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WO1990002175A1 - Procede de production de polypeptides par culture de cellules eukaryotiques en presence d'inhibiteurs de protease - Google Patents

Procede de production de polypeptides par culture de cellules eukaryotiques en presence d'inhibiteurs de protease Download PDF

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Publication number
WO1990002175A1
WO1990002175A1 PCT/DK1989/000194 DK8900194W WO9002175A1 WO 1990002175 A1 WO1990002175 A1 WO 1990002175A1 DK 8900194 W DK8900194 W DK 8900194W WO 9002175 A1 WO9002175 A1 WO 9002175A1
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WIPO (PCT)
Prior art keywords
medium
cells
fviii
phe
polypeptide
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Application number
PCT/DK1989/000194
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English (en)
Inventor
Karen Hansen
Jan Møller MIKKELSEN
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Novo Nordisk A/S
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from DK457788A external-priority patent/DK457788D0/da
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Publication of WO1990002175A1 publication Critical patent/WO1990002175A1/fr

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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
    • C12P21/02—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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes

Definitions

  • a method of producing polypeptides by culturing eukaryotic cells in the presence of protease inhibitors.
  • the present invention refers to a method of producing a desired polypeptide or protein, such as coagulation inhibitors, fibrinolytic proteins or coagulation factors, among which Factor VIII, FVIII derivatives, FVII, TPA and TPA analogs.
  • a desired polypeptide or protein such as coagulation inhibitors, fibrinolytic proteins or coagulation factors, among which Factor VIII, FVIII derivatives, FVII, TPA and TPA analogs.
  • Factor VIII is a polypeptide, normally present in blood. Lack of FVIII will reduce or prevent the ability of the blood to coagulate, thus causing hemophilia A. Patients with this disease are treated with preparations contain ⁇ ing FVIII.
  • Hemophilia is an X-chromosome-linked inherited disease which normally afflicts 1-2 males per 10,000. The defect is manifested as a bleeding disorder due to the absence of clot formation arising from the lack of Factor X acti ⁇ vation. FVIII by itself is also activated by proteolytic processing. The main enzyme responsible for this activati is thrombin.
  • Factor VIII for therapeutic use has so far been recovered from blood. However, concerns about the transmission of hepatitis and AIDS have intensified activity towards providing alternative supplies. Factor VIII can now also be produced as a recombinant protein in mammalian cells (EP 160457, WO 85/01961, EP 150735).
  • Mammalian cells are usually grown in medium containing serum from various species. Normally the medium contains 10% serum. The recombinant protein is secreted into the culture medium and has to be separated from the serum components afterwards. Serum contains a high amount of protein which impede the purification and diminishes the yield. Therefore purification of FVIII from a less protein rich culture would be preferable. A number of cells will grow in serum free medium. In the absence of serum in the medium the yield and stability of FVIII: are diminished to one tenth to one fifth of the yield obtained in a medium containing serum (WO 87/04197).
