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 Â 10 7 cells ml À1 and a final saCBMFX concentration of 4 mg l À1 . 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 À1 . The cellretention 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 Â 10 7 Sf9 cells ml À1 were obtained with a viability greater than 80%. With a doubling of viable cell concentration from 1.5 to 3 Â 10 7 cells ml À1 the saCBMFX production rate was doubled to 6 mg l À1 day À1 . The saCBMFX volumetric productivity of the perfusion system was higher than the batch fermentations (0.6 mg l À1 day À1 ) by an order of magnitude.

Perfusion Processes

“…A detailed review of the acoustic settler has been published by Shirgaonkar et al (2004). 6 cells/mL and 40 Â 10 6 cells/mL have been reported using the acoustic settler (Zhang et al 1998;Gorenflo et al 2004) and Gorenflo obtained 10 Â 10 6 CHO cells/mL in 100 L scale (Gorenflo et al 2002). An improvement of the technology was brought by installing an air-driven system able to suck the cell broth into the settler chamber and to back flush the cells into the bioreactor, avoiding the use of a peristaltic pump and enabling to empty the chamber completely (Gorenflo et al 2003).…”

Scale-down studies for assessing the impact of different stress parameters on growth and product quality during animal cell culture

“…Perfusion culture has been frequently studied and often involves the use of pumps to recirculate the cells. Pump damage was found to reduce the viability of a variety of animal cell types, including insect (Merten, 2000;Gorenflo et al, 2004), hybridoma (LaPorte et al, 1996) and CHO cells (Kim et al, 2008). Yet, in perfusion culture, the rate of pumping is far lower than here, circulation rates being about 1 to 10 bioreactor volumes/day (Merten, 2000;Kim et al, 2008) compared to ~ 100 to ~ 200 per day here.…”

High cell density fed batch and perfusion processes for stable non‐viral expression of secreted alkaline phosphatase (SEAP) using insect cells: Comparison to a batch Sf‐9‐BEV system

“…In the case of stably transformed insect cells, baculovirus infection is no longer necessary for expression of the recombinant protein. The hypothesis is that in the cases of stably transformed insect cells the potential of the high cell density culture methods such as fed batch and perfusion (Gorenflo et al, 2004) can be fully realized. High cell density culture methods which can result in significant increases in cell densities increase the potential for bioprocess intensification (Griffiths, 1990) resulting in a subsequent increase in the volumetric productivity without increasing the size of the reactor, and a concurrent decrease in the requirements for liquid handling and overall downstream unit operations (Chisti and Moo-Young, 1996;Smith et al, 1991).…”

“…Earlier workers have described perfusion culture methods for recombinant protein expression using Sf-9-BEVS (Jäger, 1996;Zhang et al, 1998); the system however, is better suited for cells which express a secreted protein in a stable continuous manner, allowing the product to be harvested for prolonged periods of time. Gorenflo et al (2004) studied the expression of a recombinant protein saCBMFX in stably transformed Sf-9 insect cells in the perfusion system and reported that the protein produced had a higher activity than that expressed in a mammalian expression system. Stably transformed Drosophila S2 cells have also been used for the expression of several human proteins including erythropoietin, interleukin-2 (hIL-2), and human transferrin (hTF).…”