EP1554389A2 - Procede de fabrication de proteines recombinantes - Google Patents

Procede de fabrication de proteines recombinantes

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Publication number
EP1554389A2
EP1554389A2 EP03797190A EP03797190A EP1554389A2 EP 1554389 A2 EP1554389 A2 EP 1554389A2 EP 03797190 A EP03797190 A EP 03797190A EP 03797190 A EP03797190 A EP 03797190A EP 1554389 A2 EP1554389 A2 EP 1554389A2
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EP
European Patent Office
Prior art keywords
seq
mar
mar elements
expression
factor
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
EP03797190A
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German (de)
English (en)
Inventor
Ruth Buemann Simesen
Jan Eric Jensen
Anette Amstrup Pedersen
Steffen Faisst
Dietmar Weilguny
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Novo Nordisk AS
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Novo Nordisk AS
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Publication of EP1554389A2 publication Critical patent/EP1554389A2/fr
Withdrawn legal-status Critical Current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)
    • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21021—Coagulation factor VIIa (3.4.21.21)
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/46—Vector systems having a special element relevant for transcription elements influencing chromatin structure, e.g. scaffold/matrix attachment region, methylation free island
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

  • S/MARs positively interact with enhancers, form loop domains and often are located at the borders of transcrip- tionally active domains have led to the idea of using S/MARs as flanking elements around transgenes, forming so called mini-domains, in order to protect transgenes or expression cassettes from transcriptional silencing and the effects of surrounding heterochromatin (tran- scriptionally inactive chromatin) as well as possibly increase gene expression.
  • S/MARs in a flanking position can strongly stimulate expression of transgenes, as well as reduce expression variability between cell clones (position effects).
  • expression should be independent from the integration site.
  • the present invention is based on the discovery of new S/MAR elements from hamster-derived CHO and BHK cells, which may be used for increasing and stabilizing the expression yield of recombinant proteins in mammalian cells.
  • the S/MAR elements are thought to increase the genetic stability of nearby transcription cassettes and to inhibit gene silencing tby interfering with mechanisms such as DNA methylation and histone deacetylation.
  • the presence of S/MAR elements is thought to decrease clone-to-clone variability through decreasing position effects, thereby minimalizing the need of screening work to identify high-yielding producer cell clones.
  • the vector comprises two S/MAR elements flanking the DNA en- coding Factor VII or a Factor Vll-related polypeptide and the expression control regions.
  • the S/MAR elements may be selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, functional fragments thereof and sequences being at least 70% homologous thereto as determined by pair wise DNA sequence alignment using matching methods like the BLAST (Basic Local Alignment Search Tool) algorithm (Altschul, S. F. et al., 1990, "Basic local alignment search tool". J. Mol. Biol. 215:403-410; and Altschul, S. F. et al., 1997, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”. Nucl. Acids Res. 25:3389-3402).
  • BLAST Basic Local Alignment Search Tool
  • the S/MAR elements will be at least 75%; 80%, 85%, 90% or 95% homologous to SEQ ID NO:1 or 2, respectively as determined by pair wise DNA se- quence alignment using matching methods like the BLAST (Basic Local Alignment Search Tool) algorithm (Altschul, S. F. et al., 1990, "Basic local alignment search tool”. J. Mol. Biol. 215:403-410; and Altschul, S. F. et al., 1997, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”. Nucl. Acids Res. 25:3389-3402).
  • BLAST Basic Local Alignment Search Tool
  • S/MAR sequences homologuos to SEQ ID NO:1 and 2 are SEQ ID NO:3; 4 and 5.
  • the S/MAR elements may be the same or different and may be located in a distance of from 0 to10 kb from the DNA encoding Factor VII or a Factor Vll-related polypeptide and the expression control regions.
  • the vector comprises two S/MAR elements which are different from each other with respect to base pairs and numbers.
  • the pair of S/MAR elements can be any combination of SEQ ID NO:1; 2; 3; 4; and 5.
  • the present invention is related to an isolated DNA molecule having a nucleotide sequence selected from the group consisting of SEQ ID NO:1 and 2 and sequences being at least 70% homologous thereto or sequences that hybridize to the isolated DNA under stringent conditions.
