WO2010063845A1

WO2010063845A1 - Liquid substance circulation device comprising a compartment for an impeller having a sloped upper portion

Info

Publication number: WO2010063845A1
Application number: PCT/EP2009/066460
Authority: WO
Inventors: Jose Castillo; Jean-Christophe Drugmand
Original assignee: Artelis S.A.
Priority date: 2008-12-05
Filing date: 2009-12-04
Publication date: 2010-06-10

Abstract

A liquid substance circulation device includes a lower portion with an upstanding side wall and an upper portion (3202) associated with the lower portion. The upper portion and the upstanding side wall of the lower portion cooperatively delimit at least a portion of a compartment. An impeller is located within the compartment, The upper portion has at least one axial inlet (3204) to the impeller. The upper portion has a slope sufficient to reduce likelihood of solid-phase settling on the upper portion. At least one outlet (3208) from the compartment is formed in at least one of the upper portion and the upstanding side wall of the lower portion. An exemplary apparatus includes a liquid container and a liquid substance circulation device of the kind described located therein.

Description

LIQUID SUBSTANCE CIRCULATION DEVICE COMPRISING A COMPARTMENT FOR AN IMPELLER HAVING

A SLOPED UPPER PORTION

Cross-Referenee Io Related Applications

5 This PCT patent application ciaims the benefit of, and priority to, United States

Provisional Patent Application Serial No. 61/120,169 filed on December 5, 2008 and entitled "Bioreactor With Features To Enhance Ceil Density And Viability." The disclosure of the aforementioned Provisional Patent Application Serial No. 61/120,169 is expressly incorporated herein by reference in its entirety for all purposes to the full extent 10 permitted by law.

Field of the Invention

The present invention relates generally to the mechanical and biotechno logical arts, and, more particularly, to bioreactors and the like.

15

Background of the Invention

The mixing (and/or suspension) of solutions is required in many technical fields, such as biotechnology, pharmaceuticals, and medical. For example, in the field of the biotechnology and pharmaceutical industry, it is often necessary to prepare and to

20 complement solutions, buffers, culture medium, suspensions, and the like (referred to in genera] terms hereinafter as liquid substances). More specifically, some specific applications of circulation/mixing devices include dilution, dissolution and/or adjusting pH, salinity conditions, concentration, osmolality, and/or temperature of a liquid substance of any kind (buffer, culture medium, saline solution, and so on).

25 To be safe and effective for their intended use, solutions of culture media, buffers, reagents, and the like used in these fields must be pure and sterile. Accordingly, the mixing tank, mixing device, and all other reusable components that contact the solution must be carefully cleaned after use to avoid any cross contamination with subsequent batches of solutions. The cleaning of the structural components is labor-intensive, time-

30 consuming, and costly,

In order to avoid these cleaning steps, single use "mixing bags" have been developed. These are disposable flexible bags provided with a mixing device and

1851-09 PCT - 1 - intended to be supported in a rigid container. Mixing bags are already widespread in the bio-production market (mostly for storage applications). The rigid containers used to handle them are designed to be stacked and fork lifted (which are two requirements for automated stock management). In most cases, the user is already equipped with a large number of standard containers.

Summary of the Invention

Principles of the present invention provide techniques for a bioreactor with features to enhance cell density and viability. In one aspect, an exemplary liquid substance circulation device includes a lower portion with an upstanding side wall; and an upper portion associated with the lower portion. The upper portion and the upstanding side wall of the lower portion cooperatively delimit at least a portion of a compartment. An impeller is located within the compartment. The upper portion has at least one axial inlet to the impeller. The upper portion has a slope sufficient to reduce likelihood of solid-phase settling on the upper portion. At least one outlet from the compartment is formed in at least one of the upper portion and the upstanding side wall of the lower portion.

In another aspect, an exemplary apparatus includes a liquid container and a liquid substance circulation device of the kind described located therein. As used herein, "facilitating" an action includes performing the action, making the action easier, helping to carry the action out, or causing the action to be performed. Thus, by way of example and not limitation, instructions executing on one processor might facilitate an action carried out by instructions executing on a remote processor, by sending appropriate data or commands to cause or aid the action to be performed. These and other features and advantages of the invention will become apparent from the following detailed description of illustrative embodiments thereof, which is to be read in connection with the accompanying drawings.

1851-09 PCT - 2 - Brief Description of the Drawings

FIGS. 1-12 refer to embodiments of a liquid substance circulation device that can be used in a mixing system with several ways of facilitating alignment; for example, a "precise location" or a "free version"; in particular: FIG, IA is a top view of an upper portion of a particularly preferred embodiment of a liquid substance circulation (mixing) device according to an aspect of the invention;

FIG. IB is a bottom view of the upper portion of FIG. IA;

FIG. 2 is a top view of a preferred rotary magnetic element of the liquid substance circulation (mixing) device according to another aspect of the invention; FlG. 3 is a top view of a bearing element of the liquid substance circulation

(mixing) device;

FIG. 4 is a top view of the bottom portion of the liquid substance circulation (mixing) device;

FIG. 5 is an exploded side view of the liquid substance circulation (mixing) device;

FIG. 6 is a top view of an alternative rotary magnetic element for the liquid substance circulation (mixing) device, according to still another aspect of the invention;

FIG. 7 is a view illustrating the liquid substance circulation (mixing) device integrated in a graduated rigid container; FIG. 8 is a perspective view of the liquid substance circulation (mixing) device integrated in a prismatic container, such as a parallelepiped (flexible bag or pouch of rigid container);

FIG. 9 is a view illustrating the liquid substance circulation (mixing) device integrated in a flexible container such as a pouch or a bag; FIG. 10 is a view illustrating the liquid substance circulation (mixing) device integrated in a flexible container such as a pouch or a bag and comprising an internal reservoir;

FIG. 11 is a top view of the upper portion of an alternative embodiment of the liquid substance circulation (mixing) device according to yet another aspect of the invention;

1851-09 PCT - 3 - FIG. 12 is a top view of the upper portion of another alternative embodiment of the liquid substance circulation (mixing) device according to a further aspect of the invention;

FIGS. 13 and 14 show two embodiments of a "free positioning" embodiment; FIGS. 15 and 16 relate to a magnetic impeller with a particular shape;

FIG. 17 relates to an embodiment of the invention where the mixing device has no bottom;

FIG. 18 shows exemplary shapes of some wall portions of the mixing device according to some embodiments of the invention (mixing device with deflector element(s));

FIG. 19 shows mixing devices with axial output instead of radial;

FIGS. 20-24 depict hardware which is compatible with both the "precise location" and the "free location" embodiments; in particular:

FIG. 20 shows a trolley suitable both for the "precise location" version and for the "free aligning" version;

FIG. 21 shows an intermediate frame adapted to work with the trolley;

FIGS. 22 and 23 show the trolley and an intermediate frame mounted on a rigid container;

FIG. 24 shows schematic views of some rigid containers used in the frame; FlG. 25 is a perspective view of a "flat" embodiment of a liquid substance circulation (mixing) device according to a still further aspect of the invention, suitable for use, for example, with a bag having a relatively large volume;

FIG. 26 depicts an exemplary embodiment of a so-called "volcano" bioreactor with features to enhance cell density and viability, according to an additional aspect of the invention;

FIG. 27 depicts an exemplary embodiment of a so-called "crater" bioreactor with features to enhance cell density and viability, according to another additional aspect of the invention;

FIGS. 28 and 29 depict an exemplary embodiment of an ellipsoidal or spherical cap bioreactor with features to enhance cell density and viability, according to yet another additional aspect of the invention;

1851-09 PCT - 4 - FIG. 30 depicts exemplary data for cell density versus time;

FIG. 31 depicts exemplary data for cell viability versus time;

FIG. 32 depicts exemplary plots for cell density versus time;

FIG. 33 depicts exemplary plots for cell viability versus time; FIG. 34 depicts the complete "volcano" bioreactor of FIG. 26 including a lower portion and with a close-fitting mixing vessel;

FIG. 35 depicts the complete "crater" bioreactor of FIG. 27 including a lower portion and with a close-fitting mixing vessel; and

FIG. 36 depicts an alternative embodiment of the "crater" bioreactor, according to still another additional aspect of the invention.

In the drawings, the same reference signs have been allotted to the same or analogous elements, unless otherwise noted.

Detailed Description of Preferred Embodiments In a first aspect, embodiments of the present invention relate to a mixing system including a flexible bag; to a specific flexible bag intended therefore and to specific hardware related thereto.

As noted, the mixing (and/or suspension) of solutions is required in many technical fields such as biotechnology, pharmaceuticals and medical. For example, in the field of the biotechnology and pharmaceutical industry, it is often necessary to prepare and to complement solutions, buffers, culture medium, suspensions, and so on (referred to in general terms hereinafter as liquid substances). More specifically, some specific applications of circulation/mixing devices include dilution, dissolution and/or adjusting pH, salinity conditions, concentration, osmolality, and/or temperature of a liquid substance of any kind (buffer, culture medium, saline solution, and the like).

