KR20220039841A - Centrifuge system for separating cells in suspension - Google Patents

Abstract

The device for separating cell suspension material into filtrate and concentrate includes disposable structures 178 , 240 , 250 releasably positioned in a cavity within a solid walled rotatable ball 172 . The ball and a portion of the disposable structure rotate about an axis 174 . A stationary inlet feed tube 184 , a filtrate outlet tube 212 and a concentrate outlet tube 230 extend along the axis of the rotating, disposable structure. The filtrate centripetal pump 208 is in fluid communication with the filtrate drain tube. The concentrate centripetal pump 216 is in fluid communication with the concentrate discharge tube. The controller 274 regulates the flow rates of the retentate pump 272 and the filtrate pump 266 in accordance with the sensors 264 and 270 in the respective filtrate and retentate discharge lines 262 and 268 to control the cell retentate and generally It works to create a flow of cell-free filtrate.

Description

Centrifugation system for separating cells in suspension

The present disclosure relates to a process for centrifugal separation of materials. Exemplary embodiments relate to an apparatus for separating cells from suspension via a centrifugation process.

Apparatus and methods for centrifuging cells in suspension are useful in many technical settings. Such systems may benefit from improvements.

It is an object of the present invention to provide an apparatus and method for centrifuging cells in large-scale cell cultures with high cell concentrations using pre-sterilized disposable fluid pathway components.

Exemplary embodiments described herein include apparatus and methods for centrifuging cells in large-scale cell cultures with high cell concentrations using pre-sterilized disposable fluid pathway components. Exemplary centrifuges discussed herein may be solid wall centrifuges using pre-sterilized disposable components and capable of handling cell suspensions with high cell concentrations.

Exemplary embodiments use rotationally fixed feed and discharge components. The disposable component includes a flexible membrane mounted to a rigid frame with an enlarged diameter core. The disposable component may further comprise at least one centripetal pump. The disposable structure may be supported in a multi-use rigid ball having the shape of a truncated cone. These structures allow the exemplary system to maintain angular velocities high enough to produce sedimentation rates suitable for efficiently processing highly concentrated cell culture streams. Features that minimize feed turbidity and other features that allow continuous or semi-continuous release of cell concentrates increase overall production rates above achievable rates. Exemplary structures and methods provide effective operation and reduce the risk of contamination.

1 is a schematic diagram of an exemplary embodiment of a centrifugal separation system including a single-use component and a multi-use component;
Fig. 2 is an enlarged view of the upper flange area of the centrifugal separator of Fig. 1, showing a method of sealing the flexible chamber material to the flange surface;
3 is an isometric cross-sectional view of a core and upper flange of a disposable component of the centrifugal separation system embodiment of FIG. 1 ;
FIG. 4 is a schematic diagram of the embodiment shown in FIG. 1 , wherein the pump chamber of the centrifugal separation system comprises accelerator pins;
5 is a top isometric view of a pump chamber of an exemplary embodiment of the centrifugal separation system shown in FIG. 4 ;
6 is an isometric cross-sectional view of the core, upper flange and lower flange of a disposable centrifugal separation system with an enlarged core diameter (to make a shallow pool centrifuge) and feed accelerator.
Fig. 7 is an isometric view of the feed accelerator of Fig. 6;
8 is an isometric cross-sectional view of the core and top flange of a disposable centrifugal separation system using standard core diameters and a feed accelerator with curved vanes and elliptical balls.
Fig. 9 is an isometric view of the feed accelerator of Fig. 8;
10 is a schematic diagram of a portion of a continuous concentrate discharge centrifugal separation system.
11 is a schematic diagram of a portion of a second embodiment comprising a continuous concentrate discharge centrifugal separation system;
12 is a schematic diagram of a continuous concentrate discharge centrifugal separation system injecting diluent.
13 is a schematic diagram of a portion of a third exemplary embodiment of a continuous concentrate discharge system having a throttle mechanism for a centripetal pump;
14 is an isometric cross-sectional view of a core and upper flange of a single-use centrifugal separation system with a core and a feed accelerator with straight vanes;
Figure 15 is an isometric view of the feed accelerator of Figure 14;
16 is an isometric cutaway view of an alternative continuous concentrate discharge centrifugation system.
17 is an exploded isometric view of an alternative centripetal pump;
18 is an isometric view of a plate of an alternative centripetal pump including a vortex passageway therein;
19 is a schematic diagram of a centrifugal separation system operating to maintain positive pressure in the centrifuge core cavity.
20 is a schematic diagram illustrating a simplified exemplary logic flow executed by at least one control circuit of the system shown in FIG. 19 ;
21 is a schematic cross-sectional view of an alternative continuous filtrate and concentrate discharge centrifugal separation system.
22 is a cross-sectional schematic view of a further alternative continuous filtrate and concentrate discharge centrifugal separation system.
23 is a cross-sectional schematic view of a further alternative continuous filtrate and concentrate discharge centrifugal separation system.
24 is a schematic diagram of a control system for an exemplary continuous filtrate and concentrate discharge centrifugal separation system.
25 is a schematic diagram of a logic flow associated with the example control system of FIG. 24 ;
26 is a cross-sectional view of an exemplary upper portion of a disposable centrifugal separation structure comprising a retentate and filtrate dam in a separation chamber.
27 is a cross-sectional view of an exemplary upper portion of a disposable structure including vanes within a filtrate pump chamber and a thickening pump chamber for the purpose of controlling the radial position of an air/liquid interface.
28 is a perspective view of a chamber surface of an exemplary concentrate or filtrate pump chamber including a plurality of chamber vanes.
29 is a cross-sectional view of an exemplary upper portion of a disposable structure similar to that shown in FIG. 27 showing the location of the air/liquid interface;
30 is a cross-sectional view of an exemplary upper portion of a disposable structure including an air passageway for retaining pressurized air in an air pocket;
31 is a schematic diagram of an exemplary system for controlling a centrifugal separation system including filtrate flow backpressure control.

In the field of cell culture applied to bio-pharmaceutical processes, it is necessary to separate cells from a fluid medium, such as the fluid in which the cells are grown. The desired product from the cell culture may be a molecular species that the cell excretes into the medium, a molecular species that remains within the cell, or the cell itself. On a mass production scale, the initial steps of a cell culture process are usually made in a bioreactor that can operate in batch mode or continuous mode. Variants such as an iterative batch process may also be implemented. The desired product must be finally separated from other process components prior to final purification and product formulation. Cell harvesting is a general term applied to the isolation of these cells from other process components. Clarification is a term denoting cell separation in which the cell-free supernatant (or centrate) is the objective. Cell recovery is a term often applied to separations in which cell concentrates are the goal. Exemplary embodiments of the present application relate to cell harvest separation in large scale cell culture systems.

Methods for cell harvest separation include batch, intermittent, continuous and semi-continuous centrifugation, tangential flow filtration (TFF) and depth filtration. Historically, centrifuges for cell harvesting of large-scale cell cultures at production scale have required clean-in-place (CIP) and steam-in-place (SIP) technologies to provide a sterile environment to prevent contamination by microorganisms. It is a complex multi-use system. At laboratory scale and for continuous cell harvesting processes, smaller systems can be used. The UniFuge centrifugal separation system manufactured by Pneumatic Scale Corporation, which is described in published US patent application US2010/0167388, has a cell capacity of up to about 2000 liters in the range of 3-30 liters/min and using intermittent treatment. Successfully process culture batches for harvest. The entire contents of this document are incorporated herein by reference. See also, U.S. Patent Application Serial Nos. 15/886,382, filed on February 1, 2018; and US Patent No. 9,222,067 owned by Pneumatic Scale Corporation, the assignee of the present application, are incorporated herein in their entirety. Intermittent processing usually requires periodic stopping of the feed flow and rotation of the centrifuge ball to drain the concentrate. This approach generally works well in low concentration, high viability cultures. Large batches can be processed in this culture and the cell concentrate is drained relatively quickly and completely.

Sometimes the material supplied contains high concentrations of cells and cellular debris, and it is necessary to harvest the cells from a high concentration and/or low viability cell culture, also referred to as a “high turbidity feed”. there is. This high turbidity feed can slow processing in some centrifugal separation systems for the following reasons:

1. It is necessary to make the feed flow rate slower to increase the residence time in the centrifuge to separate the small cell debris particles.

2. The high concentration of cells and cell debris can quickly fill the vessel with the cell concentrate, which makes it necessary to stop the vessel to drain the concentrate.

These combined factors can result in reduced net processing rates and unacceptably long cell harvest processing times. In addition to the increased cost that may be associated with longer processing times, the increased time in the centrifuge can also result in higher levels of product contamination and loss in low viability cell culture harvests.

High concentrations of cells and cell debris in the material feed can result in very viscous cell concentrates. This can make it more difficult to completely drain the cell concentrate from the centrifuge, even with increased draining cycles. In some cases, additional buffer rinse cycles may be added to fully drain the concentrate. If one or both of these adjustments to the shedding cycle have to be made, processing time is additionally increased, which can be more complex and expensive to process large volumes of cell cultures.

Extending the size of the system by increasing the ball size to increase the length of the feed portion of the intermittent treatment cycle is not practical as it sometimes results in a proportionally longer drain cycle for cell concentrates. Another limitation that may hinder simple geometric scale-up is the scaling change of the relevant fluid dynamic factors. The maximum throughput rate of any centrifuge depends on the settling rate of the particles being separated. The sedimentation rate is given by modifying the Stokes law defined in Equation 1.

방정식 1

Equation 1

where v = sedimentation rate, Δρ is the solid-liquid density difference, d is the particle diameter, r is the radial position of the particle, ω is the angular velocity, and μ is the liquid viscosity. Regarding the scale-up shape, changing the radius of the ball changes the maximum radial position r that the particle can occupy. Thus, if the other parameters of Equation 1 are held constant, increasing the ball radius increases the average sedimentation rate, increasing the throughput for a given separation efficiency. However, as the radius increases, it becomes more difficult to maintain the ball's angular velocity due to increased material strength and other engineering limitations that may be required. If the decrease in angular velocity is greater than the square root of the proportional increase in radius, both the average sedimentation velocity and the gain in throughput (proportional to the radius) decrease.

One of the engineering limitations that must be considered is that the angular velocity required to rotate a larger ball may be difficult to achieve in practice. This is due to the need for a larger and more expensive centrifugal drive platform.

Also, if the angular velocity remains constant as the radius increases, the force pushing the cells towards the centrifuge wall also increases. When the ball rotates at an angular velocity high enough to produce the desired processing efficiency, additional stress is created on the walls of the vessel and the cells that accumulate there. As for cells, this can lead to cell damage due to excessively high concentration of the cells. Cell damage is a disadvantage for applications where cell viability must be maintained and can lead to contamination of products present in solution in the filtrate. Higher viscosities due to excessively high cell concentrations are sometimes also a drawback for complete drainage of cell concentrates.

Exemplary embodiments include apparatus and methods for continuous or semi-continuous centrifugation of low viability cell suspension cultures containing high concentrations of cells and cell debris at rates suitable for processing large volumes of cell suspensions on a commercial scale. . Some exemplary centrifuges are pre-sterilized, single-use designs and are capable of processing such cell suspensions at flow rates in excess of 20 liters per minute. This flow capacity enables a total running time of a 2000 liter bioreactor in the range of 2-3 hours. Exemplary embodiments of the disposable centrifugal separation system can process about 300 to 2,000 liters of fluid while operating at a rate of about 2 to 40 liters per minute.

1 discloses a disposable centrifuge structure 1000 . Centrifuge structure 1000 is a core structure comprising a core 1510 , an upper flange 1300 , a lower flange 1200 , and a flexible liner 1100 sealed to both the upper and lower flanges 1300 and 1200 . 1500 (best shown in FIG. 3). The centrifugal separator structure 1000 also includes a centripetal pump 1400 that includes a rotating mechanical seal 1700 and a pair of stationary fairing disks 1410 in a rotating pump chamber 1420 .

The centrifugal separator structure 1000 also includes a feed/discharge assembly 2000 . The feed/discharge assembly 2000 includes a plurality of concentric tubes around an axis of rotation 1525 (shown in FIG. 12 ) of the centrifuge 1000 . The innermost portion of the feed/discharge assembly 2000 includes a feed tube. A plurality of additional tubes concentrically surround feed tube 2100 , filtrate drain 2200 , concentrate drain 2500 (see, eg, FIG. 12 ) or diluent supply 5000 (see, eg, FIG. 12 ). ) to allow for tubing or fluid pathways. Each portion of the supply/discharge connection may be in fluid communication with a portion of the interior of the centrifuge 1000 and a collection or supply chamber (not shown) through appropriate fluid connections to remove filtrate, concentrate, or diluent from or to the system; It may include additional tubes in fluid communication with the concentric tube for addition.

As shown in FIG. 1 , upper and lower flanges 1300 , 1200 include conical balls axially aligned with and concave towards core 1510 . The core 1510 includes a generally cylindrical body having a hollow cylindrical center large enough to receive a feed tube 2100 having an axis 1525 (marked in FIG. 12 ). The upper flange 1300 , the core 1510 and the lower flange 1200 may be of a single structure to provide a stronger support structure for the flexible liner 1100 , alternatively referred to herein as a membrane. In other embodiments, the core structure 1500 may be formed from a plurality of components. In further embodiments, lower flange 1200 comprises separate components and core 1510 and upper flange 1300 comprise a single component, or upper flange 1300 comprises separate components and core 1510 ) and lower flange 1200 may comprise a single component.

An embodiment of a single core 1510 and upper flange 1300 is illustrated in FIG. 3 . This single component will be coupled to the lower flange 1200 to create the internal support structure 1500 of the disposable component of the centrifugal separator 1000 . This structure secures the flexible liner 1100 at both the top and bottom around the fixed inner rigid or semi-rigid support structure 1500 . When the centrifugal separation system is in use, the flexible liner 1100 is also supported externally by the walls and cover of the multi-use structure 3000 .

Exemplary separation chamber 1550 is approximately cylindrical in shape, with outer surface 1515 of core 1510 and flexible liner 1100 and upper surface 1210 of lower flange 1200 and lower surface of upper flange 1300 . It is an open chamber bounded approximately by (1310). The separation chamber 1550 is in fluid communication with the feed tube 2100 through an aperture 1530 extending from the central cavity 1520 of the core 1510 to the outer surface 1515 of the core 1510 . Separation chamber 1550 is in fluid connection with pump chamber 1420 through similar apertures 1540 through core structure 1500 . In this example, aperture 1540 is angled upward towards pump chamber 1420 , opening into separation chamber 1550 just below the junction between core 1510 and upper flange 1300 . 12 , apertures 1420 or 4420 may enter the pump chamber at angles other than upward, including horizontal or downward angles. Also, in some embodiments apertures 1420 and 4420 may be replaced with slits or gaps between accelerator pins.

FIG. 1 also shows a feed/discharge assembly 2000 comprising a feed tube carrier 2300 through which the feed tube 2100 extends to the position shown in FIG. 3 proximate the bottom of the centrifuge structure 1000 . do. In this position, the feed tube 2100 can perform both feed and discharge functions without moving. The feeding process by carefully designing the spacing between the nozzle 2110 of the feed tube 2100 and the upper surface 1210 of the lower flange 1200, the diameter of the nozzle 2110 of the feed tube 2100, and the angular velocity of the centrifuge. shear force can be minimized. U.S. Patent No. 6,616,590 (the disclosure of which is incorporated herein by reference in its entirety) describes a method of selecting an appropriate relationship to minimize shear forces. Other suitable feed tube designs that minimize the shear forces associated with feeding liquid cell cultures to rotary centrifuges known to those skilled in the art may also be used.