  • One object of the invention is to increase the yield of a polypeptide produced by eukariotic cells, such as mammalian cells.
  • the specific object of this invention is to obtain an increased yield of FVIII, produced in mammalian cells.
  • Still another object of the invention is to simplify the isolation and purification of FVIII or another re ⁇ combinant protein from culture medium.
  • the invention provides for a medium suitable for cul- turing mammalian cells that produce biosynthetic poly ⁇ peptides, such as FVIII.
  • the invention is based on the surprising discovery that the presence of some protease inhibitors in the culture medium will stabilize FVIII or another recombinant prote and thus increase the yield. It is also discovered that this stabilizing effect makes it possible to use a maxim free of serum for the production of FVIII in high yield.
  • the coagulation activity of FVIII can be increased con ⁇ siderably compared to the level observed in serum free medium without inhibitor.
  • the level of FVIII:C in serum free medium with inhibitors is up to the level observed in serum containing medium when calculated on a per ml or per cell base.
  • the activity calculated as m ⁇ FVIII:C per mg of protein in the medium is 5 times higher in serum free medium with protease inhibitors than in medium containing 10% newborn calf serum.
  • VWF von Willebrand Factor
  • WO 87/04187 WO 87/04187
  • the yield of FVIII may be increase by adding lipoproteins to the medium (DK Patent Applica ⁇ tion No. 3594/87). Lipoproteins are not protease inhibi ⁇ tors, however, and adding a lipoprotein fraction does not increase the yield of FVIII in CHO cells.
  • the present invention of adding one or a mixture of pro ⁇ tease inhibitors, such as a specific polypeptide provides a simple versatile and inexpensive solution to the problem of stabilizing FVIII in serum free medium.
  • the protease inhibitor may be a peptide having a func- tional group, e.g. a chloromethylketone, an aldehyde, diazomethyl ketone, fluoromethyl ketone, sulfonium salts, or an diazomethane group.
  • a polypeptide like antithrombin III, leupeptin or l 2 -macroglobulin having no such func ⁇ tional group might also provide the protease inhibitoring effect.
  • non-peptide inhibitors such as 3 ,4-dichloroiso- coumarin, L-chloro-3 (4-tosylamido)-7-amino-2-heptanone- HC1 or (4-amidinophenyl )methane sulfonyl fluoride might have a similar effect.
  • protease inhibitors can stabilize full length, frag ⁇ ments as well as subunits of Factor VIII.
  • the inhibitors can be added to the cell culture medium once or several times or continuously during the fermen ⁇ tation period.
  • the preferred production cell usually is a mammalian cell line, such as CHO, COS or cell lines derived from tissue made of secreting cells. These cell lines may be of parotid gland origin e.g. CASA-1, kidney e.g. CAKI-1, liver e.g. SK-HEP-1, lung CALU-2 or endothelian origin.
  • Yeast cells may be used for the production of a subunit of FVIII without coagulation activity to treat inhibitor patients, and this subunit might also be stabilized by using an inhibitor in accordance with the invention. Other cells may be used, however, such as a microorganism.
  • Any known method for cell culture may be used, e.g. suspen ⁇ sion culture, roller bottle, microcarriers or hollow fiber.
