KR102568314B1 - Centrifuge system for separating cells in suspension - Google Patents

Abstract

An apparatus for separating cell suspension material into filtrate and retentate includes a disposable structure (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, filtrate outlet tube 212 and concentrate outlet tube 230 extend along the axis of the rotating disposable structure. A filtrate centrifugal pump 208 is in fluid communication with the filtrate discharge tube. Concentrate centrifugal pump 216 is in fluid communication with the concentrate discharge tube. Controller 274 adjusts the flow rate of concentrate pump 272 and filtrate pump 266 according to sensors 264 and 270 in respective filtrate and concentrate outlet lines 262 and 268 to obtain cell concentrate and generally It operates to create a flow of cell-free filtrate.

Description

Centrifugal separation system for separating cells in suspension {CENTRIFUGE SYSTEM FOR SEPARATING CELLS IN SUSPENSION}

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

Devices and methods for centrifuging cells in suspension are useful in many technical settings. Such systems could 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 path 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 path components. Exemplary centrifuges discussed herein may be solid wall centrifuges that use pre-sterilized disposable components and are capable of handling cell suspensions with high cell concentrations.

Exemplary embodiments use rotationally fixed supply and discharge components. The disposable component includes a flexible membrane mounted on a rigid frame containing a core of enlarged diameter. The disposable component may further include at least one centrifugal pump. The disposable structure may be supported in a multi-use rigid ball shaped like a truncated cone on the inside. These structures allow the exemplary system to maintain angular velocities high enough to produce settling velocities suitable for efficiently processing highly concentrated cell culture streams. Features that minimize feed turbidity and other features that allow for continuous or semi-continuous discharge of the cell concentrate increase overall production rates beyond achievable rates. Exemplary structures and methods provide for effective operation and reduce the risk of contamination.

1 is a schematic diagram of an exemplary embodiment of a centrifugal separation system that includes a disposable component and a multi-use component.
FIG. 2 is an enlarged view of the upper flange area of the centrifuge of FIG. 1 showing how to seal the flexible chamber material to the flange surface.
3 is an isometric cross-sectional view of the core and top flange of the disposable component of the embodiment of the centrifugal separation system of FIG. 1;
FIG. 4 is a schematic diagram of the embodiment shown in FIG. 1 in which the pump chamber of the centrifugal separation system includes 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 system with an enlarged core diameter (to make a shallow pool centrifuge) and a feed accelerator.
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.
Figure 9 is an isometric view of the feed accelerator of Figure 8;
10 is a schematic diagram of a portion of a continuous retentate discharge centrifugal separation system.
11 is a schematic diagram of a portion of a second embodiment comprising a continuous retentate discharge centrifugal separation system.
12 is a schematic diagram of a continuous concentrate discharge centrifugal system with diluent injection.
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 the core and top flange of a disposable centrifugal separation system with a core and a feed accelerator with straight vanes.
15 is an isometric view of the feed accelerator of FIG. 14;
16 is an isometric cut away view of an alternative continuous concentrate discharge centrifugal separation system.
17 is an isometric exploded view of an alternative centripetal pump.
18 is an isometric view of a plate of an alternative centripetal pump including vortex passages therein;
19 is a schematic diagram of a centrifugal system operating to maintain a positive pressure in the centrifuge core cavity.
20 is a schematic diagram showing a simplified exemplary logic flow executed by at least one control circuit of the system shown in FIG. 19;
21 is a cross-sectional schematic of an alternative continuous filtrate and retentate discharge centrifugal separation system.
22 is a cross-sectional schematic of a further alternative continuous filtrate and retentate discharge centrifugal separation system.
23 is a cross-sectional schematic of a further alternative continuous filtrate and retentate discharge centrifugal separation system.
24 is a schematic diagram of a control system for an exemplary continuous filtrate and retentate discharge centrifugal separation system.
25 is a schematic diagram of the logic flow associated with the exemplary control system of FIG. 24;
26 is a cross-sectional view of an exemplary upper portion of a disposable centrifugal separation structure including retentate and filtrate dams in separation chambers.
27 is a cross-sectional view of an exemplary upper portion of a disposable structure including vanes in the filtrate pump chamber and the concentrate pump chamber for the purpose of controlling the radial position of the air/liquid interface.
28 is a perspective view of a chamber surface of an exemplary concentrate or filtrate pump chamber comprising 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 air passages for maintaining pressurized air in air pockets.
31 is a schematic diagram of an exemplary system for controlling a centrifugal separation system that includes filtrate flow back pressure control.

In the field of cell culture applied in bio-pharmaceutical processing, it is necessary to isolate cells from a fluid medium, such as the fluid in which they are grown. The desired product from the cell culture may be a molecular species excreted by the cells into the medium, a molecular species remaining within the cells, or the cells themselves. On a large-scale production scale, the initial steps of the cell culture process are usually in bioreactors that can be operated in either batch mode or continuous mode. Variations such as an iterative batch process can also be implemented. The desired product must be finally separated from other process components prior to final purification and formulation of the product. Cell harvesting is a general term applied to the separation of these cells from other process components. Clarification is a term denoting the separation of cells in which a cell-free supernatant (or filtrate) is the goal. Cell recovery is a term often applied to isolations where a cell concentrate is the goal. Exemplary embodiments of the present application relate to cell harvest isolation 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 mass cell cultures at production scale required clean-in-place (CIP) and steam-in-place (SIP) technology 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, described in published US patent application US2010/0167388, ranges in volume from 3-30 liters/minute and uses intermittent processing to hold up to about 2000 liters of cells. The culture batch for harvesting is processed successfully. The entire contents of this document are incorporated herein by reference. See also US Patent Application Serial No. 15/886,382, filed February 1, 2018; and U.S. Patent No. 9,222,067 owned by Pneumatic Scale Corporation, the assignee of this application, are also incorporated in their entirety into this application. Intermittent treatment usually requires periodic stopping of the feed flow and rotation of the centrifuge bowl to discharge the concentrate. This approach usually works well in low concentration, high viability cultures. Large batches can be processed in this culture and the cell concentrate is discharged relatively quickly and completely.

Sometimes the feed material contains high concentrations of cells and cell debris, and cells must be harvested from highly concentrated and/or low viability cell cultures, also referred to as "high turbidity feeds". there is. These high turbidity feeds can slow throughput in some centrifugal systems because:

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

2. High concentrations of cells and cell debris can cause the vessel to fill rapidly with cell concentrate, which may require a stop to drain the concentrate from the vessel.

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

A high concentration of cells and cell debris in the material feed may result in a very viscous cell concentrate. This can make it more difficult to completely drain the cell concentrate from the centrifuge, even with increased drain cycles. If desired, an additional buffer rinse cycle may be added to sufficiently completely drain the concentrate. If one or both of these adjustments to the discharge cycle have to be made, processing time will be additionally increased, which can be more complex and costly to process large cell cultures.

Scaling up the system by increasing the ball size to lengthen the feed portion of the intermittent treatment cycle is sometimes impractical as it results in a proportionately longer discharge cycle for the cell concentrate. Another limitation that can prevent simple geometrical scale-up is the scaling change of the relevant fluid dynamic factors. The maximum throughput of any centrifuge depends on the settling rate of the particles being separated. The settling rate is given by modifying the Stokes law defined in Equation 1.

Equation 1

where v = settling velocity, 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 in Equation 1 are held constant, increasing the ball radius increases the average settling velocity, increasing the throughput for a given separation efficiency. However, as the radius increases, maintaining the angular velocity of the ball becomes more difficult due to the 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 settling velocity and the throughput gain (proportional to radius) decrease.

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

Also, if the angular velocity is held constant as the radius increases, the force pushing the cells against the centrifuge wall also increases. As the balls rotate at angular velocities high enough to achieve the desired throughput, additional stresses are created on the walls of the vessel and on the cells that accumulate there. As for cells, this can result in cells being packed at excessively high concentrations, resulting in cell damage. Cell damage is a disadvantage in applications where cell viability must be maintained and can lead to contamination of products present in solution in the filtrate. Higher viscosity due to excessively high cell concentration is sometimes also a drawback for complete evacuation of the cell concentrate.

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 amounts of cell suspensions on a commercial scale. . Some exemplary centrifuges are pre-sterilized, disposable 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 operating time of a 2000 liter bioreactor in the range of 2 to 3 hours. An exemplary embodiment of a 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 . The 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 centrifuge structure 1000 also includes a centrifugal pump 1400 that includes a pair of stationary fairing disks 1410 and a rotating mechanical seal 1700 in a rotating pump chamber 1420 .

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

As shown in FIG. 1 , upper and lower flanges 1300 and 1200 include conical balls axially aligned with core 1510 and concave toward core 1510 . Core 1510 includes a generally cylindrical body with a hollow cylindrical center large enough to receive feed tube 2100 having shaft 1525 (indicated in FIG. 12 ). Upper flange 1300, core 1510, and lower flange 1200 may be a unitary structure to provide a stronger support structure for flexible liner 1100, alternatively referred to herein as a membrane. In other embodiments, core structure 1500 may be formed from a plurality of components. In further embodiments, lower flange 1200 includes separate components and core 1510 and upper flange 1300 include a single component, or upper flange 1300 includes 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 is to be coupled to the lower flange 1200 to create the internal support structure 1500 of the disposable component of the centrifuge 1000. This structure secures the flexible liner 1100 at both the top and bottom around a fixed internal rigid or semi-rigid support structure 1500. When the centrifugal separation system is in use, flexible liner 1100 is also externally supported by the walls and cover of reusable structure 3000 .

Exemplary separation chamber 1550 is approximately cylindrical, with outer surface 1515 of core 1510 and upper surface 1210 of flexible liner 1100 and lower flange 1200 and lower surface of upper flange 1300. It is an open chamber roughly bounded by 1310. Separation chamber 1550 is in fluid communication with feed tube 2100 through aperture 1530 extending from central cavity 1520 of core 1510 to outer surface 1515 of core 1510 . Isolation chamber 1550 is in fluid communication with pump chamber 1420 via similar aperture 1540 throughout core structure 1500 . In this example, aperture 1540 slopes upward toward pump chamber 1420, opening into isolation chamber 1550 just below the junction between core 1510 and upper flange 1300. As shown in FIG. 12, aperture 1420 or 4420 may enter the pump chamber at an angle other than upward, including horizontal or downward angle. Additionally, in some embodiments holes 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 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. Feeding process by carefully designing the spacing between the nozzle 2110 of the feeding tube 2100 and the upper surface 1210 of the lower flange 1200, the diameter of the nozzle 2110 of the feeding tube 2100 and the angular velocity of the centrifuge. The shear force can be minimized at US Patent No. 6,616,590 (the disclosure of which is incorporated herein by reference in its entirety) describes how to select an appropriate relationship to minimize shear forces. Other suitable feed tube designs that minimize the shear forces associated with feeding a liquid cell culture into a spin centrifuge known to those skilled in the art may also be used.