1 further includes a centripetal pump 1400 for discharging the filtrate through the filtrate discharge path 2200 . 1 , the filtrate pump 1400 is located within the pump chamber 1420 above the upper flange 1300 . The pump chamber 1420 is a chamber defined by the upper surface 1505 of the core 1510 and the inner surfaces 1605 , 1620 of the centrifuge cover 1600 . The centrifuge cover 1600 may include a cylindrical wall 1640 and a coupling cap portion 1610 shaped as a generally circular disk (shown in FIG. 5 ). The centrifuge cover 1600 may be formed as a single piece or as a separate component.

As discussed in more detail below, in other embodiments, the shape and location of the filtrate pump chamber 1420 may vary. Chamber 1420 is generally located near the upper end of core structure 1500 in fluid communication with separation chamber 1550 via a hole or slit 1530 extending into filtrate pump chamber 1420 from the adjacent exterior of core 1515 . It becomes an axisymmetric chamber. In some embodiments, as most clearly shown in FIGS. 11 and 12 , the filtrate pump chamber 1420 may be located in a recess within the chamber 1550 .

Exemplary filtrate pump 1400 includes a pair of fairing disks 1410 . Fairing disk 1410 is two thin circular disks (plates) aligned axially with axis 1525 of core structure 1500 . 1-5 , fairing disks 1410 are held against centrifuge structure 1000 and are separated from each other by fixed gaps 1415 (indicated by reference numeral 1415 in FIG. 10 ). . The gap 1415 between the fairing disks 1410 forms part of a fluid connection for removing filtrate from the centrifuge 1000 , such that the filtrate surrounds the feed tube carrier 2300 between the fairing disks 1410 . And let it flow to the hollow cylindrical filtrate discharge path (2200) that ends at the filtrate outlet (2400).

Exemplary disposable centrifuge structure 1000 is included within a multi-use centrifuge structure 3000 . The multi-use centrifuge structure 3000 includes a ball 3100 and a cover 3200 . The walls of the centrifuge ball 3100 support the flexible liner 1100 of the centrifuge structure 1000 while the centrifuge rotates. To this end, the external structure of the disposable structure 1000 and the internal structure of the multi-use structure are matched with each other. Similarly, the upper surface of the upper flange 1200, the outer portion of the upper portion of the core 1510, and the lower portion of the wall 1640 of the centrifuge cover 1600 are also applied to the inner surface of the utility container cover 3200 and Consistently, they provide support during rotation. The features of the disposable ball 3100 and ball cover 3200, discussed in more detail below, are designed such that shear forces do not tear the liner 1100 free from the disposable centrifuge structure 1000 . In some cases, an existing multi-use structure 3000 may be retrofitted for single-use processing by selecting a compliant disposable structure 1000 . In other instances, the disposable structure 3000 may be specifically designed for use with the disposable structure insert 1000 .

2 is an illustration of a top flange 1300 , a plastic liner 1100 and a cover 3200 of a multi-use centrifugal separation structure 3000 to illustrate sealing the flexible liner 1100 to the top flange 1300 . It shows a part of the structural structure. The flexible liner 1100 may be a thermoplastic elastomer, such as polyurethane (TPU) or other stretchable, strong, non-tear, bio-compatible polymer, and the upper and lower flanges 1300 and 1200 may be polyetherimide, polycarbonate, or poly It can be made of hard polymers such as sulfones. The flexible liner 1100 is a thin sleeve or sheath that extends and seals between the upper and lower flanges 1300 , 1200 and forms the outer wall of the separation chamber 1550 . The configurations of the liner 1100 and the upper flange 1300 and the lower flange 1200 and the core 1510 described herein are only examples. A person skilled in the art can substitute suitable materials with properties similar to those proposed or known or may be known.

A thermal bond attachment process may be used to bond dissimilar materials in the area indicated in FIG. 2 . The thermal bond 1110 is formed by preheating the flange material, placing an elastomeric polymer over the heated flange, and applying heat and pressure to the elastomeric film liner 1100 at a temperature above the softening point of the film. The plastic liner 1100 is adhered to the lower flange 1200 in the same manner. Although thermal junction 1110 is described herein, this is by way of example only. Other means of creating a similarly strong, relatively permanent bond between the flexible film and the flange material may be substituted, such as temperature, chemical, adhesive or other bonding means.

Exemplary disposable components are pre-sterilized. Thermal bonding 1110 remains sterile within the disposable chamber during removal of these components from their protective packaging and installation in a centrifuge. The stretchable flexible liner 1100 conforms to the walls of the reusable ball 3100 in use. The reusable ball 3100 provides sufficient support and the flexible liner 1100 is resilient enough that the disposable structure 1000 allows the larger radius centrifuge 1000 to transfer liquid cell cultures or other cell suspensions. It rotates at an angular velocity sufficient to allow it to withstand the increased rotational forces generated when filling and to reach a processable settling rate at a rate of about 2-40 liters per minute.

In addition to thermal bonding 1110, sealing ridges or “nubbins” may be present over ball cover 3200 to compress the thermoplastic elastomeric film against rigid top flange 1300 to form an additional seal. there is. The same compression seal at the bottom of the ball 3100 may be used to seal the thermoplastic elastomeric film against the rigid lower flange 1200 . This compression seal supports the thermal coupling region 1110 by isolating it from the shear forces created by the hydrostatic pressure generated during centrifugation when the chamber is filled with liquid. The combination of thermal bonding 1110 and compression nub 3210 sealing was tested at 3000 x g, which corresponds to a hydrostatic pressure of 97 psi at the ball wall. The lining should be thick and compressible enough to allow the nub 3210 to compress and grip the flexible liner 1100 , but should minimize the risk of tearing near the thermal bond 1110 or compression nub 3210 . In one embodiment, the flexible TPU liner is sealed to a thickness of 0.010 inches without tearing or leaking.

Embodiments corresponding to the examples in FIGS. 1 and 2 were tested in a 5.5 inch diameter ball. At 2000 x g, the hydraulic power was greater than 7 liters/min and successfully separated mammalian cells with 99% efficiency at a rate of 3 liters/min.

In most cases, the upper and lower flanges 1300 and 1200 may have a shape similar to that shown in FIG. 1 , but in some cases the upper surface of the disposable centrifuge structure may be different as shown in FIGS. 10 and 11 . can have a form. 10 and 11, both the upper flange and the ball cover are relatively disk-to-match to conform to a generally conical upper flange 1300, rather than having a generally conical ball cover 3200. is my brother One of ordinary skill in the art will be able to apply the sealing techniques described herein for use with sealing surfaces of various shapes.

4 and 5 show exemplary embodiments with features for improving the efficiency of the centripetal pump 1400 . As detailed in FIG. 5 , this embodiment of an internal structure for a disposable component similar to that shown in FIGS. 1 and 2 is above the interior surface 1620 of the cap portion 1610 of the pump chamber 1420 . It includes a plurality of radial fins 1630 . 5 shows the inner surface 1620 of the cap portion 1610 of the centrifuge cover 1600 . The radial fins 1630 may be thin, generally rectangular, and radial plates that extend perpendicularly from the interior surface 1620 of the cap portion 1610 . In the exemplary embodiment, six pins 1630 are shown, although other embodiments may include fewer or more pins 1630 . In this embodiment, the fins 1630 form a portion of the inner surface of the cap 1620 , but in other embodiments the fins 1630 , which may take a form other than the cap 1610 , the upper surface 1620 of the pump chamber 1420 . may include When the centrifugal separation system 1000 is in use, a pin 1630 is positioned within the chamber 1420 over the fairing disk 1410 of the centrifugal pump 1400 . These pins 1630 transfer the angular rotation of the centrifuge 1000 to the filtrate in the pump chamber 1420 .

This increases the efficiency of the centripetal pump 1400 , stabilizes the gas and liquid interface in the pump chamber 1420 above the fairing disk 1410 , and increases the size of the gas barrier. The gas barrier is a generally cylindrical gas column that extends from the outside of the supply/discharge mechanism 2000 outward into the pump chamber 1420 to the inner surface of the center of rotation. The reason for the increase in the size of the barrier is that the increase in the angular velocity of the filtrate pushes the filtrate towards the centrifuge wall. When the rotating filtrate in the pump chamber 1420 contacts the fixed fairing disk 1410 , the resulting friction can reduce the efficiency of the pump 1400 . The addition of a plurality of radial pins 1630 rotating at the same angular velocity as the filtrate overcomes the decrease in velocity that may occur due to the encounter between the rotating filtrate and the stationary fairing disk 1410 .

6 depicts an exemplary embodiment of an improved core structure 1500 for use where the turbidity of the feed is high. Core structure 1500 includes a core 1510 , an upper flange 1300 , and a lower flange 1200 . The core 1510 has a cylindrical central cavity 1520 configured such that the feed tube 2100 can be inserted into the central cavity 1520 . The distance from the central axis 1525 to the outside of the core 1515 (the core width, indicated by dashed line 6000 in FIG. 6 ) is greater than the corresponding distance of the embodiment shown in FIG. 3 . The larger diameter core 1510 reduces the depth of the separation chamber 1550 (indicated by dashed line 6010 ), allowing the centrifuge 1000 to operate as a shallow pool centrifuge. Depth 6010 of separation chamber 1550 is generally the distance between flexible liner 1100 and the exterior of core 1510 indicated in FIGS. 1 and 12 . A shallow pool centrifuge is one that has a small depth 6010 relative to the diameter of the centrifuge. As can be seen in the exemplary embodiment shown in FIG. 12 , to facilitate the removal of cell concentrates, a shallow pool depth 6010 is provided at the bottom of the isolation chamber 1550 from shallower to a depth of the isolation chamber 1550 . It can vary from upper to slightly deeper. In some embodiments shown herein, the ratio of the average separation pool depth 6010 to the core width is 1:1 or less. An example of a shallow pool centrifuge is provided as an optional model of the ViaFuge centrifugation system manufactured by Pneumatic Scale Corporation. The advantage of shallow pool centrifuges is that they allow separation at higher feed flow rates. This is achieved by a higher average g-force for a given inner ball diameter, resulting in a higher settling velocity at a given angular velocity. The resulting improved separation performance is useful when separating highly turbid feeds containing high concentrations of cellular debris.

The exemplary embodiment of the core structure 1500 shown in FIG. 6 also includes accelerator vanes 1560 as part of the lower flange 1200 . Accelerator vane 1560 (as shown in FIG. 12 ) has a central cavity 1520 in core 1510 , rather than hole 1530 through solid core 1510 (as shown in FIGS. 10 and 11 ). ) and an alternative embodiment of a fluidic connection between the separation chamber 1550 .

In the exemplary embodiment of the core structure 1500 shown in FIG. 6 , the accelerator vane 1560 is a radially generally rectangular spaced thin plate 1580 extending upwardly from the upper conical surface of the lower flange 1200 . includes a plurality of Plate 1580 extends perpendicular to the base of core 1510 . Plate 1580 extends generally radially outward from near axis 1525 of core 1510 . In the exemplary embodiment, there are twelve plates 1580 as most clearly shown in FIG. 7 . In other embodiments, fewer or more plates 1580 may be present. Also, in other embodiments, plate 1580 may be curved in the direction of rotation of centrifuge 1000 as shown in the exemplary embodiment of FIG. 9 . The inner surface of the lower flange 1200 forms an elliptical accelerator ball 1590 from which an upwardly curved plate may be modified to extend. These embodiments are intended to be exemplary, and those skilled in the art will be able to combine these plates in different ways to further benefit from haze reduction or modify these embodiments to create the lower flange 1200 and/or embedded accelerator balls. can

Additional features of an exemplary embodiment of a disposable centrifugal separator 1000 designed to operate continuously or semi-continuously are shown in FIGS. 10-12 . The exemplary embodiment shown in FIG. 10 includes a second centripetal pump 4400 for removing cell concentrates. The centripetal pump 4400 for removing the cell concentrate is located above the centripetal pump 1400 for removing the filtrate. The centripetal pump 4400 includes a pump chamber 4420 and a fairing disk 4410 . A plurality of apertures or series of slits 4540 extend from the upper periphery of the separation chamber 1550 into the pump chamber 4420 to provide a fluid connection from the outer portion of the separation chamber 1550 to a second pump chamber 4420). do. Like pump chamber 1400 , pump chamber 4400 may have a shape other than that shown in FIGS. 10-12 , but is generally near the top of core structure 1500 in fluid communication with separation chamber 1550 . It will be an axisymmetric chamber. As with the pump chamber 1400 , the pump chamber may be partially or wholly recessed within the core structure 1500 . When the filtrate pump chamber 1400 is near the top of the core structure 1500 , the cell concentration pump chamber 4400 is generally located above it. The pump chamber 4400 for removing the cell concentrate has a hole or slit 4540 extending from the proximal outer upper wall of the separation chamber 1550 to collect the heavy cell concentrate that is compressed by centrifugal force. may be in fluid communication with the separation chamber 1550 through the

In the embodiment shown in FIG. 10 , the fairing disk 4410 used in the concentrate drain pump 4400 is approximately the same radius as that used in the filtrate drain pump 1400 and is rotationally fixed. In other embodiments, such as that shown in FIG. 11 , the fairing disk 4410 of the concentrate discharge pump 4400 is larger than the fairing disk of the filtrate discharge pump 1400 , as the pump chamber 4420 becomes larger. It can have a large radius. A variety of medium diameter fairing discs may also be used. The optimal diameter depends on the nature of the cell concentrate being expelled. Fairing discs with larger diameters have higher pumping capacity but higher shear forces.

1 , 4 and 10 , the fairing disk 4410 of the concentrate discharge pump 4400 is rotationally fixed. In other embodiments, such as that shown in FIG. 11 , the fairing disk 4410 may be configured to rotate at an angular velocity between zero and the angular velocity of the centrifuge 1000 . The desired angular velocity can be controlled by several mechanisms known to those skilled in the art. An example of a control means is an external slip clutch that allows the fairing disk 4410 to rotate at an angular velocity that is part of the angular velocity of the centrifugal separator 1000 . Other means of controlling the angular velocity of the fairing disk will be apparent to those skilled in the art.

1 , 4 , 10-12 , the gaps 1415 , 4415 between the fairing disks 1410 and 4410 are fixed. In other embodiments, such as the embodiment of FIG. 13 , the gaps 1415 , 4415 between the fairing disks 1410 and 4410 may be adjustable to control the flow rate at which the filtrate or concentrate is removed from the centrifuge 1000 . . One of each pair of fairing disks 1410 and 4410 is attached to move the throttle tube 6100 in a vertical direction. The throttle tube 6100 may be moved up and down to narrow or widen the spacing 1415 , 4415 between each pair of the fairing disks 1410 , 4410 . Additionally, an external peristaltic pump 2510 (not shown) may be added to the concentrate removal line 2500 (not shown) to aid in concentrate removal. This pump 2510 may be controlled by a sensor 4430 in the pump chamber 4420 . A sensor 4430 (not shown) may be used to control the diluent pump 5150 to synchronize concentrate removal with the addition of diluent.