  • the present invention is related to mammalian cell lines transfected with an expression vector comprising a nucleic acid molecule encoding Factor VII or a Factor Vll-related polypeptide, expression control regions operatively linked to thereto and at least one S/MAR element.
  • Figure 1 shows plasmid pSEAP2-Hygro-MCS
  • Figure 3 shows plasmid pB4B1-SEAP2-B4B1 ;
  • Figure 4 shows plasmid pLRCA2-SEAP2-LRCA2
  • Figure 5 shows plasmid delta-LRCA2-SEAP2-LRCA2
  • Figure 8 shows plasmid B4B1-FVII-HYG-B4B1
  • S/MAR elements were isolated from BHK cells and CHO cells, e.g. CHO cell line DG44 (Urlaub, G. et al., 1983, "Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells". Cell 33:405-412). Methods for identifying S/MAR elements are computer prediction using MAR-
  • DNA wherein the nucleotide sequence is least 70% homologous to a defined sequence measured by pairwise DNA sequence alignment using matching methods like the BLAST (Basic Local Alignment Search Tool) algorithm (Altschul, S. F. et al., 1990, "Basic local alignment search tool”. J. Mol. Biol. 215:403-410; and Altschul, S. F. et al., 1997, "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”. Nucl. Acids Res. 25:3389-3402).
  • BLAST Basic Local Alignment Search Tool
  • the term functional fragments of SEQ ID NO:1 and 2 as used herein means fragments of said sequences of a size large enough to have the desired effect on expression yields.
  • the functional fragments will typically contain at least 300 bp of the full size of SEQ ID NO:1 and 2.
  • the fragments will preferably be a consecutive sequence of the original sequence. Examples of such fragments are SEQ ID NO:3 from bp 543 to bp 2545 of SEQ ID NO:1; SEQ ID NO:4 from bp 437 to bp 2715 of SEQ ID NO:1 and SEQ ID NO:5 having a 12 base pair deletion in the GT rich region from bp 1551 to 1604 in SEQ ID NO:1.
  • control elements will comprise the usual regulatory elements such as transcrip- tional promoters, enhancers, RNA polymerase binding sites, polyadenylation sites, translation initiation signals, and termination signals and may be readily accomplished by one of ordinary skill in the art.
  • the polypeptide or protein may be any polypeptide or protein, e.g.
  • the protein may be structurally similar to the native protein and may be derived from the native protein by addition of one or more amino acids to either or both the C- and N-terminal end of the native protein, substitution of one or more amino acids at one or a number of different sites in the native amino acid sequence, deletion of one or more amino acids at either or both ends of the native protein or at one or several sites in the amino acid sequence, or insertion of one or more amino acids at one or more sites in the native amino acid sequence.
  • Factor Vll polypeptides polypeptides having the amino acid sequence disclosed in U.S. Patent No. 4,784,950 (wild-type Factor Vll).
  • Factor Vll or “Factor Vll polypeptide” encompasses wild-type Factor Vll, as well as variants of Factor Vll exhibiting substantially the same or improved biological activity relative to wild-type Factor Vll.
  • the term “Factor VII” is intended to encompass Factor Vll polypeptides in their uncleaved (zymogen) form, as well as those that have been proteolytically processed to yield their respective bioactive forms, which may be designated Factor Vila.
  • Factor Vll is cleaved between residues 152 and 153 to yield Factor Vila.
  • Vector Vll-related polypeptides encompasses polypeptides, including variants, in which the Factor Vila biological activity has been substantially modified or reduced relative to the activity of wild-type Factor Vila.
  • These polypeptides include, without limitation, Factor Vll or Factor Vila that has been chemically modified and Factor Vll variants into which specific amino acid sequence alterations have been introduced that modify or disrupt the bioactivity of the polypeptide.
  • Factor Vila The biological activity of Factor Vila in blood clotting derives from its ability to (i) bind to tissue factor (TF) and (ii) catalyze the proteolytic cleavage of Factor IX or Factor X to produce activated Factor IX or X (Factor IXa or Xa, respectively).
  • Factor Vila biological activity may be quantified by measuring the ability of a preparation to promote blood clotting using Factor Vll-deficient plasma and throm- boplastin, as described, e.g., in U.S. Patent No. 5,997,864.
  • Factor Vila biological activity is expressed as the reduction in clotting time relative to a control sample and is converted to "Factor Vll units" by comparison with a pooled human serum standard con- taining 1 unit/ml Factor Vll activity.
  • Factor Vila biological activity may be quantified by (i) measuring the ability of Factor Vila to produce of Factor Xa in a system comprising TF embedded in a lipid membrane and Factor X.