To be safe and effective for their intended use, solutions of culture media, buffers, reagents, and so on used in these fields must be pure and sterile. Accordingly, the mixing tank, mixing device, and all other reusable components that contact the solution must be carefully cleaned after use to avoid any cross contamination with subsequent batches of solutions. The cleaning of the structural components is labor-intensive, time-consuming, and costly.

1851-09 PCT - 5 - In order to avoid these cleaning steps, single use "mixing bags" have been developed. These are disposable flexible bags provided with a mixing device and intended to be supported in a rigid container. Mixing bags are already widespread in the bio-production market (mostly for storage applications). The rigid containers used to handle them are designed to be stacked and fork lifted, which are two requirements for automated stock management. In most cases, the user is already equipped with a large number of standard containers. Aspects of the invention advantageously provide the user with a flexible bag mixing solution to allow preparation directly in the storage bag,

In these mixing bags, the mixing device preferably is a magnetic stirring device which is driven by an external driver positioned outside of the bag and that may be part of or fixed to the rigid container. By doing so, there is little or no risk of contamination of the contents of the bag from the outside. Even more advantageously, the device may include a magnetic impeller rotating around a fixed rotational axis and not a mere stir bar (so that it can rotate at higher speed with less risk of decoupling from its driver). The motive device or driver and the impeller of the magnetic stirring device can both include permanent magnets designed to have the best coupling and thus the higher torque transmission. Beside the design of the permanent magnets, the alignment is a significant factor for the coupling. While the design of the permanent magnets can be as good as possible, if the magnets of the motive device are not aligned with those of the impeller, torque can not be properly transmitted between both. Certain desirable aspects for improved mixing systems can be summarized as follows, it being understood that all embodiments of the invention do not necessarily exhibit all aspects:

^■ Achieve alignment without specific holes or any type of relieves (plural of relief, i.e., removal of material from a surface or projection of material above a surface) in the rigid container to make the use of containers as standard as possible. Allow the use of a commercial standard magnetic driver without customization.

^■ Achieve compatibility with automated stock management, stacking and forklift.

■ Allow spray sanitization of the system. Materials, as well as design are selected to face hard chemical conditions during sanitization of clean rooms (by using formaldehyde or hydrogen peroxide).

1851-09 PCT - 6 - ^■ Achieve integration of the motive device and control unit in an integrated system.

^■ Allow the driver to be removable from its functional location once the mixing job is finished for short or even long term storage. There is typically no point in storing the motive device with the container and it would require a large number of motive devices.

^■ Prevent tubing from touching the floor underneath the tank: tubing security.

Patent US 5,270,207 discloses circulatory culture equipment. A culture equipment for bio-organisms such as animal cells and plant cells, having substratum and a circulation-inducing chamber, accommodating the material for substratum made of fibrous, or porous, or layered substance, such as of ceramics, plastics, resin or skin having a number of spaces in the substratum for adhesion or immobilization of microorganisms and medium permeation in the lid-containing vessel, and circulating the culture medium by a rotater device in the circulation- generating room is described in the such patent. The gas exchange of the culture medium is performed in the upper part of the vessel in the course of circulation. Growth and maintenance of micro-organism cells in mass and at a high density can be attained.

Patent application US 2005/0002274 discloses a way to align a magnetic impeller located in a bag supported in a rigid container with its driver, the way consisting in providing a locator projection to a rigid portion which is part of the bag (sealed on an opening in the bag) and providing the driver with a matching cavity for receiving the projection. This way of coupling doesn't allow standard equipment (container and driver) to be used since the container must comprise an opening for the projection and since the driver must comprise a cavity or a corresponding relief to match the one of the projection. Patent application WO 2006/002091 also describes a system which allows alignment between a magnetic impeller located in a bag supported in a rigid container and its driver. This system includes a connecting piece (interface) between a locator projection extending from the bag close to the impeller and a part of the driver through an opening in the rigid container. It also includes a wheeled cart guided by rails in order to position the driver relative to the interface. Potential issues of that system are the following:

1851-09 PCT - 7 - ^■ The rigid container has to be opened on its bottom for coupling through it. This container can't be used for other applications, as for example the use of standard storage bags. The motive device might also need a customization. " These containers cannot be stacked on another and in addition their form is not well adapted to be transported by an elevator. There are also very bulky.

^■ No tubing security

^■ Two fixation operations are required: fixing the intermediate piece to the bag and then, fixing the driver to the piece.

At least some embodiments of the invention address at least some of these problems by providing a system where the driver is removable easily so that the bag and its container are storable apart; where the rigid container is standard and hence, compatible with automated stock management, and doesn't require an opening to be made for mechanical coupling between the mixing device and its driver, or at least one part of the bag, the container and/or the driver; and finally, it is a solution which is simple, requires oniy one fixation operation and allows easy integration of tubing security. It is based on the finding that working without mechanical engagement (either direct or indirect using intermediate connecting piece) is possible and even with high precision of alignment of the driver towards the magnetic impeller. It can namely be achieved by either locating both very precisely relative to each other and/or relative to the rigid container or by allowing the mixing device to auto-align itself with the driver.

Accordingly, some embodiments of the present invention relate to a mixing system comprising a flexible bag with a mixing device comprising a magnetic impeller; and an alignment facilitation device adapted to facilitate alignment between the magnetic impeller and a magnetic driver located external to the system. According to a preferred embodiment, the mixing system includes a rigid container in which the flexible bag can be retained, the container having no specific aperture that functions to aid in the coupling between the mixing device and the driver. Preferably, the alignment is not merely accomplished by mechanical engagement or mating between the bag and the external magnetic driver or some connecting portion disposed there between. Instead, it is

1851-09 PCT - 8 - preferably achieved by either locating both very precisely relative to the rigid container or by allowing the mixing device to auto align itself with the driver.

According to some aspects of the invention, the term "flexible bag" designates a bag or pouch made of walls of similar structure preferably assembled by welding. These walis may be made of a mono- or multilayer film including, or not, a barrier layer based on a barrier polymer like EVOH (ethylene vinyl alcohol polymer). Generally, these films may have an inner layer (in contact with the contents of the bag when filled) based on a polyolefin, preferably an ULDPE (ultra-low density polyethylene, preferably medical grade). The bag may be of cylindrical shape although cylindrical flexible bags are not easily baffled and difficult to manufacture. A bag with a cubic or parallel-piped shape is preferable namely because it works as a baffled tank.

The flexible bag according to some embodiments of the present invention is equipped with a magnetic mixing device. Although any kind of mixing device including a magnetic impeller may be used, preferably, it is a device wherein liquid flow is well organized i.e. the liquid enters through specific opening(s) and leaves through other specific oρening(s), the former being located, in some cases, centrally and the latter, in some cases, peripherally.

"Specific" means that a given opening only acts as inlet or outlet opening and not as both. The terms "central" and "peripheral" are to be construed as being complementary i.e. "peripheral" means decentralized.

Preferably, the mixing device includes a rotary magnetic impeller located in a compartment delimited by a wall, the mixing device further comprising at least one liquid inlet opening located in a central area of the wall; at least one liquid outlet opening located in a peripheral area of the wall; and one or more deflector elements that substantially alter the natural rotational direction of the liquid exiting the outlet opening. Normally (without such deflecting), when the liquid exits the compartment of the mixing device, its flow path is tangential to the wall of the compartment at the outlet point. The deflector element(s) of this aspect of the invention act to shift this liquid outflow path from a given angle after the liquid has impacted on the deflector element(s) so that the flow path tends to become more radial (i.e., the flow direction when the liquid leaves the deflectors tends to become more parallel to the radial direction at the outlet point).

1851-09 PCT - 9 - 9 066460

This angle shift is preferably of at least 5°, more preferably of at least 10° and even more preferably of at least 20°. It preferably allows directing the liquid outflow in any preferred direction; for instance the corners of the parallelepiped-shaped containers in general and flexible bags in particular. These corners are well known to persons of ordinary skill in the art as being dead zones in a mixing process. By this angle shift at each of the deflectors, the global flow pattern created by the mixer within its action area is changed from a circular path to several divergent jets, the number of jets being equal to the number of outlets.

This directional change of the velocity vector occurs directly when the flow leaves (is no longer in contact with) the deflectors. This permits mixing of the substance away from the mixing device and throughout the bag in a more efficient manner. It allows avoiding dead zones, for instance, in the corners of the container (bag) to be mixed.

