CA2252972A1 - Serum-free production of recombinant proteins and adenoviral vectors - Google Patents

Serum-free production of recombinant proteins and adenoviral vectors Download PDF

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Publication number
CA2252972A1
CA2252972A1 CA002252972A CA2252972A CA2252972A1 CA 2252972 A1 CA2252972 A1 CA 2252972A1 CA 002252972 A CA002252972 A CA 002252972A CA 2252972 A CA2252972 A CA 2252972A CA 2252972 A1 CA2252972 A1 CA 2252972A1
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Prior art keywords
serum
cell line
free
recombinant proteins
recombinant
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Granted
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CA002252972A
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French (fr)
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CA2252972C (en
Inventor
M. T. Johanne Côte
Amine A. Kamen
Bernard Massie
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National Research Council of Canada
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National Research Council of Canada
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Priority to CA2252972A priority Critical patent/CA2252972C/en
Publication of CA2252972A1 publication Critical patent/CA2252972A1/en
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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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10351—Methods of production or purification of viral material

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a method of serum-free production of recombinant proteins and adenoviral vectors; cell lines viable in a serum-free medium and method of obtaining same.

Claims (14)

1. A serum-free medium viable cell line, which comprises an immortalized animal cell line for the expression of recombinant proteins and/or adenoviral vectors in a serum-free medium.
2. The cell line of claim 1, wherein said animal is mammal.
3. The cell line of claim 2, wherein said mammal is human.
4. The cell line of claim 3, wherein said human cell line is a human embryonic kidney cell line.
5. The cell line of claim 4, wherein said cell line is 293SF-3F6 having ATCC accession number ATCC
CRL-12585.
6. Use of the cell lines of claims 1 to 5 for establishing stable transfected cell line or for production of adenoviral vectors required for gene therapy.
7. A method of serum-free production of recombinant proteins, which comprises the steps of:
a) transfecting a serum-free cell line of claims 1 to 6 with a cDNA coding for a recombinant protein to obtain transformants;
b) isolating a stable transformant of step a);
c) cultivating said isolated transformant of step b) in suspension in a serum-free medium to produce said recombinant proteins; and d) isolating said recombinant proteins of step c).
8. The method of claim 7, wherein said recombinant protein is selected from the group consisting of cyto-kines, G-protein coupled receptors, hormones and enzymes.
9. The method of claim 7, wherein step b) is effected using a selection marker.
10. A method of serum-free production of recombinant adenoviral vectors, which comprises the steps of:
a) transforming a serum-free cell line of claims 1 to 6, which is complementing defective of an adenoviral vector, with a recombinant adenoviral vector comprising a promoter operatively linked upstream to a marker gene and/or a therapeutic gene sequence relative to the direction of transcription in an adenoviral vector, wherein said promoter is controlling transcription of said marker gene and/or said therapeutic gene, wherein said cell line is complementing said recombinant adenoviral vector;
b) isolating a stable cell line of step a) using said marker; and c) cultivating said isolated cells of step b) in suspension in a serum-free medium to produce said recombinant adenoviral vector.
11. The method of claim 10, wherein said marker is green fluorescent protein, .beta.-galactosidase or luciferase.
12. The method of claim 11, wherein said vector of step a) is Ad5 CMV-LacZ wherein LacZ gene is replaced by S65T GFP mutant gene, thereby having an increased emission signal intensity detectable in single cells by fluorescent microscopy or FCM
13. A method of obtaining cell lines for the expression of recombinant proteins and/or adenoviral vectors and which are viable in a serum-free medium, which comprises the steps of:
a) incubating immortalized cells secreting recombinant proteins and/or adenoviral vectors in a serum-free medium and comparing production of recombinant proteins and/or adenoviral vectors as compared to serum-containing medium;
b) gradually adapting the immortalized serum-free cells of step a) to a low-calcium version of said serum-free medium, thereby reducing cell aggregation; and c) screening and cloning cells of step b) based on their ability to grow in a serum-free medium at a rate and an extent comparable to parental cells in serum-containing medium as single cells.
14. The method of claim 13, wherein the cells of step c) are screened for their ability to grow at the highest specific cell growth rate and cell density while forming least and smallest cell aggregates in shake flask suspension culture.
CA2252972A 1998-11-26 1998-11-26 Serum-free production of recombinant proteins and adenoviral vectors Expired - Lifetime CA2252972C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CA2252972A CA2252972C (en) 1998-11-26 1998-11-26 Serum-free production of recombinant proteins and adenoviral vectors