  • Factor Vll variants having substantially the same or improved biological activity relative to wild-type Factor Vila encompass those that exhibit at least about 25%, pref- erably at least about 50%, more preferably at least about 75% and most preferably at least about 90% of the specific activity of wild-type Factor Vila that has been produced in the same cell type, when tested in one or more of a clotting assay, proteolysis assay, or TF binding assay as described above.
  • Factor Vll variants having substantially reduced biological activity relative to wild-type Factor Vila are those that exhibit less than about 25%, preferably less than about 10%, more preferably less than about 5% and most preferably less than about 1% of the specific activity of wild-type Factor Vila that has been produced in the same cell type when tested in one or more of a clotting assay, proteolysis assay, or TF binding assay as described above.
  • Factor Vll variants having a substantially modified biological activity relative to wild-type Factor Vll include, without limitation, Factor Vll variants that exhibit TF-independent Factor X proteolytic activity and those that bind TF but do not cleave Factor X.
  • FVII variants as disclosed in PCT/DK02/00189; and FVII variants exhibiting increased proteolytic stability as disclosed in WO 02/38162 (Scripps Research Institute); FVII variants having a modified Gla-domain and ex- hibiting an enhanced membrane binding as disclosed in WO 99/20767 (University of Minnesota); and FVII variants as disclosed in WO 01/58935 (Maxygen ApS).
  • the vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extra chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is preferably an expression vector in which the encoding DNA sequence is operably linked to additional segments required for transcription of the DNA.
  • suitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39), HEK 293 (ATCC CRL 1573) or CHO (ATCC CCL 61) cell lines.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the encoding DNA in mammalian cells are the SV40 promoter (Subramani, S. et al., 1981 ,
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g. the SV40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
  • polyadenylation signals e.g. from SV40 or the adenovirus 5 Elb region
  • transcriptional enhancer sequences e.g. the SV40 enhancer
  • translational enhancer sequences e.g. the ones encoding adenovirus VA RNAs
  • the vector may comprise a DNA sequence enabling it to replicate in the host cell in question.
  • a DNA sequence enabling it to replicate in the host cell in question.
  • An example of such a sequence in a mammalian cell is the SV40 origin of replication.
  • Separation of the expressed product from the cell culture may be achieved by any method known in the art, including, without limitation, removal of cell culture medium containing the desired product from an adherent cell culture; centrifugation or filtration to remove non-adherent cells; and the like. Purification of the crude product may be achieved using any method known in the art, including, without limitation, affinity chromatography. In the case of Factor Vll or FVII related products one or more of the following methods may be used: an anti-Factor Vll antibody column (see, e.g., Wakabayashi et al., J. Biol. Chem. 261:11097, 1986; and Thim et al., Biochem.
  • Factor Vll and Factor Vll-related polypeptides may be activated by proteolytic cleavage, using Factor Xlla or other proteases having trypsin-like specificity, such as, e.g., Factor IXa, kallikrein, Factor Xa, and thrombin. See, e.g., Osterud et al., Biochem. 11:2853 (1972); Thomas, U.S. Patent No. 4,456,591 ; and Hedner et al., J. Clin. Invest. 71 :1836 (1983).
  • Factor Vll may be activated by passing it through an ion-exchange chromatography column, such as Mono Q® (Pharmacia) or the like. The resulting activated Factor Vll may then be formulated and administered as described below.
  • a 2.7 kb fragment was cloned from EcoRV digested CHO DNA (CHO cell line DG44) by use of DNA oligonucleotides CLC394 AAAACTGGGAAC- CATTTGTG (SEQ ID NO:9) and CLC56LCTGCAGAAGAGGCGACAG (SEQ ID NO: 10) and the PCR-Select kit (CLONTECH).
  • CLC394L and CLC56L are complementary to the CHO cyclophilin cDNA sequence (Genbank Accession no. X17105).
  • Example 2 Cloning and characterization of SEQ ID NO: 2, B4B1 A recombinant Baby Hamster Kidney cell line expressing a recombinant protein was analyzed for the chromosomal DNA encompassing the integrated plasmid DNA.
  • chromosomal DNA of the recombinant BHK cell line has been isolated by standard methods. The isolated DNA became subsequently subject of restriction enzyme digest. The restriction enzymes used were characterized as enzymes that did not digest sequences present within the integrated plasmid DNA. Such a restriction digest will give rise to DNA fragments containing all of the integrated plasmid and both up- and downstream flanking chromosomal DNA.