In the mixing device, there are preferably several outlet openings, the number of which depends in fact on the size and shape of the container it is designed for. These outlets are preferably located on a side portion of the wall of the mixer. They are preferably all identical. Advantageously, they are all equipped with deflector elements and even more preferably, with identical deflector elements so that the flow pattern is symmetrical. The terms "equipped with" mean in fact that there are some deflector elements somewhere in the flow leaving the outlet opening but not necessarily directly at the opening.

The wall of the mixing device may be a continuous surface without angles like a bell which would be fixed to or lie on a bag wall by the open end thereof. In that embodiment, the rotational axis of the magnetic impeller may be fixed to the top of the bell so that there is no bottom surface needed,

Alternatively, the wall may be a kind of box comprising different walls defining angles between each other and/or comprising a cover and side walls which may be welded and/or otherwise assembled to each other (for instance by a snap fit system). It may also comprise a bottom surface although this is not mandatory if the rotational axis of the impeller is fixed to the cover (top of the wall).

1851-09 PCT - 10 - 60

Hence, in a preferred embodiment, the mixing device includes a wall delimiting a compartment with a top surface where the magnetic impeller is located, the wall comprising no bottom surface and having a rotational axis for the impeller fixed to its top surface. It is worth noting that although the above embodiment (of a mixing device without bottom surface and having its impeller fixed at the top surface) has been described in the frame of the present invention, it is advantageous in any magnetic mixing system driven from outside for the reason mentioned below (maximum torque transmission because the driver can be put closer to the impeller). In another preferred embodiment, which may be combined with the former one, the mixing device includes sidewall portions with or forming one or more outlets, the side wall portions preferably including deflector portions positioned and shaped to deflect liquid substance as it exits the outlets. The wall (portions) of the mixing device are preferably made of plastic and even more preferably, made of a plastic compatible with the contents of the bag and their use (e.g., a medical grade of a chemically resistant polymer like HDPE). Preferably, this plastic is rigid in order to allow pressure build up without deformation. If the mixing device is to be welded to the bag, the parts of the wall(s) that must be welded on it are preferably of a material compatible with it.

Considering the layout of the industrial films set forth above, a polymer of ethylene is a good choice. Preferably, a HDPE (high density polyethylene) is used because other polymers like EVA (Ethylene Vinyl Acetate copolymer) or LDPE are not rigid enough. The magnetic impeller in the mixing device according to the invention is a part comprising magnet(s) and which rotates in a compartment around a central axis which is either fixed on/part of the bottom of the compartment (in the case it has one) or of the top thereof. This axis may comprise two separate pins extending from a ring (as disclosed in US 4,162,855, the content of which is expressly incorporated by reference herein in its entirety for all purposes, to the extent permitted by law). Alternatively, it may be a single axis passing through a hole in the rotary magnetic element.

This impeller may merely be a stir bar (so an impeller with two straight arms only) or it may comprise at least three arms which may be straight or curved (although this may not allow rotation in both directions) or may comprise curved portions. They

1851-09 PCT - 11 - may also have sections in the shape of parallelograms. It may also be in the shape of a cross (or plus sign) or of a disc in which magnet(s) are embedded symmetrically versus the rotation axis. This disc preferably wears an upper relief in the form of paddles which are preferably straight (not curved) so that the disc can rotate in both directions with identical performance. In fact, the invention applies to all kinds of impeller geometries.

Whatever its shape, the decoupling of the impeller from its rotational axis (at high rotation speed or during shipping and handling) can be avoided by providing an adequate securing member on the axis. The securing member may simply be an adequate relief of the end of the axis (such as a cross shaped portion for instance, the axis being inserted in the hole of the element by its other extremity). The securing member may also be a plug (blocking plate with fixation element) fixed on top of the axis after the axis has been inserted into the hole of the rotary element.

The rotational axis preferably comprises a protrusion from the bottom of the mixing device or of the bag (if the mixing device has no bottom) or from the top of the mixer_s the protrusion being equipped with a bearing which fits in a corresponding hole of the rotary magnetic element. In that embodiment, the protrusion preferably comprises embossings (generally in the number of three or six, six being preferred) or has the shape of a cross to provide a tension plugging and a perfect fitting between protrusion and bearing and to block rotation of the bearing. In order not to lose magnetic forces and hence, torque transmission, the magnetic impeller is preferably located as close as possible relative to the bottom of the bag without touching it however (to avoid friction). Hence, it is preferably positioned at less than 10 mm of the bag bottom. In that regard, the embodiments described earlier where the wall of the mixing device has no bottom surface are advantageous because it suppresses the bottom wall of the mixing device, which may lead to a gain of up to 2 mm. In a preferred embodiment of the invention, the flexible bag comprises at least two facing walls and the mixing device is located inside of it on one of the walls, the other facing wall comprising a protective cover having a cavity matching the external shape of the mixing device. This cover is preferably fixed (preferably by welding) on an opening of the wall.

1851-09 PCT - 12 - The term "matching" means in fact that the mixing device fits (can be inserted) inside the cover and can be removed therefrom i.e, that the mixing device has smaller dimensions and an adequate shape to be able to be received (preferably completely) inside the cover. This embodiment prevents the bag from being damaged owed to the presence of a rigid mixing device inside of it. It also allows the mixing device to be kept in place during shipping and handling when the mixing device is not fixed to the bag (see above).

The mixing system of some embodiments of the invention generally comprises a rigid container which retains (supports) the flexible bag. This container may be of any shape provided the bag fits therein (i.e. can lie on its bottom and press on its side walls when it is filled with liquid). Preferably, it is a container with at least five plane walls (one bottom and four side walls) which may have openings. Preferably, the inner size of the container matches substantially the outer size of the flexible bag. "Substantially in this context means close enough such that hydrostatic pressure can be supported by its walls. It is preferably made of a metal like stainless steei, stainless steel 316 being particularly suitable. Nevertheless it could be made of plastics as well.

Generally, the container comprises a bottom plate to which four side walls are assembled. Generally, bottom plate and side walls are sheets of about less than 5 mm thickness (the thickness being adapted to the size and hence, volume of the container). These walls may be a single sheet folded in a cubic or parallelepiped shape. They advantageously have a folded back portion at their top in order to increase the resistance in torsion of the assembly. The bottom plate may be in the shape of a frame on which an additional sheet will rest to from the bottom of the container. Hence, the container generally has a beam extending downwards at its periphery. According to some aspects of the invention, the magnetic driver used to induce rotation of the magnetic impeller of the system is located outside the system, generally below it (i.e. below the bottom of the rigid container if there is one). Any kind of commercial magnetic driver can be used provided it has enough torque transmission (preferably up to 1 Newton-meter) and speed possibilities (preferably up to 1500 rpm). Its size and shape are preferably such that it can fit underneath the container without modifying it or the container. This driver may be fixed to, or part of, the system.

1851-09 PCT - 13 - However, as explained above, it preferably is removable therefrom so that the system can be stored separately. "Removable" means that it is not permanently fixed on it but on the contrary: that it is positioned there in a reversible way so that it can be removed from the container and put back again as many times as required and this very easily, without substantial effort and without damaging anything.

According to some aspects of the invention, the container and the bag have no hole or other relief matching at least part of the driver or of a connecting piece between the driver and the mixing device. In other words: there is no mechanical engagement whatsoever between the driver and the mixing device. By doing so, there is no need to provide the bag and the container with an opening or other relief for communication purposes, no need to manufacture an additional connecting piece so that the container is standard and can be used for any purpose.

In a preferred embodiment, which is quite simple, the mixing device is merely lying and/or fixed on the bottom of the bag which merely rests on a plane part of the container. Hence, in that embodiment, a portion of the flexible bag is sandwiched between at least a portion of the mixing device and a portion of the rigid container, these portions being generally part of the bottom wall of the bag and of the rigid container respectively. "Sandwiched" means that the portions are directly in contact and pressed against each other by gravity (if the mixing device rests on the bottom of the mixing bag and the container) and/or by any other techniques which preferably do not perforate the bag and the container (additional magnets for instance).

