Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Insect cell – per se
Patent
1996-02-20
1998-03-17
Naff, David M.
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Insect cell, per se
435383, 435384, 435405, C12N 100
Patent
active
057285802
ABSTRACT:
Non-carboxylated sulfated polyanions have been successfully used to rapidly obtain and maintain stable single-cell suspension of BTI-TN5B1-4 cells, a cell line which has a high intrinsic capacity for the expression of recombinant protein, but which clumps severely in suspension reducing its effectiveness as a host for foreign protein production with the baculovirus expression vector system. The three most effective polyanions for inducing a single-cell suspension were dextran sulfate, polyvinyl sulfate, and pentosan sulfate. The cost of dextran sulfate treatment is low compared to heparin treatment, which required a 20-fold higher lever to induce single-cell suspension. More importantly, dextran sulfate does not block vital infection at MOI.gtoreq.1 whereas heparin is known to seriously inhibit infection. To overcome this effect, the cells can be subcultured to a fresh culture medium without the sulfated polyanion, or the sulfated polyanion is neutralized with a polycation, then the cells are inoculated with the baculovirus. Once the cells are infected with the baculovirus, the sulfate polyanion is added back to the fresh culture medium. Inducing single-cell suspension with dextran sulfate, a highly sulfated polyanion, resulted in an increased volumetric yield of recombinant protein. Examples chosen for experimentation included, human secreted alkaline phosphatase, and b-galactosidase. Optimum volumetric yield of 6 to 11 fold higher than attached cells for the production of alkaline phosphatase in BTI-TN5B1-4 dextran sulfate cells under elevated oxygen. More importantly, cells can be infected at high density without complications from aggregation, which has important implications for scale-up.
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Dee Kennie U.
Shuler Michael L.
Cornell Research Foundation Inc.
Naff David M.
Ware Deborah K.
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