Also shown in FIG. 13 is an embodiment in which the filtrate pump 1400 is located at the base of the centrifugal separator 1000 . In the embodiment shown in FIG. 13 , a centrate well 1555 is created between the pump chamber 1420 and the flexible liner 1100 . An orifice 1530 extends from the core 1510 to the filtrate well 1555 below the pump chamber 1420 . Also, in the exemplary embodiment shown, apertures 1540 extend from separation chamber 1550 adjacent outer surface 1515 of core 1510 into pump chamber 1420 , using filtrate pump 1400 . Allow the filtrate to be removed. An orifice 4540 also extends between the separation chamber 1550 and the pump chamber 4420 in the vicinity of the outer upper surface so that the cell concentrate to be removed using the centripetal pump 4400 flows into the pump chamber 4420 . make it possible

As noted above, in the illustrated embodiment, the gaps 1415, 4415 between the fairing disks 4410 and 1410 provide a throttle tube 6100 connected to one of the pairing disks 4410, 1410 of each pair. can be adjusted using The throttle tube 6100 and an attached one of each pair of fairing disks 4410 , 1410 can move up or down to narrow or widen the gaps 1415 , 4415 . In the exemplary embodiment shown, throttle tube 6100 is attached to the lower and upper fairing disks of pair of fairing disks 4410 and 1410, respectively. In other embodiments, this attachment may be reversed and used to throttle a single centrifugal pump, or both in parallel (not vice versa as shown in FIG. 13 ).

As can be seen in the embodiment shown in FIGS. 10-12 , the walls of the solid disposable ball 3100 are configured to support the disposable centrifugal separation structure 1000 having a smaller radius at the bottom than at the top. Thicker at the base than at the top to create a truncated cone shape on the inside. This larger radius at the top of the separation chamber 1550 moves the denser cell concentrate towards the upper outer portion of the separation chamber 1550 and into the centripetal pump chamber 4420 . In the embodiment shown, the truncated cone shape is created by the disposable ball 3100 having a thicker wall at the base than at the upper portion. Those of skill in the art will recognize that disposable ball 3100 having a truncated conical shape may also include walls of uniform thickness, and that there may be other variations to create a desired interior shape for disposable ball 3100 . something to do.

10-12, the feeding mechanism 2000 also includes additional pathways for removal of cells or cell concentrates. In the embodiment shown in FIG. 1 , a cylindrical path 2200 around the feed tube 2100 is used to remove the filtrate. The embodiment shown in FIGS. 10-12 also includes a concentric cylindrical path for removal of cells or cell concentrates, referred to as cell drain tube 2500 . A cell drain tube 2500 surrounds a centrifugation path 2200 . If the centrifuge is used with a concentrate that is expected to be very viscous, an additional concentric cylindrical fluid path 5000 is added around the feed tube 2100 so that the diluent pumps the cell concentrate to reduce the viscosity of the concentrate. may be introduced into the chamber 4420 . In the exemplary embodiment shown in FIG. 12 , the diluent pathway 5000 includes concentric tubes surrounding the cell exit pathway, and at the bottom above the fairing disk 4410 draining near the outer edge of the fairing disk 4410 . is opened to a thin disk-shaped fluid path 5100 of the <RTI ID=0.0> By this means the diluent is injected and at this point it is limited to mixing with the concentrate and draining with the concentrate rather than being introduced into the filtrate where the diluent may be unnecessary in some applications. In alternative embodiments, the diluent may be introduced directly to the upper surface of the fairing disk and spread out radially outward or over a separate disk positioned above the fairing disk.

The choice of diluent depends on the purpose of the separation process and the nature of the cell concentrate to be diluted. In some cases, simple isotonic buffer or deionized water may be used as the diluent. In other cases, diluents specific to the nature of the cell concentrate may be advantageous. For example, in production-scale batch cell cultures operating at low cell viability, flocculants are usually added to the culture when fed to a centrifuge to cause cells and cell debris to aggregate or aggregate into larger particles. They become segregated as their sedimentation rate increases. Because both cells and cell debris have a negative surface charge, the compounds used as flocculants are usually cationic polymers that carry multiple positive charges, such as polyethyleneimine. Thanks to their multiple positive charges, these coagulants can link negatively charged cells and cellular debris into large clumps. An undesirable consequence of the use of such flocculants is a further increase in the viscosity of the cell concentrate. Thus, particularly useful diluents in this application are anti-binding agents that increase the viscosity of the cell concentrate. Examples of peptizing agents include high salt buffers such as sodium chloride solutions having a concentration range of 0.1 M to 1.0 M. Other peptizing agents that may be useful in reducing the viscosity of cell concentrates are anionic polymers such as acrylic acid polymers.

For cell concentrates where cell viability is to be maintained, a diluent that is a shear protecting agent such as Dextran or Pluronic F-68 may be selected. The use of shear protective agents with isotonic buffers improves the viability and viability of cells exiting the centrifuge.

The exemplary centrifugal separator shown in FIG. 4 operates as described below. During the feed cycle, feed suspension flows through feed tube 2100 into the rotating ball assembly. As the feed suspension enters the central cavity 1520 of the core 1510 near the lower flange 1200 , the feed suspension is urged outwardly along the upper surface of the lower flange 1200 by centrifugal force, the hole in the core 1510 . Pass through 1530 into separation chamber 1550 .

The filtrate is collected in a separation chamber 1550, a generally cylindrical space below the upper flange 1300 surrounding the core 1510. The filtrate flows from the inlet to the separation chamber through hole 1530 until it meets hole 1540 between separation chamber 1550 and pump chamber 1420 in the upper portion of separation chamber 1550 adjacent to core 1410. flows upwards Particles that are denser than the liquid move towards the outer wall of the separation chamber 1550 by sedimentation (particle concentrate) and away from the aperture 1530 . When the centrifuge 1000 stops rotating, the particle concentrate moves under the influence of gravity to the nozzle 2110 of the feed tube 2100 for removal via the combined feed/discharge mechanism 2000 .

During rotation, the filtrate enters the filtrate pump chamber 1420 through an orifice 1540 . Within the pump chamber 1420 , the rotating filtrate encounters a stationary fairing disk 1410 that converts the kinetic energy of the rotating liquid into pressure, and the converted pressure causes the filtrate to pass through the filtrate discharge path within the supply/discharge mechanism 2000 . It is discharged upward through 2200 and is pressed to be discharged past the filtrate discharge tube 2400 .

The efficiency of the centripetal pump 1400 is increased by adding a radial fin 1630 on the inner surface 1620 of the cap portion 1610 of the rotating pump 1400 . These pins 1630 impart angular momentum of the rotating assembly to the filtrate in the pump chamber 1420 . Otherwise, the rotating filtrate may be slowed down due to friction as it meets the stationary fairing disk 1410 . The centripetal pump 1400 provides an improved means of filtrate evacuation over mechanical sealing due to the gas-liquid interface within the pump chamber 1420 . The gas in the pump chamber 1420 is isolated from contamination by the external environment by the rotation seal 1700 . Since the filtrate discharged between the fairing disks 1410 does not come into contact with air during the feeding or discharging process, excessive foaming occurring when air is introduced into the cell culture during the discharging process is prevented.

In the centrifuge 1000 embodiment shown in FIGS. 4 and 5 , the cell concentrate periodically stops ball rotation and feed flow, then pumps the collected cell concentrate along the outer wall of the separation chamber 1550 . discharged by doing This process is known as intermittent treatment. When the volumetric capacity of the separation chamber 1550 is reached, the centrifuge stops rotating. The cell concentrate moves down towards nozzle 2110 of feed tube 2100 , where the concentrate is pumped out through feed tube 2100 . A suitable valve (not shown) external to centrifuge 1000 is used to direct the concentrate to a collection vessel (not shown). If the entire bioreactor batch has not yet been fully processed, ball rotation and feed flow will resume, followed by additional feed and discharge cycles until the entire batch has been processed.

As mentioned above, if the cell culture is concentrated or contains significant cell debris, the process described above is slowed down because the residence time must be increased to capture the small debris particles. This requires a slower feed flow rate and the separation chamber 1550 fills and rotates quickly, with frequent and repeated stops for each culture batch. Also, the cell concentrate becomes more viscous and takes longer as gravity does not work efficiently to drain the cell concentrate to the bottom of the centrifuge 1000, and in some cases washing may be required to remove the remaining cells. .

As modified in the exemplary embodiment shown in FIGS. 6-13 , the disposable centrifuge produces a higher average sedimentation velocity without increasing angular velocity, and the centrifuge 1000 operates continuously or semi-continuously. and allows a diluent to be added to the cell concentrate during the cell removal process, making cell removal easier and more complete.

The embodiment of the disposable centrifugal separator structure 1000 shown in FIGS. 6-12 operates as discussed herein. The feed suspension enters the disposable centrifuge structure 1000 via feed tube 2100 . As the feed suspension encounters accelerator vanes 1560 , vanes 1560 impart an angular velocity to the feed suspension that approximates the angular velocity of disposable centrifuge 1000 . Using vanes 1560 rather than apertures 1530 allows a larger amount of feed suspension to enter the separation chamber 1550 at a less radial velocity, resulting in a hole with a smaller cross-sectional opening than the opening between the vanes 1560 ( 1530) to avoid jetting that occurs when the feed suspension is forced. Reducing the velocity of the feed stream as it enters the separation zone or pool allows for more efficient settling by minimizing disruption of the liquid contents of the pool.

As the centrifugal separator 1000 rotates, particles denser than the filtrate are pushed out of the separation chamber 1550 , and the filtrate without particles remains near the core 1510 . The centrifuge ball 3100 has the shape of an inverted cone, and has a wider radius at the top than at the bottom. The centrifugal force causes the particles to collect in the upper and outer portions of the chamber. The centrifuge 1000 may operate with semi-continuous discharge of the concentrate. The filtrate drain generally operates as described with respect to FIG. 4 . The cell concentrate evacuation works similarly, wherein the cell concentrate is collected near the upper outer portion of the separation chamber 1550 and the concentrate exhaust pump chamber 4400 through an orifice 4540 adjacent the upper outer wall of the separation chamber 1550. ) is introduced into

The feed rate and rotational angular rate of the suspension may be monitored using a vibration sensor system such as that described in US Pat. No. 9,427,748, which is incorporated herein by reference in its entirety. This sensor system fills the centrifuge at a lower speed until vibration indicates that the centrifuge is nearly full, and then allows the feed rate and angular velocity to be adjusted accordingly based on this information. Typically, when the centrifuge is near full, the feed rate is reduced or stopped, the angular velocity is increased to increase the settling rate, and the cycle repeats when settling and discharging are essentially complete. If the system is optimized using the additional features described here to reduce the need to interrupt the process, the system can be operated continuously or nearly continuously at the angular velocity required for stability.

As the concentrate is discharged semi-continuously, the suspension is continuously fed to the centrifugal separator 1000 using a concentration pump 4400 operating intermittently to remove the concentrate. The operation of the concentrate pump 4400 may be controlled by an optical sensor in the concentrate discharge line that indicates the presence or absence of the concentrate being discharged. Instead of the thickening pump 4400, the evacuation cycle may be managed electronically using controllers and sensors that determine when to open and close the valves for the most efficient treatment of the fluid suspension.

The average ejection rate can be further controlled using a centrifuge 1000 with an adjustable gap between the fairing disks 4410 and 1410. It may be desirable or necessary to have only one set of pairing disks 4410 and 1410 adjustable. The gap between the fairing disks 4410 , 1410 (which forms part of the fluid path outside the centrifuge 1000 ) may open or close to allow flow to act as an internal valve to block flow. It may also be useful to widen or narrow the gaps 4415 and 1415 between the fairing disks 4410 and 1410 depending on the desired product or characteristics of the product. Changing the spacing affects both the pumping and shear rates associated with the fairing disk.

The rate of removal of the concentrate and filtrate from the centrifuge 1000 and the viability of the removed concentrate can be further controlled using a number of features of the exemplary embodiments shown in FIGS. 4-13 . Accelerator pins 4630 similar to those in centrifugal pump chamber 1420 may be added to the thickening pump chamber 4420 . The addition of accelerator pins 4630 increases the rate at which the concentrate can be removed by overcoming some of the deceleration due to friction. In addition to the accelerator fins 4630 on the upper surface of the pump chamber 4420, these fins 4630 may also be added to the lower surface of the pump chamber 4420 to increase its effectiveness. An additional feature may be the replacement of holes 1540 and 4540 with slits, which minimize shear of material entering the pump chambers 1420 and 4420.

If viability of the concentrate is a concern, a rotatable separation disk 4410 may be included in the pump chamber 4420 , which reduces the shear transmitted to the concentrate when in contact with the surface of the fairing disk 4410 . . The rotational speed of the fairing disk 4410 may be adjusted to some degree between the stop and rotational speeds of the separation chamber 1550 to balance the retentate viability and the discharge rate. The desired angular velocity can be controlled by a number of mechanisms known to those skilled in the art. An example of a control means is an external slip clutch that allows the fairing disk to rotate at an angular velocity that is part of the centrifugal separator. The use of slip clutches is well known to those skilled in the art. Also, means other than the slip clutch for adjusting the angular velocity are apparent to those skilled in the art.

A peristaltic pump 2510 may be used to make removal of the concentrate more efficient and reliable, particularly when using highly concentrated feed suspensions. The use of a peristaltic pump 2510 allows the user to more precisely control the flow rate of the concentrate from the centrifugal separator 1000 rather than relying solely on the centripetal pump 4400, which means that the speed of the centripetal pump is equal to that of the peristaltic pump 2510. Because it can't be adjusted as easily as speed.

In addition, in order to reduce the viscosity of the concentrate, a diluent such as sterile water or a buffer may be pumped into the concentration pump chamber 4420 through the dilution path 5000 using the dilution pump 5150 . A more complete discussion of useful diluents can be found above. The rate at which one or both of the peristaltic pump 2510 or the diluent pump 5150 operates may be controlled by an automated controller (not shown) responsive to a concentration sensor 4430 located at the concentrate discharge connection 2500 . can The controller is responsive to concentration sensor 4430 to independently link pump rates for diluent addition and concentrate removal depending on the particle concentration of the concentrate, in conjunction with a standard feed/discharge cycle, to remove concentrate based on particle concentration in the concentrate. It can be programmed to start, stop, or adjust pump speed for both and diluent supply.

16 shows an alternative exemplary embodiment of a core used in connection with a centrifugal separator that provides a continuous separation process to continuously produce a concentrate and filtrate feed. The core 10 is similar to that previously discussed configured to be positioned in a rotatable ball of a centrifuge. The centrifuge ball and core rotate about an axis 12 during processing. The device includes a stationary assembly 14 and a rotatable assembly 16 .

As in the previous embodiment, the securing assembly 14 includes a feed tube 18 . The feed tube 18 is coaxial with the shaft 12 and terminates in an opening 20 adjacent the cavity 22 of the core or bottom of the separation chamber. The stationary assembly further includes a filtrate centripetal pump (24). The exemplary embodiment of the filtrate centripetal pump 24 described in greater detail below includes an inlet opening 26 and an annular outlet opening 28 . The annular outlet opening is in fluid communication with the filtrate tube 30 . The filtrate tube extends coaxially and surrounding the feed tube 18 .

In this exemplary embodiment, the filtrate centripetal pump 24 is located in the filtrate pump chamber 32 . The filtrate pump chamber provides an inlet opening 26 for the filtrate centripetal pump that is defined by a wall that is part of the rotatable assembly and is exposed to the liquid filtrate pool during operation.

The exemplary embodiment further includes a concentrate centripetal pump 34 . The concentrate centripetal pump 34 of the exemplary embodiment may also be of a similar configuration as discussed in detail later. The centrifugal centrifugal pump 34 in the exemplary arrangement includes an inlet opening 36 located in the wall bounding the annular perimeter of the centripetal pump. It should be noted that the centrifugal centrifugal pump 34 has a larger peripheral diameter than that of the centrifugal pump. The concentrate pump further comprises an annular outlet opening 38 . The annular outlet opening 38 is in fluid communication with the concentrate outlet tube 40 . The concentrate outlet tube extends coaxially with the central tube 30 .