According to the first embodiment of the invention, the mixing device and the driver are located very precisely relative to the rigid container. In that embodiment, preferably, the mixing device is secured to the bag; and the alignment facilitating device comprises structure for positioning the driver at a precise location relative to the device. Preferably, the mixing device is securely fixed to one of the bag's walls, preferably to its bottom wall or a side wall, preferably in a place close to the bottom. It may be fixed there by welding or by mechanical coupling (welding, clipping, and so on) with drain(s) or other part(s) fixed into the bag. It is preferably welded to the flexible bag by at least part of its bottom (if it has one) or by a "basement" disc if it has no bottom (preferred embodiment described earlier). This basement disc can merely be a prolongation of the

1851-09 PCT - 14 - wall of the mixing device. The welding of the mixing device to the bag (the case being) is preferably performed using common industrial devices like heat impulse welding devices which work by pressing the layers to be welded together against each other using heating stamps. The level of pressure and pressure uniformity are relevant for obtaining a good sealing. The welding temperature and the width of the welding zone are also relevant thereto. Accordingly, all these parameters should be optimized for each practical case. It is worth noting that the same welding technique is generally used to manufacture the bag by assembling the above mentioned films (walls of the bag) and to weld the mixing device therein. Alternatively to welding, the precise positioning may be obtained through at least one of the following: magnets in extension arms susceptible to work with corresponding magnets disposed in the rigid container; and at least two extension arms welded (or mechanically fixed in any other way) to at least two drains or two other rigid components of the bag, In this "precise location" embodiment, the flexible bag is received in the container in a way such that the mixing device always occupies the same location relative to it, which means that its center should be aligned with a given point of the container with preferably only up to 1 mm possible offset. This point generally is near or at the center of the bottom of the container.

This precise location can be achieved by adapting the tolerances of the outer size of the bag and the inner size of the container and/or by fixing the bag by at least one of its accessories (for instance a drain) at a fixed location of the container. Inserting at least two of the bag drains through two matching holes in the container and securing them there gives good results. In fact, all the rigid parts present on the bag could be used to attach the bag precisely into the container. Hence, instead of drains, sampling ports, inlet/outlet ports, the external tubing of a sparger system, and the like, could be used.

In this embodiment, the driver should also be located very precisely relative to the rigid container, which means that it should also always occupy the same location relative to it, with the same definition as above. In order not to lose magnetic forces and hence, torque transmission, the driver is preferably at least partially in contact with the rigid container, preferably at least in the zone where the magnet(s) are located.

1851-09 PCT - 15 - 009/066460

The precise location of the driver relative to the rigid container can be achieved by making this driver specific to the container; i.e., non removable. However, as explained above, in order to limit the number of drivers (expensive parts) for a given park of containers, the driver is preferably removable. Accordingly, the container is preferably provided (directly or indirectly, via an intermediate part like a frame for instance) with guiding members insuring that the driver will be located at the same place each time it will be fixed on the container. Depending on the kind of guiding members and their location, the driver couid be fixed directly (through direct manual action) on the container or the intermediate part, or it could be assisted by a trolley, roller plate or the like. Guiding members allowing direct manual fixation would be for instance any frame, open compartment or other support fixed underneath the container (or on a part where the container is resting like a roller plate or simply: the floor), which is within the reach of an operator and in which the driver can be firmly secured at a precise location. To that end, clips, locking castors or pins cooperating with matching holes, or the like could be used,

They could also include four fixation parts able to receive and fix the arms of a shaft bearing the driver and which an operator could slide underneath the container until the end of the arms are in their fixation part. He could even eventually manually assist the fixation if required (for instance by inserting a pin through matching holes). Finally, they could include a kind of drawer underneath the bottom of the container, the drawer comprising blocking member(s) to keep the driver at a given location therein. Provided the drawer is completed closed by the operator, the driver inside of it can be located very precisely relative to the container in that way too.

Alternatively to direct manual fixation, a trolley, roller plate or the like (which will be called "trolley" further on for sake of simplicity) may be used in conjunction with a guide to move the trolley and bring the driver at its precise location. There, the driver could be fixed automatically (for instance by using locking castors) or it could be fixed manually (if the fixation points are within the reach of an operator). Also, the driver could be fixed alone (or on a support) on the container and the trolley removed thereafter. Alternatively, the trolley could remain on the container and help fixing the driver there. In a first embodiment, the guiding members for the trolley are directly fixed to the rigid

1851-09 PCT - 16 - container. They may comprise at least one tail in which the trolley is first engaged and then moved (for instance using a wheeled cart or trolley) to a precise location where it will be fixed (like in the above cited WO 2006/002091 application). This rail may be able to cooperate with at least one roller or wheel (preferably at least two) located at one end of the trolley. The other end of the trolley may be provided with a cross member (or any other hanging member) allowing to fix the trolley (with the driver on it) to the container.

In a second embodiment, the guiding member(s) for the trolley can be part of an intermediate support structure on which the container rests. This intermediate support structure may be a roller plate used to move the container around; it may also be an intermediate frame inserted in between the roller plate and the container. The latter is preferred because it allows using existing containers and their roller plates. This embodiment also may use rail(s)) which is/are then integrated to the intermediate support. This support preferably has fixations (like hollow upstanding extensions) compatible with the feet of standard containers (so that the container may merely be rested on the frame by its feet). Tt may also comprise a chamber or other protection shield for tubing security (i.e. for preventing tubing extending from the bag from touching the floor underneath the tank). As described in the above mentioned prior art document (WO 2006/002091), the rail(s) mentioned above may be straight. However, if the bottom of the container comprises a small beam extending downwards at its periphery (i.e, if the bottom is supported on a frame), it would be required to first roll the trolley underneath the fixation location and then, to completely lift the driver before fixing it. Hence, preferably, the rail(s) is/are inclined in which case the trolley will be lifted progressively while its wheel(s) are climbing the rail(s). When it reaches the end of the raii(s), its lower extremity can be lifted up for bringing the driver in contact with the container bottom. In that embodiment, the trolley is preferably equipped on one end with at least one wheel (preferably 2 wheels) resting on the floor when the trolley is not hung up on the container; somewhere in between both ends, with at least one foldable foot bearing a wheel which rests on the floor when the trolley is disengaged from the rails and which folds back when the trolley is engaged with the container and starts climbing the rail(s); and on the other end, with at least one wheel (preferably two wheels) which roll(s) on the rail(s) when the foldable foot is folded back. Once the driver has been correctly located or

1851-09 PCT - 17 - preferably, at the end of the location process, it is being fixed to the container or to the intermediate frame by any suitable members. These members may comprise locking castors located at one end of the rail and able to cooperate with corresponding holes in the trolley in order to automatically fix the trolley at the end of the rails when it arrives at that location. In this embodiment, the trolley is preferably equipped with a hanging device (for instance, hooks matching with the shape of the beam of the container) so that when the operator feels that the locking castors have engaged with the holes, he merely has to engage these members.

For solving the problem of going beneath a peripheral beam of the container, an alternative to the inclined rail could be to provide the trolley and the container with members allowing the driver and/or at least part of the trolley (the one bearing the driver) to move up and down so that it can retract (get closer to the floor) while passing the beam and lift up again after having passed the beam. These members might be electrical (like a small elevator for instance) or purely mechanical (like dashpot(s) or spring(s) on the driver and/or trolley cooperating with a deflector on the container).

Another alternative for solving the "beam" problem could be to remove a part from the frame so that it acquires approximately the shape of a "U" and that the trolley can be rolled underneath the container by the open end of the "U". All these solution may of course be used in combination. A feature which is preferably present in all the above mentioned embodiments is the fact of putting the driver on a support provided with members allowing the driver to be pushed against the bottom of the container while allowing it to move a little vertically in order to be able to address the problem of the deformation of the bottom owed to fatigue and of other reasons for non flatness of the bottom (for instance if it is angled or convex). Dashpots, springs and the like can be used for that purpose. Alternatively, the support itself may be flexible (either by its nature (if it is made of rubber for instance) and/or by its dimensions).

Hence, the present application also concerns a locating system as described above and allowing the location of a driver below a rigid container, as close as possible to its bottom and in a place where the container has no hole or other relief matching at least part of the driver or of a connecting piece between the driver and the mixing device, the

1851-09 PCT - 18 - P2009/066460

bottom being not necessarily flat. This locating system comprises member(s) for guiding the driver to the location and for fixing it there, together with a support allowing to fix the driver in the location, the support comprising member(s) for pushing the driver against the bottom of the container in a place where the container has no hole or other relief matching at least part of the driver or of a connecting piece between the driver and the mixing device. In one embodiment, the support of the driver comprises member(s) allowing it to move a little vertically relatively to the container so that non fiat bottoms can be dealt with.

In another embodiment, to solve the same problem, the support itself is flexible. In yet another embodiment, the locating system comprises at least one inclined rail adapted to be fixed to a standard rigid container (directly or through an intermediate support) and a trolley suitable to support a magnetic driver and to be engaged at least partly under the container when bearing the driver, the trolley comprising a body with two ends suitable for supporting the driver; at one end of the body, at least one wheel intended to roll on the floor; at least one foldable foot bearing a wheel which rolls on the floor when the trolley is disengaged from the container and which folds back when the trolley is engaged with the container; and at the other end of the body, at least one wheel which never contacts the floor but which rolls on the rail when the trolley is engaged with the container. At the end close to the wheel rolling on the floor, the trolley preferably comprises a handle in order to be able to move it easily. At the same end, it also preferably comprises hanging member(s) for hanging the end to the container in order to put and keep the driver at its right location, pushed against the bottom of the container. These hanging member(s) may have the shape of hooks collaborating with springs. Preferably, the inclined rail is part of an intermediate frame as described above.

