METHODS AND CULTURE MEDIA FOR INDUCING SINGLE CELL SUSPENSION IN INSECT CELL LINES

Patent No. 5,728,580

Issued: March 17, 1998

Inventor: Michael L. Shuler, Kennie U. Dee, Ithaca, NY

Assignee: Cornell Research Foundation, Inc., 20 Thornwood Drive, Ithaca, NY 14850


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 recombinant protein production but 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 single-cell formation were dextran sulfate, polyvinyl sulfate, and pentosan sulfate - all highly sulfated. The cost of dextran sulfate treatment is low compared to heparin which required a 20-fold higher level to induce single-cell suspension. More importantly, dextran sulfate does not block viral infection at MOI 1 whereas heparin is known to seriously inhibit infection, an attribute shared by polyvinyl sulfate. 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 a four-fold increase in volumetric yield of the recombinant glycosylated protein, human secreted alkaline phosphatase, and a two-fold increase in volumetric yield of the recombinant cytoplasmic protein, b-galactosidase. High yields of 82 U/ml (ca. 110 mg/L) for alkaline phosphatase, and 705 U/ml (ca. 2.3 g/L) for b-galactosidase under elevated oxygen have been obtained. The optimum volumetric yield of alkaline phosphatase in BTI-TN5B1-4 dextran sulfate cells under elevated oxygen but unsupplemented medium is 6 to 11-fold higher than attached cultures, and 3-fold higher than the best yield obtained for SF21 cells in suspension at elevated oxygen and with nutrient supplementation. More importantly, cells can be infected at high density without complications from aggregation, which has important implications for scale-up.


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