Gla proteins (TMGPs), and muteins thereof.
  • the polypeptide is selected from the blood coagulation factors II, VII, IX and X or a mutein thereof, in particular the polypeptide is factor VII or factor Vila.
  • the polypeptide may comprise additional N- or C-terminal sequences, including but not limited to the natural export signal peptide or tags for facilitating purification.
  • the amino acid sequences of the naturally occurring polypeptides may be found in public or commercial databases such as BLAST or SwissProt.
  • the host cell having ⁇ -glutamyl carboxylase activity which is used in the method is a mammalian cell, especially a human cell, such as an immortalized human blood cell which may be of myeloid leukemia origin.
  • Suitable host cells are disclosed, for example, in the PCT application WO 2008/028686, herein incorporated by reference.
  • Vertebrate cells generally naturally contain ⁇ -glutamyl carboxylase activity.
  • other host cells may be used wherein the v- glutamyl carboxylase is introduced recombinantly.
  • the ⁇ -carboxylated polypeptide may be recombinantly produced in said host cell.
  • the v- carboxylated polypeptide is secreted by the host cells.
  • the host cell may further comprise a promoter functionally linked to the nucleotide sequence coding for the ⁇ -carboxylated polypeptide.
  • the promoter could be selected from viral promoters, housekeeping host promoters or tissue specific promoters known in the art.
  • the nucleotide sequence coding for the ⁇ - carboxylated polypeptide and optionally the promotor functionally linked thereto are present on a vector. Suitable vectors can be obtained by routine cloning.
  • the vector may contain additional regulatory sequences or sequences for selection markers. In order to achieve secretion of the polypeptide, the vector may additionally contain secretory sequences.
  • the nucleotide sequence coding for the ⁇ -carboxylated polypeptide may be introduced into the host cell by transfection.
  • the host cell may be stably or transiently transfected with the above vector using conditions known in the art.
  • the ⁇ -carboxylated polypeptide may be obtained from the cell culture, e.g. by disrupting the cells or by harvesting the secreted ⁇ -carboxylated polypeptide from the culture medium.
  • the method further comprises the step of purifying the ⁇ -carboxylated polypeptide from the culture medium.
  • the product preferably is secreted by the cells.
  • the method for producing a ⁇ -carboxylated polypeptide comprises the steps of
  • the method may further comprise the step (d) of isolating the ⁇ -carboxylated polypeptide.
  • the ⁇ -carboxylated polypeptide is secreted by the host cells, the ⁇ -carboxylated polypeptide is isolated from the culture medium.
  • the present invention provides the use of cyclodextrin in a method for recombinant expression of a ⁇ -carboxylated polypeptide.
  • the features and embodiments described above with respect to the method according to the invention likewise apply to the use of cyclodextrin in a method for recombinant expression of a ⁇ -carboxylated polypeptide.
  • the present invention provides a cell culture medium comprising cyclodextrin and vitamin K.
  • the cell culture medium may further comprise further components selected from the group consisting of glucose, one or more amino acids, one or more vitamins and one or more trace elements.
  • the cell culture medium can be used for culturing host cells which produce a ⁇ -carboxylated polypeptide.
  • Figure 1 shows (A) cell vitality and (B) factor VII titer of host cells expressing ⁇ - carboxylated factor VII using vitamin K concentrations in the culture medium of 1/5/10/20/50 ⁇ g/ml in complex with cyclodextrin or 5 ⁇ g/ml without cyclodextrin.
  • Figure 2 shows cell vitality and factor VII titer of host cells expressing ⁇ -carboxylated factor VII using vitamin K concentrations in the culture medium of 0.05/0.1/0.2/0.4/0.8 ⁇ g/ml in complex with cyclodextrin or 5 ⁇ g/ml without cyclodextrin.
  • Figure 3 shows a Western blot analysis of the obtained factor VII using (A) a primary antibody against factor VII or (B) a primary antibody against ⁇ -carboxylated polypeptides.
  • Lane 1 molecular weight standard
  • lane 2 control cell culture w/o factor VII
  • lane 3 1 st commercially available factor VII
  • lane 4 1 ⁇ g/ml VitK-CyD
  • lane 5 5 ⁇ g/ml VitK w/o CyD
  • lane 6 0.05 ⁇ g/ml VitK-CyD
  • lane 7 0.1 ⁇ g/ml VitK-CyD
  • lane 8 0.2 ⁇ g/ml VitK-CyD
  • lane 9 0.4 ⁇ g/ml VitK-CyD
  • lane 10 2 nd commercially available factor VII.
  • Cyclodextrin produced by CycloLab (Budapest, Hungary) were loaded with vitamin K1 of Sigma. Best results were obtained with a composition comprising of 0.87% (w/w) vitamin K. Higher loading ratios were less effective.
  • a stock solution of 2.5 mg/ml vitamin K was prepared by dissolving 23 g of the cyclodextrin/vitamin K composition in 80 ml water.
  • a clone of an immortalized human blood cell line producing factor VII was 6x inoculated at 2x10 5 cells/ml in a 6-well plate in cell culture medium comprising 1/5/10/20/50 ⁇ g vitamin K/ml in cyclodextrin (VitK-CyD) or comprising 5 ⁇ g/ml vitamin K without cyclodextrin (conventional method). After 2 days, the cultures were split 1 :10. The cells cultures with the lower vitamin K concentrations showed improved cell viability and factor VII production (see Figure 1 ).

