Production of a Secreted Glycoprotein from an Inducible Promoter System in a Perfusion Bioreactor

Abstract

The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.

Perfusion Processes

“…A heat exchanger to decrease the cell broth temperature to room temperature is advantageously implemented to reduce heat convection problems in the settler and reduce the cell metabolism. Lipscomb et al (2004) used an inclined settler where the bottom plate was chilled (4 C) to reduce the cell adhesion as well as back-pulsing was daily applied to prevent cell build-up in the drain port. Batt reported cell density of 10 Â 10 6 hybridoma cells/mL (Batt et al 1990).…”

Breaking the bottleneck: Eukaryotic membrane protein expression for high-resolution structural studies

“…While this is taken for granted in microbial systems, most of the promoters used for transient transfection in mammalian systems have been constitutive, which can lead to cell death if the protein is toxic. However, a wide variety of inducible systems are now available (Tate, 2001;Neddermann et al, 2003;Lipscomb et al, 2004;Weber et al, 2004;Ivanova et al, 2005;Bach et al, 2007), which may also prove important for tuning the level of expression to avoid overwhelming protein processing systems (see below).…”

“…As a result, bioreactors may prove much more favorable for membrane protein expression than batch incubations. Operated in fed batch or perfusion mode, they offer precise control of the nutrient supply, and, at least in perfusion mode, also permit the removal of waste products and dead cells (Lipscomb et al, 2004).…”

A two-stage perfusion fibrous bed bioreactor system for mass production of embryonic stem cells

“…These problems can be alleviated by using a perfusion culture system, in which fresh medium is continuously fed and spent medium withdrawn at the same rate. Both cell growth and protein production can thus be improved significantly in the perfusion system [70] . Oh and Choo described a perfusion system for ESC cultures, in which the culture medium was perfused to a multi-well system.…”