1 further includes a centrifugal pump 1400 for discharging the filtrate through the filtrate discharge path 2200. In the embodiment shown in FIG. 1 , the filtrate pump 1400 is positioned within the pump chamber 1420 above the upper flange 1300 . Pump chamber 1420 is a chamber defined by upper surface 1505 of core 1510 and inner surfaces 1605 and 1620 of centrifuge cover 1600 . The centrifuge cover 1600 may include a cylindrical wall 1640 and an engagement cap portion 1610 generally shaped like a circular disc (shown in FIG. 5 ). The centrifuge cover 1600 may be formed in a single piece or may be formed 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 top of core structure 1500 in fluid communication with separation chamber 1550 through a hole or slit 1530 extending from the adjacent exterior of core 1515 into filtrate pump chamber 1420. It becomes an axially symmetrical chamber. In some embodiments, as shown most clearly in FIGS. 11 and 12 , filtrate pump chamber 1420 may be located in a recess within chamber 1550 .

The exemplary filtrate pump 1400 includes a pair of fairing disks 1410 . Fairing disks 1410 are two thin circular disks (plates) axially aligned with axis 1525 of core structure 1500. In the embodiment shown in FIGS. 1-5 , fairing disks 1410 are retained relative to the centrifuge structure 1000 and are separated from each other by a fixed gap 1415 (indicated by reference numeral 1415 in FIG. 10 ). . The gaps 1415 between the fairing disks 1410 form part of the fluidic connection for removing filtrate from the centrifuge 1000, which surrounds the feed tube carrier 2300 between the fairing disks 1410. and into the hollow cylindrical filtrate discharge path 2200 terminating at the filtrate outlet 2400.

Exemplary disposable centrifuge structure 1000 is included within multi-use centrifuge structure 3000 . The multi-use centrifugal separator 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 outer structure of the disposable structure 1000 and the inner 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 similar to the inner surface of the multipurpose vessel cover 3200 to which they are also applied. Coincidentally, it provides support during rotation. The features of the disposable bowl 3100 and bowl cover 3200, discussed in more detail below, are designed so 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 can be adapted for disposable processing by selecting a compliant disposable structure 1000. In other cases, the multi-use structure 3000 may be specifically designed for use with the disposable structure insert 1000.

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

A thermal bond attachment process may be used to join dissimilar materials in the area indicated in FIG. 2 . The thermal bond 1110 is formed by preheating the flange material, placing an elastomer over the heated flange, and applying heat and pressure to the elastomer 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 a thermal bond 1110 is described herein, this is merely an example. Other means that create a similarly strong and 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. While removing these components from their protective packaging and installing them into the centrifuge, the thermal bond 1110 remains sterile within the disposable chamber. The stretchable flexible liner 1100 conforms to the walls of the ball 3100, which is reusable when in use. The reusable ball 3100 provides sufficient support and the flexible liner 1100 is sufficiently resilient that the disposable structure 1000 allows the larger radius centrifuge 1000 to be used with liquid cell culture 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 of about 2-40 liters per minute.

In addition to the thermal bond 1110, a sealing ridge or “nubbin 3210” may be present over the ball cover 3200 to compress the thermoplastic elastomer film against the rigid upper flange 1300 to form an additional seal. there is. The same compression seal may be used at the bottom of the ball 3100 to seal the thermoplastic elastomer film against the rigid lower flange 1200. This compression seal supports thermal bonding area 1110 by isolating it from shear forces generated by the hydrostatic pressure that occurs during centrifugation when the chamber is filled with liquid. The combination of a thermal bond (1110) and compression nubbin (3210) seal was tested at 3000 x g, which corresponds to a hydrostatic pressure of 97 psi at the ball wall. The lining should be sufficiently thick and compressible so that the nubbin 3210 can compress and grip the flexible liner 1100, but minimize the risk of tearing near the thermal bond 1110 or compression nubbin 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 of FIGS. 1 and 2 were tested in a 5.5 inch diameter ball. At 2000 x g, the hydraulic power was over 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 top surface of the disposable centrifuge structure may have a different shape as shown in FIGS. 10 and 11 . can have a shape. 10 and 11 , rather than having a generally conical ball cover 3200, to accommodate a generally conical upper flange 1300, both the upper flange and the ball cover are relatively disc-shaped. older brother One skilled in the art will be able to adapt the sealing techniques described herein for use with sealing surfaces of various shapes.

4 and 5 show an exemplary embodiment having features for improving the efficiency of the centripetal pump 1400. As shown in detail in FIG. 5 , this embodiment of an internal structure for a disposable component similar to that shown in FIGS. A plurality of radial fins 1630 are included. 5 shows an inner surface 1620 of a cap portion 1610 of a centrifuge cover 1600 . Radial fin 1630 may be a thin, generally rectangular, radial plate extending perpendicularly from inner surface 1620 of cap portion 1610 . In the exemplary embodiment, six pins 1630 are shown, but other embodiments may include fewer or more pins 1630. In this embodiment, the pins 1630 form part of the inner surface of the cap 1620, but in other embodiments the top surface 1620 of the pump chamber 1420 may take a form other than the cap 1610. can include When centrifugal system 1000 is in use, pins 1630 are positioned within chamber 1420 above fairing disk 1410 of 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-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 column of gas 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 filtrate rotating within the pump chamber 1420 contacts the stationary fairing disk 1410 , the resulting friction can reduce the efficiency of the pump 1400 . Adding a plurality of radial fins 1630 rotating at the same angular velocity as the filtrate overcomes any reduction in speed that may occur due to an encounter between the rotating filtrate and the stationary fairing disk 1410.

6 shows an exemplary embodiment of an improved core structure 1500 for use where the feed has high turbidity. The core structure 1500 includes a core 1510 , an upper flange 1300 and a lower flange 1200 . Core 1510 has a cylindrical central cavity 1520 configured such that a feed tube 2100 can be inserted into central cavity 1520 . The distance from the central axis 1525 to the outside of the core 1515 (core width, indicated by dotted line 6000 in FIG. 6 ) is greater than the corresponding distance in 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. The depth 6010 of the separation chamber 1550 is generally the distance between the exterior of the core 1510 and the flexible liner 1100 as shown 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 removal of the cell concentrate, the shallow pool depth 6010 varies from being shallower at the bottom of the separation chamber 1550 to that of the separation chamber 1550. It can vary from top to slightly deeper. In some embodiments shown herein, the ratio of average isolation pool depth 6010 to core width is less than or equal to 1:1. An example of a shallow pool centrifuge is provided as an option model for the ViaFuge centrifugal 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, yielding 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 cell debris.

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

In the exemplary embodiment of core structure 1500 shown in FIG. 6 , accelerator vanes 1560 are radially generally rectangular spaced thin plates 1580 extending upwardly from the upper conical surface of 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 12 plates 1580 as shown most clearly in FIG. 7 . In other embodiments, fewer or more plates 1580 may be present. Also, in other embodiments, the plate 1580 may be curved in the direction of rotation of the centrifuge 1000 as shown in the exemplary embodiment of FIG. 9 . The inner surface of the lower flange 1200 may be modified to form an elliptical accelerator ball 1590 from which an upwardly curved plate extends. These embodiments are intended to be illustrative, and those skilled in the art may modify these embodiments or combine these plates in different ways to further benefit from turbidity reduction to create 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 to remove the cell concentrate. The centrifugal pump 4400 for cell concentrate removal is positioned above the centrifugal pump 1400 for filtrate removal. The centripetal pump 4400 includes a pump chamber 4420 and a fairing disk 4410. A plurality of holes 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 the second pump chamber 4420. do. Like pump chamber 1400, pump chamber 4400 may have a different shape than that shown in FIGS. It will be an axisymmetric chamber. As with pump chamber 1400 , the pump chamber may be partially or wholly recessed within core structure 1500 . If the filtrate pump chamber 1400 is near the top of the core structure 1500, the cell enrichment 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 an adjacent portion in the outer upper wall of the separation chamber 1550 to collect the heavy cell concentrate pressed down by the centrifugal force. will be in fluid communication with the separation chamber 1550 via

In the embodiment shown in FIG. 10 , the fairing disk 4410 used in the concentrate exhaust pump 4400 has approximately the same radius as that used in the filtrate exhaust pump 1400 and is rotationally fixed. In another embodiment, 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, with the pump chamber 4420 being larger. It can have a large radius. Fairing discs of various medium diameters may also be used. The optimal diameter depends on the characteristics of the cell concentrate to be drained. Larger diameter fairing discs have higher pumping capacity but greater shear forces.

In the embodiment shown in FIGS. 1 , 4 and 10 , the fairing disk 4410 of the concentrate discharge pump 4400 is rotationally stationary. In another embodiment, such as that shown in FIG. 11 , fairing disk 4410 may be configured to rotate at an angular velocity between zero and the angular velocity of 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 fairing disk 4410 to rotate at an angular velocity that is a fraction of the angular velocity of centrifuge 1000 . Other means of controlling the angular velocity of the fairing disk will be apparent to those skilled in the art.

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

13 shows 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 aperture 1530 extends from the core 1510 to a filtrate well 1555 under the pump chamber 1420 . Also, in the illustrated exemplary embodiment, an aperture 1540 extends from the separation chamber 1550 adjacent the outer surface 1515 of the core 1510 into the pump chamber 1420, using the filtrate pump 1400 to Allow the filtrate to be removed. An aperture 4540 also extends between the separation chamber 1550 and the pump chamber 4420 proximate the outer upper surface so that the cell concentrate to be removed using the centripetal pump 4400 will flow into the pump chamber 4420. make it possible

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

As can be seen in the embodiments shown in FIGS. 10 to 12, the wall of the solid multi-use ball 3100 is for supporting the disposable centrifugal separation structure 1000 having a smaller radius at the bottom than at the top. It is thicker at the base than at the top to create the shape of a truncated cone 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 illustrated embodiment, the truncated cone shape is created by the ball 3100 having thicker walls at the base than at the top portion. Those skilled in the art will recognize that a ball 3100 having a conical shape with a truncated interior may also include walls of uniform thickness, and there may be other variations that create the desired internal shape for the ball 3100. something to do.