  • the medium is preferably a serum free formulation but the inhibitor may also have a stabilizing effect in serum containing medium if the Factor VIII produced is not fully stabilized already, e.g. if the medium contains less than 10% serum.
  • a CHO cell line expressing full length human FVIII was cultured in suspension in D-MEM (Biochrome) supplemented with 1% ITS + (Coll. Res.) and 5% TP (Gibco) to a concen ⁇ tration of 10 cells per ml.
  • the cells were centrifuged and resuspended in 100 ml. of each of the media listed in Table 1.
  • the peptide added was Phe-Phe-Arg-CK (Cal- biochem) . After 24 hours the No. of cells and the FVIII activity (Kabi Coatest chromogene-assay) were determined.
  • FVIII determined as mU per mg protein is much higher in serum free medium because of the high protein content of NCS, approx. 8 mg per ml medium with 10% NCS. In serum free medium there is only 1.26 mg protein per ml.
  • a CHO cell line expressing full lentgh human FVIII was cultured in T-25 flasks in 5 ml of D-MEM containing a low percentage of NCS and supplemented with 10 ,ug/ml of each of 3 peptides: Glu-Gly-Arg-CK, Phe-Phe-Arg-CK, Phe-Pro-Arg-CK.
  • the cells were grown to confluency, the media was changed and the peptides added. After 24 hours the No. of cells and the FVIII activity (Kabi Coatest chromogene-assay) were determined.
  • the low concentration of serum is insufficient to protect the FVIII against degradation and from Table 2 it can be observed that even a low concentration of peptides provides a 50% increase in the FVIII activity.
  • COS-7 cells were transfected with full length FVIII plas- mid pSVF8-303 (a psv 7d (Truett et al., DNA 4:333-349, 1985) derivative encoding the wild type FVIII protein) and the cells were grown to confluency in 5 cm petri dishes at 37°C, the media changed and the peptide Phe- Phe-Arg-CK added in a concentration of 40 / ug/ml. After 24 hours the FVIII activity (Kabi Coatest chromogene- assay) was measured.
  • FVIII plas- mid pSVF8-303 a psv 7d (Truett et al., DNA 4:333-349, 1985) derivative encoding the wild type FVIII protein
  • COS-7 cells were transfected with full length FVIII plas- mid pSVF8-303 (a psv 7d (Truett et al . , DNA 4:333-349, 1985) derivative encoding the wild type FVIII protein) and the cells were grown to confluency in 5 cm petri dishes at 37°C, the medium was changed and the peptide Phe-Phe-Arg-CK was added in a concentration of 40 / ug/ml. After 24 hours the FVIII activity (Kabi Coatest chromo- gene-assay) was measured.
  • FVIII plas- mid pSVF8-303 a psv 7d (Truett et al . , DNA 4:333-349, 1985) derivative encoding the wild type FVIII protein
  • COS-7 cells were transfected with pPR60 (DK Patent Appli ⁇ cation No. 3422/87) and cultured in serum containing medium. The cells were grown to confluency in 5 cm petri dishes at 37°C, the medium was changed and the peptide 5 Phe-Phe-Arg-CK was added in a concentration of 40 / ug/ml. After 24 hours the FVIII activity (Kabi Coatest chromo ⁇ gene-assay) was measured.
  • COS-7 cells were transfected with pPR60 (DK Patent Appli ⁇ cation No. 3422/87) and cultured in serum free medium. The cells were grown to confluency in 5 cm petri dishes 20 at 37°C, the medium was changed and the peptide Phe-Phe- Arg-CK was added in a concentration of 40 ,ug/ml. After 24 hours the FVIII activity (Kabi Coatest chromogene- assay) was measured. TABLE 6
  • COS-7 SV40 transformed African Green monkey kidney cell line Phe-Phe-Arg-CK - D-Phenylalanyl-L-Phenylalanyl-L-Arginin