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PCT/EP2016/063107 2015-06-09 2016-06-09 IMPROVED METHOD FOR PRODUCTION OF γ-CARBOXYLATED POLYPEPTIDES WO2016198499A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070968A2 (en) 2000-03-22 2001-09-27 Octagene Gmbh Production of recombinant blood clotting factors in human cell lines
WO2003048313A2 (en) * 2001-11-30 2003-06-12 Invitrogen Corporation Cell culture media
WO2008028686A2 (en) 2006-09-10 2008-03-13 Glycotope Gmbh Use of human cells of myeloid leukaemia origin for expression of antibodies
WO2009146490A1 (en) * 2008-06-03 2009-12-10 John Ray Biffin A method for increasing bone density and/or reducing any osteochondral defects in an animal and a composition including vitamin k

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070968A2 (en) 2000-03-22 2001-09-27 Octagene Gmbh Production of recombinant blood clotting factors in human cell lines
WO2003048313A2 (en) * 2001-11-30 2003-06-12 Invitrogen Corporation Cell culture media
WO2008028686A2 (en) 2006-09-10 2008-03-13 Glycotope Gmbh Use of human cells of myeloid leukaemia origin for expression of antibodies
WO2009146490A1 (en) * 2008-06-03 2009-12-10 John Ray Biffin A method for increasing bone density and/or reducing any osteochondral defects in an animal and a composition including vitamin k

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1980, SZEJTLI J ET AL: "STABILIZATION OF FAT SOLUBLE VITAMINS WITH BETA CYCLO DEXTRIN", XP002252121, retrieved from BIOSIS Database accession no. PREV198171055441 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1988, HORIUCHI Y ET AL.: "Improvement of bioavailability of menaquinone-4 by dimethyl-beta-cyclodextrin complexation following oral administration", XP002750901, Database accession no. PREV198987074177 *
HORIUCHI Y ET AL: "Improvement of bioavailability of menaquinone-4 by dimethyl-[beta]-cyclodextrin complexation following oral administration", YAKUGAKU ZASSHI, vol. 108, no. 11, 1 January 1988 (1988-01-01), pages 1093 - 1100, ISSN: 0331-6903 *
MASROORI N ET AL: "High-level expression of functional recombinant human coagulation factor VII in insect cells", BIOTECHNOLOGY LETTERS, SPRINGER NETHERLANDS, NL, vol. 32, no. 6, 1 June 2010 (2010-06-01), pages 803 - 809, XP002750900, ISSN: 0141-5492, [retrieved on 20100307], DOI: 10.1007/S10529-010-0227-7 *
PRESNELL; STRAFFORD, THROMB HAEMOST, vol. 87, 2002, pages 937 - 946

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