In the exemplary embodiment shown in FIGS. 10-12 , the feeding mechanism 2000 also includes an additional path for removal of cells or cell concentrates. In the embodiment shown in FIG. 1 , a cylindrical pathway 2200 around feed tube 2100 is used to remove filtrate. The embodiment shown in FIGS. 10-12 includes a concentric cylindrical pathway for removal of cells or cell concentrate, also referred to as cell exit tube 2500 . A cell exit tube 2500 surrounds the centrifugal removal 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 to allow diluent to flow through the cell concentrate centrifuge to reduce the viscosity of the concentrate. It may be introduced into the pump chamber 4420. In the exemplary embodiment shown in FIG. 12 , the diluent pathway 5000 includes concentric tubes surrounding the cell exit pathway and above the fairing disk 4410 exiting near the outer edge of the fairing disk 4410 at the bottom. of the thin disc-shaped fluid pathway 5100 to provide fluid communication with the pump chamber 4420. This means injects the diluent and at this location limits the diluent to mixing with the concentrate and exiting with the concentrate rather than being introduced into the filtrate, which may be unnecessary in some applications. In an alternative embodiment, the diluent may be introduced directly to the upper surface of the fairing disk and spread radially outward or spread 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 a diluent. In other cases, diluents specific to the properties 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 as it is fed to the centrifuge to cause the cells and cell debris to aggregate or aggregate into larger particles. They become separated as their sedimentation rate increases. Since both cells and cellular 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 flocculants can connect negatively charged cells and cellular debris into large clumps. An undesirable result of the use of such flocculants is a further increase in the viscosity of the cell concentrate. Thus, a particularly useful diluent in this application is a deflocculant that hinders binding that increases the viscosity of the cell concentrate. Examples of peptizing agents include high-salt buffers such as sodium chloride solution in the concentration range of 0.1 M to 1.0 M. Other deflocculants that may be useful in reducing the viscosity of the cell concentrate 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 can be selected. The use of shear protection agents in combination with isotonic buffer improves the survival and viability of cells exiting the centrifuge.

The exemplary centrifugal separator shown in FIG. 4 operates as described below. During the feed cycle, the feed suspension flows through the feed tube 2100 and 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 forced outward along the upper surface of the lower flange 1200 by centrifugal force, and the bore of the core 1510 Passes through 1530 into separation chamber 1550.

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

During rotation, filtrate enters filtrate pump chamber 1420 through 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, which converts the filtrate into a supply/discharge mechanism 2000 into the filtrate discharge path. It is discharged upwards through 2200 and is forced to exit past 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 spinning filtrate may be slowed down by friction as it encounters the stationary fairing disk 1410. The centripetal pump 1400 provides an improved filtrate discharge means compared to mechanical seals 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 rotational seal 1700. Since the filtrate discharged between the fairing discs 1410 does not come into contact with air during the supply or discharge process, excessive foaming that occurs when air is introduced into the cell culture during the discharge process is prevented.

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

As mentioned above, if the cell culture is enriched or contains significant cellular debris, the process described above is slowed down because the residence time must be increased to capture small debris particles. This requires a slower feed flow rate and requires the separation chamber 1550 to fill and rotate quickly, stopping frequently and repeatedly for each culture batch. In addition, the cell concentrate becomes more viscous, so gravity does not work as efficiently to discharge the cell concentrate to the bottom of the centrifuge 1000, so it takes longer and in some cases washing may be required to remove the remaining cells. .

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

The embodiment of the disposable centrifuge structure 1000 shown in FIGS. 6-12 operates as discussed herein. The feed suspension enters the disposable centrifuge structure 1000 through the feed tube 2100. When the feed suspension encounters the accelerator vanes 1560, the vanes 1560 impart an angular velocity to the feed suspension that approximates the angular velocity of the disposable centrifuge 1000. By using the vanes 1560 rather than the holes 1530, a larger amount of the feed suspension is introduced into the separation chamber 1550 at a smaller radial velocity, resulting in a hole with a smaller cross-sectional opening than the opening between the vanes 1560 ( 1530) to prevent jetting that occurs when the feed suspension is forced. Reducing the velocity of the feed stream as it enters the separation zone or pool minimizes disruption of the liquid content of the pool, allowing for more efficient settling.

As the centrifuge 1000 rotates, particles denser than the filtrate are pushed out of the separation chamber 1550, leaving a particle-free filtrate near the core 1510. The centrifuge ball 3100 has the shape of an inverted truncated cone, with 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 centrifugal separator 1000 may operate with a semi-continuous discharge of retentate. The filtrate drain generally operates as described with respect to FIG. 4 . The cell concentrate exhaust operates similarly, the cell concentrate is collected near the upper outer portion of the separation chamber 1550 and passed through an aperture 4540 adjacent to the upper outer wall of the separation chamber 1550 to the concentrate exhaust pump chamber 4400. ) is introduced into

The feed rate and rotational angular velocity of the suspension can be monitored using a vibration sensor system such as that described in US Pat. No. 9,427,748, which is hereby incorporated by reference in its entirety. These sensor systems allow the centrifuge to fill at a lower speed until vibrations indicate that the centrifuge is nearly full, then adjust the feed rate and angular velocity appropriately based on this information. Typically, when the centrifuge is nearly full, the feed rate is reduced or stopped, the angular velocity is increased to increase the rate of settling, and the cycle is repeated when settling and ejection 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 operate continuously or near-continuously at the angular velocity required to stabilize.

When the concentrate is discharged semi-continuously, the suspension is continuously fed to the centrifuge 1000 using the concentrate pump 4400 operating intermittently to remove the concentrate. Operation of the concentrate pump 4400 may be controlled by an optical sensor in the concentrate discharge line indicating the presence or absence of concentrate being discharged. Instead of the concentrate pump 4400, the discharge cycle can be electronically managed using controllers and sensors that determine when valves open and close for the most efficient treatment of the fluid suspension.

The average discharge rate can be additionally controlled using centrifuge 1000 with an adjustable gap between fairing disks 4410 and 1410. It may be desired or necessary that only one set of pairing disks 4410, 1410 be adjustable. The gap between fairing disks 4410 and 1410 (which form part of the fluid path external to centrifuge 1000) can be opened or closed to allow flow to act as an internal valve to block flow. It may also be useful to widen or narrow the spacing 4415, 1415 between the pairing disks 4410, 1410 depending on the desired product or product characteristics. Changing the spacing affects both the pumping and shear rates associated with the fairing disk.

The rate of removal of the retentate and filtrate from the centrifuge 1000 and the viability of the removed retentate may be further controlled using a number of features of the exemplary embodiments shown in FIGS. 4-13 . An accelerator pin 4630 similar to that in the centrifugal pump chamber 1420 may be added to the concentrate pump chamber 4420. The addition of accelerator pins 4630 increases the rate at which concentrate can be removed by overcoming some of the slowdown 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 their effectiveness. An additional feature may be the replacement of holes 1540 and 4540 with slits, which minimize shear of material entering pump chambers 1420 and 4420.

If the viability of the concentrate is a concern, a rotatable separation disk 4410 can be included in the pump chamber 4420, which reduces the shear imparted to the concentrate when in contact with the surface of the fairing disk 4410. . The rotational speed of fairing disk 4410 can be adjusted to some degree between the stationary and rotational speed of separation chamber 1550 to balance concentrate viability and 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 which allows the fairing disc to rotate at an angular velocity that is part of the centrifuge. 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 obvious to those skilled in the art.

A peristaltic pump 2510 may be used to make removal of the concentrate more efficient and reliable, especially when using very concentrated feed suspensions. The use of the peristaltic pump 2510 allows the user to more precisely control the flow rate of the concentrate from the centrifuge 1000 than relying solely on the centripetal pump 4400, since the speed of the centrifugal pump is the same as that of the peristaltic pump 2510. This is because it is not as easy to adjust as speed.

In addition, 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 to reduce the viscosity of the concentrate. A more complete discussion of useful diluents can be found above. The rate at which one or both of peristaltic pump 2510 or diluent pump 5150 operates may be controlled by an automated controller (not shown) responsive to concentration sensor 4430 located at concentrate discharge connection 2500. can The controller responds to the concentration sensor 4430 to adjust the pump speeds for diluent addition and concentrate removal independently, in conjunction with standard feed/discharge cycles, depending on the particle concentration in the concentrate, together with the concentrate removal, depending on the particle concentration in the concentrate. It can be programmed to start, stop or adjust pump speed for both pump and diluent feed.

16 shows an alternative exemplary embodiment of a core used in conjunction with a centrifuge that provides continuous separation processing to continuously produce retentate and filtrate feeds. The core 10 is similar to that previously discussed, configured to be placed in a rotatable ball of a centrifuge. The centrifuge balls and cores rotate about an axis 12 during processing. The device includes a stationary assembly (14) and a rotatable assembly (16).

As in the foregoing embodiments, the fixation 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 the bottom of the separation chamber. The holding assembly further includes a filtrate centrifugal pump (24). An exemplary embodiment of the filtrate centrifugal pump 24 described in more 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 around the supply tube 18 .

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

The exemplary embodiment further includes a concentrate centrifugal pump 34 . The concentrate centrifugal pump 34 of the exemplary embodiment may also be of a similar construction to that discussed in detail later. In the exemplary arrangement, the concentrate centrifugal pump 34 includes an inlet opening 36 located in the wall bordering the annular periphery of the centrifugal pump. It should be noted that the concentrate centrifugal pump 34 has a larger peripheral diameter than that of the centrifugal pump. The concentrate pump further includes an annular outlet opening (38). An annular outlet opening (38) is in fluid communication with the concentrate outlet tube (40). The concentrate outlet tube extends coaxially around the central tube (30).

In the exemplary embodiment, the inlet opening 36 of the concentrate centrifugal pump is located in the concentrate centrifugal pump chamber 42 . The concentrate centripetal pump chamber is defined by the walls of the rotatable assembly (16). During operation, the inlet opening 36 of the concentrate centrifugal pump is exposed to the concentrate in the concentrate centrifugal pump chamber 42 . Concentrate centrifugal pump chamber 42 is vertically bound by upper portion 44. At least one fluid seal (46) extends between the outer circumference of the outlet tube (40) and the upper portion (44). The exemplary seal 46 is configured to reduce the risk of fluid escaping inside the separation chamber and prevent contaminants from entering the outer region of the inner core.

During operation of the centrifugal separator, the balls and cores containing the cavities or separation chambers are rotated about the axis 12 in the direction of rotation. Rotation in the direction of rotation operates to separate the cell suspension drawn through the feed tube 18 into a filtrate that exits through the filtrate tube 30 and a retentate that exits through the concentrate outlet tube 40 .

The cell suspension enters the separation chamber 22 through a tube opening 20 in the bottom of the separation chamber. The cell suspension moves outward through centrifugal force and a plurality of accelerating vanes (48). As the suspension moves outward by the accelerating vanes, the cell suspension material is acted upon by centrifugal force, causing the cell material to move outward toward the annular tapered wall 50 bordering the outside of the separation chamber. The concentrated cellular material is forced to move outwardly and upwardly as shown against the tapered wall 50 and through the plurality of concentrate slots 52 . The concentrate material travels over the concentrate slot and upwards by the concentrate centrifugal pump 34 into the concentrate pump chamber 42 from which the concentrate is discharged.