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CA2252972A CA2252972C (en) 1998-11-26 1998-11-26 Serum-free production of recombinant proteins and adenoviral vectors

Publications (2)

Publication Number Publication Date
CA2252972A1 true CA2252972A1 (en) 2000-05-26
CA2252972C CA2252972C (en) 2012-09-18

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Family Applications (1)

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CA2252972A Expired - Lifetime CA2252972C (en) 1998-11-26 1998-11-26 Serum-free production of recombinant proteins and adenoviral vectors

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CA (1) CA2252972C (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6447995B1 (en) 2000-10-04 2002-09-10 Genvec, Inc. Utilizing intrinsic fluorescence to detect adenovirus
CN101233238A (en) * 2005-06-30 2008-07-30 奥克塔法马股份有限公司 Serum-free stable transfection and production of recombinant human proteins in human cell lines
US9260695B2 (en) 2004-03-05 2016-02-16 Dpx Holdings B.V. Process for cell culturing by continuous perfusion
US9833505B1 (en) 2016-07-18 2017-12-05 Variation Biotechnologies Inc. Vaccine compositions for treatment of Zika virus
WO2021048081A1 (en) 2019-09-09 2021-03-18 Glaxosmithkline Biologicals Sa Immunotherapeutic compositions

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6630299B2 (en) 2000-10-04 2003-10-07 Genvec, Inc. Fluorescence detection
US6447995B1 (en) 2000-10-04 2002-09-10 Genvec, Inc. Utilizing intrinsic fluorescence to detect adenovirus
US9260695B2 (en) 2004-03-05 2016-02-16 Dpx Holdings B.V. Process for cell culturing by continuous perfusion
US9273325B2 (en) 2005-06-30 2016-03-01 Carola Schroeder Serum-free stable transfection and production of recombinant human proteins in human cell lines
US8871439B2 (en) 2005-06-30 2014-10-28 Octapharma Ag Serum-free stable transfection and production of recombinant human proteins in human cell lines
RU2453597C2 (en) * 2005-06-30 2012-06-20 Октафарма Биофармасьютикал ГмбХ Method for producing immortalised human cell, stable transfected immortalised human cell, method for recombinant production of target human protein, application of transfection vector
CN101233238A (en) * 2005-06-30 2008-07-30 奥克塔法马股份有限公司 Serum-free stable transfection and production of recombinant human proteins in human cell lines
EP1896590B1 (en) * 2005-06-30 2016-03-23 Octapharma AG Serum-free stable transfection and production of recombinant human proteins in human cell lines
CN105755041A (en) * 2005-06-30 2016-07-13 奥克塔法马股份有限公司 Serum-free stable transfection and production of recombinant human proteins in human cell lines
US9796986B2 (en) 2005-06-30 2017-10-24 Octapharma Ag Serum-free stable transfection and production of recombinant human proteins in human cell lines
NO343221B1 (en) * 2005-06-30 2018-12-10 Octapharma Ag Serum-free stable transfection and production of recombinant human proteins in human cell lines
US9833505B1 (en) 2016-07-18 2017-12-05 Variation Biotechnologies Inc. Vaccine compositions for treatment of Zika virus
WO2018014113A1 (en) 2016-07-18 2018-01-25 Variation Biotechnologies Inc. Vaccine compositions for treatment of zika virus
US10456460B2 (en) 2016-07-18 2019-10-29 Variation Biotechnologies Inc. Vaccine compositions for treatment of Zika virus
WO2021048081A1 (en) 2019-09-09 2021-03-18 Glaxosmithkline Biologicals Sa Immunotherapeutic compositions

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Publication number Publication date
CA2252972C (en) 2012-09-18

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