Application of a Cell-Once-Through Perfusion Strategy for Production of Recombinant Antibody from rCHO Cells in a Centritech Lab II Centrifuge System

Abstract

Based upon the results of scale-down intermittent perfusion processes, a cell-once-through (COT) perfusion concept was applied to a dual bioreactor system coupled to a Centritech Lab II centrifuge for culture of recombinant Chinese hamster ovary (rCHO) cells for monoclonal antibody production. In this new culture mode, i.e., the COT perfusion process, total spent medium was transferred to the centrifuge and a fixed percentage was removed. Approximately 99% of the viable cells are transferred to another bioreactor filled with fresh medium by single operation of the Centritech Lab II centrifuge system for about 30 min. Accordingly, a significant reduction of the cell-passage frequency to the centrifuge led to minimization of cell damage caused by mechanical shear stress, oxygen limitation, nutrient limitation, and low temperature outside the bioreactor. The effects of culture temperature shift and fortified medium on cell growth and recombinant antibody production in the COT perfusion process were investigated. Although the suppressive effects of low culture temperature on cell growth led to a loss of stability in a long-term COT perfusion culture system, the average antibody concentration at 33 degrees C was 157.8 mg/L, approximately 2.4-fold higher than that at 37 degrees C. By the use of a fortified medium at 37 degrees C, rCHO cells were maintained at high density above 1.2 x 10(7) cells/mL, and antibody was produced continuously in a range of 260-280 mg/L in a stable long-term COT perfusion culture. The proposed new culture mode, the COT perfusion approach, guarantees the recovery of rCHO cells damaged by lowered temperature or high lactate and ammonium concentration. It will be an attractive choice for minimization of cell damage and stable long-term antibody production with high cell density.

Perfusion Processes

“…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.…”

Long-term operation of depth filter perfusion systems (DFPS) for monoclonal antibody production using recombinant CHO cells: Effect of temperature, pH, and dissolved oxygen

“…Kim et al [19,20] achieved a monoclonal antibody concentration of 250 mg/L by operating a commercial Centritech Lab II Centrifuge System semi-continuously. The concentrations of the same monoclonal antibody produced by rCHO CS13*-1.00 cells ranged from 70~80 mg/L in the single stage DFPS and 250 mg/L in the Centritech Lab II system, which is equivalent to that of a fed-batch system [17].…”

Two-stage depth filter perfusion culture for recombinant antibody production by recombinant Chinese hamster ovary cell

“…While batch or fed-batch approaches have been well established for animal cell culture [13], they suffer from low productivity. Thus, it is time to look more closely at production systems other than conventional fedbatch cultures [14,15]. Owing to well-developed membrane filtration technology, it is not difficult to build a trouble-free cell recycling system for animal cell culture [16,17].…”