title: Inducing single-cell suspension of BTI-TN5B1-4 insect cells: I. The use of sulfated polyanions to prevent cell aggregation and enhance recombinant protein production.
abstract: 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 efficacy of inducing single-cell suspension correlated positively with the increase in sulfation of the added polyanion. Unsulfated polyanions, neutral polymers, polycations, disaccharides, and monosaccharides were ineffective in inducing single-cell suspension.Elimination of clumping in suspension culture by adding a dispersing agent can lead to enhanced recombinant protein production. 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, beta-galactosidase. High yields of 82 U/ml (ca. 110 mg/L) for alkaline phosphatase, and 705 U/mL (ca. 2.3 g/L) for beta-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.
authors.affiliation: School of Chemical Engineering, Cornell University, 340 Olin Hall, Ithaca, New York 14853-5201, USA.
authors.forename: K U
authors.initials: KU
authors.lastname: Dee
authors.forename: M L
authors.initials: ML
authors.lastname: Shuler
authors.forename: H A
authors.initials: HA
authors.lastname: Wood