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Dans le procédé amélioré décrit, qui sert à produire des polypeptides, des cellules eukaryotiques, qui contiennent une structure d'ADN codant pour le polypeptide même, sont cultivées dans un milieu en présence d'un ou de plusieurs inhibiteurs de protéase, tel qu'un polypeptide comportant des groupes fonctionnels. Le milieu doit être de préférence exempt de sérum. Un tel procédé peut en principe être utilisé pour la production de n'importe quel polypeptide de valeur, tel que le facteur VIII:C et des sous-unités, des fragments ou des dérivés du FVIII:C. Un meilleur rendement est ainsi obtenu.
PCT/DK1989/000194 1988-08-16 1989-08-15 Procede de production de polypeptides par culture de cellules eukaryotiques en presence d'inhibiteurs de protease WO1990002175A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DK457788A DK457788D0 (da) 1988-08-16 1988-08-16 Fremgangsmaade til fremstilling af et oensket polypeptid
DK4577/88 1988-08-16
US31207189A 1989-02-16 1989-02-16

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WO1990002175A1 true WO1990002175A1 (fr) 1990-03-08

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993013125A1 (fr) * 1991-12-16 1993-07-08 Novo Nordisk A/S Production de proteines
WO1996040866A1 (fr) * 1995-06-07 1996-12-19 Novartis Ag Milieux sans serum pour des cellules hematopoietiques primaires et procedes d'utilisation
WO1997043436A1 (fr) * 1996-05-14 1997-11-20 Pharmacia & Upjohn Ab Procede pour produire un polypeptide recombine comprenant l'addition d'un inhibiteur de proteases metal-dependantes ou de chymotrypsines au milieu de culture cellulaire
WO1998013475A1 (fr) * 1996-09-25 1998-04-02 Academisch Ziekenhuis Leiden Procedes destines a la culture de cellules
JPH10509144A (ja) * 1994-11-14 1998-09-08 フアーマシア・アンド・アツプジヨン・アー・ベー ▲ｖｉｉｉ▼因子の精製方法
EP1318198A1 (fr) * 1996-05-14 2003-06-11 Biovitrum Ab Procédé pour produire un polypeptide recombine comprenant l'addition d'un inhibiteur de chymotrypsines au milieu de culture cellulaire
US6579698B1 (en) 1996-09-24 2003-06-17 The Procter & Gamble Company Stabilized proteinaceous protease inhibitors and variants thereof
WO2008102923A1 (fr) 2007-02-23 2008-08-28 Sk Chemicals Co., Ltd. Méthode de production et de purification du facteur viii et de ses dérivés
WO2008135501A1 (fr) 2007-05-04 2008-11-13 Novo Nordisk A/S Amélioration de titres de polypeptide du facteur viii dans des cultures cellulaires
WO2012045769A1 (fr) 2010-10-05 2012-04-12 Novo Nordisk Health Care Ag Procédé de production de protéine
WO2018194413A1 (fr) 2017-04-21 2018-10-25 Yuhan Corporation Procédé de production de protéines à double fonction et ses dérivés
US11136364B2 (en) 2015-10-28 2021-10-05 Yuhan Corporation Dual function proteins comprising FGF21 mutant protein and pharmaceutical composition comprising same
US11142557B2 (en) 2015-10-28 2021-10-12 Yuhan Corporation Long-acting FGF21 fusion proteins and pharmaceutical composition comprising same
US11179440B2 (en) 2016-11-10 2021-11-23 Yuhan Corporation Pharmaceutical composition containing FGF21 mutant fusion protein and method for treating hepatitis, hepatic fibrosis, and hepatic cirrhosis

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0133070A2 (fr) * 1983-06-29 1985-02-13 Choay S.A. Procédé de production d'un activateur tissulaire du plasminogène et activateur obtenu par ce procédé
EP0140127A1 (fr) * 1983-09-20 1985-05-08 F. Hoffmann-La Roche Ag Procédé pour la préparation d'interféron-immun
WO1987001389A1 (fr) * 1985-09-06 1987-03-12 Codon Procedes de recuperation d'un activateur de plasminogene des tissus
EP0306968A2 (fr) * 1987-09-10 1989-03-15 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Procédé de production de facteur VIII:C humain recombinant

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0133070A2 (fr) * 1983-06-29 1985-02-13 Choay S.A. Procédé de production d'un activateur tissulaire du plasminogène et activateur obtenu par ce procédé
EP0140127A1 (fr) * 1983-09-20 1985-05-08 F. Hoffmann-La Roche Ag Procédé pour la préparation d'interféron-immun
WO1987001389A1 (fr) * 1985-09-06 1987-03-12 Codon Procedes de recuperation d'un activateur de plasminogene des tissus
EP0306968A2 (fr) * 1987-09-10 1989-03-15 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Procédé de production de facteur VIII:C humain recombinant