The materials used for all the above described equipment are preferably able to withstand sanitization and the wheels of the trolleys are preferably "pharma" compliant. The trolleys preferably include the control unit of the driver and all electrical wiring thereof. All metals preferably are submitted to passivation in order to obtain the surface smoothness required (and the absence of staining plus ease of cleaning associated). In the second embodiment mentioned above, rather than fixing and/or securing the mixing

1851-09 PCT - 19 - 66460

device to the bag tightly and fixing the driver firmly to the container (or to an intermediate support structure fixed to it), the mixing device and the driver are free to auto align themselves through the magnetic forces they exert on each other. This can be achieved by giving at least one of these devices a degree of freedom, Le, , allowing it to make small displacements in at least one direction of space (but preferably two, or even three) so that it can move through the magnetic forces between both elements and align itself with the other one. Preferably, it is the mixing device which is allowed to move.

In order to allow the mixing device to move, the flexible bag may comprise a "free" positioning mechanism to position and maintain the mixing device only approximately at a given location (i.e. the device is not secured to its walls but free to move a little relative to it) when the bag is inserted inside the rigid container (but without the mixing device being coupled with the driver, of course, because then, the location is precise through self alignment). This "free" positioning mechanism may include welding tabs, bridges or any other fixation part(s) (like a double annular wall for instance) fixed on the bag wherein the mixing device is retained but can move freely (relative to the bag) in at least one direction of space (and preferably, in the three directions of space) while remaining in a given perimeter (surrounding the driver when the bag is inserted in the rigid container). Preferably, in this embodiment, the alignment facilitating device comprises member(s) for allowing the mixing device to move relative to the bag while remaining in a given perimeter thereof so that when the bag is located proximate to the driver, the mixing device will align itself with the driver.

This is advantageous because the mixing device will align itself automatically with its driver (through the magnetic forces they both exert on each other) so that there is no need for a perfect location of the driver on the rigid container. This embodiment is also advantageous because there is no need for the welding of a (potentially) large circumferential part, what present quality issues as far as leakages are concerned. This embodiment allows decreasing constraints on the manufacturing of both the flexible bag, by avoiding all the different parts needed for the precise relative positioning of the turbine inside the bag, and the stainless steel parts, mainly by avoiding precision on the relative positioning of the magnetic driver on the trolley, and on the relative positioning of the trolley versus the container and this for the several designs of container - driver -

1851-09 PCT - 20 - trolley - intermediate frame (or not) disclosed above for the "precise location" version. Hence, the same hardware (driver locating system) may be used for both solutions although precision and/or tolerances may be a little less constraining in the case of the "free" version. In a second aspect, embodiments of the present invention concern a flexible bag equipped with a liquid substance circulation device, a particular device of that kind, and a liquid substance circulation system.

The mixing (and/or suspension) of solutions is required in many technical fields such as biotechnology, pharmaceuticals, and medical. Liquid substance circulation devices are particularly useful for the mixing of one or more liquid, gaseous or solid substances in the presence of a liquid substance. There exists a requirement in industry and in research units for a liquid substance circulation device which is effective and inexpensive. This is because all the existing devices have various drawbacks as explained in more detail below. For example, in the field of the biotechnology industry, it is often necessary to prepare and to complement solutions, buffers, culture medium, suspensions, and the like (referred to in general terms hereinafter as liquid substances). More specifically, in the field of biotechnology, some specific applications of circulation devices include dilution, dissolution and/or adjusting pH, salinity conditions, concentration, osmolality, and/or temperature, of a liquid substance of any kind (buffer, culture medium, saline solution, and so on).

Many devices are known for circulating a liquid substance ranging from the simple magnetic stir bar designed to stir a solution in a beaker, for example when preparing the solution, to appreciably more sophisticated devices involving the circulation of the liquid substance by an industrial pump, and the like. To be safe and effective for their intended use, solutions of culture media, buffers, reagents, and so on used in these fields must be pure and sterile. Accordingly, the mixing tank, mixing device, and all other reusable components that contact the solution must be carefully cleaned after use to avoid any cross contamination with subsequent batches of solutions. The cleaning of the structural components is labor-intensive, time-consuming, and costly. In order to avoid these cleaning steps, single use "mixing bags" have been developed. These are disposable flexible bags provided with a stirrer and intended to be

1851-09 PCT - 21 - supported in a rigid container. Advantageously, as disclosed in US Patent 3,647,397, the stirrer can be a magnetic stirrer (and more particularly a magnetic stir bar) and is driven by an external driver positioned outside of the bag and may be part of or fixed to the rigid container. By doing so, there is little or no risk of contamination of the contents of the bag from the outside. However, because the magnetic stir bar is simply disposed on a bottom surface of the bag, it has the propensity to decouple and, perhaps, fly off from the magnetic hold of the external driver, particularly at high rotation rates, and it also has the tendency to wear off the bottom surface by friction.

Several inventions have attempted to address this issue. For example, US Patent 7,153,021 discloses one such container system with a mixing dish assembly that holds a magnetic stir bar. The magnetic stir bar, when engaged with an external drive, rotates in the mixing dish which is welded to the edge of an opening in the bottom of the flexible bag. The mixing dish, with its "substantially frustoconical side wall" and its welded position below the bottom surface of the bag acts to maintain the stir bar in position. The mixing dish is preferably disclosed as being provided with a retention plate for the stir bar and comprising several openings for putting the inside of the mixing dish in liquid communication with the inside of the bag.

Another attempt to address the issue of securing a magnetic stir bar at the bottom of a flexible container is disclosed in US 2004/0062140. This publication discloses a "rod containment disk" that is disposed on the bottom of a flexible container to contain a magnetic rod or stir bar and prevent its exiting the magnetic field formed by the external driver. The rod containment disk includes an upper ring and a lower ring connected together with bolts and including spacer posts to allow enough room for the stir bar to rotate. While such prior art devices succeed in some manner to retain the stir bar, they have limited mixing efficiency due to the resulting undirected and non-uniform liquid flow in the bag. Even if the mixing efficiency of such systems is somewhat increased due to the presence of the retention devices which allow higher bar rotation speeds than classical systems, such systems bring about a turbulent agitation of the liquid substance. The turbulent agitation of the liquid substance creates rotation of the liquid substance, thereby creating several dead zones.

1851-09 PCT - 22 - Because, in the first prior art publication, the retention plate comprises simple drain-like openings (holes) across most of its surface and, in the second prior art publication, the openings formed in the upper ring and between the spacer posts are large compared with the surface area of the containment disk material, fluid exchange between the dish and/or rod containment disk cavity and the inside of the bag is disorganized and random, the openings allowing the liquid to flow in both directions depending upon the mixing conditions.

Indeed, for the preparation of a liquid substance, when the volume is large, a magnetic stir bar alone does not afford homogenization of the solution, because, as from a certain volume, it is generally considered that the magnetic bar puts the liquid substance in rotation without homogenizing it. Even in smaller volumes, devices that use magnetic agitators tend to put the substance into rotation rather than distributing the substances to be mixed in a manner that achieves uniform, quick and efficient mixing. Such reduced homogenization leads generally to unmixed zones and to inaccurate conditions within the liquid substance, since a parameter measured over time does not really reflect the exact value of the parameter of the prepared liquid substance.

Some embodiments of the invention address at least some of these drawbacks by providing a flexible mixing bag with a liquid substance circulation (hereinafter "mixing") device that provides uniform and efficient circulation (mixing) of the liquid substance by avoiding global rotation of the substance and by reducing the presence of dead zones when mixing one or more liquid substances and/or one or more solid substances in the presence of a liquid substance. One or more embodiments also provide a mixing device, adaptable to any type of containers designed to contain a liquid substance to be homogenized, maintained in suspension, prepared or even for culturing cells or microorganisms, not requiring any investment with regard to equipment and which is inexpensive.

The bag and device are also preferably inexpensive, affording a considerable saving in time during the preparation of the liquid substance, homogenization, maintaining is suspension. The bag and device can therefore be considered as being designed to be disposable if it is so wished in order to avoid any cross contamination. Moreover, the liquid substance circulation (mixing) device preferably ensures a real

1851-09 PCT - 23 - mixing by circulating and homogenizing the liquid substance and does not bring the liquid substance in turbulent movement or in rotation which would unavoidably result in several dead zones.

Aspects of the invention provide a flexible bag equipped with a very efficient liquid substance circulation (mixing) device. This liquid substance circulation (mixing) device is preferably based on the principal of a centrifugal pump (i.e. with liquid flow organization) which can be magnetically driven from the outside (with a magnetic driver) and which is able to accumulate (build up) sufficient pressure in order to maximize the action area of the device. Accordingly, some embodiments of the present invention relate to a flexible mixing bag equipped with a liquid substance circulation (mixing) device comprising a rotary magnetic element located in a compartment delimited by a wall, the mixing device further comprising at least one liquid inlet located in the central area of the wall; and at least one liquid outlet located in a peripheral area of the wall.

