Limited Use of Centritech Lab II Centrifuge in Perfusion Culture of rCHO Cells for the Production of Recombinant Antibody

Abstract

Perfusion cultures of recombinant Chinese hamster ovary cells, producing recombinant antibody against the S surface antigen of Hepatitis B virus, were carried out in continuous and intermittent mode using a Centritech Lab II Centrifuge. In the continuous perfusion process, despite the absence of shear stress from the pump head, long-term operation was not possible because of continuously repeated exposure to oxygen limitation and low temperature, as well as shear stress from centrifugal force. In the intermittent perfusion processes, the frequency of cell-passage through the centrifuge was substantially reduced, compared with the continuous perfusion mode; however, the degree of reduction could not guarantee stable long-term operation. Although various operating parameters were applied in the intermittent perfusion cultures, high cell densities could not be maintained stably. In a single bioreactor culture system, a specific cell that is returned from the centrifuge to the bioreactor could be transferred from the bioreactor to the centrifuge again in the next cycle. These repetitive damages, caused by shear stress from the pump head and centrifugal force, as well as exposure to suboptimal conditions such as oxygen limitation and low temperature below 37°C, were more serious at higher perfusion rates. Subsequently, damaged cells and dead cells were continuously accumulated in the bioreactor. Culture temperature shift from 37 to 33°C increased antibody concentrations but showed inhibitory effects on cell growth. The negative effects of lowering culture temperature on cell growth overwhelmed the positive effects on antibody production. To protect cells from shear stress, Pluronic F-68 was 2-fold concentrated in the culture medium; nevertheless, a significantly higher concentration of Pluronic F-68 (2 g/L) may have inhibitory effects on cell growth.

Perfusion Processes

“…The cell damage increases with the rotor speed and the applied g force. From studies supervised by T. Björling at Pharmacia-Upjohn, it was concluded that this effect was not due to the pumps as claimed by Kim et al (2008). It is quite probably not due to the acceleration it-self but rather associated to a wall shear stress in the insert bag.…”

“…Observe that the total residence time is not longer in the large centrifuge compared to the small one since larger diameter tubes are used at larger scale. Several studies have reported a damageable effect of the Centritech on the cells (Johnson et al 1996;Chotteau et al 2002;Kim et al 2007Kim et al , 2008. The cell damage increases with the rotor speed and the applied g force.…”

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.…”

Metabolomic study of baculovirus infected insect cells to identify metabolite based strategies for improving virus yields

“…1A and B). The quenching and washing procedures involved multiple centrifugation and cell pellet resuspension steps, but since the latter was performed by gentle tube inversion the shear stresses encountered by cells were mainly from the centrifugal forces [55]. The ability of PF68 to protect animal cells from fluid-mechanical damage may derive from a fast-acting biological mechanism involving direct interaction between the surfactant molecules and the cell plasma membrane, resulting in a more rigid and shear-resistant membrane [52,56].…”

“…This system is important for the production of a biopesticide to target the major heliothis caterpillar pest. A preliminary study showed that the metabolite extraction protocol developed was efficient for infected HzAM1 until [48][49][50][51][52][53][54][55][56][57][58][59][60] hpi. As found for infected Sf9 cells, many intracellular metabolite concentrations for infected HzAM1 also significantly reduced with increasing ICD.…”