In the exemplary device, during operation, the cell-free filtrate is positioned proximate the vertical annular wall 54 bordering the interior of the separation chamber 22 . The filtrate material travels upward through the filtrate aperture 56 of the annular base structure bordering the filtrate pump. The filtrate travels upward through the filtrate aperture 56 and forms a pool of liquid filtrate in the filtrate chamber. From the filtrate chamber, filtrate is moved through the action of filtrate centrifugal pump 24 and delivered from the core through filtrate tube 30.

In the exemplary embodiment of FIG. 16 , the concentrate and filtrate pumps may have a configuration generally as shown in FIG. 17 . In FIG. 17 , the filtrate centrifugal pump 24 is represented in an isometric exploded view. As shown in FIG. 17 , the exemplary centrifugal pump has a disk-shaped body composed of a first plate 58 and a second plate 60 . During operation, the first and second plates are releasably held together 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 walls bounding three sides of the curved vortex passage 64 . It should be understood that the centrifugal pump in the exemplary device includes a pair of generally opposed vortex passages 64 . In other devices, other numbers and configurations of vortex passages may be used.

In the exemplary device, the first and second plates constitute a disc-shaped body of a centrifugal pump with an annular vertically extending wall 67 defining an annular circumference 66 . An inlet opening 68 to the swirl passage 64 extends around the annular shape. An annular collecting chamber 70 extends from the body radially outwardly from the shaft 12 and is fluidly connected to the vortex passage. An annular collection chamber 70 receives the material entering the inlet opening 68 . An annular collection chamber 70 is in fluid communication with an annular outlet opening coaxial with shaft 12 . In an exemplary arrangement for a filtrate centrifugal pump, the annular discharge opening is an annular space extending between the outer wall of the supply tube 18 and the inner wall of the second plate 60, the outlet of which is fluidly connected to the central discharge tube 30.

Each vortex passage 64 in the exemplary arrangement is configured such that the vortex passage curves toward the direction of rotation of the ball and separation chamber, which direction of rotation is indicated by arrow R in FIG. 17 . In the exemplary device, the vertically extending walls 74 bounding the vortex passages and facing the direction of rotation are each curved in the direction of rotation. The curved configuration of the wall 74 horizontally bordering the vortex passage provides enhanced pumping characteristics of the exemplary device. Also, the opposing boundary walls 76 of each vortex passage in the exemplary device have a similar curved configuration. The curved configuration of the vertically extending walls horizontally bordering the vortex passages provides a constant cross-sectional area of each vortex passage 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 walls 74 and 76 and bounding the vortex passage 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 disc-shaped body of the centripetal pump. In this exemplary device, face 80 serves to vertically bound the sides of both vortex passages 64 of the centripetal pump.

Of course, it includes a pair of plates, one of the plates of one phase including a walled recess bounding three of the four sides of the curved vortex passage and the other bounding the other side of the vortex passage. It should be understood that this example device, including a surface, is illustrative. It should be understood that other configurations and structures may be used in other devices.

In the exemplary centripetal pump structure shown in FIG. 16, a centripetal pump structure is used and can move more liquid than a similarly sized paired disc-type centrifugal pump. Also, the exemplary configuration generates less heating of the liquid than comparable fairing disks.

Also, in the previously discussed exemplary device, the annular circumference of the filtrate centrifugal pump 24 has a smaller outer diameter than the circumference of the concentrate centrifugal pump 34 . In the exemplary apparatus, this configuration is used to prevent the filtrate centrifugal pump from removing too much liquid from the pool of liquid filtrate that forms 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 centrifugal pump. Wave formation from less than sufficient liquid filtrate can cause vibration and other undesirable properties of the centrifuge and core.

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

In a further exemplary embodiment, using a centrifugal pump with the described configuration, the characteristics and flow characteristics 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 centrifugal pump can be sized to achieve optimum characteristics for a particular processing activity. For example, the larger the diameter of the circumference of a centrifugal pump, the greater the flow rate and pressure at the outlet. Moreover, larger diameters tend to produce greater mixing than relatively small diameters. However, a larger diameter results in greater heating than a smaller peripheral diameter of the centripetal pump. A smaller diameter periphery can thus be used to achieve less heating. It should also be appreciated that different sizes, areas and numbers of inlet openings and different vortex passage configurations may be used to vary the flow and pressure characteristics as desired for the purpose of a particular separation process.

19 schematically depicts an exemplary system used to ensure a positive pressure within a separation chamber, alternatively referred to collectively in this application, during processing of a cell suspension. As discussed in relation to previous exemplary embodiments, it is generally desirable to always ensure a positive pressure above atmospheric pressure within the separation chamber. This reduces the risk of contaminants entering the separation chamber through one or more fluid seals operably extending between the stationary assembly and the rotatable assembly of the core. Also as discussed above, it is generally desirable to maintain the air at a positive pressure within the separation chamber in contact with the inner surface of the fluid seal. The presence of air pockets adjacent to the seal prevents the seal from contacting the material being processed and reduces the risk of contaminants entering the processed material and the risk of material escaping from the separation chamber.

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

As shown schematically in FIG. 19 , the centrifugal separator includes a rotatable ball 82 . The centrifuge ball may be rotated about an axis 84 by a motor 86 or other suitable rotational 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 the other embodiments described above, the exemplary core includes a holding assembly comprising a suspension inlet feed tube 92 having an inlet opening 94 located adjacent to the bottom region of the cavity. The stationary assembly further includes at least one centrifugal pump (96). The centripetal pump of the exemplary embodiment includes a disk-shaped body having at least one pump inlet 98 adjacent to its periphery and a pump outlet 100 adjacent to the center of the centripetal pump. The pump outlet is in fluid communication with the filtrate outlet tube (102). The filtrate outlet tube extends coaxially around the suspension inlet tube in a manner similar to that previously discussed. The fluid rotatable upper portion 104 containing the separation chamber is operatively connected with at least one seal 106 operative to fluidically seal the cavity of the core to the inlet and outlet tubes. At least one seal 106 is formed 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. It is operatively extended in a sealing 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 for pumping a 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 pump 108 to act as a feed pump to pump cell suspension from inlet line 110 to the inlet tube at a controlled rate. In a further exemplary device, pump 108 may operate as a concentrate removal or discharge pump after the cell concentrate has been separated by the centrifugal action. In performing this function, pump 108 operates to pump cell concentrate from the separation chamber by reversing the flow of material in inlet tube 92 as the cell suspension is supplied to the separation chamber. The cell concentrate is then pumped to concentrate line 112. As shown in FIG. 19 , inlet line 110 and concentrate 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 outlet tube 102 is fluidly connected to the filtrate outlet 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 pump, the flow rate of which can be optionally adjusted. For example, in some exemplary devices, pump 120 may include a peristaltic pump that includes a motor whose speed may be controlled to selectively increase or decrease flow rate through the pump. The outlet of the filtrate discharge pump delivers the treated filtrate to an appropriate collection chamber or other processing device.

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

On the opposite side of reservoir 122 is upper port 126 . The top port is exposed to air pressure. In the exemplary device, the upper port is exposed to air pressure from a source of elevated air pressure indicated schematically at 128 . In exemplary embodiments, 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 within a range necessary for system operation. Air from the high pressure source 128 is passed through a sterilization filter 130 to remove impurities. Regulator 132 operates to maintain a generally constant air pressure level above atmospheric at the upper port of the pressure decay reservoir. In the exemplary device, the air pressure regulator includes an electronic fast acting regulator to ensure that a generally constant air pressure is maintained at a desired level. The exemplary fast-acting regulator 132 operates to rapidly increase the pressure acting on the top port 126 when the pressure drops below a desired level, and through the regulator when the pressure acting on the top port is above a set value. alleviate quickly

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 schematically shown in phantom. In this exemplary device, the outlet pressure of the regulator acting on the upper port 126 of the reservoir is also above the air pocket inside the separation chamber which extends down through the air line 143 to the cavity level above the centripetal pump inlet; It acts radially from a region inside at least one seal 106 and proximate axis 84 to an upper interior wall inside top 104 . In the exemplary arrangement, line 143 is routed through at least one separate passageway extending through a fixed structure of an assembly comprising filtrate outlet tube 102 and inlet feed tube 92 into a separation chamber under at least one seal. Apply positive pressure to the area. At least one exemplary isolated passage of air line 143 applies air pressure to the interior of 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 the outlet tube 102 , above the inlet 98 to the centripetal pump, and below the at least one seal 106 . Of course, it should be understood that the lines providing positive air pressure inside the at least one seal and the air pockets of the isolation chamber are exemplary, and that other structures and approaches may be used in other embodiments.

In the exemplary arrangement of the pressure decay reservoir 122, the upper liquid level sensor 134 is configured to sense liquid filtrate within the interior of the pressure decay reservoir. The upper liquid level sensor operates to sense liquid at the upper level. A 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 high liquid level sensor is arranged to detect an unacceptably high level of liquid to indicate an abnormal condition that requires shutting down the system or taking other appropriate safety measures. In the exemplary device, the liquid level sensors 134, 136 and 138 include capacitive proximity sensors suitable for sensing the level of liquid filtrate adjacent to and within the pressure decay 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 as may be appropriate to operation of the system. These other components may include other valves, lines, pressure connections, or other suitable components for performing treatment and handling of suspensions, filtrates, and concentrates suitable for the particular system. This may include additional valves, such as valve 140 schematically shown, to control the open and closed state of filtrate discharge 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. The exemplary at least one control circuit 142 includes one or more processors 144 . The processor is in operative connection with one or more data stores 146 indicated schematically 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 through a processor executing instructions to do so. 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, the 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 functions in the manner of an electronic computing device. Data storage may also be one or more of volatile or non-volatile memory devices such as RAM, flash memory, hard drives, solid state devices, CDs, DVDs, optical memory, magnetic memory, or other circuit readable media or media. should understand Computer executable instructions and/or data may be stored.

Circuit execution instructions may include instructions in any of a number of programming languages and formats, including but not limited to routines, subroutines, programs, threads of execution, objects, methodologies, and functions that perform operations such as those described herein. can The architecture for the control circuit includes, corresponds to, and can be utilized the principles described in the book Microprocessor Architecture, Programming, and Applications with the 8085 by Ramesh S. Gaonker (Prentice Hall, 2002). It is to be understood, of course, that these control circuit structures are exemplary and that other circuit structures for storing, processing, resolving, and outputting information may be used in other embodiments.

In the exemplary device, at least one control circuit 142 is operatively connected to at least one sensor, such as sensors 134, 136 and 138, through a suitable interface. 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 in operative connection with other devices such as motor 86, pump 108, regulator 132, pneumatic source 128, fluid control valves and other devices. there is.