In the exemplary embodiment the inlet opening 36 of the concentrate centripetal pump is located in the concentrate pump chamber 42 . The concentrate pump chamber is defined by the walls of the rotatable assembly 16 . In operation, the inlet opening 36 of the concentrate centrifugal pump is exposed to the concentrate in the concentrate pump chamber 42 . The concentrate pump chamber 42 is vertically bounded by an upper portion 44 . At least one fluid seal 46 extends between the outer periphery of the outlet tube 40 and the upper portion 44 . Exemplary seal 46 is configured to reduce the risk of fluid escaping inside the separation chamber and to prevent entry of contaminants from areas outside of the inner core.

During operation of the centrifuge, the ball and core comprising the cavity or separation chamber are rotated about the axis 12 in the direction of rotation. Rotation in the direction of rotation operates to separate the cell suspension entering through feed tube 18 into filtrate exiting through filtrate tube 30 and concentrate exiting through concentrate outlet tube 40 .

The cell suspension enters the separation chamber 22 through a tube opening 20 at the bottom of the separation chamber. The cell suspension moves outward through centrifugal force and a plurality of acceleration vanes (48). As the suspension moves outward by the accelerating vanes, the cell suspension material acts by centrifugal force to cause the cell material to move outwardly towards the annular tapered wall 50 bounding the outside of the separation chamber. The concentrated cellular material is forced to move outward and upward as shown against the tapered wall 50 and through a plurality of retentate slots 52 . The concentrate material moves upwards over the concentrate slot and into the concentrate pump chamber 42 where the concentrate is discharged by the concentrate centripetal pump 34 .

In the exemplary apparatus, the cell-free filtrate during operation is positioned proximate to a vertical annular wall 54 bounding the interior of the separation chamber 22 . The filtrate material travels upward through the filtrate apertures 56 of the annular base structure bordering the filtrate pump. The filtrate travels upward through the filtrate apertures 56 and forms a pool of liquid filtrate in the filtrate chamber. From the filtrate chamber, the filtrate is moved through the actuation of a filtrate centripetal pump (24) and delivered from the core through a filtrate tube (30).

In the exemplary embodiment of FIG. 16 , the concentrate and filtrate pumps may generally have a configuration as shown in FIG. 17 . In Figure 17, the filtrate centripetal pump 24 is represented in an isometric exploded view. As shown in FIG. 17 , the exemplary centripetal pump has a disc-shaped body comprised of a first plate 58 and a second plate 60 . In operation, the first plate and the second plate are held releasably engaged via fasteners represented by screws 62 . Of course, it should be understood that other configurations and fastening methods may be used in other embodiments.

In the exemplary arrangement, the second plate 60 includes a wall delimiting three sides of the curved vortex passage 64 . It should be understood that the centripetal pump in the exemplary arrangement generally includes a pair of opposed vortex passageways 64 . In other devices, other numbers and configurations of vortex passageways may be used.

In the exemplary arrangement, the first and second plates constitute a disk-shaped body of the centripetal pump having an annular vertically extending wall 67 defining an annular perimeter 66 . An inlet opening 68 to the vortex passage 64 extends around the annular perimeter. An annular collection chamber 70 extends in the body radially outward from axis 12 and is fluidly connected to the vortex passageway. The annular collection chamber 70 receives the material entering the inlet opening 68 . The annular collection chamber 70 is in fluid communication with an annular outlet opening coaxial with the shaft 12 . In the exemplary arrangement for the filtrate centripetal pump, the annular outlet opening is an annular space extending between the outer wall of the feed tube 18 and the inner wall of the second plate 60 at which the outlet is fluidly connected to the central outlet tube 30 .

Each vortex passageway 64 in the exemplary device is configured such that the vortex passageway curves toward a direction of rotation of the ball and separation chamber, the direction of rotation being indicated by arrow R in FIG. 17 . In the exemplary arrangement, the vertically extending walls 74 bounding the vortex passageways and facing the direction of rotation are each curved toward the direction of rotation. The curved configuration of the wall 74 horizontally delimiting the vortex passageway provides improved pumping characteristics of the exemplary device. Also, in the exemplary arrangement the opposing boundary walls 76 of each vortex passage have a similar curved configuration. The curved configuration of the vertically extending walls horizontally delimiting the vortex passageways provides a constant cross-sectional area of each vortex passageway from each inlet to the collection chamber. This consistent cross-sectional area is further achieved through the use of a generally flat wall 78 extending between the walls 74 and 76 and delimiting the vortex passageway vertically on one side. Also in the exemplary embodiment, the first plate 58 includes a generally planar circular surface 80 on its side that faces inward when the plate is assembled to form the disk-shaped body of the centripetal pump. In this exemplary arrangement, face 80 serves to vertically demarcate the sides of both vortex passageways 64 of the centripetal pump.

Of course, including a pair of plates, wherein one of the plates on one comprises a walled recess bounding three of the four sides of the curved vortex passageway, and the other comprising a walled recess bordering the other side of the curved vortex passageway. It should be understood that this exemplary device, including a surface, is exemplary. It should be understood that in other devices, other configurations and structures may be used.

In the exemplary centripetal pump structure shown in Fig. 16, a centripetal pump structure is used, which can move more liquid than a fairing disk-type centripetal pump of a similar size. In addition, the exemplary configuration generates less heating of the liquid than comparable fairing disks.

Also, in the example arrangement previously discussed, the annular perimeter of the filtrate centripetal pump 24 has a smaller outer diameter than the perimeter of the concentrate centripetal pump 34 . In the exemplary apparatus, this configuration is used to prevent the filtrate centripetal pump from removing too much liquid from the pool of liquid filtrate formed in the filtrate pump chamber 32 . Ensuring that there is a sufficient amount of liquid filtrate in the filtrate pump chamber helps to avoid the formation of waves in the filtrate around the inlet of the filtrate centripetal pump. The formation of ripples resulting from less than sufficient liquid filtrate can cause vibration and other undesirable properties of the centrifuge and core.

The larger annular perimeter of the concentrate pump of the exemplary device allows material to preferentially flow out of the core through the concentrate centripetal pump. In the exemplary apparatus, the flow of retentate downstream of the retentate discharge tube 40 may be controlled to control the ratio of the filtrate flow to the retentate flow from the core.

In a further exemplary embodiment, using a centrifugal pump having the described configuration, the properties and flow properties of the centrifugal separator can be tailored to the specific materials and requirements of the separation process being performed. Specifically, the diameter of the annular circumference of the centripetal pump can be sized to achieve optimum characteristics for a particular processing activity. For example, the larger the perimeter diameter of the centripetal pump, the greater the flow and pressure at the outlet. Moreover, larger diameters tend to produce larger mixes than relatively smaller diameters. However, the larger diameter results in greater heating than the smaller peripheral diameter of the centripetal pump. Thus a smaller diameter perimeter can be used to achieve less heating. It should also be understood that different sizes, areas and numbers of inlet openings and different vortex passage configurations may be utilized to vary the flow and pressure characteristics as desired for the purposes of a particular separation process.

19 schematically depicts an exemplary system used to ensure positive pressure within a separation chamber, collectively alternatively referred to herein, during processing of a cell suspension. As discussed in connection with the previous exemplary embodiments, it is generally desirable to ensure a positive pressure above atmospheric pressure in the separation chamber at all times. This reduces the risk of contaminants entering the separation chamber through one or more fluid seals operatively extending between the stationary assembly and the rotatable assembly of the core. Also as previously discussed, it is generally desirable to maintain the air at a positive pressure in the separation chamber in contact with the inner surface of the fluid seal. Having an air pocket adjacent to the seal prevents the seal from coming into contact with the material being processed and reduces the risk of contaminants entering the treated material and material escaping from the separation chamber.

The exemplary system described with respect to FIG. 19 serves to maintain a consistent amount of pressure in the separation chamber and to reduce the risk of ingress of contaminants and of outflow of treated material.

As schematically shown in FIG. 19 , the centrifuge includes a rotatable ball 82 . The centrifuge ball may be rotated about an axis 84 by a motor 86 or other suitable rotating device.

The exemplary centrifuge structure shown includes a rotatable, disposable core 88 surrounding a cavity 90 , also referred to herein as a separation chamber.

As with other embodiments described above, the exemplary core includes a securing assembly comprising a suspension inlet feed tube 92 having an inlet opening 94 positioned adjacent the bottom region of the cavity. The stationary assembly further includes at least one centripetal pump 96 . The centripetal pump of the exemplary embodiment includes a disk-shaped body having at least one pump inlet 98 adjacent its periphery and a pump outlet 100 adjacent the center of the centripetal pump. The pump outlet is in fluid communication with the filtrate outlet tube 102 . The filtrate outlet tube extends around and coaxially with the suspension inlet tube in a manner similar to that previously discussed. The rotatable upper portion 104 of the fluid containing the separation chamber is operatively connected with at least one seal 106 operative to fluidly seal the cavity of the core with respect to the inlet tube and the outlet tube. The at least one seal 106 is between the outer annular surface of the fixed central outlet tube 102 and the rotatable upper portion 104 of the core having an upper inner wall internally surrounding the cavity 90 as shown. operatively extended in a sealed relationship.

In the exemplary device inlet tube 92 is fluidly connected to pump 108 . In the exemplary device, pump 108 is a peristaltic pump effective to pump the cell suspension without causing damage to the cell suspension. Of course, it should be understood that these types of pumps are exemplary and that other types of pumps may be used in other devices. Also in the exemplary device the pump 108 is reversible. This allows the pump 108 to act as a feed pump to pump the cell suspension from the inlet line 110 to the inlet tube at a controlled rate. In a further exemplary device, the pump 108 may operate as a retentate removal or drain pump after the cell retentate has been separated by a centrifugal action. In performing this function, the pump 108 operates to pump the cell concentrate from the separation chamber by reversing the flow of material in the inlet tube 92 as the cell suspension is supplied to the separation chamber. The cell concentrate is then pumped into a concentrate line 112 . 19, inlet line 110 and retentate line 112 may be selectively opened and closed by valves 114 and 116, respectively. In an exemplary embodiment, valves 114 and 116 include pinch valves that open and close flow through the flexible line or tube. Of course, it should be understood that this approach is exemplary and that other approaches may be used in other devices.

In the exemplary system, the filtrate drain tube 102 is fluidly connected to the filtrate drain line 118 . The filtrate discharge line is fluidly connected to the filtrate discharge pump 120 . In the exemplary device, the filtrate discharge pump 120 is a variable flow rate pump, which can selectively adjust the flow rate. For example, in some example devices, pump 120 may include a peristaltic pump including a motor whose speed may be controlled to selectively increase or decrease the flow rate through the pump. The outlet of the filtrate drain pump delivers the treated filtrate to an appropriate collection chamber or other treatment device.

In the exemplary apparatus schematically shown in FIG. 19 , the pressure damping reservoir 122 is fluidly connected to a filtrate drain line 118 intermediate the filtrate drain tube 102 and the pump 120 . In the exemplary device, the pressure damping reservoir includes a vertically extending vessel having an interior region configured to retain the liquid filtrate in a generally vertically fluid tight relationship. The pressure damping reservoir includes a bottom port 124 fluidly connected to a filtrate discharge line 118 .

Opposite the reservoir 122 is the top port 126 . The upper port is exposed to pneumatic pressure. In the exemplary device, the upper port is exposed to atmospheric pressure from a source of elevated atmospheric pressure, schematically indicated at 128 . In an exemplary embodiment, the source of elevated pressure may include a compressor, air storage tank, or other suitable device for providing a source of elevated air pressure above atmospheric pressure to the extent necessary for system operation. Air from the high pressure source 128 is passed through a sterile filter 130 to remove impurities. Regulator 132 operates to maintain a generally constant atmospheric pressure level above atmospheric pressure at the upper port of the pressure damping reservoir. In an exemplary arrangement, the air pressure regulator includes an electronic high speed actuation regulator to ensure that a generally constant air pressure is maintained at a desired level. Exemplary high-speed actuation regulator 132 operates to rapidly increase the pressure acting at top port 126 when the pressure drops below a desired level, and pressure through the regulator when pressure acting at top port is higher than a set value. to quickly relieve

In some embodiments, the regulator outlet may also be in working fluid communication with the interior of the upper portion 104 of the separation chamber via an air line 143 shown schematically and schematically. In this exemplary arrangement, the outlet pressure of the regulator acting on the upper port 126 of the reservoir is above a pocket of air inside the separation chamber that also extends down through an air line 143 to a cavity level above the centripetal pump inlet; Acts radially within the at least one seal 106 and from a region proximate to the axis 84 to an upper inner wall inside the upper portion 104 . In the exemplary device line 143 may flow into the separation chamber under at least one seal through at least one separate passageway that extends through the anchoring structure of the assembly including the filtrate outlet tube 102 and the inlet feed tube 92 . Apply positive pressure to the area. At least one exemplary isolated passage of the air line 143 applies air pressure to the interior of the upper portion 104 through at least one air opening 145 to the separation chamber. Exemplary at least one opening 145 is located outside the outer surface of outlet tube 102 , above inlet 98 to the centripetal pump, and below at least one seal 106 . Of course, it should be understood that the air pockets of the separation chamber and the lines providing positive air pressure inside the at least one seal are exemplary, and that in other embodiments, other structures and approaches may be used.

In the exemplary arrangement of the pressure damping reservoir 122 , the upper liquid level sensor 134 is configured to sense the liquid filtrate within the interior of the pressure damping reservoir. The upper liquid level sensor operates to sense liquid at the upper water level. The lower liquid level sensor 136 is positioned to sense liquid in the reservoir at a lower liquid level. A high liquid level sensor 138 is positioned to sense a high level in the reservoir above the upper liquid level. A sensor with a high liquid level is arranged to detect an unacceptably high liquid level to indicate an abnormal condition that requires shutting down the system or taking other appropriate safety measures. In the exemplary arrangement, liquid level sensors 134, 136 and 138 include capacitive proximity sensors suitable for sensing the level of liquid filtrate in and adjacent to the pressure damping reservoir. Of course, it should be understood that these types of sensors are exemplary and that other sensors and approaches may be used in other devices.

Exemplary embodiments further include other components that may be suitable for operation of the system. These other components may include other valves, lines, pressure connections, or other suitable components for carrying out the processing and handling of suspensions, filtrates and concentrates suitable for the particular system. It may include additional valves, such as the schematically shown valve 140 , to control the open and closed states of the filtrate drain line 118 . Additional lines, valves, connections or other items included may vary depending on the nature of the system.

The exemplary system of FIG. 19 further includes at least one control circuit 142 , which may alternatively be referred to as a controller. Exemplary at least one control circuit 142 includes one or more processors 144 . The processor is operatively connected to one or more data stores 146 , schematically indicated at 146 . As used herein, a processor processes data stored within one or more data stored or received from an external source to resolve information and provide output that can be used to control other devices or perform other operations. Refers to any electronic device configured to operate via processor-executed instructions for The one or more control circuits may be implemented as hardware circuits, software, firmware, or applications that operate to enable the control circuits to receive, store, or process data and perform other operations. For example, control circuitry may include one or more of a microprocessor, CPU, FPGA, ASIC, or other integrated circuit or other type of circuit capable of performing a function in the manner of an electronic computing device. Data storage may also correspond to one or more of a volatile or non-volatile memory device, such as RAM, flash memory, hard drive, solid state device, CD, DVD, optical memory, magnetic memory, or other circuit readable media or media. should understand Computer executable instructions and/or data may be stored therein.

Circuit execution instructions may include instructions in any of a plurality of programming languages and formats including, but not limited to, routines, subroutines, programs, threads of execution, objects, methodologies and functions that perform operations as described herein. can The structure for the control circuit can include, respond to, and utilize the principles described in the book Microprocessor Architecture, Programming, and Applications with the 8085 by Ramesh S. Gaonker (Prentice Hall, 2002). Of course, it should be understood that these control circuit structures are exemplary and that other circuit structures for storing, processing, solving, and outputting information may be used in other embodiments.