The terms "flexible bag" designate a bag or pouch made of walls of similar structure preferably assembled by welding. These walls may be made of a mono- or multilayer film including a barrier layer based on a barrier polymer like EVOH (ethylene vinyl alcohol polymer). Generally, these films may have an inner layer (in contact with the contents of the bag when filled) based on a polyolefin, preferably an ULDPE (ultra- low density polyethylene, preferably medical grade). The bag may be of cylindrical shape although cylindrical flexible bags are not easily baffled and difficult to manufacture. A bag with a cubic or parallel-piped shape is preferable namely because it works as a baffled tank. The flexible bag is preferably equipped with a liquid substance circulation (mixing) device and is therefore referred to as a mixing bag.

The mixing device preferably includes sidewall portions with or forming one or more outlets, the side wall portions including deflector portions positioned and shaped to deflect liquid substance as it exits the outlets.

The terms "equipped with a mixing device" mean that the bag contains a mixing device inside the space defined by its walls or communicating with the space. This mixing device may be fixed to one of its walls, preferably to the bottom wall or a side wall, preferably in a place close to the bottom. It may be fixed there by welding or by mechanical coupling (welding, clipping, and the like) with drain(s) or other part(s) fixed

1851-09 PCT - 24 - into the bag. It may for instance be fixed by welding its periphery (or a specific flange provided therefor) to the periphery of an opening into the bag.

When the mixing device is welded on an opening of the bag, its bottom may be provided with at least one portion in relief (generally a cavity) matching a corresponding portion in relief (generally a protrusion) on a driver (generally part of and/or fixed on a rigid container supporting the bag, the driver being a magnetic one capable of driving the rotary magnetic element) or on a rigid container used to handle the flexible bag. These complementary portions in relief enable to align mixing device and driver very easily. It is preferable that the rotary magnetic element is well located upon the driver for optimum torque transmission, avoiding decoupling and avoiding wear concentration (good balance of the element around its rotation axis).

The welding of the mixing device to the bag (the case being) is preferably performed using common industrial devices like heat impulse welding devices which work by pressing the layers to be welded together against each other using heating stamps. The level of pressure and pressure uniformity are relevant for obtaining a good sealing. The welding temperature and the width of the welding zone are also relevant thereto. Accordingly, all these parameters should preferably be optimized for each practical case. It is worth noting that the same welding technique is generally used to manufacture the bag by assembling the above mentioned films (walls of the bag) and to weld the mixing device therein. In an alternative embodiment, rather than the mixing device being fixed/secured to the tank walls, the bag may comprise a positioning mechanism to position and maintain the mixing device at a given location when the bag is inserted inside a rigid container equipped with a magnetic driver able to drive the rotary magnetic element, the mixing device being merely located inside the bag without being secured to its walls. The positioning mechanism mentioned above may be at least one of the following: magnets in extension arms susceptible to work with corresponding magnets disposed in the rigid container; at least two extension arms welded (or mechanically fixed in any other way) to at least two drains or two other rigid components of the bag; and/or welding tabs, bridges or any other fixation part (like a double annular wall for instance) fixed on the bag wherein the mixing device is retained but can move freely (relative to the bag) in at least one direction of space (and preferably, in the three

1851-09 PCT - 25 - directions of space) while remaining in a given perimeter (surrounding the driver when the bag is inserted in the rigid container). This embodiment is advantageous because the mixing device will align itself automatically with its driver (through the magnetic forces they both exert on each other) so that there is no need for a perfect location of the driver on the rigid container and such that a single, non sophisticated driver can easily be used for several rigid containers. This embodiment is also advantageous because there is no need for the welding of a (potentially) large circumferential part, what present quality issues as far as leakages are concerned.

According to one or more embodiments of the invention, the mixing device comprises a compartment delimited by a wall. This wall may be a continuous surface without angles like a bell which would be fixed to or lie on a bag wall by the open end thereof. It could also be maintained in tabs, bridges or inside a double annular wall allowing it to move freely a few millimeters in at least one direction of space (as explained above). In that embodiment, the rotational axis of the magnetic element (if there is one: see below) may be fixed to the top of the bell so that there is no bottom surface needed. Alternatively, the wall may be a kind of box comprising different walls defining angles between each other and/or comprising a cover and side walls which may be welded and/or otherwise assembled to each other (for instance by a snap fit system). It may also comprise a bottom surface although this is not mandatory if there is no rotation axis for the rotary element or if the axis is fixed to the cover (top of the wall).

In any event, the surface (wall) being continuous or not, rounded or with angles, it is preferred that the height (H) and the diameter (D) of the compartment (provided it has a circular section, which is preferred) is such that it has an H/D ratio of less than 0.5 and even more preferably of less than 0.3. If the compartment comprises a dome (or raised ceiling), mixing will be less efficient due to the lessened pressure build up capacity.

In a preferred embodiment, the height of the compartment leaves enough space in height for the rotating element to rotate freely but not too much space however in order to allow sufficient pressure build up (which will be explained later on). It is also worth noting that having all height occupied by the rotating element could also increase the pressure too much and increase risk of leakage (in the case of a welded mixer) and decoupling of the rotary element, A good compromise is obtained when the rotary magnetic element

1851-09 PCT - 26 - occupies at least one quarter of the height between the bottom and the top the compartment (substantially in the center of both), preferably at least one third of the height. Preferably, it does not occupy more than 90% of the height and even more preferably, not more than 75% thereof. Another design factor that has an influence on the performance of the mixing device is the ratio between the rotary element length and the compartment diameter. The ratio is preferably at least 0.5 and preferably at least 0.75 and even at least 0.90. The wall(s) of the mixing device are preferably made of plastic and even more preferably, made of a plastic compatible with the contents of the bag and their use (e.g., a medical grade of a chemically resistant polymer like HDPE). Preferably, this plastic is rigid in order to allow pressure build up without deformation. If the mixing device is to be welded to the bag, the parts of the wall(s) that must be welded on it are preferably of a material compatible with it. Considering the layout of the industrial films set forth above, a polymer of ethylene is a good choice. Preferably, a HDPE (high density polyethylene) is used because other polymers like EVA (Ethylene Vinyl Acetate copolymer) or LDPE are not rigid enough.

The rotary magnetic element comprised in the mixing device may be a simple magnetic stir bar rotating on the bottom of the compartment (if it has one, or on the bottom of the bag in the case of a bell embodiment for instance). However, preferably (in order to avoid friction and hence, provide optimum torque transmission between the element and its driver), this element is a magnetic impeller that rotates around a central axis which is either fixed on and/or part of the bottom of the compartment (in the case it has one) or of the top thereof. This axis may comprise two separate pins extending from a ring (as disclosed in US 4,162,855, the content of which is expressly incorporated by reference herein in its entirety for all purposes to the extent permitted by law). Alternatively, it may be a single axis passing through a hole in the rotary magnetic element. More details on this variant are given below, in relation with a specific embodiment of the invention. These details apply to at least some other aspects the invention as well. Instead of being in the shape of a bar, the rotary magnetic element may have the shape of an impeller having at least three arms which may be straight or curved (although

1851-09 PCT - 27 - this may not allow rotation in both directions) or may comprise curved portions. They may also have sections in the shape of parallelograms. The rotary magnetic element may also be in the shape of a cross (or plus sign). The rotary magnetic element may also have the shape of a disc in which magnet(s) are embedded symmetrically versus the rotation axis. This disc preferably wears an upper relief in the form of paddles which are preferably straight (not curved) so that the disc can rotate in both directions. Whatever the shape of the rotary magnetic element, its decoupling from its rotation axis (at high rotation speed or during shipping and handling) can be avoided by providing adequate securing member(s) on the axis. The securing member(s) may be an adequate relief of the end of the axis (such as a cross shaped portion for instance, the axis being inserted in the hole of the element by its other extremity). The securing member(s) may also be a plug (blocking plate with fixation element) fixed on top of the axis after the axis has been inserted into the hole of the rotary element.

As will be described in more details later on, if the rotational axis is a physical one, it preferably comprises a protrusion from the bottom of the mixing device or of the bag (if the mixing device has no bottom) or from the top of the mixer, the protrusion being equipped with a bearing which fits in a corresponding hole of the rotary magnetic element. In that embodiment, the protrusion preferably comprises embossings (generally in the number of 3 or 6, 6 being preferred) or has the shape of a cross to provide a tension plugging and a perfect fitting between protrusion and bearing and to block rotation of the bearing.