  • TAAGCTTGATCTGACGGTTCACTAAAC (SEQ ID NO:8) were used to generate a 0.6 kb
  • pB4B1-SEAP2 and pSEAP2-B4B1 were cut with Hind ⁇ and Age ⁇ and the B4B1 containing DNA fragments from these digests were ligated to generate pB4B1-SEAP2-B4B1.
  • the plasmids express a therapeutic protein coagulation factor Vll (FVII), from an expression cassette that is or is not flanked by S/MAR ele- ments.
  • FVII therapeutic protein coagulation factor Vll
  • the modified ⁇ LRCA2-SEAP2-LRCA2 vector was afterwards digested with Apa ⁇ and Nhel and the 7.0 kb LRCA2 fragment ligated to the 4.4 kb Nhel/Apal fragment from vector pFVII-CMVp ( Figure 6) thus generating vector ⁇ LRCA2-FV I I-HYG-LRCA2 ( Figure 7).
  • CHO-K1 cells ATCC CCL-61
  • growth medium Dulbecco's modified Eagle's medium, 10% fetal calf serum, 100 IU penicillin and streptomycin, non-essential amino acids, and 5 mg/l vitamin K1
  • Fugene 6 transfection reagent as per manufacturer's instructions (Roche, Basel, Switzerland).
  • Stable pools of transfectants were obtained by Hygromycin selection as per manufacturer's instructions (Invitrogen, Carlsbad, CA).
  • FVII protein yields in the culture medium were determined by standard sandwich ELISA technique (Novo Nordisk), well known to persons skilled in the art. From the pools of transfectants, a limiting dilution cloning was performed.
  • LRCA2 leads to decreased variation in expression levels between different founder clones.
  • the median values of the FVII expression levels are also shown where the expression levels obtained from the pCMV-FVII-HYG construct was set to 100% and the others calculated in relation to that. These values show that flanking of the Factor FVII expression construct by either B4B1 or LRCA2 leads to increased FVII expression levels.
  • Chinese Hamster Ovary (CHO) DG44 cells maintained in MEM Alpha medium (Invi- trogen, Cat # 22571) supplemented with 5% heat inactivated fetal bovine serum (Invitrogen), 108 mg/L L-proline (Sigma), and penicillin (100 units/ml)/streptomycin (100 ⁇ g/ml) (Invitro- gen) at 37°C and 5% C0 2 were transfected using the GeneJammer transfection agent (Stratagene) according to the manufactures instructions. Briefly, cells seeded in 6-weII cell culture plates were approximately 40-50% confluent on the day of transfection and transfected with 2 ⁇ g of linearized plasmid DNA.
  • each cell culture was washed with PBS, trypzined and seeded to approximately 30% confluency in 200 ⁇ l growth medium in five wells in a 96-well cell culture plate. After 24 hours of growth at 37°C and 5% C0 2 100 ⁇ l culture supernatant was withdrawn from each micro titer plate well and used for assaying Secreted Alkaline Phosphatase (SEAP) activity. In order to be able to measure specific SEAP production levels each well was assayed for relative cell numbers using Alamar Blue (BioSource International) cell proliferation assay according to the manufactures instructions.
  • SEAP Secreted Alkaline Phosphatase
  • SEAP levels were measured fluorometrically using 4-methylumbelliferyl as substrate. Briefly, the harvested medium was incubated at 65°C for 15 min to inactivate endogenous alkaline phosphatase and the medium was clarified by centrifugation at 4000 x g for 5 min at 4°C. 50 ⁇ l of culture supernatant and 2- to 10-fold diluted samples was pippeted into 96-well plates and development in fluorescence (Ex 36o n m and Em 450 folkm) was followed as a function of time in a FLUOstar Galaxy (BMG Labtechnologies, Germany) fluorescence mi- croplate reader after addition of 225 ⁇ l of 4-methylumbelliferyl phosphate liquid substrate (Sigma).
  • the following method can be used to assay Factor Vila bioactivity.
  • the assay is car- ried out in a microtiter plate (MaxiSorp, Nunc, Denmark).
  • the absorbance at 405 nm is measured continuously in a SpectraMaxTM 340 plate reader (Molecular Devices, USA).
  • the ab- sorbance developed during a 20-minute incubation, after subtraction of the absorbance in a blank well containing no enzyme, is used to calculate the ratio between the activities of a test and a reference Factor Vila.
  • Example 7 In Vitro Proteolysis Assay The following method can be used to assay Factor Vila bioactivity. The assay is carried out in a microtiter plate (MaxiSorp, Nunc, Denmark). Factor Vila (10 nM) and Factor X (0.8 microM) in 100 ⁇ l 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 5 mM CaCI 2 and 1 mg/ml bovine serum albumin, are incubated for 15 min. Factor X cleavage is then stopped by the addition of 50 ⁇ l 50 mM Hepes, pH 7.4, containing 0.1 M NaCI, 20 mM EDTA and 1 mg/ml bovine serum albumin.