In the exemplary arrangement, the at least one control circuit 142 is operatively connected to at least one sensor, such as sensors 134 , 136 and 138 , via a suitable interface. The at least one control circuit is also operatively connected with the variable flow discharge pump 120 . Also in some demonstrative embodiments, the at least one control circuit may also be operatively connected with other devices such as motors 86 , pumps 108 , regulators 132 , pneumatic sources 128 , fluid control valves, and other devices. there is.

Exemplary at least one control circuitry is operative to receive data and control such devices according to circuit execution instructions stored in data store 146 . In the exemplary device, the fluid level 147 of the fluid damping reservoir is a characteristic corresponding to the pressure in the filtrate drain tube 102 . In one exemplary implementation that does not use the air line 143 , the fact that the pressure in the filtrate outlet tube is characteristic of the pressure in the upper portion 104 of the core and the pressure in the separation chamber adjacent the seal 106 is , used to control the operation of discharge pumps and other components. As previously discussed, it is desirable to maintain a positive pressure above atmospheric pressure with a pocket of air adjacent to at least one seal within the separation chamber to prevent entry of contaminants into the separation chamber, which may occur due to negative pressure. However, if the fluid level within the separation chamber becomes too high, the pressure and suspension material being processed can overflow the seal, resulting in undesirable exposure to potential contamination and loss of material being processed. This can occur in situations where the back pressure on the filtrate line connected to the outlet of the centripetal pump is too high.

In the exemplary arrangement, the ball velocity produces a corresponding pumping force and pump output pressure level of the centripetal pump. This pump output pressure level of the centripetal pump depends on the rotational speed of the ball and core. An exemplary apparatus that does not use the air line 143 provides a controlled back pressure on the filtrate drain tube. The back pressure is provided by controlling the speed of the motor that operates the pump 120 and the liquid level 147 in the pressure damping reservoir. The backpressure is maintained below the pump output pressure level (so that the centripetal pump can transmit centrifugal force in the separation chamber), but is maintained at a positive pressure above atmospheric pressure to prevent contaminants from penetrating past the seal and into the separation chamber, A pressure is maintained inside the separation chamber adjacent the seal to isolate the seal from the components of the suspension being processed.

In the exemplary arrangement, the elevated pressure applied to the upper port 126 of the pressure damping reservoir is maintained by a regulator 132 . also maintaining a liquid level 147 between an upper liquid level and a lower liquid level 136 sensed by the sensor 134 by at least one control circuit 142 controlling the speed of the pump 120 , and separating The filtrate flow out of the separation chamber is controlled so that the pressure in the upper region of the chamber is maintained at a desired constant value and the filtrate does not contact the seal or overflow the seal.

In an alternative embodiment using an air line 143 , the positive pressure level of the regulator acts on both the fluid in the reservoir 122 and the separation chamber region above the centripetal pump inlet. Because the positive pressure level of the air applied at both points is the same, the back pressure in the filtrate discharge line (the pressure applied over the fluid in the reservoir) is in fact always equal to the pressure in the air pockets at the top of the separation chamber. This allows the centripetal pump to operate without the net effect of both pressures.

In this exemplary embodiment, the pump 120 and other system components are controlled in response to the at least one control circuit 142 to ensure that there is an appropriate amount of air within the interior of the reservoir 122 at all times during filtrate production. do. This ensures that the reservoir provides a variable damping effect of the filtrate discharge line pressure that may be caused by the pumping action of the pump 120 . This is done by maintaining the liquid in the reservoir 122 no higher than the upper liquid level sensed by the sensor 134 . Further, the liquid level in the reservoir is controlled to remain above the underlying liquid level as sensed by the sensor 136 . This ensures that the centripetal pump does not pump air and does not aerate the filtrate.

In the exemplary apparatus, the filtrate flow from the separation chamber is controlled through actuation of at least one control circuit. Exemplary control circuitry may operate the system during processing conditions to maintain an influent flow of cell suspension introduced by pump 108 into separation chamber 90 at a generally constant rate, wherein the separation process is configured to concentrate the filtrate and cells. This is done with a motor 86 operating to keep the ball speed constant to separate the water. The exemplary device maintains an ideally constant backpressure in the filtrate discharge line from the centripetal pump, while drawing air in the separation chamber to the lower level of the air pockets to isolate at least one seal 106 from the filtrate and concentrate material being processed. It works to keep it higher.

In the exemplary apparatus, the pressure maintained through actuation of the regulator in the pressure damping reservoir is set to about 2 kpa (0.29 psi) above atmospheric pressure. In the exemplary system, this pressure has been found to be suitable to ensure that seal integrity and isolation are maintained during all steps of cell suspension processing. Of course, these values are exemplary, and it should be understood that other pressure values and pressure damping reservoir configurations, sensors, and other features may be utilized in other devices.

20 schematically illustrates exemplary logic implemented through actuation of the filtrate drain tube and at least one control circuit 142 related to maintaining a desired pressure level within the upper portion of the separation chamber. It should be understood that in some demonstrative embodiments, the control circuitry may perform many additional or different functions than those represented. In addition to the pressure control functions described above, these functions may include overall control of the various processes and steps for operating the centrifuge. 20 , in an initial subroutine step 148 , the at least one control circuit 142 is operative to determine whether centrifugal operation is currently in a mode in which filtrate is withdrawn from the separation chamber. If so, the at least one control circuit is operative to cause the filtrate drain pump 120 to operate to drain the filtrate delivered through the filtrate drain line 118 . This can be done by causing the motor of the pump to operate. In the exemplary arrangement, the flow rate of the pump 120 may be an initially set value or may be changed during the process according to specific operating conditions as determined through control circuit operation. Operation of the filtrate drain pump is indicated by step 150 .

The at least one control circuit is then operative to determine at step 152 whether liquid is sensed at a high level of the high liquid level sensor 138 . If so, this indicates an undesirable condition. When liquid is sensed at the level of sensor 138 , the control circuitry operates to take steps to resolve this condition. This may include activating the pump 120 to increase its flow rate and making a subsequent determination as to whether the level will drop within a period of time while the centrifuge continues to operate. Alternatively or additionally, the at least one control circuit may reduce the speed of the pump 108 to reduce the flow of incoming material. If these measures do not cause the level to drop within a certain amount of time, an additional step is taken. These steps may also include slowing or stopping rotation of the ball 182 . This operation may also include shutting down the pump 108 to prevent more suspended material from being introduced into the separation chamber. This step, generally referred to as terminating the normal operation of the system, is indicated by step 154 .

If no liquid is sensed at the level of the high level sensor 138 , then at least one control circuit is activated to determine if liquid is sensed at the upper liquid level of the sensor 134 . This is represented by step 156 . When liquid is sensed at the upper liquid level sensor, the at least one circuit increases the speed in response to the stored command, thereby increasing the flow rate of the discharge pump 120 . This is done, in an exemplary embodiment, by increasing the speed of the motor that is part of the pump. This is represented by step 158. Increasing the flow rate of the pump causes the liquid level 147 in the pressure damping reservoir to begin to drop as more liquid is moved by the pump 120 .

If in step 156 no liquid is sensed at the upper liquid level of the sensor 134 , the at least one control circuit is operative to make a determination as to whether no liquid is sensed at the lower liquid level of the sensor 136 . This is represented by step 160 . If the liquid level is not at the sensor 136 level, the control circuit operates according to its programming to control the pump 120 to reduce the flow rate. This is done, in an exemplary embodiment, by slowing down the motor. This is represented by step 162 . In the exemplary arrangement, slowing the flow rate of the pump 120 causes the liquid level 147 to begin to rise in the pressure damping reservoir. If the level does not rise within the reservoir within a given amount of time, the control circuitry may operate according to programming to cause further actions, such as those associated with shut down step 154 previously discussed. The control circuitry of the exemplary embodiment operates to vary the pumping rate of pump 120 to maintain level 147 in the pressure damping reservoir at a generally constant level between the levels of sensors 134 and 136 while producing filtrate. can do.

In the exemplary apparatus, maintaining a generally constant elevated pressure of sterile air above the liquid in the pressure damping reservoir helps to ensure that similar elevated pressures in the seal and filtrate outlet lines within the separation chamber are consistently maintained. Also in the exemplary arrangement, the pressure may be controlled at a desired level during different operating conditions of the centrifuge while the balls are rotating at different speeds. Examples include conditions in which the separation chamber is initially filled at a relatively high speed through introduction of the cell suspension and the centrifuge is rotated at a relatively low speed. The pressure may be maintained during subsequent conditions of final filling in which the cell suspension flow into the separation chamber occurs at a slower rate and the rotational speed of the ball increases to a higher rotational speed. In addition, positive pressure is maintained as previously described while feeding the suspension into the bowl and discharging the filtrate from the separation chamber. In a further exemplary embodiment, the at least one control circuit may be operable to maintain a positive pressure for a period in which the concentrate is removed by being pumped out of the separation chamber. Maintaining a positive pressure within the separation chamber under all these conditions reduces the risk of contamination and other undesirable conditions that can arise from negative (sub-atmospheric) conditions.

Of course, it is to be understood that these features, components, structures, and control methodologies are exemplary, and that other approaches may be used in other arrangements. Additionally, although exemplary apparatus includes systems operating in batch mode rather than in a mode in which the filtrate and retentate are processed continuously, the principles herein are applicable to these other types of systems as well.

A pressure damping reservoir is useful in exemplary embodiments to help ensure that a desired pressure level is maintained in the outlet tube and separation chamber, although other approaches may also be used in other exemplary embodiments. For example, in some devices, pressure may be directly sensed and/or applied at an outlet tube, separation chamber, or other location corresponding to the pressure in the separation chamber. In some configurations, the flow rate of the evacuation pump may be controlled to maintain an appropriate pressure level. In another configuration, the exemplary control circuitry is operable to control both the pump supplying the suspension and the discharge pump to the core and/or to an appropriate valve or other flow control device to maintain an appropriate pressure level. This alternative approach may be desirable depending on the particular centrifugation device used and the type of material being processed.

21 schematically depicts an alternative centrifugation system 170 specifically configured to separate cells in a cell culture batch into cell filtrate and cell concentrate on a continuous or semi-continuous basis. The exemplary system shows a rigid centrifuge ball 172 that can rotate about an axis 174 . The ball includes a cavity 176 configured to releasably receive a disposable structure 178 therein. The rigid ball includes an upper opening 180 . An annular retaining ring or other schematically indicated retaining structure 182 may releasably secure the disposable structure 178 within the ball cavity.

The exemplary disposable structure 178 of this exemplary embodiment includes an axially extending feed tube 184 . As will be discussed later, the feed tube is used to transport the cell culture batch material to the interior region 186 of the disposable structure 178 . A supply tube 184 extends from the top of the first axial end 188 of the disposable device to an opening 190 in the lower interior region of the second axial end 192 . The disposable structure 178 includes a substantially disk-shaped portion 194 adjacent the first axial end. Exemplary disk-shaped portion 194 is generally rigid, meaning that the disk-shaped portion is rigid or semi-rigid, and includes an annular perimeter 196 . The annular perimeter is configured to abut the upper annular boundary wall 198 of the centrifuge ball cavity 176 . The annular perimeter of the disk-shaped portion 194 is configured to engage the rigid ball 172 such that the disposable structure rotates therewith.

Exemplary disposable structure 178 further includes a hollow rigid or at least semi-rigid cylindrical core 200 . The core 200 is operatively coupled to the disk-shaped portion 194 and is rotatable therewith. The core 200 is axially aligned with the disk-shaped portion and extends axially midway between the upper and lower portions of the disposable structure. The core 200 includes an upper opening 202 and a lower opening 204 through which a feed tube 184 extends.

The disk-shaped portion 194 includes a substantially circular filtrate centripetal pump chamber 206 . A filtrate centripetal pump 208 is located within the chamber 206 . The substantially annular filtrate opening 210 is in fluid communication with the filtrate pump chamber 206 . Substantially annular means that the opening may consist of a continuous opening as well as discrete openings of an annular structure. The filtrate centripetal pump 208 is in fluid communication with the filtrate drain tube 212 . The filtrate drain tube 212 extends in a coaxial circumferential relationship of the feed tube 184 . The discharged filtrate passes through a substantially annular opening at the periphery of the filtrate centripetal pump and through an annular space in the filtrate discharge tube 212 external to the feed tube.

The disk-shaped portion 194 further includes a concentrate centripetal pump chamber 214 . The concentrate centripetal pump chamber 214 is a substantially circular chamber located above the filtrate centripetal pump chamber 206 . The concentrate centripetal pump chamber 214 has a concentrate centripetal pump 216 positioned therein. The concentrate centripetal pump is in fluid communication with the concentrate discharge tube 220 . The concentrate discharge tube 220 annularly surrounds the central discharge tube 212 and extends. The concentrate passes through a substantially annular opening at the periphery of the concentrate centripetal pump and an annular space outside the central discharge pipe of the concentrate discharge conduit 220 .

The substantially annular concentrate opening 218 is in fluid communication with the concentrate pump chamber 214 . In the exemplary device, the substantially annular concentrate opening and the substantially annular central opening are concentric coaxial openings with the concentrate opening disposed radially outward of the filtrate opening. Of course, these structures are exemplary and other approaches and configurations may be used in other embodiments.

Exemplary disposable structure 178 further includes a flexible outer wall 222 . The flexible outer wall 222 is a fluid tight wall that extends in an operatively supported engagement with the wall bounding the rigid ball cavity 176 in the operative position of the exemplary disposable structure 178 . In the exemplary device, the flexible outer wall 222 is operatively coupled in a fluid tight connection with the disk-shaped portion 194 . The flexible outer wall has a truncated cone shape with a smaller inner radius adjacent the bottom of the disposable structure adjacent the second axial end 192 .

An exemplary flexible outer wall 222 extends surrounding at least a portion of the core 200 . A wall 222 further surrounds the annular separation chamber 224 . The separation chamber 224 extends radially between the outer wall of the core 200 and the flexible outer wall 222 . The substantially annular retentate opening 218 and the substantially annular filtrate opening 210 are each in fluid communication with the separation chamber 224 .

In the exemplary device, the flexible outer wall 222 has a textured outer surface 226 . The textured outer surface is configured to permit air passage out of the space between the flexible outer wall 222 and the surface surrounding the cavity of the rigid ball 172 . In the exemplary device, the textured outer surface may contact the rigid ball substantially the entire area of the flexible outer wall. In an exemplary device, the textured exterior surface may include one or more patterns of outwardly extending protrusions or dimples 228 having spaces or recesses therebetween to facilitate passage of air. Air may pass out of the ball cavity 176 when the disposable structure 178 is positioned therein through the upper opening 180 or the lower opening 230 . In an exemplary arrangement, the protrusion may comprise a resilient deformable material whose height may be reduced by the force of the liner against the rigid wall of the ball. The textured outer surface of the flexible outer wall 222 reduces the risk of air pockets becoming trapped between the rigid balls of the centrifuge and the disposable structure. These air pockets can create imbalances or cause irregularities in the wall contour that can alter the contour of the separation chamber in a way that adversely affects the separation process. Of course, it should be understood that the air release structures described are exemplary and that other air release structures may be used in other embodiments.

