A Multiplexed Plastic Spiral Inertial Microfluidic Platform for the Rapid and High-Recovery Clarification of Lentiviral Vectors from HEK 293 Cells

Technology

The multiplex spiral microfluidic device incorporates a stack of N layers of spiral microfluidic channels positioned between an inlet layer and an outlet layer. Each layer includes a spiral microchannel with an inlet, an inward outlet, and an outward outlet. The inlet pump delivers fluid containing a mixture of particles from the inlet reservoir to the inlet port of the microfluidic device, providing controlled flow that ensures consistent distribution across all spiral microchannels within the multiplexed stack. Within the spiral channels, larger particles (e.g., viral producer cells) experience size-dependent lift forces that equilibrate them near the inner wall side of the spiral microchannel. Smaller particles (e.g., viral vectors) are minimally affected by lift forces and instead follow Dean forces, remaining unaffected within the channel. At the outlet, a retentate stream enriched in larger particles flows from the inward outlet port back into the inlet reservoir, while a harvest stream containing the smaller particles flows from the outward outlet port of the device into the harvest reservoir. A media source, such as dilution buffer, is connected to the cell retention vessel to allow controlled additions of the media to regulate cell density and replace removed volume. The cycle can be continued repeatedly until the target particle concentration, background‑fluid reduction, or separation of larger and smaller particle populations has been achieved.

Problems Addressed

An important step in viral vector manufacturing is clarification, in which producer cells are removed from the viral vectors they generate. Traditional clarification methods employ membrane-based cell retention devices, but these systems are prone to challenges such as membrane clogging, fouling, and undesired retention of dead cells and other products. Additionally, conventional spiral devices have limited throughput capacity, processing only small sample volumes with correspondingly low harvest outputs. To support larger industry-scale batch processes or perfusion bioreactors involving several liters to hundreds of liters, enhanced processing capacity is required. The present invention addresses these needs by providing improved microfluidic devices, systems, and methods that enable high-recovery clarification while addressing scalability challenges for industrial applications.

Advantages

• Scalable throughput enabled by stacking multiple spiral layers, allowing processing of expanded volumes to support industrial applications
• Continuous operation allows processing until target level of separation is achieved
• Label‑free, passive, and gentle separation avoids cell fouling and clogging associated with centrifugation and membrane‑based clarification
• Supports automated operation, reducing manual handling and risk of contamination