In the mixing device of the present invention, liquid flow is preferably well organized, i.e., the liquid enters though specific opening(s) and leaves through other specific opening(s), the former being located centrally and the latter, peripherally. "Specific" means that a given opening only acts as inlet or outlet opening and not as both. The terms "central" and "peripheral" are to be construed as being complementary i.e. "peripheral" means decentralized.

According to one or more embodiments of the invention, the mixing device comprises at least one inlet opening and at least one outlet opening. Preferably, there are at least two outlet openings and even more preferably, at least four or even, up to six if

1851-09 PCT - 28 - the container to be mixed has big dimensions. When there are four, the outflow directions are preferably pointing at the corners of the bag. There are however preferably not more than 8 outlet openings in order to avoid increasing the head losses too much (which would reduce pressure build-up and hence, mixing efficiency). Also, the outlet openings are preferably designed hydrodynamicaily. This will be discussed more in detail later on. In order to favor pressure build up, the outlet openings are preferably located on the side wall of the mixer. Even more preferably, they occupy 40% or iess of the side wall surface area, preferably 30% or less and even more preferably less than 20% of the side wall surface area. In this embodiment, the wall defining the compartment is discontinuous and preferably comprises a substantially flat top (roof) where there only is/are inlet opening(s) and a side wall where there only are outlet openings. Even more preferably, the side wall is substantially perpendicular to the bottom of the bag.

Also, in this embodiment, the outlet openings preferably are in the shape of vertical slots which extend substantially over the entire height of the side wall. If there is an obstruction on their bottom, solids will not be well agitated on the bottom of the bag near these slots and complete emptying will not be allowed and if there is an obstruction on their top, gas bubbles can accumulate which will tend to render the system unstable and reduce mixing efficiency. Although the openings through which liquid enters and/or exits the mixing device are located on the wall(s) of the mixer, this does not exclude the fact that they could be "extended" by tubes (perforated or not), baffles or the like which may extend from the compartment into (and even out of) the bag. It is desirable that the mixing device be able to centrifuge the liquid, i.e., to put the liquid in circulation from a central point of its compartment towards a radial direction by rotating at high speed (for instance a few hundreds of rpm or higher, for instance up to 1000 rpm or higher). Hence, in the case there would be a space between the bottom of the mixing device and the bottom of the bag, the inlet and/or outlet openings could also be located in the bottom wall of the mixer. This may be the case if the mixing device is welded on the bottom of the bag through welding feet (so that it looks like if it were built on piles inside the bag).

1851-09 PCT - 29 - Also, it is preferred that the side wall of the mixing device is not continuous but instead, includes parts of given geometry (slices of an annular wail; parallelograms; triangles; curved half moons, and so on), This is described with more detail in the frame of a specific embodiment later on. These parts need not be identical nor do they need to be disposed symmetrically versus the rotation axis of the magnetic element, although they preferably are. Preferably, their shape is rounded such that the outlet slots are shaped hydrodynamically. This reduces pressure drop as explained above. In the case the side wall comprises six identical parts separated by six identical slots and is put into a parallelepiped bag, good results (in terms of mixing homogeneity) are obtained when two of the parts are traversed in the middle by the small median of the bag bottom, This is also preferably the case when there are four such parts and associated slots,

In one embodiment, to increase the vertical action distance of the mixer, the height between inlet and outlet openings is increased by fixing a tube of a certain length on the inlet opening. To be compatible with injection molding, this tube is generally not longer than 10 cm. It is worth noting however that longer flexible tubes, for instance obtained by extrusion, could be fixed to the central area of the mixer.

In at least some cases, the outlet flow directions also characterize the action area of the mixer:

- axial outlet flow (generally through openings in the top of the mixing device wall) generally limits the action area horizontally and doesn't allow homogenization of liquids heavier than water and solid suspensions which tend to accumulate on the bottom; however, it may be required in the case the mixing device is located outside the bag and welded to the periphery of an opening into the bottom of the bag (unless tubes are provided to deflect the liquid radially after it exits the top of the compartment) or if the flexible bag has a height to diameter ratio (H/D) of 2 or more;

- radial outlet flow (generally through openings in the side wall of the mixer) generally limits the action area vertically but allows heavier liquids and solid suspensions to be mixed; hence, radial expulsion with the mixing device being located in the bottom centre of the bag is preferred when the mixing device is

1851-09 PCT - 30 - located inside the bag but it implies multiple exits in order to cover the entire bottom area.

Preferably, there are several outlet openings and the outlet flow directions are in a plane which is either parallel or perpendicular to the rotational axis of the magnetic element (which may be real or virtual). Preferably, the outflow directions are all parallel to the bottom of the bag, and so perpendicular to the rotation axis. This allows a better homogenization of solids and/or liquids at the bottom.

As mentioned above, pressure build up is a preferred feature of the mixing device according to the invention. Hence, the design of the mixing device is preferably adapted to obtain a pressure inside the compartment of at least 10 mbar, preferably at least 20 mbar and even more preferably, of at least 50 mbar when the rotary magnetic element is rotated at about 1000 rpm (which is a quite classical rotation speed). However, in order to limit the risk of leakage and decoupling of the rotary element, the pressure is preferably not in excess of 500 mbar, even more preferably not exceeding 300 mbar and in some instances, even not exceeding 150 mbar.

In order to limit the vortex effect mentioned previously, it may be advantageous to provide the inlet opening (or the open end of the tube, the case being) with anti-vortex member(s) as will be described below. Alternatively, there could be several inlet openings located in the central area of the wall. The presence of anti- vortex member(s) allows reduces the risk of decoupling of the magnetic rotary element from the driver, The presence of a physical rotation axis where the rotary element is retained also helps in reducing this risk,

In one embodiment, the mixing device comprises an opening for bubbling gas inside of it. It then preferably also comprises an internal baffle around the rotating element in order to prevent the gas bubbles from interacting with the liquid flow in that area. This baffle should of course leave some space for the liquid to flow out, in the space between the baffle and the wall of the mixer, to reach the outlet opening(s). Preferably, in order to achieve that, the baffle has a restricted height allowing the liquid to pass over it. In another embodiment, the bag is provided with baffles external to the mixing device but located close to its outlet openings in order to guide the liquid flow (preferably

1851-09 PCT - 31 - in several directions) and to promote the homogeneity of the mixing effect. Some of these baffles may be linked to and/or prolong parts of the mixing device wall. Tn yet a further embodiment, the flexible bag comprises at least two facing walls and a mixing device located inside of it on one of the walls, the other facing wall comprising a cover having a cavity matching the external shape of the mixing device. This cover is preferably fixed (preferably by welding) on an opening of the wall and more precisely: the lower border of the cover is welded to the outer periphery of the opening i.e, on the outer surface of the bag.

The term "matching" means in fact that the mixing device fits (can be inserted) inside the cover and can be removed therefrom easily, i.e., that the mixing device has smaller dimensions and an adequate shape to be able to be received (preferably completely) inside the cover. This embodiment prevents the bag from being damaged owing to the presence of a rigid mixing device inside of it. It also allows the mixing device to be kept in place during shipping and handling when the mixing device is not fixed to the bag (see above).

Some embodiments of the invention also relate to a liquid substance circulation (mixing) device provided for being placed in a container comprising at least a bottom surface and a rotary magnetic element for rotating around a central axis extending vertically from a bottom plate, being optionally the same as the bottom surface of the container, provided to be driven by a motor external to the container. The liquid substance circulation (mixing) device according to this embodiment of the invention is characterized in that that the device further comprises a compartment delimited by an upper plate, provided with an inlet in the central area; the bottom plate, and a peripheral wall extending vertically from the bottom plate and comprising a plurality of side wall portions, each side wall portion being separated from each other side wall portion by an outlet slot, and each outlet slot and each side wall portion being respectively disposed symmetrically to other outlet slots and to the other side wall portion.

The presence of a first compartment with an upper plate comprising a liquid substance inlet in the central area allows the device to axially aspirate the liquid substance. The presence of the plurality of outlet slots being each a liquid substance outlet allows the device to radially bring the liquid substance out of the circulation device

1851-09 PCT - 32 - in a parallel manner with the bottom surface of the container. Therefore, the liquid substance is circulated throughout the container, thereby reducing the presence of dead zones. Indeed, the liquid substance is displaced along the bottom of the container and the complete bottom surface of the container is perfectly swept and stirred before being guided by the container wall(s) upwards and being again aspirated by the liquid substance inlet.