The exemplary disposable structure shown in FIG. 21 further includes a lower rigid or semi-rigid disk-shaped portion 232 . A rigid or semi-rigid material operates to maintain its shape during operation. In the exemplary device, the lower disk-shaped portion 232 has a conical shape and is operatively connected to the lower end of the core 200 by a vertically extending wall portion or other structure. A plurality of angularly spaced fluid passages 234 extend between an upper surface of the disk-shaped portion 232 and a radially outer lower portion of the core. The fluid passageway 232 extends radially outwardly and upwardly with respect to the bottom of the second axial end 192 , and provides access to the cell culture batch material entering the interior region 186 through the opening 190 of the feed tube 184 . Allow cells to pass radially outward and upwardly into the separation chamber 224 .

In the exemplary device, the flexible outer wall 222 extends below the lower disc-shaped portion 232 at the second axial end 192 of the disposable structure. The flexible outer wall 222 extends midway through the wall surface of the rigid ball 172 bounding the lower disc-shaped portion 232 and the cavity in which the disposable structure is located.

In the exemplary apparatus, the feed tube 184, the filtrate drain tube 212 and the concentrate drain tube 220, as well as the filtrate centripetal pump 208 and the concentrate centripetal pump 216 remain stationary while the centrifugal separator Ball 172 and upper disk-shaped portion 194, lower disk-shaped portion 232 and flexible outer wall 222 rotate with respect to the ball. At least one annular resilient seal 236 extends in an operably sealing engagement between the upper disc-shaped portion 194 and the outer surface of the concentrate discharge tube 220 . The at least one seal 236 may maintain an airtight seal in a manner as previously discussed, such that a pocket of air may be maintained in the interior region 186 during cell processing to isolate the seal from the cell culture batch material being processed. Air pockets retained within the interior region of the disposable structure are configured to maintain the filtrate centripetal pump 208 and the concentrate centripetal pump 216 in fluid communication with the cell culture batch material. In a manner as previously discussed, positive pressure may be maintained within the interior region to ensure that a pocket of air is present to properly separate the at least one seal 236 from the cell culture batch material being processed. Alternatively, other approaches may be used to maintain isolation of the seal from the material being processed.

Exemplary system 170 operates in a manner as previously discussed. Cells within the cell culture batch material are introduced into the interior region 186 of the disposable structure 178 via the feed tube 184 . Cells enter the interior region 186 through a feed tube opening 190 at the lower axial end of the disposable structure. The centrifugal force causes the cells to move outward through the opening 234 and into the separation chamber 224 . The outwardly and upwardly tapering outer walls 222 allow the cells or cellular material comprising the cell concentrate to gather adjacent the radially outer and upper regions of the separation chamber 224 . Typically, the cell-free filtrate is collected radially inward adjacent to the outer wall of the core 200 in the separation chamber.

In the exemplary device, the cell filtrate travels upward through the substantially annular filtrate opening to the filtrate pump chamber. The filtrate passes inward through the substantially annular opening of the filtrate centripetal pump and then upwards through the filtrate outlet tube 212 . At the same time, the cell retentate passes through a substantially annular retentate opening 218 and enters the retentate centripetal pump chamber 214 . The concentrate passes inward through the substantially annular opening of the concentrate centripetal pump 216 and then upwards through the concentrate discharge tube 220 . This example configuration allows the example system 170 to operate continuously or semi-continuously. The operation of system 170 can be controlled in a manner as discussed later to facilitate reliable extended operation of the system and delivery of a desired cell concentrate and, generally, cell-free filtrate in a separate output fluid stream. there is.

22 shows an alternative centrifugal separation system generally designated 238 . System 238 has a disposable structure 240 . Disposable construction 240 is similar in most respects to disposable construction 178 previously described. Some of the structures and features of disposable structure 240 that are generally identical to those described with respect to disposable structure 178 are denoted by the same reference numbers used to describe disposable structure 178 .

Disposable structure 240 differs from disposable structure 178 in that it includes a rigid or semi-rigid lower disc-shaped portion 242 . The lower disc-shaped portion 242 is a generally conical structure in operative connection with the lower end of the core 200 . A plurality of radially outwardly and upwardly extending fluid passageways 244 extend between the lower end of the core 200 and the lower disc-shaped portion 242 . Exemplary lower disc-shaped portion 242 also includes a plurality of angularly spaced radially extending vanes 246 . In this exemplary arrangement, vanes 246 extend upwardly from a bottom portion of disk-shaped portion 242 and at least a portion operatively engages the core at a radially outer portion thereof. In the exemplary device, vanes 246 accelerate cell culture deployment to facilitate movement and separation within the interior region of the disposable structure.

An alternative exemplary embodiment of a centrifugal separation system 248 is shown in FIG. 23 . This exemplary embodiment includes a disposable structure 250 . Disposable structure 250 is similar in many respects to disposable structure 178 previously described. Some of the structures and features similar to those of the previously described disposable structure 178 are labeled with the same reference numerals on the disposable structure 250 .

Exemplary disposable structure 250 differs from disposable structure 178 in that it includes a lower disc-shaped portion 252 . The lower disc-shaped portion 252 is a rigid or semi-rigid cone-shaped structure in an operatively attached connection with the core 200 through a wall portion or other suitable structure. The lower disc-shaped portion 252 includes a plurality of angularly spaced plurality of accelerator vanes 254 extending radially outward. Accelerator vanes 254 extend down from the lower conical side of disk-shaped portion 252 . Each immediately adjacent pair of angularly adjacent vanes 254 has a fluid passageway extending therebetween. In this exemplary device, the flexible outer wall 222 extends in an intermediate relationship between the lower end of the vane 254 and the wall of the rigid ball 172 bounding the cavity 176 . This exemplary configuration provides an immersion accelerator operative to accelerate cell culture batch material to facilitate separation within the interior region of the disposable structure. Of course, it should be understood that the features of the disposable constructions described herein may be combined into different constructions to facilitate separation of different types of substances and substances having different properties and to achieve a desired output fluid stream.

26 shows an alternative disposable structure 304 . Disposable construction 304 is similar to disposable construction 178 previously described except as otherwise noted herein. Elements that are identical to those of the disposable structure 178 are indicated using the same reference numerals in FIG. 26 .

The disposable structure 304 includes a continuous annular concentrate dam 306 . The concentrate dam 306 extends downwardly from the separation chamber 224 and is disposed radially inward of the substantially annular concentrate opening 218 . The exemplary annular concentrate dam, shown in cross section, extends below the concentrate opening and includes a tapered outwardly facing surface 308 that extends outwardly and towards the opening 218 in axial cross section.

The disposable structure 304 further includes a continuous annular filtrate dam 310 . The filtrate dam 310 extends downward in the separation chamber 224 below the substantially annular filtrate opening 210 . The filtrate dam 310 is disposed radially outward from the filtrate opening 210 . In the exemplary structure, the distance at which the concentrate dam 306 and the filtrate dam 310 extend downward in the separation chamber 224 is substantially the same. However, other configurations may be used for other exemplary devices. Also in other exemplary devices, the centrifugal separator structure may include a concentrate dam or a filtrate dam, but not both.

An annular recess 312 in the separation chamber extends radially between the filtrate dam and the retentate dam. An exemplary annular recess extends upwardly between the filtrate dam and the concentrate dam to form an annular pocket between the filtrate dam and the concentrate dam.

In an exemplary embodiment, the concentrate dam 306 is an upper portion bounding the separation chamber 224 for primarily cellular material or other solids to be separated to reach the concentrate opening 218 and the concentrate centripetal pump chamber 214 . It helps to ensure that it can pass outwards along the part. Filtrate dam 310 also allows primarily cell-free filtrate material to pass along an upper surface bordering separation chamber 224 and into a substantially annular filtrate opening 210 to reach filtrate pump chamber 206 helps to ensure that It should be understood that various configurations of concentrate dams and filtrate dams may be used in various exemplary apparatuses depending on the nature of the material being treated and the specifications for handling such material.

24 is a schematic diagram of an exemplary control system for providing generally continuous processing of cell culture material to generally produce a stream of cell free filtrate and cell concentrate. The centrifugal separation system 170 previously discussed in an exemplary apparatus is shown. However, it should be understood that the exemplary system features may be used with various types of materials and centrifugal separation systems and structures such as those discussed herein.

In the exemplary embodiment shown, centrifuge ball 172 is rotated by motor 256 about axis 174 at a selected speed. Feed tube 184 is operatively connected to cell culture feed line 258 through which the cell culture batch material is received. The feed line is operatively connected to the feed pump 260 . Feed pump 260 in the exemplary device may be a peristaltic pump or other suitable pump for delivering the cell culture to a disposable structure at a selected flow rate.

The filtrate drain tube 212 is in fluid communication with the filtrate drain line 262 . The filtrate optical density sensor 264 is operatively connected with the interior region of the filtrate discharge line 262 . In the exemplary device the filtrate optical concentration sensor is an optical sensor currently operative to determine the density of cells in the filtrate passing from the disposable structure. In an exemplary embodiment, this is done by receiving the light intensity output by the transmitter with at least a portion of the filtrate flow passing between the transmitter and the receiver into the receiver and measuring the reduced amount. The amount of light from the transmitter that the receiver receives decreases as the cell density in the filtrate increases. Of course, this is only one example of a sensor that may be used to determine the density or amount of cells present in the filtrate, and other types of sensors may be used in other devices. For example, the light may be near infrared or other visible or invisible light. Other types of electromagnetic, sonic or other types of signals may be used for sensing in other sensing devices. The filtrate drain line is also in operative connection with a filtrate pump 266 . In exemplary embodiments, the filtrate pump may include a peristaltic pump or other variable speed pump suitable for pumping the filtrate material.

In the exemplary arrangement, the concentrate discharge tube 220 is operatively connected with the concentrate discharge line 268 . The concentrate optical density sensor 270 is operatively connected with at least a portion of the interior region of the concentrate discharge line 268 . The exemplary concentrate optical density sensor may operate in the same manner as the filtrate optical density sensor previously discussed. Of course, it should be understood that the concentrate optical density sensor may include different structures or properties, and that different types of cell density sensors may be used in other exemplary embodiments. A concentrate discharge line 268 is operatively connected to a concentrate pump 272 . In an exemplary embodiment, the concentrate pump 272 may include a peristaltic pump or other variable speed pump suitable for pumping the concentrate without causing damage. Of course, it should be understood that these structures and components are exemplary and that other systems may include different or additional components.

The exemplary control system includes control circuitry 274, also referred to herein as a controller. In an exemplary embodiment, the control circuitry may include one or more processors, schematically indicated by reference numeral 276 . The control circuitry may also include one or more data stores, indicated schematically at 278 . The one or more data stores may include one or more tangible tangible media holding executable instructions and data that, when executed by the controller, cause the controller to perform operations such as those discussed herein below. Such media may include, for example, solid state memory, magnetic memory, optical memory, or other suitable non-transitory media for carrying circuit executable instructions and/or data. The control circuit may include structures such as those discussed above.

The operations performed by the exemplary controller 274 will now be described with respect to a schematic representation of the logic flow shown in FIG. 25 . In the exemplary device, the controller 274 is generally operative to control the operation of the components of the system so as to maintain delivery of a simultaneous output flow of cell-free filtrate and retentate. This generally involves installing optical density sensors within each filtrate and retentate discharge line to detect the cell density (or turbidity) of the output feed and to adjust the operation of system components to maintain the output within the desired range. is performed using

In use of the exemplary control system, the concentration of cells in the cell culture material to be treated is separately measured prior to starting operation of the system. The desired axial rotation speed of the centrifugal separator is determined as the operating speed of the feed pump 260 . In the exemplary device, the rotation speed of the centrifuge and the feed rate of the cellular material by the feed pump are generally maintained at a constant set value by a controller. Of course, alternative approaches may be used in which the rate and feed rate may be adjusted by the controller during processing of cells in other devices and systems.

In the exemplary device, based on the determined cell concentration, the discharge rate (flow rate) of the external concentrate pump 272 is set to an initial value referred to herein as a “prime value”. Also in the exemplary embodiment is a "prime period" corresponding to the period during which the preset external concentrate pump 272 initially operates at a prime value. This period allows the disposable structure 178 to be partially filled. Also in the exemplary system a “base rate” is set for the concentrate pump based on the cell density and the feed rate from the feed pump 260 . The base speed of the concentrate pump is the rate (corresponding to the flow rate) at which the concentrate pump will operate after the prime period. In the exemplary apparatus, a set base rate generally corresponds to a concentrate pump speed that will produce a filtrate having a cell density below a desired set limit and a cell concentrate having a cell concentration above a further desired set limit, generally It is usually expected to be The set values and limits are received by the controller in response to input via an appropriate input device and stored in at least one data store.

In the exemplary logic flow shown in FIG. 25 , operation of the concentrate pump 272 at the initial prime speed is represented by step 280 . A determination is made at step 282 by the controller as to whether the concentrate pump is operating at prime speed during a period corresponding to a prime period operative to at least partially fill the disposable structure 178 .

If the concentrate pump is operating at prime speed during the prime period, the controller increases the concentrate pump speed to the base speed as expressed in step 284. The controller 274 operates to monitor the cell density in the filtrate as sensed by the sensor 264 . The controller operates to determine if the optical density is above the desired set point, as indicated in step 286 . If the optical density of the filtrate is not higher than the set point, the logic returns to step 284 as there are not enough cells or cellular material in the filtrate such that this measurement will not cause a controller change in the operating speed of the concentrate pump.

If at step 286 it is determined that the optical density in the filtrate is above the set point, then the logic proceeds to step 288 . In step 288, the controller operates to increase the speed of the thickening pump by a set incremental step amount. This rate step increase is usually to reduce the optical density of the filtrate by reducing the number of cells in the filtrate.

After the speed of the concentrate pump 272 is increased in step 288, the controller operates in response to the sensor 264 in step 290 to incrementally increase the speed (flow rate) of the concentrate pump and increase the optical density of the filtrate at a set time. It works to determine if the set point is still exceeded. In that case, the controller continues to monitor the optical density of the center until it is not higher than the set point. In the exemplary apparatus the command is that the filtrate optical density must not be higher than the set point before the concentrate pump speed controller determines whether adjustment of the base speed is sufficient to maintain the optical density of the filtrate at or below a desired set point. Includes a set period. Step 292 consistently checks whether increased concentrate pump speed for a stored set period of time corresponding to reaching a programmed waiting time or generating an output of the cell-free filtrate is consistently sufficient to maintain the optical density of the filtrate below a set point. Represents the controller that decides. Upon generating sufficient aromatic cell-free filtrate for a desired period of time or upon reaching a programmed waiting time, the controller operates, in step 294, to adjust the base speed value of the concentrate pump to a value corresponding to the increased base speed. The controller sets the new base speed, and the logic returns to step 284. If the filtrate optical density is still above the set point as determined in step 286, then the concentrate pump speed is again adjusted.

The exemplary controller also simultaneously monitors the optical density of cells in the exiting retentate flow. This is done by monitoring the optical density detected by the sensor 270 . As represented by step 296, the controller is operative to determine if the optical density of the concentrate is below a desired set point. If the concentrate optical density is detected at or above the desired set point value stored in the data store, then the cellular concentration of the exiting concentrate flow is above the desired level, and logic returns to step 284. However, if the optical density of the concentrate is below the desired set point, it means that the cellular level of the concentrate is lower than desired, so the controller moves to step 298. In step 298 the speed of the concentrate pump is reduced by a predetermined amount of incremental steps. Reducing the speed of the concentrate pump decreases the output flow and generally increases the amount of cells in the concentrate output flow, increasing the optical density of the concentrate output flow.

The controller then operates the thickening pump 272 at a new reduced speed as shown in step 300 . As shown in step 302, the controller writes to the data store so that the concentration of cells in the output retentate flow can increase before determining whether the rate reduction is sufficient. Operate the thickening pump at this reduced speed for a set time corresponding to the stored set point. If it is determined in step 302 that the period of time has elapsed, the controller returns to step 284 where the logic flow is repeated to determine if further speed adjustments are necessary.

