Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

Abstract

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min -1 at 100 g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 x 10(6) cells ml -1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process. (Himmelfarb et al. 1969; Feder and Tolbert 1983) usually have the problem of filter clogging, and perfusion culture processes with gravitational cell settling (Tokashiki and Arai 1989) are limited in scale-up because of low separation efficiency. Recently centrifugal separation of cells from culture medium has been used for perfusion culture processes (Hamamoto et al. 1989; Tokashiki et al. 1990; Apelman 1992; Jäger 1992). This process essentially has a high separation efficiency and can avoid further operational problems, such as filter clogging, by the type of filtration. To investigate the scale-up feasibility of the centrifuge culture system, we have developed a new type of centrifuge with four layers of spiral cell-settling zones to increase the separation area, and a novel cell separation and withdrawal method using a perfluorocarbon.

Potential of cell retention techniques for large‐scale high‐density perfusion culture of suspended mammalian cells

“…Several investigators designed custom centrifuges for perfusion culture, working either continuously (Tokashiki et al, 1990) or semicontinuously (Hamamoto et al, 1989;Takamatsu et al, 1996). During the perfusion cultures using a centrifuge for cell retention, no report was made concerning a detrimental effect of centrifugation on animal cells.…”

A novel scale‐down mimic of perfusion cell culture using sedimentation in an automated microbioreactor (SAM)

“…For optimal perfusion operation 40 min of sedimentation can be used to obtain an almost cell free harvest (99% separation efficiency) for both GEX ® and a CHO cell line, which is comparable to other cell retention methods. 9,[11][12][13][14][15] Cell growth (Figure 2a) and viability (not shown) were unaffected by the sedimentation method. Figure 2c,e,f shows that glucose, glutamine and glutamate levels do not differ between the culture conditions with continuous stirring and sedimentation.…”

“…Centrifugal methods have been found to retain 95-100% 9,11,12 while laboratory-scale experiments with acoustic cell separation yields 90-98% cell retention. [13][14][15] The separation efficiency of cell retention devices based on gravity like external settlers or sedimentation inside the culture vessel as presented in this work can be a function of the perfusion rate and reactor volume.…”

In situ cell retention of a CHO culture by a reverse‐flow diafiltration membrane bioreactor

“…The main applied physical separation criteria in cell retention devices are size and density. 8 Cell retention devices based on density are for example hydrocyclones 9,10 and centrifuges, [11][12][13] both working with centrifugal forces, settlers, [14][15][16][17] using sedimentation properties, and acoustic filters, [18][19][20] where an ultrasonic field enhances the sedimentation. Separation by means of size exclusion is conducted most often using filtration.…”