At least one exemplary control circuit is operative to receive data and control such a device according to circuit execution instructions stored in data store 146 . In the exemplary device, the fluid level 147 in the fluid attenuation reservoir is a characteristic corresponding to the pressure in the filtrate discharge tube 102 . In one exemplary implementation that does not use an air line 143, the fact that the pressure in the filtrate discharge tube characterizes the pressure in the upper portion 104 of the core and the pressure in the separation chamber adjacent to the seal 106. , 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 and an air pocket adjacent to at least one seal within the separation chamber to prevent entry of contaminants into the separation chamber that may result from negative pressure. However, if the fluid level in the separation chamber becomes too high, the pressure and suspension material to be processed may overflow the seal, resulting in undesirable exposure to potential contaminants and loss of the material to be processed. This can occur in situations where the back pressure on the filtrate line connected to the outlet of the centrifugal 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. The pump output pressure level of a centripetal pump depends on the rotational speed of the ball and core. An exemplary device that does not use air line 143 provides controlled back pressure on the filtrate discharge tube. Back pressure is provided by controlling the speed of the motor that drives the pump 120 and the liquid level 147 in the pressure damping reservoir. The back pressure is maintained below the level of the pump output pressure (so that the centrifugal pump can transmit the centrifugal force in the separation chamber), but at a positive pressure above atmospheric to prevent contaminants from penetrating past the seal and into the separation chamber, and at an elevated level of air. Pressure is maintained inside the separation chamber adjacent to the seal to isolate the seal from the components of the suspension being processed.

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

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

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

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

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

20 schematically illustrates exemplary logic implemented through the operation of at least one control circuit 142 related to maintaining a desired pressure level within the upper portion of the filtrate discharge tube and separation chamber. It should be understood that in some exemplary embodiments, the control circuitry may perform many additional or different functions than those represented. These functions may include overall control of various processes and steps for centrifuge operation in addition to the pressure control function described above. As shown in FIG. 20 , in initial subroutine step 148, at least one control circuit 142 is operative to determine whether centrifuge operation is currently in a mode in which filtrate is discharged from the separation chamber. If so, at least one control circuit operates to cause the filtrate discharge pump 120 to operate for filtrate discharge delivered through the filtrate discharge line 118 . This can be done by causing the pump's motor to run. In the exemplary arrangement, the flow rate of pump 120 may be at an initially set value or may be changed in accordance with specific operating conditions determined through control circuit operation during processing. Operation of the filtrate discharge 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. If liquid is sensed at the level of sensor 138, the control circuitry is operative to take steps to remedy this condition. This may include activating the pump 120 to increase its flow rate and making a subsequent determination as to whether the level has fallen 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 actions do not cause the level to drop within a certain amount of time, an additional step is performed. This step may also include slowing down or stopping rotation of the ball 182 . This action 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 ending normal operation of the system, is indicated by step 154.

If liquid is not sensed at the level of the high level sensor 138, at least one control circuit is then actuated 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, 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 a 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 decay reservoir to begin to drop as more liquid is displaced by the pump 120 .

If liquid is not sensed at the upper liquid level of sensor 134 at step 156, at least one control circuit is operative to make a determination as to whether liquid is not sensed at the lower liquid level of 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 decay 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 additional actions, such as those associated with shutdown 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 pressure decay reservoir at a generally constant level between the levels of sensors 134 and 136 during filtrate production. can do.

In the exemplary apparatus, maintaining a constant overall elevated pressure of sterile air over the liquid in the pressure damping reservoir helps ensure that similar elevated pressures are consistently maintained in the seals in the separation chamber and in the filtrate discharge line. Also in the exemplary arrangement, the pressure may be controlled at a desired level during different operating conditions of the centrifugal separator while the balls rotate at different speeds. For example, conditions are included where the separation chamber is initially filled at a relatively high rate through introduction of the cell suspension and the centrifuge is rotated at a relatively low rate. The pressure may be maintained during subsequent conditions of final filling where the cell suspension flow rate into the separation chamber occurs at a slower rate and the rotational speed of the balls increases to a higher rotational rate. In addition, positive pressure is maintained as previously described during the feeding of the suspension into the bowl and during the discharge of the filtrate from the separation chamber. In a further exemplary embodiment, the at least one control circuit may operate to maintain a positive pressure during a period in which the retentate is removed by being pumped out of the separation chamber. Maintaining a positive pressure within the isolation chamber under all these conditions reduces the risk of contamination and other undesirable conditions that can result from negative (sub-atmospheric) conditions.

Of course, it should be understood that these features, components, structures and control methodologies are exemplary and that other approaches may be used in other devices. Further, exemplary devices include systems that operate in a batch mode rather than a mode in which the filtrate and retentate are processed continuously, but the principles herein can be applied to these other types of systems as well.

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

21 schematically depicts an alternative centrifugation system 170 specially configured to separate cells in a cell culture batch into a cell filtrate and a 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 retaining structure 182 schematically indicated may releasably secure the disposable structure 178 within the ball cavity.

The exemplary disposable structure 178 of this exemplary embodiment includes an axially extending feeding tube 184 . As will be discussed later, the feeding tube is used to transport the cell culture batch material to the inner region 186 of the disposable structure 178 . The feeding 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 disc-shaped portion 194 is generally rigid, meaning that the disc-shaped portion is rigid or semi-rigid, and includes an annular circumference 196 . The annular circumference is configured to abut the upper annular boundary wall 198 of the centrifuge ball cavity 176 . The annular circumference of the disk-shaped portion 194 is configured to engage the rigid ball 172 such that the disposable structure rotates with it.

The exemplary disposable structure 178 further includes a hollow rigid or at least semi-rigid cylindrical core 200 . The core 200 is operatively coupled with and rotatable with the disk-shaped portion 194 . The core 200 is axially aligned with the disc-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 the feed tube 184 extends.

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

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

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

The exemplary disposable structure 178 further includes a flexible outer wall 222 . The flexible outer wall 222 is a fluid-tight wall that extends in 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 to the disk-shaped portion 194 in a fluid tight connection. The flexible outer wall has a truncated cone shape with a smaller inner radius adjacent the lower portion of the disposable structure adjacent the second axial end 192 .

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

In the exemplary device, flexible outer wall 222 has a textured outer surface 226 . The textured outer surface is configured to allow air to pass out of the space between the flexible outer wall 222 and the surface surrounding the cavity of the rigid ball 172 . In an exemplary device, the textured outer surface may allow substantially the entire area of the flexible outer wall to contact the rigid ball. In an exemplary device, the textured exterior surface may include one or more patterns of outwardly extending protrusions or dimples 228 with spaces or recesses therebetween to facilitate passage of air. Air may pass out of the cheek cavity 176 when the disposable structure 178 is placed therein through either the upper opening 180 or the lower opening 230 . In an exemplary device, the protrusion may include a resilient, deformable material that can decrease in height by 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 being trapped between the rigid ball of the centrifuge and the disposable structure. These air pockets can create imbalances or cause wall contour irregularities that can change 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 structure described is 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 . Rigid or semi-rigid materials act to retain their shape during operation. In the exemplary device, the lower disc-shaped portion 232 has a conical shape and is operatively attached and 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 the upper surface of the disc-shaped portion 232 and the radially outer lower portion of the core. A fluid passageway 232 extends radially outward and upward relative to the bottom of the second axial end 192 and passes through an opening 190 of the supply tube 184 into an interior region 186 of cell culture batch material. Allow the cells to pass radially outward and upward into the separation chamber 224 .

In the exemplary device, the flexible outer wall 222 extends below the lower disk-shaped portion 232 at the second axial end 192 of the disposable structure. A flexible outer wall 222 extends halfway to the wall surface of the rigid ball 172 bordering the lower disk-shaped portion 232 and the cavity in which the disposable structure is located.

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

Example system 170 operates in the same manner as previously discussed. Cells in the cell culture batch material are introduced into the interior region 186 of the disposable structure 178 via a feed tube 184 . Cells enter interior region 186 through feed tube opening 190 at the lower axial end of the disposable structure. The centrifugal force causes the cells to migrate outward through the aperture 234 and into the separation chamber 224 . Outwardly and upwardly tapered exterior walls 222 allow cells or cellular material, including cell concentrate, to congregate adjacent to the radially outward and upper regions of 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 into the filtrate pump chamber. The filtrate passes inward through the substantially annular opening of the filtrate centrifugal pump and then upwards through the filtrate discharge tube 212 . At the same time, the cell concentrate passes through the substantially annular concentrate opening 218 into the concentrate centrifugal pump chamber 214 . The concentrate passes internally through the substantially annular opening of the concentrate centrifugal pump 216 and then upwards through the concentrate discharge tube 220 . This exemplary configuration allows exemplary system 170 to operate continuously or semi-continuously. Operation of system 170 may be controlled in a manner discussed later to facilitate reliable extended operation of the system and delivery of the desired cell concentrate and generally cell-free filtrate in a separate output fluid stream. there is.

22 shows an alternative centrifugal separation system, generally indicated at 238 . System 238 includes a disposable structure 240 . Disposable structure 240 is similar in most respects to previously described disposable structure 178 . Some of the structures and features of disposable structure 240 that are generally the same as those described with respect to disposable structure 178 are denoted by the same reference numerals 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-shaped structure in operative connection with the lower end of the core 200 . A plurality of radially outward and upwardly extending fluid passages 244 extend between the lower end of the core 200 and the lower disk shaped portion 242 . The exemplary lower disk-shaped portion 242 also includes a plurality of angularly spaced radially extending vanes 246 . In this exemplary device, the vanes 246 extend upwardly from the bottom portion of the disk-shaped portion 242 and operatively engage the core at least partially at their radially outer portions. Vane 246 in the exemplary device accelerates cell culture placement to facilitate movement and separation within the inner 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 previously described disposable structure 178 . Disposable structure 250 is labeled with the same reference number with some of the similar structures and features to those of disposable structure 178 previously described.

The exemplary disposable structure 250 differs from the disposable structure 178 in that it includes a lower disk-shaped portion 252 . Lower disc shaped portion 252 is a rigid or semi-rigid conical shaped structure in operably attached connection with core 200 via a wall portion or other suitable structure. The lower disc-shaped portion 252 extends radially outward and includes a plurality of angularly spaced accelerator vanes 254 . Accelerator vanes 254 extend down from the lower conical side of disk-shaped portion 252 . Each immediately angularly adjacent pair of 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 that operates to accelerate cell culture batch materials to facilitate dissociation within the inner region of the disposable structure. Of course, it should be understood that the features of the disposable structures described herein may be combined in different structures to achieve a desired output fluid stream and facilitate the separation of different types of materials and materials having different properties.

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

Disposable structure 304 includes a continuous annular concentrate dam 306 . A concentrate dam 306 extends down from the separation chamber 224 and is disposed radially inwardly of the substantially annular concentrate opening 218 . An exemplary annular concentrate dam, shown in cross section, extends below the concentrate opening and includes a tapered outward facing surface 308 extending towards and out of the opening 218 in an axial cross section.

Disposable structure 304 further includes a continuous annular filtrate dam 310 . A filtrate dam 310 extends downwardly from 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 configuration, the distance that the concentrate dam 306 and the filtrate dam 310 extend downwardly in the separation chamber 224 is substantially the same. However, other configurations may be used with other exemplary devices. Also in other exemplary devices the centrifuge structure may include a concentrate dam or a filtrate dam, but not both.