Of course, it should be understood that this schematic simplified logic flow is exemplary and in other embodiments other logic flows and/or additional operating parameters of system components may be monitored and adjusted to achieve the desired output flow of filtrate and retentate. . For example, in another exemplary device, the speed of the filtrate drain pump and thus the filtrate drain flow is varied, at least in part, by the response of the controller to an optical density detected by a filtrate optical density sensor corresponding to a level of cells in the filtrate. can do For example, if it is detected that the level of cells in the filtrate is above a set limit, the controller may act to reduce the flow rate of the filtrate pump. This may be performed by the controller as an alternative to or in combination with controlling the concentrate discharge flow rate. The controller may change the filtrate flow rate as appropriate such that the level of cells in the filtrate remains below a set limit or within a set range.

Alternatively or additionally, the controller may also control the flow rate of the cell suspension entering the disposable construct. This may be done in conjunction with varying the flow rates of the filtrate and retentate exiting the disposable construct to maintain the cellular level of concentration within a programmed set limit stored in a memory associated with the controller. The controller can also operate programmatically to vary the ball rotation speed, diluent introduction and diluent introduction rates, and other process parameters to maintain the properties of the filtrate and concentrate at desired throughput rates within programmed limits. In still other exemplary embodiments, other properties or parameters may be monitored and adjusted by the control system to achieve a desired product.

27 shows a cross-section of another alternative disposable centrifuge structure 314 . Disposable structure 314 is generally similar to disposable structure 178 previously discussed except where specifically noted. The disposable structure 314 includes elements extending from the disposable structure and operative to ensure that the air/liquid interface of the air pockets isolating the seal 236 from the material being processed remains more stable in the desired radial direction. do.

In the disposable configuration 314 , the filtrate pump 208 is located in the filtrate pump chamber 316 . The filtrate pump chamber 316 is bordered vertically at the bottom by a circular lower filtrate pump chamber surface 318 . The filtrate pump chamber 316 is vertically bounded at the top by a circular upper filtrate pump chamber surface 320 .

The lower filtrate pump chamber surface 318 extends radially outward from the lower filtrate centripetal pump chamber opening 322 . In the exemplary apparatus the lower filtrate centripetal pump chamber opening 322 extends through the circular top of the core 200 and corresponds to the upper opening 202 discussed previously. A feed tube 184 extends through the lower filtrate centripetal pump chamber opening.

The upper filtrate centripetal pump chamber surface 320 extends radially outward from the circular upper filtrate centripetal pump chamber opening 324 . Feed tube 184 and filtrate outlet tube 212 extend axially through the upper filtrate centripetal pump chamber opening.

A plurality of angularly spaced upwardly extending lower filtrate chamber vanes 326 extend on the lower filtrate centripetal pump chamber surface 318 . Each lower filtrate chamber vane 326 starts from a lower filtrate centripetal pump chamber opening 322 and extends radially outward. The lower filtrate chamber vane 326 , shown in greater detail in FIG. 28 , extends radially outward from the lower central vane distance V from the axis of rotation 174 . In the exemplary apparatus, the lower filtrate chamber vane 326 extends upward in a circular recess on the lower filtrate centripetal pump chamber surface 318 . However, it should be understood that these structures are exemplary and that other structures may be used in other embodiments. For example, the radial length of the vanes, the vane height, the depth and diameter of the recesses can be varied to achieve the desired fluid pressure characteristics.

A plurality of angularly spaced downwardly extending upper filtrate chamber vanes 328 extend from the upper filtrate centripetal pump chamber surface 320 . Each upper filtrate chamber vane 328 starts from an upper filtrate centripetal pump chamber opening 324 and extends radially outward. The upper filtrate chamber vanes extend radially outward from the upper filtrate vane distance from the axis of rotation 174 . The upper filtrate vane distance in the exemplary apparatus substantially corresponds to the lower central vane distance V. In the exemplary apparatus, the upper filtrate chamber vanes extend downward in a circular recess on the upper filtrate centripetal pump chamber surface having a configuration as shown in FIG. 28 , but in a reversed direction.

In the exemplary arrangement shown, the filtrate centripetal pump 208 includes a substantially annular filtrate centripetal pump opening 330 . The substantially annular filtrate centripetal pump opening 330 is disposed radially outward from the axis of rotation 174 which is the filtrate pump opening distance. The filtrate pump opening distance at which the filtrate centripetal pump opening 330 is located is a greater radial distance than the lower filtrate vane distance and the upper filtrate vane distance for reasons discussed later.

In the exemplary arrangement of the disposable construction 314 , the concentrate centripetal pump 216 is located in the concentrate pump chamber 332 . The concentrate pump chamber 332 is bounded vertically at the bottom by a circular lower concentrate centripetal pump chamber surface 334 . Concentrate pump chamber 332 is bordered vertically on the upper side by a circular upper concentrate centripetal pump chamber surface 336 .

The lower concentrate centripetal pump chamber surface 334 extends radially outward from the lower concentrate centripetal pump chamber opening 338 . In the exemplary apparatus, the lower concentrate centripetal pump chamber opening corresponds in size to and is continuous with the upper filtrate centripetal pump chamber opening 324 . Feed tube 184 and centrifugal discharge tube 212 extend through lower concentrate centrifugal pump chamber opening 338 .

A plurality of angularly spaced upwardly extending lower concentrate chamber vanes 340 extend on the lower concentrate centripetal pump chamber surface 334 . The lower concentrate chamber vane 334 starts from the lower concentrate centripetal pump chamber opening 338 and extends radially outward. The lower concentrate chamber vane 334 extends radially outward from the axis of rotation at the lower concentrate vane distance. In the exemplary apparatus, a lower concentrate chamber vane 334 extends over a circular recessed portion of the lower concentrate centripetal pump chamber surface similar to the previously discussed upper and lower filtrate chamber vanes. Of course, it should be understood that this configuration is exemplary.

The upper concentrate centripetal pump chamber surface 336 extends radially outward from the upper concentrate centripetal pump chamber opening 342 . Feed tube 184 , filtrate drain tube 212 , and concentrate drain tube 220 extend coaxially through upper concentrate centripetal pump chamber opening 342 . A plurality of angled, spaced-apart upper concentrate chamber vanes 344 extend downward from surface 336 . The upper concentrate chamber vanes extend radially outwardly from the upper concentrate centripetal pump chamber opening 342 to the upper concentrate vane distance. The upper concentrate chamber vane extends in an upwardly extending circular recess in the surface of the upper concentrate centripetal pump chamber. In the exemplary apparatus, the upper concentrate chamber vanes are constructed in a manner similar to the previously discussed lower concentrate chamber vanes and the upper and lower central chamber vanes. Of course, it should be understood that these approaches are exemplary and that other embodiments may use other approaches.

The concentrate centripetal pump 216 includes a substantially annular concentrate pump opening 346 . The concentrate pump opening is disposed radially from the axis of rotation 174 by the thickening pump opening distance. The upper and lower concentrate vane distances in the exemplary apparatus are less than the concentrate pump opening distance. Of course, this configuration is exemplary and it should be understood that other embodiments may use other approaches.

In the exemplary disposable structure 314 , upper and lower concentrate chamber vanes 344 , 340 and upper and lower filtrate chamber vanes 326 , 328 are annular air/liquid interface 348 of centrifugal pump chamber 330 and Acts to stabilize and radially position the air/liquid interface 350 of the concentrate pump chamber 332 . 28, the air/liquid interface 348 is positioned radially midway along the radial length of the filtrate chamber vane. It is radially inward from the filtrate pump opening 330 . The radially extending filtrate chamber vanes operate to provide a centrifugal pumping force that maintains the annular air/liquid interface 348 in radial positions both above and below the radially disposed centrifugal pump. The vanes in the exemplary device further help stabilize the air/liquid interface to maintain a coaxial circular configuration both above and below the filtrate pump. In a further exemplary arrangement, the radial position of the interface relative to the axis of rotation may be controlled, as discussed later, such that the filtrate pump opening 330 remains consistently centered in the liquid and not exposed to air.

Upper retentate chamber vane 344 and lower retentate chamber vane 340 operate in a similar manner to the filtrate chamber vane. The concentrate chamber vanes maintain the circular air/liquid interface 350 of the concentrate pump chamber 332 at a radial distance substantially inside the annular concentrate pump opening 346 . This configuration ensures that the concentrate pump opening is continuously exposed to the concentrate but not to air. It should also be understood that in the illustrated embodiment the filtrate centripetal pump and the concentrate centripetal pump are substantially the same size, and other structures allow the centripetal pump to have different sizes. In this situation, the radial distance from the axis of rotation the filtrate chamber vane and the concentrate chamber vane extend may be different. Also, the radial position of the air/liquid interface with respect to the axis of rotation in the filtrate pump chamber and the concentrate pump chamber may be different. Various vane configurations and arrangements may be utilized depending on the particular relationship between the components making up the disposable device and the particular material being processed through the disposable structure.

30 shows an upper portion of another alternative disposable structure 352 . Disposable structure 352 is similar to disposable structure 304 unless otherwise discussed. The disposable structure 352 includes an air tube 354 extending coaxially surrounding the concentrate exhaust tube 220 . Air tube 354 communicates with opening 356 within the disposable structure. An opening 356 extends from the interior of the air tube over the concentrate centripetal pump 216 of the concentrate pump chamber 332 . In this exemplary device, as schematically shown, seal 236 operatively engages the air tube, the concentrate exhaust tube, the filtrate drain tube, and the feed tube to maintain a hermetic bond with air. As will be appreciated, an air tube may be used to selectively maintain a level of air pressure within an air pocket within the disposable structure. Such structures may be utilized in conjunction with systems such as those previously discussed or other systems, where an external supply of compressed air isolates the seal of the centrifuge structure from the material being processed and maintains the air/liquid interface in a desirable position. used to Of course, it should be understood that this structure is exemplary and that other approaches may be used in other embodiments.

31 schematically depicts a system 358 that can be used to sequentially separate a cell suspension into a substantially cell-free filtrate and a concentrate. System 358 is similar to system 170 previously discussed except as otherwise noted herein. In the exemplary device, system 358 operates using a disposable structure similar to disposable structure 352 . The controller 274 of the system 358 controls the position of the air/liquid interface within the disposable structure to ensure that the air/liquid interface remains radially inward with respect to the axis of rotation from each of the filtrate pump opening and the thickening pump opening. operates to control

In the exemplary device, flow back pressure regulator 360 is in fluid communication with filtrate outlet line 262 . In the exemplary arrangement, flow back pressure regulator 360 is fluidly intermediate between filtrate outlet tube 212 and filtrate pump 266 . Exemplary system 358 includes a source of pressurized air, shown schematically at 362 . A pressurized air source 362 is connected to a pilot pressure control valve 364 . The control valve is operatively connected with the controller 274 . A signal from the controller 274 selectively generates a variable pressure in the pilot line 366 . Pilot line 366 is in fluid connection with back pressure regulator 360 . The pressure applied by the pilot pressure control valve 264 in the pilot line 366 operates to control the filtrate flow back pressure applied by the filtrate fluid and consequently flow back pressure regulator 360 .

Pressure control valve 368 in the exemplary device is in fluid communication with a pressurized air source 362 . Control valve 368 is also in operative connection with controller 274 . In this exemplary arrangement, the control valve 368 is controlled to selectively apply precise pressure to the air tube 354 and air pockets within the upper portion of the disposable structure 352 .

In the exemplary arrangement, the controller 274 operates according to stored executable instructions to control the operation of the system 358 in a manner as previously discussed with respect to the system 170 . Also in the exemplary arrangement, the controller 274 operates to control the pilot pressure valve 364 to vary the back pressure applied to the filtrate drain tube 212 by the back pressure regulator 360 . Controller 274 also operates to control valve 368 . The controller operates to maintain and selectively vary the pressure applied to the air pockets in the upper portion of the interior of the disposable structure. The controller operates programmatically to change the back pressure and/or air pocket pressure of the filtrate flow to thereby change the air/liquid interface of the air pocket at a radial distance from the axis of rotation inward from the filtrate pump opening 330 and the concentrate pump opening 346 . Vary the back pressure of the filtrate flow and/or the pressure of the air pockets to maintain With the action of the filtrate chamber vanes and concentrate chamber vanes in the exemplary embodiment, these pressure changes in both filtrate flow back pressure and air pocket pressure maintain stability and are introduced into the filtrate and retentate exiting the disposable structure. Keep the radially outer extent of the air/liquid interface constant so that air is minimal. Additionally, the ability to selectively vary filtrate flow and backpressure can affect the optical density perceived at the cell level and correspondingly discharged concentrate. Accordingly, the controller selectively changes all of the retentate flow rate, filtrate back pressure and flow rate, internal air pocket pressure, the feed rate of the cell suspension in the disposable structure and other other parameters of the centrifugation process, and in association with the controller at least one data store It can operate programmatically to maintain filtrate and concentrate properties within set limits and/or ranges stored in the . The exemplary apparatus also enables separation of different types of materials and different flow rates, while maintaining reliable control of the separation process. Of course, it should be understood that control of the position of the air/liquid interface is described with respect to features of system 170, but such control may be utilized in other types of systems including other types of processing elements.

Accordingly, the novel centrifugal separation system and method of the exemplary embodiments achieve at least some of the above-mentioned objects, obviate the difficulties encountered in the use of conventional apparatus and systems, solve the problems and achieve the desirable results described herein. achieve

Although specific terminology has been used in the above description for the sake of brevity, clarity, and understanding, such terminology is for the purpose of description and is intended to be interpreted broadly and should not suggest unnecessary limitation. Moreover, the description and illustrations herein are for the purpose of example and not to limit the invention to the precise details shown and described.

In the claims below, all features described as means for performing a function are to be construed as including any means known to one of ordinary skill in the art as applied for being capable of performing the stated function, and to the structure indicated herein or equivalent thereto. not limited

It describes the features, discoveries and principles of new and useful features, the manner in which they are constructed, used, and operated, and the advantages and useful results obtained, and describes new and useful structures, devices, elements, arrangements, parts, combinations, systems, equipment, operations and The interrelationships are set forth in the appended claims.

