In dense cell perfusion cultures, the surfactant Pluronic F-68 (PF-68) is often added to protect cells from the damage caused by shear stress (such as agitation or bursting gas bubbles). Extremely high levels of PF-68 in cell culture media, however, are toxic, while concentrations that are too low offer inadequate protection.
However, “Alternating tangential filtration (ATF) hollow fibers with small pore sizes of 50 kD or less may retain and concentrate PF-68 in the bioreactors, resulting in extremely high concentrations. This may also, eventually, damage process performance,” Haiyang Yu, PhD, director of cell culture process development (process development department) at WuXi Biologics tells GEN.
Writing in a recent paper, Yu and colleagues outline a framework to optimize PF-68 concentrations and, thus, process performance. While effective for concentrated fed-batch cultures, its greatest value may be for intensified cell culture strategies involving perfusion.
ATF pore size is key
By adjusting the PF-68 feeding schedule based on the pore size of the ATF hollow fibers, WuXi Biologics scientists increased viable cell densities by 20–30% and productivity by approximately 30%.
Yu and colleagues cultured Chinese hamster ovary (CHO) cells in both concentrated fed-batch (CFB) and the intensified perfusion culture (IPC) modes to produce monoclonal antibodies. After seeing lower productivity and negligible amounts of PF-68 concentrations in the IPC mode, they began periodically feeding PF-68 into the cell culture. This significantly improved cell viability and mAb productivity without harming product quality. They determined a minimum effective PF-68 concentration of 5 g/L was required for cell cultures as dense as 100 x 106 cells/mL undergoing micro-sparging.
In their studies, PF-68 passed through ATF hollow fibers with of 0.2 µm pores, which reduced the effective concentration of the PF-68 and left the cells subject to damage during micro-sparging. With the much smaller 50 kD pores typically used with CFB cultures, however, enough PF-68 was retained to protect cells during micro-sparging.
WuXi Biologics is applying this PF-68 feeding strategy now to its large-scale biologics manufacturing activities to maximize the output of drug substance productivity and minimize cost of goods.
“We are fully confident this technology can be widely applied to perfusion cell cultures, especially for ultra-intensified strategies,” Yu says. That includes WuXiUp™ and its most recent innovation, the WuXiUI ultra-intensified fed-batch process strategy with limited perfusion cycles. “The principles of cell culture remain similar, no matter what cell types or process modes are used,” so the use of PF-68 is applicable to other mammalian cells, such as HEK293, NSO, T cells and stem cells as well as to CHO cells.