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

In the exemplary embodiment, the concentrate dam 306 is primarily formed in an upper portion bordering the separation chamber 224 for the cellular material or other solids to be separated to reach the concentrate opening 218 and the concentrate centrifugal pump chamber 214. It helps to ensure that it can pass outward along the part. The filtrate dam 310 also allows the primarily cell-free filtrate material to pass along the upper surface bordering the separation chamber 224 and into the substantially annular filtrate opening 210 to reach the filtrate pump chamber 206. help to ensure that It should be understood that various configurations of concentrate dams and filtrate dams may be used in the various exemplary devices 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 produce streams of generally cell-free filtrate and cell concentrate. The centrifugal separation system 170 previously discussed in the exemplary device 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 illustrated exemplary embodiment, centrifuge ball 172 is rotated about axis 174 by motor 256 at a selected speed. Supply tube 184 is operatively connected to cell culture supply line 258 through which cell culture batch material is received. The supply line is operatively connected to the supply pump 260 . Feed pump 260 in the exemplary device may be a peristaltic pump or other suitable pump for delivering a cell culture in a disposable configuration at a selected flow rate.

The filtrate outlet tube 212 is in fluid communication with the filtrate outlet line 262 . A filtrate optical density sensor 264 is in operative connection with the inner region of the filtrate discharge line 262 . The filtrate optical concentration sensor in the exemplary device is an optical sensor that operates to determine the cell density in the filtrate currently 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 received by the receiver decreases as the cell density increases in the filtrate. Of course, this is just one example of a sensor that can be used to determine the density or quantity of cells present in the filtrate, other types of sensors may be used in other devices. For example, the light may be near infrared or other visible or invisible light. In other sensing devices, other types of electromagnetic, acoustic or other types of signals may be used for sensing. The filtrate discharge 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 filtrate material.

In the exemplary arrangement, the concentrate outlet tube 220 is operatively connected with the concentrate outlet line 268. Concentrate optical density sensor 270 is in operative communication with at least a portion of an interior region of concentrate discharge line 268 . The exemplary concentrate optical density sensor can operate in the same way as the previously discussed filtrate optical density sensor. Of course, it should be understood that the concentrate optical density sensors may include different structures or properties, and that different types of cell density sensors may be used in other exemplary embodiments. Concentrate discharge line 268 is in operative communication with concentrate pump 272 . In an exemplary embodiment, concentrate pump 272 may include a peristaltic pump or other variable speed pump suitable for pumping 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, indicated schematically at 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 media that hold executable instructions and data that, when executed by the controller, cause the controller to perform operations as discussed below. Such medium may include, for example, solid state memory, magnetic memory, optical memory, or other suitable non-transitory medium for holding circuit executable instructions and/or data. The control circuitry may include structures as discussed above.

The operations performed by the example controller 274 are now described in conjunction with the schematic representation of the logic flow shown in FIG. 25 . In the exemplary device, controller 274 is generally operative to control the operation of components of the system to maintain delivery of simultaneous output flows of cell-free filtrate and retentate. This typically involves an optical density sensor within each filtrate and concentrate discharge line to detect the cell density (or turbidity) of the output feed and adjust the operation of system components to maintain the output within a desired range. is done using

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

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

In the exemplary logic flow shown in FIG. 25 , operation of concentrate pump 272 at an initial prime rate 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 the prime period operating to at least partially fill the disposable structure 178 .

If the concentrate pump operates at prime speed during the prime period, the controller increases the concentrate pump speed to base speed as expressed in step 284. Controller 274 is operative to monitor the cell density in the filtrate as sensed by sensor 264. The controller operates to determine if the optical density is higher than a 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 that this measurement will not cause a change by the controller in the operating speed of the concentrate pump.

If at step 286 it is determined that the optical density in the filtrate is higher than the set point, the logic proceeds to step 288. At step 288, the controller operates to increase the speed of the concentrate pump by the set incremental step amount. This speed step increase is generally intended to lower the optical density of the filtrate by reducing the number of cells in the filtrate.

After the speed of concentrate pump 272 is increased in step 288, the controller operates in response to 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 above. If so, the controller continues to monitor the optical density of the center until it is not higher than the set point. The instructions in the exemplary device are that the filtrate optical density must not be higher than the set point before the concentrate pump speed controller determines that the adjustment of the base speed is sufficient to maintain the optical density of the filtrate at or below the desired set point. Include the set period. Step 292 determines whether the increased concentrate pump speed maintains the optical density of the filtrate below the set point for a stored set period of time corresponding to consistently producing a discharge of sufficiently cell-free filtrate or reaching a programmed hold time. Indicates the controller that decides. When sufficient flavor cell-free filtrate has been produced for the desired period of time or the programmed hold time has been reached, the controller operates, at 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, the concentrate pump speed is adjusted again.

The exemplary controller also simultaneously monitors the optical density of the cells in the exiting concentrate flow. This is done by monitoring the optical density detected by 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, the cell concentration of the exiting concentrate flow is above the desired level and the logic returns to step 284. However, if the optical density of the concentrate is below the desired set point, indicating that the cellular level of the concentrate is lower than desired, the controller moves to step 298. In step 298 the speed of the concentrate pump is reduced by a predetermined incremental step amount. Reducing the speed of the concentrate pump reduces the output flow rate and generally increases the amount of cells in the concentrate output flow, thereby increasing the optical density of the concentrate output flow.

The controller then operates the concentrate pump 272 at a new reduced speed as shown in step 300. As shown in step 302, the controller stores data in a data store so that the cell concentration in the output retentate flow can be increased before determining whether the rate reduction is sufficient. Run the concentrate pump at this reduced speed for a set time corresponding to the stored set point. If it is determined at step 302 that the time period has passed, the controller returns to step 284 where the logic flow is repeated to determine if further speed adjustments are required.

Of course, it should be understood that this schematic simplified logic flow is illustrative and that other logic flows and/or additional operating parameters of system components in other embodiments may be monitored and adjusted to achieve desired filtrate and concentrate output flows. . For example, in another exemplary device, the speed of the filtrate output pump and thus the filtrate output flow is varied at least in part by the controller's response to an optical density detected by the filtrate optical density sensor corresponding to a cellular level within the filtrate. can do For example, if it is detected that the cell level in the filtrate is above a set limit, the controller can act to reduce the flow rate of the filtrate pump. This may be performed by a controller as an alternative to, or in combination with, controlling the concentrate discharge flow rate. The controller can change the filtrate flow rate as appropriate to keep the cell level in the filtrate 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 structure. This may be done in conjunction with changing the flow rates of filtrate and concentrate exiting the disposable structure to maintain cellular levels of concentration within programmed set limits stored in a memory associated with the controller. The controller can also operate under programming to vary ball rotation speed, diluent introduction and diluent introduction rates, and other process parameters to maintain filtrate and concentrate properties at desired throughput rates within programmed limits. In yet other exemplary embodiments, other characteristics 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 previously discussed disposable structure 178 except where noted. The disposable structure 314 includes elements that operate to ensure that the air/liquid interface of the air pockets extending from the disposable structure and isolating the seal 236 from the material being processed will remain more stable in the desired radial direction. do.

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

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

Upper filtrate centrifugal pump chamber surface 320 extends radially outward from circular upper filtrate centrifugal pump chamber opening 324 . Feed tube 184 and filtrate discharge tube 212 extend axially through the upper filtrate centrifugal pump chamber opening.

A plurality of angularly spaced, upwardly extending lower filtrate chamber vanes 326 extend over the lower filtrate centripetal pump chamber surface 318 . Each lower filtrate chamber vane 326 extends radially outward starting from the lower filtrate centripetal pump chamber opening 322 . The lower filtrate chamber vane 326 , shown in more detail in FIG. 28 , extends radially outward from a lower center vane distance V from the axis of rotation 174 . In the exemplary device the lower filtrate chamber vane 326 extends upward from 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 vane, the vane height, and the depth and diameter of the recess may be varied to achieve a desired fluid pressure characteristic.

Extending from the upper filtrate centripetal pump chamber surface 320 is a plurality of angularly spaced, downwardly extending upper filtrate chamber vanes 328 . Each upper filtrate chamber vane 328 extends radially outward starting from the upper filtrate centripetal pump chamber opening 324 . The upper filtrate chamber vane extends radially outward from the upper filtrate vane distance from the axis of rotation 174 . In the exemplary apparatus the upper filtrate vane distance corresponds substantially to the lower center vane distance V. In the exemplary arrangement, the upper filtrate chamber vanes extend downward in circular recesses on the upper filtrate centripetal pump chamber surface having the configuration shown in FIG. 28 , but in an inverted direction.

In the illustrated exemplary device, filtrate centrifugal pump 208 includes a substantially annular filtrate centrifugal pump opening 330 . The substantially annular filtrate centrifugal 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 centrifugal 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.

Concentrate centrifugal pump 216 is located in concentrate centrifugal pump chamber 332 in the exemplary device of disposable structure 314 . Concentrate centrifugal pump chamber 332 is bounded vertically at the bottom by a circular lower concentrate centrifugal pump chamber surface 334 . Concentrate centrifugal pump chamber 332 is vertically bounded on its upper side by a circular upper concentrate centrifugal pump chamber surface 336 .

A lower concentrate centrifugal pump chamber surface 334 extends radially outwardly from the lower concentrate centrifugal pump chamber opening 338 . In the exemplary arrangement, the lower concentrate centrifugal pump chamber opening corresponds in size to and is continuous with the upper filtrate centrifugal 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 340 extends radially outward starting from the lower concentrate centripetal pump chamber opening 338 . The lower concentrate chamber vane 334 extends radially outward from the axis of rotation at a lower concentrate vane distance. In the exemplary arrangement, lower concentrate chamber vanes 334 extend over circular recessed portions of the lower concentrate centrifugal 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.

An upper concentrate centrifugal pump chamber surface 336 extends radially outward from the upper concentrate centrifugal pump chamber opening 342 . The feed tube 184 , the filtrate exhaust tube 212 and the concentrate exhaust tube 220 extend coaxially through the upper concentrate centrifugal pump chamber opening 342 . A plurality of angularly spaced upper concentrate chamber vanes 344 extend down from surface 336 . The upper concentrate chamber vane extends radially outward from the upper concentrate centripetal pump chamber opening 342 the upper concentrate vane distance. Upper concentrate chamber vanes extend from upwardly extending circular recesses in the upper concentrate centrifugal pump chamber surface. In the exemplary arrangement, the upper concentrate chamber vanes are configured 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 centrifugal 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 concentrate pump opening distance. In the exemplary arrangement, the upper and lower concentrate vane distance is less than the concentrate pump opening distance. Of course, it should be understood that this configuration is exemplary and 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 form an annular air/liquid interface 348 of centrifugal pump chamber 330 and It acts to stabilize and radially position the air/liquid interface 350 of the concentrate centrifugal pump chamber 332. As shown in FIG. 28, the air/liquid interface 348 is located radially midway along the radial length of the filtrate chamber vanes. It is radially inward from the filtrate pump opening 330 . The radially extending filtrate chamber vanes operate to provide a centrifugal pumping force that holds the annular air/liquid interface 348 in a radial position 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 consistent in the liquid center and is not exposed to air.