Claims (21)

A disposable structure configured for use in a centrifugal separation system comprising a multi-use rotatable centrifugal ball, wherein the disposable structure is releasably positioned within the bowl and wherein the cells in the cell culture batch within an interior region of the structure are separated from the cell concentrate and a disposable construct configured to dissociate into cell filtrate;
The structure in the operating position comprises:
upper disc-shaped part,
a lower portion located below the upper disc-shaped portion;
a cylindrical core intermediate the upper disk-shaped portion and the lower portion;
a separation chamber radially surrounding the core;
With the outer wall, the upper disc-shaped part and the outer wall can rotate about a vertical axis in the ball, the outer wall comprising:
extending in liquid-tight operating relation with the upper disc-shaped portion and surrounding the separation chamber;
extending around the core;
an outer wall extending radially outward from the core in the shape of a truncated cone with an inner radius smaller than that of a neighboring lower portion;
a supply tube extending in the vertical direction;
a filtrate drain tube extending in a vertical direction;
filtrate pump chamber;
As a filtrate centripetal pump, the filtrate centripetal pump comprises:
aligned axially with the core,
located coaxially with the supply tube,
in fluid communication with the filtrate drain tube;
located within a filtrate pump chamber, wherein the filtrate pump chamber is in fluid communication with the separation chamber;
a filtrate centripetal pump;
An apparatus, characterized in that while the ball rotates, the upper disc-shaped part and the outer wall rotate with respect to each of the feed tube, the filtrate discharge tube and the filtrate centripetal pump. Device according to claim 1, characterized in that the filtrate pump chamber is in the upper disc-shaped part. 2. The apparatus of claim 1, wherein the exterior wall comprises an exterior textured surface to allow air to escape from between the exterior wall and the ball. 2. The method of claim 1, wherein the lower portion comprises a lower disc-shaped portion in fixed operative connection with the core;
The lower portion within the interior region includes a plurality of angularly spaced radially extending accelerator vanes, wherein a fluid passageway extends between the vanes;
The device of claim 1, wherein the radially extending accelerator vanes extend upwardly from the lower disk-shaped portion or downwardly from the lower disk-shaped portion. The method of claim 1 , wherein the outer wall comprises a pattern of outwardly extending protrusions, with intermediate recesses between the protrusions;
The device of claim 1, wherein the pattern of protrusions and recesses allows air to escape from between the outer wall and the ball. 2. The method of claim 1, wherein the lower portion in the inner region comprises a lower disc-shaped portion,
The lower disk-shaped portion is fixedly and operatively connected to the core,
and the outer wall extends midway between the rigid ball and the lower disc-shaped portion. According to claim 1, Disposable structure,
With a vertically extending concentrate discharge tube,
Concentrate discharge tube, wherein the upper disc-shaped part and the outer wall rotate with respect to the concentrate discharge tube while the ball rotates;
a concentrate pump chamber, wherein the upper disc-shaped portion comprises the concentrate pump chamber;
A centripetal centrifugal pump, wherein the centripetal centrifugal pump comprises:
axially aligned with the core,
located concentrically around the feed tube,
located above the filtrate centripetal pump in the vertical direction,
located within the concentrate pump chamber,
The concentrate pump chamber comprises a concentrate centripetal pump in fluid communication with the separation chamber;
A device characterized in that the upper disc-shaped part and the outer wall rotate relative to the centrifugal centrifugal pump while the ball rotates. According to claim 1, Disposable structure,
With a vertically extending concentrate discharge pump,
Concentrate discharge tube, wherein the upper disc-shaped part and the outer wall rotate with respect to the concentrate discharge tube while the ball rotates;
concentrate pump chamber,
A centripetal centrifugal pump, wherein the centripetal centrifugal pump comprises:
axially aligned with the core,
located concentrically around the feed tube,
located above the filtrate centripetal pump in the vertical direction,
in fluid communication with the concentrate drain tube;
located within the concentrate pump chamber,
The concentrate pump chamber further comprises a concentrate centrifugal pump in fluid communication with the separation chamber;
While the ball rotates, the upper disc-shaped part and the outer wall rotate against the centrifugal centrifugal pump,
a concentrate optical density sensor operatively connected with the interior of the concentrate discharge line, the concentrate discharge line operatively connected with the concentrate discharge tube;
and a controller operatively connected to the concentrate optical density sensor. According to claim 1,
a filtrate optical density sensor operatively connected with the interior of the filtrate drain line;
and a controller operatively connected to the filtrate optical density sensor. According to claim 1, Disposable structure,
With a vertically extending concentrate discharge pump,
Concentrate discharge tube, wherein the upper disc-shaped part and the outer wall rotate with respect to the concentrate discharge tube while the ball rotates;
concentrate pump chamber,
A centripetal centrifugal pump, wherein the centripetal centrifugal pump comprises:
axially aligned with the core,
located concentrically around the feed tube,
located above the filtrate centripetal pump in the vertical direction,
in fluid communication with the concentrate drain tube;
The concentrate pump chamber further comprises a concentrate centrifugal pump in fluid communication with the separation chamber;
While the ball rotates, the upper disc-shaped part and the outer wall rotate against the centrifugal centrifugal pump,
a concentrate optical density sensor operatively connected with the interior of the concentrate discharge line;
a filtrate optical density sensor operatively connected with the interior of the filtrate drain line;
operatively connected to the filtrate optical density sensor and the retentate optical density sensor, and further comprising a controller operative to control the flow of the retentate or filtrate in response at least in part to the retentate optical density sensor and the filtrate optical density sensor Device characterized in that. According to claim 1,
The filtrate pump chamber is vertically surrounded by a lower circular axially centered filtrate centrifugal pump chamber surface and an upper axially centered circular filtrate centrifugal pump chamber surface;
The lower filtrate centrifugal pump chamber surface is,
a plurality of angularly spaced, radially extending lower filtrate chamber vanes, the lower filtrate chamber vanes starting from the lower filtrate centripetal pump chamber opening and extending radially outward;
The upper filtrate centripetal pump chamber surface is,
a plurality of angularly spaced, radially extending upper filtrate chamber vanes, the upper filtrate chamber vanes including upper filtrate chamber vanes starting from an upper filtrate centripetal pump chamber opening and extending radially outward; device characterized. According to claim 1, Disposable structure,
With a vertically extending concentrate discharge pump,
Concentrate discharge tube, wherein the upper disc-shaped part and the outer wall rotate with respect to the concentrate discharge tube while the ball rotates;
concentrate pump chamber,
A centripetal centrifugal pump, wherein the centripetal centrifugal pump comprises:
axially aligned with the core,
located concentrically around the feed tube,
located above the filtrate centripetal pump in the vertical direction,
in fluid communication with the concentrate drain tube;
located within the concentrate pump chamber,
the concentrate pump chamber is in fluid communication with the separation chamber;
While the ball rotates, the upper disc-shaped part and the outer wall rotate against the centrifugal centrifugal pump,
the concentrate centripetal pump chamber is vertically surrounded by a lower circular axially centered concentrate centrifugal pump chamber surface and an upper axially oriented circular concentrate centripetal pump chamber surface;
The lower concentrate centripetal pump chamber surface is,
a lower concentrate chamber vane comprising a plurality of angularly spaced, radially extending lower concentrate chamber vanes, the lower concentrate chamber vanes extending radially outward starting from the lower concentrate centripetal pump chamber opening including those,
The upper concentrate centripetal pump chamber surface is,
an upper concentrate chamber vane comprising a plurality of angularly spaced, radially extending upper concentrate chamber vanes, the upper concentrate chamber vanes extending radially outward starting from the upper concentrate centripetal pump chamber opening A device comprising: According to claim 1,
a substantially annular filtrate opening,
further comprising an annular filtrate dam,
the separation chamber and the filtrate chamber are fluidly connected through a substantially annular filtrate opening,
The annular filtrate dam is
extending within the separation chamber;
extending downwardly below the substantially annular filtrate opening;
and located radially outward of the substantially annular filtrate opening. According to claim 1, Disposable structure,
With a vertically extending concentrate discharge pump,
Concentrate discharge tube, wherein the upper disc-shaped part and the outer wall rotate with respect to the concentrate discharge tube while the ball rotates;
concentrate pump chamber,
A centripetal centrifugal pump, wherein the centripetal centrifugal pump comprises:
axially aligned with the core,
located concentrically around the feed tube,
located above the filtrate centripetal pump in the vertical direction,
in fluid communication with the concentrate drain tube;
located within the concentrate pump chamber,
the concentrate pump chamber is in fluid communication with the separation chamber through a substantially annular concentrate opening;
While the ball rotates, the upper disc-shaped part and the outer wall rotate against the centrifugal centrifugal pump,
Further comprising an annular concentrate dam,
The annular concentrate dam,
extending within the separation chamber;
extending downwardly below the substantially annular concentrate opening;
and located radially inward of the substantially annular concentrate opening. According to claim 1, Disposable structure,
a substantially annular filtrate opening, wherein the separation chamber and the filtrate pump chamber are fluidly connected through the substantially annular filtrate opening;
a concentrate discharge tube extending vertically, wherein as the ball rotates, the upper disc-shaped portion and the outer wall rotate relative to the concentrate discharge tube;
concentrate pump chamber,
A centripetal centrifugal pump, wherein the centripetal centrifugal pump comprises:
axially aligned with the core,
located concentrically around the feed tube,
located above the filtrate centripetal pump in the vertical direction,
in fluid communication with the concentrate drain tube;
located within the concentrate pump chamber,
the concentrate pump chamber is in fluid communication with the separation chamber through a substantially annular concentrate opening;
Concentrate centripetal pump, wherein the upper disc-shaped part and the outer wall rotate relative to the centrifugal centrifugal pump while the ball rotates;
As an annular filtrate dam,
extending within the separation chamber;
extending downwardly below the substantially annular filtrate opening;
an annular filtrate dam positioned substantially radially outward of the annular filtrate opening;
As an annular concentrate dam,
extending within the separation chamber;
extending downwardly below the substantially annular concentrate opening;
further comprising an annular concentrate dam positioned substantially radially inward of the annular concentrate opening;
An annular upwardly extending recess extending radially between the annular concentrate dam and the annular filtrate dam in the separation chamber. According to claim 1,
at least one annular hermetic seal;
further comprising a pressurized air source;
the at least one annular hermetic seal extends operatively in sealing relationship between the upper disc-shaped portion and at least one radially outwardly extending at least one annular outer wall of at least one of the supply tube and the filtrate outlet tube;
the at least one annular seal is operative to retain a pocket of air within the interior region;
the pressurized air source is in fluid communication with the air pocket;
wherein the air in the air pocket isolates the at least one seal from the cell culture batch being processed, the air pocket being positioned radially inward of the substantially annular filtrate pump opening. According to claim 1, Disposable structure,
with at least one annular hermetic seal,
the at least one annular hermetic seal extends operatively in sealing relationship between the upper disc-shaped portion and at least one radially outwardly extending at least one annular outer wall of at least one of the supply tube and the filtrate outlet tube;
the at least one seal is operative to retain a pocket of air within the interior region;
the air in the air pocket further comprises at least one annular hermetic seal isolating the at least one seal from the cell culture batch being processed;
a flow back pressure regulator in fluid communication with the filtrate outlet tube and operative to selectively apply back pressure to the filtrate flow;
and a controller operatively connected to the flow back pressure regulator, the controller operative to maintain a pocket of air positioned radially inward of the substantially annular filtrate pump opening. According to claim 1, Disposable structure,
With a vertically extending concentrate discharge tube,
Concentrate discharge tube, wherein the upper disc-shaped part and the outer wall rotate with respect to the concentrate discharge tube while the ball rotates;
concentrate pump chamber,
A centripetal centrifugal pump, wherein the centripetal centrifugal pump comprises:
axially aligned with the core,
located concentrically around the feed tube,
located above the filtrate centripetal pump in the vertical direction,
in fluid communication with the concentrate drain tube;
located within the concentrate pump chamber,
a concentrate centrifugal pump in fluid communication with the separation chamber;
A device characterized in that the upper disc-shaped part and the outer wall rotate relative to the centrifugal centrifugal pump while the ball rotates. A disposable structure configured for use in a centrifugal separation system comprising a multi-use rotatable centrifugal ball, wherein the disposable structure is releasably positioned within the bowl and wherein the cells in the cell culture batch within an interior region of the structure are separated from the cell concentrate and a disposable construct configured to dissociate into cell filtrate;
wherein the structure is in an operative position extending along a vertical axis;
The structure is
upper part,
lower part,
the core in the middle of the upper part and the lower part,
an annular separation chamber radially surrounding the core;
an outer wall, wherein an upper portion and the outer wall can rotate in a ball about a vertical axis, the outer wall comprising:
operatively extending liquid-tightly with said upper portion and surrounding a separation chamber;
an outer textured surface through which air may escape from between the outer wall and the centrifugal ball;
an outer wall extending around the core,
supply tube,
filtrate drain tube,
filtrate pump chamber,
As a filtrate centripetal pump, the filtrate centripetal pump comprises:
aligned axially with the core,
located coaxially with the supply tube,
in fluid communication with the filtrate drain tube;
located within a filtrate pump chamber, wherein the filtrate pump chamber is in fluid communication with the separation chamber;
a filtrate centripetal pump;
An apparatus, characterized in that while the ball rotates, the upper disc-shaped part and the outer wall rotate with respect to each of the feed tube, the filtrate discharge tube and the filtrate centripetal pump. A disposable structure configured for use in a centrifugal separation system comprising a multi-use rotatable centrifugal ball, wherein the disposable structure is releasably positioned within the bowl and wherein the cells in the cell culture batch within an interior region of the structure are separated from the cell concentrate and a disposable construct configured to dissociate into cell filtrate;
wherein the structure is in an operative position extending along a vertical axis;
The structure is
upper part,
lower part,
the core in the middle of the upper part and the lower part,
an annular separation chamber radially surrounding the core;
an outer wall, wherein an upper portion and the outer wall can rotate in a ball about a vertical axis, the outer wall comprising:
operatively extending liquid-tightly with said upper portion and surrounding a separation chamber;
an outer wall extending around the core,
supply tube,
filtrate drain tube,
filtrate pump chamber,
As a filtrate centripetal pump, the filtrate centripetal pump comprises:
aligned axially with the core,
located coaxial with the supply tube,
in fluid communication with the filtrate drain tube;
located within a filtrate pump chamber, wherein the filtrate pump chamber is in fluid communication with the separation chamber;
The filtrate centripetal pump chamber is vertically surrounded by a lower circular axially centered filtrate centrifugal pump chamber surface and an upper axially centered circular filtrate centrifugal pump chamber surface;
the lower filtrate centrifugal pump chamber surface comprises a plurality of angularly spaced, radially extending lower filtrate chamber vanes;
wherein the upper filtrate centripetal pump chamber surface comprises a plurality of angularly spaced, radially extending upper filtrate chamber vanes;
An apparatus comprising a filtrate centripetal pump, wherein the upper disc-shaped portion and the outer wall rotate about an axis relative to each of the feed tube, the filtrate outlet tube and the filtrate centrifugal pump while the rotatable centrifugal ball rotates. A disposable structure configured for use in a centrifugal separation system comprising a multi-use rotatable centrifugal ball, wherein the disposable structure is releasably positioned within the bowl and wherein the cells in the cell culture batch within an interior region of the structure are separated from the cell concentrate and a disposable construct configured to dissociate into cell filtrate;
wherein the structure is in an operative position extending along a vertical axis;
The structure is
upper part,
lower part,
the core in the middle of the upper part and the lower part,
an annular separation chamber radially surrounding the core;
an outer wall, wherein an upper portion and the outer wall can rotate in a ball about a vertical axis, the outer wall comprising:
operatively extending liquid-tightly with said upper portion and surrounding a separation chamber;
an outer wall extending around the core,
supply tube,
filtrate drain tube,
filtrate pump chamber,
As a filtrate centripetal pump, the filtrate centripetal pump comprises:
aligned axially with the core,
located coaxially with the supply tube,
in fluid communication with the filtrate drain tube;
located within a filtrate pump chamber, wherein the filtrate pump chamber is in fluid communication with the separation chamber;
Filtrate centripetal pump;
concentrate pump chamber,
A centripetal centrifugal pump, wherein the centripetal centrifugal pump comprises:
aligned axially with the core,
located coaxially with the supply tube,
located above the filtrate centripetal pump in the vertical direction,
in fluid communication with the concentrate drain tube;
located within the concentrate pump chamber, wherein the concentrate pump chamber is in fluid communication with the separation chamber;
While the rotatable centrifugal ball rotates about its axis, the upper part and the outer wall rotate against the centrifugal centrifugal pump,
the concentrate centripetal pump chamber is vertically surrounded by a lower circular axially centered concentrate centrifugal pump chamber surface and an upper axially oriented circular concentrate centripetal pump chamber surface;
wherein the lower concentrate centripetal pump chamber surface comprises a plurality of angularly spaced, radially extending lower concentrate chamber vanes;
and wherein the upper concentrate centrifugal pump chamber surface includes a plurality of angularly spaced, radially extending upper concentrate chamber vanes.

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