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专利名称：Methods and culture media for inducing
single cell suspension in insect cell lines
发明人：Michael L. Shuler,Kennie U. Dee申请号：US08/603722申请日：19960220公开号：US05728580A公开日：19980317
摘要：Non-carboxylated sulfated polyanions have been successfully used to rapidlyobtain and maintain stable single-cell suspension of BTI- TN5B1-4 cells, a cell line whichhas a high intrinsic capacity for the expression of recombinant protein, but which clumpsseverely in suspension reducing its effectiveness as a host for foreign protein productionwith the baculovirus expression vector system. The three most effective polyanions forinducing a single-cell suspension were dextran sulfate, polyvinyl sulfate, and pentosansulfate. 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≧1 whereas heparin is known toseriously inhibit infection. To overcome this effect, the cells can be subcultured to a freshculture medium without the sulfated polyanion, or the sulfated polyanion is neutralizedwith a polycation, then the cells are inoculated with the baculovirus. Once the cells areinfected with the baculovirus, the sulfate polyanion is added back to the fresh culturemedium. Inducing single-cell suspension with dextran sulfate, a highly sulfated polyanion,resulted in an increased volumetric yield of recombinant protein. Examples chosen forexperimentation included, human secreted alkaline phosphatase, and b- galactosidase.
Optimum volumetric yield of 6 to 11 fold higher than attached cells for the production ofalkaline phosphatase in BTI-TN5B1-4 dextran sulfate cells under elevated oxygen. Moreimportantly, cells can be infected at high density without complications fromaggregation, which has important implications for scale-up.
申请人：CORNELL RESEARCH FOUNDATION, INC.
代理机构：Brown, Pinnisi & Michaels
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