Upper concentrate chamber vane 344 and lower concentrate chamber vane 340 operate in a similar manner to the filtrate chamber vanes. The concentrate chamber vanes hold 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. In the illustrated embodiment the filtrate centrifugal pump and the concentrate centrifugal pump are substantially the same size, but it should also be understood that other configurations may have different sizes of centrifugal pumps. In this situation, the radial distance from the axis of rotation at which the filtrate chamber vane and the concentrate chamber vane extend may be different. Also, the radial position of the air/liquid interface relative to the axis of rotation in the filtrate pump chamber and the concentrate centrifugal pump chamber may be different. A variety of vane configurations and arrangements may be utilized depending on the particular relationship between the components that make up the disposable device and the specific material being processed through the disposable structure.

30 shows the upper portion of another alternative disposable structure 352 . Disposable structure 352 is similar to disposable structure 304 unless otherwise discussed. Disposable structure 352 includes an air tube 354 extending coaxially around concentrate discharge tube 220 . Air tube 354 communicates with opening 356 inside the disposable structure. An aperture 356 extends from the interior of the air tube above the concentrate centrifugal pump 216 in the concentrate centrifugal pump chamber 332 . In this exemplary device, as shown schematically, seal 236 operatively engages the air tube, the concentrate exhaust tube, the filtrate exhaust tube, and the supply tube to maintain an airtight connection with the air. As will be appreciated, air tubes may be used to selectively maintain the level of air pressure in air pockets within the disposable structure. This structure may be utilized in conjunction with systems such as 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 desired 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 continuously separate a cell suspension into a substantially cell-free filtrate and retentate. 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 . Controller 274 of system 358 positions 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 filtrate pump opening and concentrate pump opening. operates to control

In the exemplary arrangement, the flow back pressure regulator 360 is in fluid communication with the filtrate discharge line 262 . In the exemplary arrangement, the flow back pressure regulator 360 is fluidically intermediate the filtrate discharge tube 212 and the filtrate pump 266 . Exemplary system 358 includes a source of pressurized air, indicated schematically at 362 . The pressurized air source 362 is connected to the pilot pressure control valve 364. The control valve is operatively connected to the controller 274. A signal from controller 274 selectively generates a variable pressure in pilot line 366. Pilot line 366 is in fluid communication 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 the flow back pressure regulator 360 .

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

In the exemplary arrangement, controller 274 operates according to the stored executable instructions to control the operation of system 358 in a manner similar to that previously discussed with respect to system 170 . Also in the exemplary arrangement, controller 274 is operative to control pilot pressure valve 364 to vary the back pressure applied to filtrate outlet tube 212 by back pressure regulator 360 . Controller 274 also operates to control valve 368. The controller operates to maintain and selectively change the pressure applied to the air pockets in the upper portion of the interior of the disposable structure. The controller operates according to the program to change the air pocket pressure and/or the back pressure of the filtrate flow to change the air/liquid interface of the air pocket at a radial distance from the axis of rotation pointing 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 pocket to maintain This pressure change, both filtrate flow back pressure and air pocket pressure, along with the action of the filtrate chamber vanes and concentrate chamber vanes in an exemplary embodiment, maintains stability and is introduced into the filtrate and concentrate exiting the disposable structure. Keep the radially outer extent of the air/liquid interface constant so that air is minimal. Also, the ability to selectively change filtrate flow and back pressure can affect the optical density perceived at the cell level and correspondingly expelled concentrate. Accordingly, the controller selectively changes the retentate flow rate, the filtrate back pressure and flow rate, the internal air pocket pressure, the single-use structure cell suspension feed rate, and all other parameters of the centrifugation process, and, associated with the controller, has at least one data store. programmable to maintain filtrate and concentrate properties within set limits and/or ranges stored in the In addition, the exemplary apparatus allows 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 in relation to a feature of system 170, but such control may be used with other types of systems that include other types of treatment elements.

Thus, the novel centrifugal separation systems and methods of the exemplary embodiments achieve at least some of the above-stated objectives, eliminate the difficulties encountered in the use of prior apparatus and systems, solve the problems and achieve the desirable results described herein. to achieve

While certain terms have been used in the above description for brevity, clarity, and understanding, such terms are for purposes of explanation and are intended to be interpreted broadly and should not imply unnecessary limitations. Further, the description and examples herein are for purposes of example only and the invention is not limited to the exact details shown and described.

Any feature recited as a means for performing a function in the claims below is to be construed as including any means known to one of ordinary skill in the art as filed to be capable of performing the recited function, and the structure shown herein or its equivalent. Not limited.

The characteristics, discoveries and principles of new and useful features, how they are constructed, used and operated, and the benefits and useful results obtained have been described; new and useful structures, devices, elements, arrangements, components, combinations, systems, equipment, operations and The reciprocal relationship is set out in the appended claims.

Claims (21)