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 108, (1988), abstract No. 101185t; & Nippon Ketsueki Gakkai Zasshi 1987, 50(6), 1239-45 (Eng). *
CHEMICAL ABSTRACTS, Vol. 110, (1989), abstract No. 191184k, MIRCHEN, J. APPL. MICROBIOL. BIOTECHNOL. 1988, 4(4), 407-14 (Eng). *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5371198A (en) * 1991-12-16 1994-12-06 Novo Nordisk A/S Method for protection of proteolysis-susceptible protein during protein production in a fluid medium
WO1993013125A1 (fr) * 1991-12-16 1993-07-08 Novo Nordisk A/S Production de proteines
JPH10509144A (ja) * 1994-11-14 1998-09-08 フアーマシア・アンド・アツプジヨン・アー・ベー ▲ｖｉｉｉ▼因子の精製方法
US5831026A (en) * 1994-11-14 1998-11-03 Pharmacia & Upjohn Ab Process for purifying factor VIII
WO1996040866A1 (fr) * 1995-06-07 1996-12-19 Novartis Ag Milieux sans serum pour des cellules hematopoietiques primaires et procedes d'utilisation
WO1997043436A1 (fr) * 1996-05-14 1997-11-20 Pharmacia & Upjohn Ab Procede pour produire un polypeptide recombine comprenant l'addition d'un inhibiteur de proteases metal-dependantes ou de chymotrypsines au milieu de culture cellulaire
US5851800A (en) * 1996-05-14 1998-12-22 Pharmacia & Upjohn Ab Process for producing a protein
EP1318198A1 (fr) * 1996-05-14 2003-06-11 Biovitrum Ab Procédé pour produire un polypeptide recombine comprenant l'addition d'un inhibiteur de chymotrypsines au milieu de culture cellulaire
US6579698B1 (en) 1996-09-24 2003-06-17 The Procter & Gamble Company Stabilized proteinaceous protease inhibitors and variants thereof
EP0834556A1 (fr) * 1996-09-25 1998-04-08 Academisch Ziekenhuis Leiden Procédés de culture de cellules
WO1998013475A1 (fr) * 1996-09-25 1998-04-02 Academisch Ziekenhuis Leiden Procedes destines a la culture de cellules
WO2008102923A1 (fr) 2007-02-23 2008-08-28 Sk Chemicals Co., Ltd. Méthode de production et de purification du facteur viii et de ses dérivés
US9441030B2 (en) 2007-02-23 2016-09-13 Sk Chemicals Co., Ltd. Process for producing and purifying factor VIII and its derivatives
US9926361B2 (en) 2007-02-23 2018-03-27 Sk Chemicals Co., Ltd. Process for producing and purifying factor VIII and its derivatives
EP3112471A1 (fr) 2007-02-23 2017-01-04 Sk Chemicals Co., Ltd. Méthode de production et de purification du facteur viii et de ses dérivés
WO2008135501A1 (fr) 2007-05-04 2008-11-13 Novo Nordisk A/S Amélioration de titres de polypeptide du facteur viii dans des cultures cellulaires
US9982033B2 (en) 2007-05-04 2018-05-29 Novo Nordisk A/S Factor VIII polypeptide titers in cell cultures
EP2957628A1 (fr) 2010-10-05 2015-12-23 Novo Nordisk Health Care AG Procédé de production de protéine
WO2012045769A1 (fr) 2010-10-05 2012-04-12 Novo Nordisk Health Care Ag Procédé de production de protéine
US10138290B2 (en) 2010-10-05 2018-11-27 Novo Nordisk Healthcare Ag Process for protein production
US11136364B2 (en) 2015-10-28 2021-10-05 Yuhan Corporation Dual function proteins comprising FGF21 mutant protein and pharmaceutical composition comprising same
US11142557B2 (en) 2015-10-28 2021-10-12 Yuhan Corporation Long-acting FGF21 fusion proteins and pharmaceutical composition comprising same
US11179440B2 (en) 2016-11-10 2021-11-23 Yuhan Corporation Pharmaceutical composition containing FGF21 mutant fusion protein and method for treating hepatitis, hepatic fibrosis, and hepatic cirrhosis
WO2018194413A1 (fr) 2017-04-21 2018-10-25 Yuhan Corporation Procédé de production de protéines à double fonction et ses dérivés
KR20180118550A (ko) 2017-04-21 2018-10-31 주식회사유한양행 이중 작용 단백질 및 그의 유도체의 제조방법
US11560416B2 (en) 2017-04-21 2023-01-24 Yuhan Corporation Method for producing dual function proteins and its derivatives

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