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Optimization of cell culture conditions for production of biologically active proteins

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Abstract

We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells. Namalwa cells (Klein, 1972), human B lymphoblastoid cells, were used for large scale production of alpha-interferon (Klein, 1979). Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 × 107 cells/ml in suspension mode by the use of a perfusion culture system, Biofermenter™, containing a cone-type cell-sedimentation column as cell separator (Sato, 1983).

Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells (Miyaji, 1990a,b,c). Some of these were produced in large quantities by use of a gene amplification method with dhfr (Miyaji, 1990c), even though the Namalwa KJM-1 cells contained endogenous dhfr genes. For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium.

Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.

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Abbreviations

AP:

Ampicillin-resistance gene

BCIP:

5-Bromo-4-Chrolo-3-Indoyl Phosphonate

BSA:

Bovine Serum Albumin

dhfr:

dihydrofolate reductase

FCS:

Fetal Calf Serum

G-CSF:

Granulocyte-Colony Stimulating Factor

GS:

Glutamine Synthetase

HEPES:

4(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IFN:

Interferon

LT:

Lymphotoxin

MCA:

4-Methylcoumaryl-7-amide

MTX:

Methotrexate

NBS:

New Born Bovine Serum

PBS:

Phosphate Buffered Saline

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Authors and Affiliations

  1. Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi, 194, Machida-shi, Tokyo, Japan

    Shinji Hosoi, Hiromasa Miyaji, Mitsuo Satoh, Nobuo Fujiyoshi, Seiga Itoh & Seiji Sato

  2. Pharmaceutical Research Laboraties, Kyowa Hakko Kogyo Co., Ltd., 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, 411, Shizuoka, Japan

    Tsukasa Kurimoto & Akira Mihara

Authors
  1. Shinji Hosoi
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  2. Hiromasa Miyaji
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  3. Mitsuo Satoh
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  4. Tsukasa Kurimoto
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  5. Akira Mihara
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  6. Nobuo Fujiyoshi
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  7. Seiga Itoh
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  8. Seiji Sato
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Hosoi, S., Miyaji, H., Satoh, M. et al. Optimization of cell culture conditions for production of biologically active proteins. Cytotechnology 5 (Suppl 2), 17–34 (1991). https://doi.org/10.1007/BF00573878

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  • DOI: https://doi.org/10.1007/BF00573878

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