A disposable structure configured for use in a centrifugal separation system comprising a multi-use rotatable centrifugal separation ball, wherein the disposable structure is releasably positioned within the ball and, within an interior region of the structure, separates cells in a cell culture batch from a cell concentrate. A disposable structure configured to separate into a cell filtrate,
The disposable structure in the operative position,
upper disc-shaped part,
a lower portion located below the upper disk-shaped portion;
A cylindrical core intermediate the upper disc-shaped portion and the lower portion,
a separation chamber radially surrounding the cylindrical core;
With the outer wall, the upper disk-shaped part and the outer wall can rotate around a vertical axis in the ball, the outer wall comprising:
Extending in liquid-tight working relationship with the upper disc-shaped portion and surrounding the separation chamber;
Surrounding and extending the cylindrical core,
an outer wall extending radially outward from the cylindrical core in the shape of a truncated cone with an inner radius smaller than that of the neighboring lower portion;
a supply tube extending in a vertical direction;
a filtrate discharge tube extending in a vertical direction;
filtrate pump chamber;
As a filtrate centrifugal pump, the filtrate centrifugal pump comprises:
axially aligned with the cylindrical core,
located coaxially with the supply tube;
in fluid communication with the filtrate discharge tube;
positioned within the filtrate pump chamber, wherein the filtrate pump chamber is in fluid communication with the separation chamber.
a filtrate centrifugal pump;
While the ball rotates, the upper disc-shaped portion and the outer wall rotate for each of the supply tube, the filtrate discharge tube and the filtrate centrifugal pump,
The apparatus of claim 1, wherein the separation chamber is bounded on the upper side by an upper disc-shaped portion, and the filtrate pump chamber is within the upper disc-shaped portion. delete 2. The device of claim 1, wherein the outer wall includes an exterior textured surface to allow air to escape from between the outer wall and the ball. The method of claim 1, wherein the lower portion includes a lower disk-shaped portion fixedly and operatively connected with the cylindrical core;
a lower portion within the inner region includes a plurality of angularly spaced radially extending accelerator vanes, with fluid passages extending between the vanes;
A device, characterized in that the radially extending accelerator vanes extend either upwardly from the lower disk-shaped portion or downwardly from the lower disk-shaped portion. 2. The method of claim 1 wherein the outer wall comprises a pattern of outwardly extending protrusions with intermediate recesses between the protrusions;
The device according to 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 disk-shaped portion,
The lower disc-shaped portion is fixedly and operatively connected to the cylindrical core,
The device, characterized in that the outer wall extends midway between the ball and the lower disc-shaped portion. The method of claim 1, wherein the disposable structure,
With a concentrate discharge tube extending vertically,
The concentrate discharge tube, wherein the upper disk-shaped portion and the outer wall rotate relative to the concentrate discharge tube while the ball rotates;
a concentrate centrifugal pump chamber, wherein the upper disc-shaped portion comprises the concentrate centrifugal pump chamber;
As a concentrate centrifugal pump, the concentrate centrifugal pump comprises:
axially aligned to the cylindrical core,
located concentrically around the supply tube;
Located above the filtrate centrifugal pump in the vertical direction,
located within the concentrate centrifugal pump chamber;
the concentrate centrifugal pump chamber comprises a concentrate centrifugal pump in fluid communication with the separation chamber;
The device characterized in that the upper disc-shaped part and the outer wall rotate relative to the concentrate centrifugal pump while the ball rotates. The method of claim 1, wherein the disposable structure,
With a concentrate discharge tube extending vertically,
The concentrate discharge tube, wherein the upper disk-shaped portion and the outer wall rotate relative to the concentrate discharge tube while the ball rotates;
concentrate centrifugal pump chamber,
A concentrate centrifugal pump, said concentrate centrifugal pump comprising:
axially aligned to the cylindrical core,
located concentrically around the supply tube;
Located above the filtrate centrifugal pump in the vertical direction,
in fluid communication with the concentrate discharge tube;
located within the concentrate centrifugal pump chamber;
the concentrate centrifugal pump chamber further comprises a concentrate centrifugal pump in fluid communication with the separation chamber;
While the ball rotates, the upper disk-shaped part and the outer wall rotate against the concentrate centrifugal pump,
a concentrate optical density sensor operably connected with the interior of the concentrate exhaust line, the concentrate exhaust line being operatively connected with the concentrate exhaust tube;
The apparatus of claim 1 , further comprising a controller in operative connection with the concentrate optical density sensor. According to claim 1,
a filtrate optical density sensor operably connected to the interior of the filtrate discharge line;
The apparatus of claim 1 , further comprising a controller in operative connection with the filtrate optical density sensor. The method of claim 1, wherein the disposable structure,
With a concentrate discharge tube extending vertically,
The concentrate discharge tube, wherein the upper disk-shaped portion and the outer wall rotate relative to the concentrate discharge tube while the ball rotates;
concentrate centrifugal pump chamber,
A concentrate centrifugal pump, said concentrate centrifugal pump comprising:
axially aligned to the cylindrical core,
located concentrically around the supply tube;
Located above the filtrate centrifugal pump in the vertical direction,
in fluid communication with the concentrate discharge tube;
the concentrate centrifugal pump chamber further comprises a concentrate centrifugal pump in fluid communication with the separation chamber;
While the ball rotates, the upper disk-shaped part and the outer wall rotate against the concentrate centrifugal pump,
a concentrate optical density sensor operatively connected to the interior of the concentrate discharge line;
a filtrate optical density sensor operably connected to the interior of the filtrate discharge line;
further comprising a controller operatively connected to the filtrate optical density sensor and the concentrate optical density sensor and operative to control the flow of the concentrate or filtrate in response at least in part to the concentrate optical density sensor and the filtrate optical density sensor. A device characterized in that. According to claim 1,
The filtrate pump chamber is surrounded in the vertical direction by a lower circular axially central filtrate centrifugal pump chamber surface and an upper axially central circular filtrate centrifugal pump chamber surface,
The lower filtrate centrifugal pump chamber surface,
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 centrifugal pump chamber surface,
a plurality of angularly spaced, radially extending upper filtrate chamber vanes, the upper filtrate chamber vanes starting from an upper filtrate centripetal pump chamber opening and extending radially outward; characterized device. The method of claim 1, wherein the disposable structure,
With a concentrate discharge tube extending vertically,
The concentrate discharge tube, wherein the upper disk-shaped portion and the outer wall rotate relative to the concentrate discharge tube while the ball rotates;
concentrate centrifugal pump chamber,
A concentrate centrifugal pump, said concentrate centrifugal pump comprising:
axially aligned to the cylindrical core,
located concentrically around the supply tube;
Located above the filtrate centrifugal pump in the vertical direction,
in fluid communication with the concentrate discharge tube;
located within the concentrate centrifugal pump chamber;
the concentrate centrifugal pump chamber is in fluid communication with the separation chamber;
While the ball rotates, the upper disk-shaped part and the outer wall rotate against the concentrate centrifugal pump,
The concentrate centrifugal pump chamber is surrounded in the vertical direction by a lower circular axially centered concentrate centrifugal pump chamber surface and an upper axially centered circular concentrate centrifugal pump chamber surface,
The lower concentrate centrifugal pump chamber surface is,
a plurality of angularly spaced, radially extending lower concentrate chamber vanes, the lower concentrate chamber vanes starting from the lower concentrate centripetal pump chamber opening and extending radially outwardly; include them,
The upper concentrate centrifugal 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 starting from an upper concentrate centripetal pump chamber opening and extending radially outwardly. 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 via a substantially annular filtrate opening;
The annular filtrate dam,
extending within the separation chamber;
extending downwardly below the substantially annular filtrate opening;
and positioned radially outside the substantially annular filtrate opening. The method of claim 1, wherein the disposable structure,
With a concentrate discharge tube extending vertically,
The concentrate discharge tube, wherein the upper disk-shaped portion and the outer wall rotate relative to the concentrate discharge tube while the ball rotates;
concentrate centrifugal pump chamber,
A concentrate centrifugal pump, said concentrate centrifugal pump comprising:
axially aligned to the cylindrical core,
located concentrically around the supply tube;
Located above the filtrate centrifugal pump in the vertical direction,
in fluid communication with the concentrate discharge tube;
located within the concentrate centrifugal pump chamber;
the concentrate centrifugal pump chamber is in fluid communication with the separation chamber through the substantially annular concentrate opening;
While the ball rotates, the upper disk-shaped part and the outer wall rotate against the concentrate 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;
The device of claim 1 , located radially inside the substantially annular concentrate opening. The method of claim 1, wherein the 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 the upper disc-shaped portion and outer wall rotate relative to the concentrate discharge tube while the ball rotates;
concentrate centrifugal pump chamber,
As a concentrate centrifugal pump, the concentrate centrifugal pump comprises:
axially aligned to the cylindrical core,
located concentrically around the supply tube;
Located above the filtrate centrifugal pump in the vertical direction,
in fluid communication with the concentrate discharge tube;
located within the concentrate centrifugal pump chamber;
the concentrate centrifugal pump chamber is in fluid communication with the separation chamber through the substantially annular concentrate opening;
A concentrate centrifugal pump, wherein the upper disc-shaped portion and the outer wall rotate relative to the concentrate 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 radially outside the substantially 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 located substantially radially inside the annular concentrate opening;
A device, characterized in that a radially, annular upwardly extending recess extends 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 source of pressurized air,
the at least one annular gastight seal operably extends in sealing relationship between the upper disc-shaped portion and at least one annular outer wall extending radially outward of at least one of the supply tube and the filtrate discharge tube;
at least one annular seal operates to retain an air pocket within the interior region;
A source of pressurized air is in fluid communication with the air pockets;
Air in the air pockets isolates the at least one seal from the cell culture batch being treated, the air pockets being located radially inside the substantially annular filtrate pump opening. The method of claim 1, wherein the disposable structure,
With at least one annular hermetic seal,
the at least one annular gastight seal operably extends in sealing relationship between the upper disc-shaped portion and at least one annular outer wall extending radially outward of at least one of the supply tube and the filtrate discharge tube;
at least one seal operates to retain an air pocket 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 treated;
a flow back pressure regulator in fluid communication with the filtrate discharge 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 an air pocket located radially inward of the substantially annular filtrate pump opening. The method of claim 1, wherein the disposable structure,
With a concentrate discharge tube extending vertically,
The concentrate discharge tube, wherein the upper disk-shaped portion and the outer wall rotate relative to the concentrate discharge tube while the ball rotates;
concentrate centrifugal pump chamber,
As a concentrate centrifugal pump, the concentrate centrifugal pump comprises:
axially aligned to the cylindrical core,
located concentrically around the supply tube;
Located above the filtrate centrifugal pump in the vertical direction,
in fluid communication with the concentrate discharge tube;
located within the concentrate centrifugal pump chamber;
further comprising a concentrate centrifugal pump in fluid communication with the separation chamber;
The device characterized in that the upper disc-shaped part and the outer wall rotate relative to the concentrate centrifugal pump while the ball rotates. A disposable structure configured for use in a centrifugal separation system comprising a multi-use rotatable centrifugal separation ball, wherein the disposable structure is releasably positioned within the ball and, within an interior region of the disposable structure, cells in a cell culture batch are cell enriched. A disposable structure configured to separate into water and cell filtrate,
the disposable structure is in an operative position extending along a vertical axis;
The disposable structure,
upper part,
lower part,
A cylindrical core in the middle of the upper part and the lower part,
an annular separation chamber radially surrounding the cylindrical core;
With the outer wall, the upper part and the outer wall can rotate in the ball about a vertical axis, the outer wall comprising:
Extends operably liquid-tight with the upper portion and surrounds the separation chamber;
an outer textured surface through which air can be expelled from between the outer wall and the distal ball;
An outer wall that surrounds and extends the cylindrical core,
feeding tube,
filtrate discharge tube;
filtrate pump chamber;
As a filtrate centrifugal pump, the filtrate centrifugal pump comprises:
axially aligned with the cylindrical core,
located coaxially with the supply tube;
in fluid communication with the filtrate discharge tube;
positioned within the filtrate pump chamber, wherein the filtrate pump chamber is in fluid communication with the separation chamber.
a filtrate centrifugal pump;
While the ball rotates, the upper disc-shaped portion and the outer wall rotate relative to each of the supply tube, the filtrate discharge tube and the filtrate centrifugal pump. A disposable structure configured for use in a centrifugal separation system comprising a multi-use rotatable centrifugal separation ball, wherein the disposable structure is releasably positioned within the ball and, within an interior region of the disposable structure, cells in a cell culture batch are cell enriched. A disposable structure configured to separate into water and cell filtrate,
the disposable structure is in an operative position extending along a vertical axis;
The disposable structure,
upper part,
lower part,
A cylindrical core in the middle of the upper part and the lower part,
an annular separation chamber radially surrounding the cylindrical core;
With the outer wall, the upper part and the outer wall can rotate in the ball about a vertical axis, the outer wall comprising:
Extends operably liquid-tight with the upper portion and surrounds the separation chamber;
An outer wall that surrounds and extends the cylindrical core,
supply tube,
filtrate discharge tube;
filtrate pump chamber;
As a filtrate centrifugal pump, the filtrate centrifugal pump comprises:
axially aligned with the cylindrical core,
located coaxially with the supply tube;
in fluid communication with the filtrate discharge tube;
positioned within the filtrate pump chamber, wherein the filtrate pump chamber is in fluid communication with the separation chamber;
The filtrate centrifugal pump chamber is surrounded in the vertical direction by a lower circular axial center filtrate centrifugal pump chamber surface and an upper axial center circular filtrate centrifugal pump chamber surface,
the lower filtrate centripetal pump chamber surface includes a plurality of angularly spaced, radially extending lower filtrate chamber vanes;
the upper filtrate centripetal pump chamber surface includes a plurality of angularly spaced, radially extending upper filtrate chamber vanes;
The device comprising a filtrate centrifugal pump, wherein during rotation of the rotatable centrifugal ball, the upper disk-shaped portion and the outer wall rotate about an axis relative to each of the supply tube, the filtrate discharge tube and the filtrate centrifugal pump. A disposable structure configured for use in a centrifugal separation system comprising a multi-use rotatable centrifugal separation ball, wherein the disposable structure is releasably positioned within the ball and, within an interior region of the disposable structure, cells in a cell culture batch are cell enriched. A disposable structure configured to separate into water and cell filtrate,
the disposable structure is in an operative position extending along a vertical axis;
The disposable structure,
upper part,
lower part,
A cylindrical core in the middle of the upper part and the lower part,
an annular separation chamber radially surrounding the cylindrical core;
With the outer wall, the upper part and the outer wall can rotate in the ball about a vertical axis, the outer wall comprising:
Extends operably liquid-tight with the upper portion and surrounds the separation chamber;
An outer wall that surrounds and extends the cylindrical core,
supply tube,
filtrate discharge tube;
filtrate pump chamber;
As a filtrate centrifugal pump, the filtrate centrifugal pump comprises:
axially aligned with the cylindrical core,
located coaxially with the supply tube;
in fluid communication with the filtrate discharge tube;
positioned within the filtrate pump chamber, wherein the filtrate pump chamber is in fluid communication with the separation chamber;
a filtrate centrifugal pump, wherein the upper portion and outer wall rotate relative to the filtrate centrifugal pump while the ball rotates about its axis;
concentrate centrifugal pump chamber,
As a concentrate centrifugal pump, the concentrate centrifugal pump comprises:
axially aligned with the cylindrical core,
located coaxially with the supply tube;
Located above the filtrate centrifugal pump in the vertical direction,
in fluid communication with the concentrate discharge tube;
positioned within the concentrate centrifugal pump chamber, wherein the concentrate centrifugal pump chamber is in fluid communication with the separation chamber;
While the rotatable centrifugal ball rotates about its axis, the upper part and outer wall rotate about the concentrate centrifugal pump,
The concentrate centrifugal pump chamber is surrounded in the vertical direction by a lower circular axially centered concentrate centrifugal pump chamber surface and an upper axially centered circular concentrate centrifugal pump chamber surface,
the lower concentrate centripetal pump chamber surface includes a plurality of angularly spaced, radially extending lower concentrate chamber vanes;
wherein the upper concentrate centrifugal pump chamber surface comprises a plurality of angularly spaced, radially extending upper concentrate chamber vanes.