Perfusion Culture of Hybridoma Cells for Hyperproduction of IgG_2a Monoclonal Antibody in a Wave Bioreactor‐Perfusion Culture System

Abstract

A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in perfusion culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale.

Experimental characterization of flow conditions in 2‐ and 20‐l bioreactors with wave‐induced motion

“…Heights measured are shown as three circles piled (5, 12, and 22 mm above the bag bottom). suspension cells, or insect cells, clearly showing the potential of these bioreactors (Lohr et al, 2009;Okonkowski et al, 2007;Tang et al, 2007;Weber et al, 2002).…”

“…Various examples can be found for the cultivation of animal cells using bioreactors with wave-induced motion, demonstrating the possibility of adapting classical processes that use either roller bottles or stirred tank bioreactors to the cellbag method (Hundt et al, 2007;Lohr et al, 2009;Okonkowski et al, 2007;Tang et al, 2007;Weber et al, 2002). For the cultivation of adherent Madin-Darby canine kidney (MDCK) cells in an influenza vaccine production process, it has been observed that higher maximum cell densities can Correspondence concerning this article should be addressed to D. Thévenin at thevenin@ovgu.de.…”

Dynamic Modeling of Apoptosis and its Interaction with Cell Growth in Mammalian Cell Culture

“…The perfusion rate depends on the type and culture behavior of the cell line, the concentration of nutrients in the feed and the level of toxicity. This mode of operation has been used for the production of monoclonal antibodies (Banik and Heath, 1995;Tang et al, 2007; and recombinant proteins (AlRubeai et al, 1999;Avgerinos et al, 1990). An example of a 500 L industrial perfusion system for monoclonal antibody production by hyridoma is described by (Deo et al, 1996) where the operation continued for 15 to 35 days to achieve commercial production of antibodies with a cell density of >10 x 10 6 cell/mL.…”

Technological progresses in monoclonal antibody production systems

“…57 It operates in different culture modes, including perfusion, which can be performed using different cell-retention mechanisms for the high-efficient production of mAbs. 58 The combined advantages of a fully closed, disposable system with process monitoring capabilities (pH and DO) make the wave bioreactor highly attractive over traditional systems for animal cell culture, such as shake flasks and STR, 57 although with limitations in culture volume.…”