Production of a self-activating CBM-factor X fusion protein in a stable transformed Sf9 insect cell line using high cell density perfusion culture

Abstract

Factor Xa is a serine protease, whose high selectivity can be used to cleave protein tags from recombinant proteins. A fusion protein comprised of a self-activating form of factor X linked to a cellulose-binding module, saCBMFX, was produced in a stable transformed Sf9 insect cell line. The activity of the insect cell produced saCBMFX was higher than the equivalent mammalian cell produced material. A 1.5 l batch fermentation reached a maximum cell concentration of 1.6 x 10(7) cells ml(-1) and a final saCBMFX concentration of 4 mg l(-1). The production of saCBMFX by this cell line was also analyzed in a 1.5 l perfusion system using an ultrasonic filter as a cell-retention device for flow rates up to 3.5 l day(-1). The cell-retention efficiency of an air backflush mode of acoustic filter operation was greater than 95% and eliminated the need to pump the relatively shear sensitive insect cells. In the perfusion system over 4 x 10(7) Sf9 cells ml(-1) were obtained with a viability greater than 80%. With a doubling of viable cell concentration from 1.5 to 3 x 10(7) cells ml(-1) the saCBMFX production rate was doubled to 6 mg l(-1) day(-1). The saCBMFX volumetric productivity of the perfusion system was higher than the batch fermentations (0.6 mg l(-1) day(-1)) by an order of magnitude.