, Volume 44, Issue 3, pp 93-102

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

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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 × 107 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 × 107 Sf9 cells ml−1 were obtained with a viability greater than 80%. With a doubling of viable cell concentration from 1.5 to 3 × 107 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.