INDUCING SINGLE-CELL SUSPENSION OF BTI-TN5B1-4 INSECT CELLS .1. THE USE OF SULFATED POLYANIONS TO PREVENT CELL-AGGREGATION AND ENHANCE RECOMBINANT PROTEIN-PRODUCTION

Sulfated polyanions have been successfully used to rapidly obtain and maintain stable single-cell suspension of BTI-TN5B1-4 cells, a cell li ne which has a high intrinsic capacity for recombinant protein product ion but clumps severely in suspension reducing its effectiveness as a host for foreign protein production with the baculovirus expression ve ctor system. The efficacy of inducing single-cell suspension correlate d positively with the increase in sulfation of the added polyanion. Un sulfated polyanions, neutral polymers, polycations, disaccharides, and monosaccharides were ineffective in inducing single-cell suspension. Elimination of clumping in suspension culture by adding a dispersing a gent can lead to enhanced recombinant protein production. Inducing sin gle-cell suspension with dextran sulfate, a highly sulfated polyanion, resulted in a four-fold increase in volumetric yield of the recombina nt glycosylated protein, human secreted alkaline phosphatase, and a tw o-fold increase in volumetric yield of the recombinant cytoplasmic pro tein, beta-galactosidase. High yields of 82 U/mL (ca. 110 mg/L) for al kaline 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 el evated oxygen but unsupplemented medium is 6 to 11-fold higher than at tached cultures, and 3-fold higher than the best yield obtained for SF 21 cells in suspension at elevated oxygen and with nutrient supplement ation. More importantly, cells can be infected at high density without complications from aggregation, which has important implications for scale-up. (C) 1997 John Wiley & Sons, Inc.