Microfluidic Cell Retention Device for Perfusion of Mammalian Suspension Culture

Abstract

Continuous production of biologics, a growing trend in the biopharmaceutical industry, requires a reliable and efficient cell retention device that also maintains cell viability. Current filtration methods, such as tangential flow filtration using hollow-fiber membranes, suffer from membrane fouling, leading to significant reliability and productivity issues such as low cell viability, product retention, and an increased contamination risk associated with filter replacement. We introduce a novel cell retention device based on inertial sorting for perfusion culture of suspended mammalian cells. The device was characterized in terms of cell retention capacity, biocompatibility, scalability, and long-term reliability. This technology was demonstrated using a high concentration (>20 million cells/mL) perfusion culture of an IgG1-producing Chinese hamster ovary (CHO) cell line for 18–25 days. The device demonstrated reliable and clog-free cell retention, high IgG1 recovery (>99%) and cell viability (>97%). Lab-scale perfusion cultures (350 mL) were used to demonstrate the technology, which can be scaled-out with parallel devices to enable larger scale operation. The new cell retention device is thus ideal for rapid perfusion process development in a biomanufacturing workflow.

Continuous removal of small nonviable suspended mammalian cells and debris from bioreactors using inertial microfluidics

“…29 We demonstrated the retention of CHO cells at a high cell concentration (>40 million cells per mL) using this. 24 Moreover, its large crosssectional area (1.7 × 10 5 μm 2 ) reduces the input pressure to achieve high input flow rates, compared with the 600 μm-wide channel (ESI † Fig. S1).…”

“…Its high separation resolution compared with the spiral channel with a rectangular cross-section was demonstrated before. 29,30 The technology has found applications in microfiltration, 21,24 blood fractionation, 30,31 bacteria detection, 32 virus recovery, 33 cell isolation (circulating tumor cells, 34 stem cells, 35,36 and immune cells 37 ), microalgae separation, 38 and sorting eggs of a nematode. 39 Fig .…”

“…The cell concentration is also known to affect the performance of the microfluidic device, as increased cell-to-cell interactions at higher cell concentrations result in broader bands of focused cells and decreased efficiency in inertial focusing. 17,21,24 CHO cell populations were flowed through the microfluidic device at concentrations of 1, 4, and 10 million cells per mL, while maintaining a flow rate of 1.5 mL min −1 , a viability of 80%, and a flow split ratio of 0.36 for each concentration condition. As expected, the microfluidic device performed worse at 10 million cells per mL in terms of viable cell retention efficiency and dead cell removal purity but not removal efficiency, (Fig.…”

“…This parameter affects the streamline boundary between the inner and outer outlet flows. 24,41,42 The flow rate was kept at 1.0 mL min −1 for all flow split ratio conditions. 10 μm and 15 μm bead trajectories were not significantly affected by changing the flow split ratio since their focusing position was close (within 100 μm) to the inner wall at a flow rate of 1.0 mL min −1 .…”

“…[21][22][23] Moreover, sorting yeast and mammalian cells at high cell concentrations in microchannels can be performed without additional buffer flows. 21,24 Chinese hamster ovary (CHO) cells, the top expression system of biological drugs, 25 were used to demonstrate our technology. Apoptosis is a main cause of cell death in bioreactor suspension cultures of mammalian cell lines during the production of biopharmaceuticals.…”