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Abstract

L'invention concerne des régions de fixation de matrice/échafaudage (S/MAR) isolée de cellules de hamster et leur utilisation dans la production industrielle de protéines et de polypeptides recombinants. Dans l'un des modes de réalisation de l'invention, un procédé de production de polypeptides de facteur VII ou lié au facteur VII consiste: a) à transfecter une cellule mammalienne avec un vecteur d'expression comprenant une molécule d'acide nucléique codant le polypeptide de facteur VII ou lié au facteur VII, des régions de commande d'expression liées fonctionnelles à ce polypeptide et au moins un élément S/MAR; b) à cultiver la cellule transfectée dans des conditions permettant l'expression du polypeptide de facteur VII ou lié au facteur VII et c) à isoler le polypeptide exprimé avec des moyens adéquats.
EP03797190A 2002-09-20 2003-09-19 Procede de fabrication de proteines recombinantes Withdrawn EP1554389A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DKPA200201384 2002-09-20
DK200201384 2002-09-20
US41656602P 2002-10-07 2002-10-07
US416566P 2002-10-07
PCT/DK2003/000607 WO2004027072A2 (fr) 2002-09-20 2003-09-19 Procede de fabrication de proteines recombinantes

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EP1554389A2 true EP1554389A2 (fr) 2005-07-20

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003290453A1 (en) 2002-12-20 2004-07-14 Chromagenics B.V. Means and methods for producing a protein through chromatin openers that are capable of rendering chromatin more accessible to transcription factors
BRPI0517380A (pt) 2004-11-08 2008-10-07 Chromagenics Bv molécula de dna, cassete de expressão, célula hospedeira, e, métodos para gerar uma célula hospedeira que expressa um polipeptìdeo de interesse e para produzir um polipeptìdeo de interesse
US8999667B2 (en) 2004-11-08 2015-04-07 Chromagenics B.V. Selection of host cells expressing protein at high levels
US20060195935A1 (en) 2004-11-08 2006-08-31 Chromagenics B.V. Selection of host cells expressing protein at high levels
US8039230B2 (en) 2004-11-08 2011-10-18 Chromagenics B.V. Selection of host cells expressing protein at high levels
EP1996705B1 (fr) 2006-03-20 2011-08-31 ChromaGenics B.V. Système d'expression permettant d'augmenter des fragments d'adn, son utilisation, et procédés permettant de le trouver
EP3382029B1 (fr) * 2017-03-31 2021-12-08 Toto Ltd. Cellules recombinantes de mammifères et procédé de production d'une substance d'intérêt

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US5888774A (en) * 1994-12-19 1999-03-30 Cangene Corporation Recombinant DNA molecules and expression vectors for erythropoietin
DE19848017A1 (de) * 1998-10-17 2000-04-20 Multigene Biotech Gmbh Episomal replizierender Vektor zur Expression eines Transgens in Säugerzellen
KR100408844B1 (ko) * 2000-07-29 2003-12-06 한국산업기술평가원 동물세포 발현벡터

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AU2003263161A8 (en) 2004-04-08
AU2003263161A1 (en) 2004-04-08
WO2004027072A3 (fr) 2004-05-13

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