Compared with cell harvesting alternatives that rely on the use of conventional centrifuges such as disc-stack or filtration
devices to separate cells from the supernatant, the FBC has several advantages over both conventional methods. First, the
FBC eliminates the need for cleaning and sterilizing cycle validations. Second, there is no risk for cross-contamination between
batches because the product-contact components are single-use only. Third, there is lower shear stress induced by the FBC
during operation compared with conventional centrifugation and filtration. Because of the establishment of the fluidized bed
during operation, FBC's g-force does not result in cell lysis because cells are not packed against the centrifuge wall. Finally,
the washing option available with the FBC provides maximal recovery of the product without diluting the centrate. In addition
to these advantages, historical clarification efficiency data from disc-stacks have been shown to be comparable with the expected
clarification efficiency performance of the FBC (7).
The FBC is based on a balancing act of two counteracting forces within the system, the centrifugal force (Fg) versus the fluid flow (VQ). The physics of the FBC follows Stokes' Law for spherical particles in a continuous fluid (Equation 1), or in the case of bioprocessing, a cell suspended in a medium:
where Vs is the settling velocity of the particle, ρp is the density of the particle, ρf is the density of the fluid, μ is the dynamic viscosity of the fluid, D is the diameter of the particle, and g is the gravitational
acceleration of the particle. The centrifugal force (Fc) and fluid flow velocity (VQ) are defined in the following equations (Equations 2 and 3):
where ω is the angular velocity, R is the radius, Q is the flow rate, and A is the cross-sectional area of the chamber. When
the settling velocity (VS) due to the centrifugal force is balanced against the fluid flow velocity (VQ), a fluidized cell bed is created within the chambers while the supernatant is discharged from the chamber as the centrate.
Figure 2 shows a schematic of the inner workings of the FBC.
Figure 2: FBC schematic demonstrating principles of FBC’s fluidized bed. (FIGURES 2–6 ARE COURTESY OF THE AUTHORS)
The overall preparation time for the FBC is relatively short compared to the cycle times for cleaning and sterilizing stainless-steel
equipment. A single-use set that contains four interconnected chambers and the valve set are first inserted into the FBC prior
to an operation. After loading the single-use set into the FBC, individual tubing ends from the single-use set can be sterile-connected
to respective vessels such as the bioreactor, buffer, centrate vessel for harvested product, and waste container. Pinch valves
and bubble sensors are built-in as part of the FBC and used to automate the cell-harvesting process. The built-in human-machine
interface (HMI) screen on the FBC allows run parameters such as centrifuge speed, flow rates, and harvest volumes to be set
in individual recipes. Cell-harvesting runs can be nearly fully automatic once the recipes are configured, but manual controls
are also available if required.
Figure 3 shows the process flow diagram of a typical harvest clarification recipe. Step one in the process is to prime the single-use
set with a buffer. All tubing and chambers are filled with the buffer to displace air from the system. Step two in the recipe
directs the system to displace the buffer initially in the single-use chamber(s) into the waste vessel. In doing so, the buffer
is not introduced into the centrate vessel and will not dilute the product. Harvest clarification begins in step three, where
cells and cell debris are retained in the chamber(s) and the clarified liquid is separated into the centrate vessel. When
the chamber(s) are nearly filled with cells, in step four, the bioreactor feed temporarily pauses, and the buffer is used
to flush the chamber(s) to recover the product that is still present in the cell bed. In step five, after the wash, pump directions
are reversed to discard the cell bed into the waste vessel. In the case where the bioreactor volume is larger than what all
four single-use chambers can handle in one batch, steps two through five can be repeated in multiple cycles to finish processing
the full volume of the bioreactor. At the end of the process, the system purges all liquids from the single-use set and prompts
the operator to seal all tubing prior to the disposal of the single-use set. The remainder of this article discusses the results
obtained from the evaluation runs conducted using the FBC for cell harvesting and clarification.
Figure 3: Process flowchart for harvest clarification using the FBC.