Moreover, the plurality of outlet slots being each a liquid substance outlet and the fact that each outlet slot and each side wall portion are respectively disposed symmetrically one to each other, acts conjointly to allow the device to accumulate a predetermined pressure without creating a decoupling of the rotary magnetic element, within the compartment. The accumulation of pressure within the compartment creates a sufficient circulation movement of the liquid substance and prevents turbulence in the liquid substance flow due to the alignment of the outlets which also reduce the presence of dead zones. The liquid substance does not rotate, is perfectly swept and presents in any point a relative speed which is different from zero,

Advantageously, the compartment has a substantially circular internal cross section and each side wall portion presents a convex external surface. The circular internal cross section acts as a first guiding member to exit the liquid substance out of the compartment while the side wall portion presenting a convex external surface acts as a second guiding member. The convex external surface allows the liquid substance to reach the corners of the container and/or the farthest portions of the bottom of the container from the circulation device. Therefore, the presence of dead zones is strongly reduced because there is no portion of the container which is not reached by the liquid substance flow exiting the circulation device. In a particular embodiment, the peripheral wall comprises four side wall portions together defining a quatrefoil circumferential shape. In this manner, the pressure accumulated in the compartment is particularly optimal and the liquid substance exiting the circulation device is also particularly well guided by the four external convex shape of the side wall portion. In a particularly advantageous embodiment, the rotary magnetic element comprises a central hole provided for receiving a bearing element provided on a

1851-09 PCT - 33 - protrusion extending perpendicularly from the bottom plate. The aforementioned feature (i.e. the physical presence of an axis) allows higher speed for the rotary element. Indeed, in order to reach a sufficient flow rate of the liquid substance when exiting the circulation device, it is desirable to accumulate a sufficient pressure inside the compartment. This pressure sometimes creates a resistance towards the rotation movement of the rotary magnetic element thereby resulting in the decoupling of this latter. The decoupling of the rotary magnetic element will lead to instability, i.e., the rotation speed of the rotary magnetic element can be affected and turbulences can occur. The presence of a central hole for receiving a central axis protruding vertically from the bottom plate counteracts the decoupling;, thereby providing a circulation device with higher efficiency by allowing the rotary magnetic element to rotate with higher speed and the accumulated pressure within the compartment to become greater, thereby increasing the exiting flow rate of the liquid substance.

In a particularly advantageous embodiment, the central hole is provided for receiving a bearing element provided on a protrusion extending perpendicularly from the bottom plate.

It is of common knowledge that two different parts of a bearing element in friction one to each other should preferably be made of different material. Indeed, the rotary magnetic element rotates on the bearing element which is fixed to the bottom plate via the protrusion, i.e. the rotation of the bearing element is prevented, for example with a truncated shape. The presence of the bearing element, being a separate element from the protrusion allows using different materials for the protrusion, for the rotary magnetic element and for the bottom plate.

In a preferred embodiment, the protrusion comprises embossings (preferably three or six) to provide a tension plugging and a perfect fitting, and the bearing element is engaged in a rotation-free manner on the protrusion by a fixing member.

Preferably, the rotary magnetic element comprises two permanent magnets connected symmetrically at each side of a medium portion, the medium portion having the central hole formed therein. This feature enables high rotation speed for the rotary magnetic element without any decoupling of this latter.

1851-09 PCT - 34 - In order to produce liquid substances according to manufacturing standards that will allow validation of the liquid substance either vis-a-vis the FDA, or GMP, GLP or other standards, a key feature is to prevent particle generation due to the friction between the rotary magnetic element and the bearing element. To this end, in a particularly preferred embodiment, the rotary magnetic element comprises an outer surface of a first polymer having self lubricating properties and being resistant to abrasion and wherein the bearing element is made of a second polymer having good wear resistance properties, the first and second polymers forming a couple having a hard partner and a soft partner.

More particularly, each polymer forming the couple is chosen from the group consisting of polytetrafluoroethylene, polyoxymethylene, high density polyethylene, polyamide, polyetheretherketone and ultra high molecular weight polyethylene and most preferably, the couple of polymers is polyetheretherketone and ultra high molecular weight polyethylene, the polyetheretherketone being the hard partner and the ultra high molecular weight polyethylene being the soft partner. Advantageously, the liquid substance circulation (mixing) device comprises guiding member(s) upstream of the liquid substance inlet with respect to a liquid substance circulation (mixing) flow, aiming to increase the efficiency of the circulation of the liquid substance. Indeed, due to the presence of the guiding member(s), for example a tubular body, the liquid substance is aspirated in the central area of the container at a higher level, thereby providing a greater circulation movement.

Preferably, the liquid substance circulation (mixing) device comprises anti-vortex member(s), which can be for example a cross shaped four wall construction extending from the central zone, increasing the circulation efficiency of the liquid substance circulation (mixing) device according to the invention. Indeed, when a commonly known stirring bar rotates in the bottom of a container, a central vortex, due to the suction siphon of the stirring bar, is created. The vortex is a dead zone in terms of mixing and circulation with undesirable turbulence. The cross shaped four wall construction extending from the central zone breaks down the vortex, thereby increasing the efficiency of the homogenization and of the circulation of the liquid substance. In a particular embodiment, the liquid substance circulation (mixing) device comprises retention member(s) provided to lock the rotary magnetic element during

1851-09 PCT - 35 - shipment and/or handling. The retention member(s) can be for example, a protrusion extending downwards from the upper plate, from the anti- vortex member(s) or from the guiding member(s). The retention member(s) can present a cross, a circular, a square shaped cross section, etc. Advantageously, in a preferred embodiment, the compartment further comprises an inlet provided for bringing a fluid into the compartment, The fluid can be a gas, for example, in order to allow adjusting the gas concentration in the liquid substance, for example, O₂, CO₂, N₂, and the like. This can be particularly advantageous when the liquid substance circulation (mixing) system is used in a cell culture system, for example in a bioreactor or in a pouch or bag or even, when the liquid substance circulation (mixing) device is used in a feeding pouch or bag perfusing a culture of cells or microorganisms in a bioreactor. The fluid can also be a liquid substance being for example a nutriment solution, an antibiotic, a buffer and the like.

In a particularly preferred embodiment, the compartment comprises sensors, in particular optical sensors provided for measuring parameters such as pH, temperature, dissolved oxygen, or existing or future sensors not requiring direct contact between the probe and the recorder.

In a variant, particularly adapted for cell culture or microorganism culture, the inlet is provided with a filter and/or a membrane preventing the passage of particles or cells, when present. Advantageously, the rotary magnetic element, having optionally an aerodynamic

(i.e., hydrodynamic) geometry, creates a liquid substance flow rate within the range from 0.06 to 40 liters/min which is particularly adapted for small scale uses.

In a variant, the rotary magnetic element, having optionally an aerodynamic (i.e., hydrodynamic) geometry creates a liquid substance flow rate within the range from 10 and 300 liters/min, in particular from 20 to 250 liters/min and preferably from 25 to 200 liters/min, which flow rate is particularly suitable for large scale applications.

Other embodiments of the device are mentioned in the annexed claims. In at least some cases, it is intended to manufacture the liquid substance circulation (mixing) device by the following process comprising the steps of:

1851-09 PCT - 36 - - injection of the selected polymer, preferably of the high density polyethylene for forming the compartment,

- injection of the selected polymer, preferably of the high density polyethylene for forming the guiding member(s), optionally comprising the anti-vortex member(s), - injection of the selected polymer, preferably of the polyetheretherketone for forming the bearing element of the bearing of the rotary magnetic element, and forming the rotary magnetic element with the central hole and both permanent magnets coated with the selected polymer, preferably with ultra high molecular weight polyethylene.

Particularly, the step of forming the rotary magnetic element can include:

forming at least two cavities, provided for accommodating each permanent magnet having a predetermined diameter, in a polymer bar having a size close to the predetermined diameter, from each end of the cylindrical polymer bar, the at least two cavities remaining separated from each other by a central part of the polymer bar.

- Machining the central part of the rotary magnetic element for forming the central hole and for giving the outline of the central part, - inserting each permanent magnet in its own cavity through a terminal end,

- forming at least two covers provided to close each terminal end, and

- welding each cover to the terminal end.

In a variant, the step of forming the rotary magnetic element comprises:

- forming at least two cavities, provided for accommodating each permanent magnet having a predetermined diameter, in at least two separate cylindrical polymer bars having a diameter close to the predetermined diameter, each cavity remaining sealed at one end and opened at the other end, - Machining a polymer bar having a diameter greater than the predetermined for forming a central part of the rotary magnetic element 1-09 PCT - 37 - - inserting each permanent magnet in its own cavity through the open end,

- placing the at least two polymer bars, with their cavities having each its own permanent magnet inside, and the central part in a mold,

- baking the at least two polymer bars together with the central part in a furnace to form a raw rotary magnetic element, and

- finishing machining of the raw rotary magnetic element to bore the central hole in the central part and to form a horizontal contact area.

This concept can be generalized to a method for manufacturing a rotary magnetic element, the method comprising the following steps:

putting at least one polymeric part or powder and at least one magnet into a mold able to mold a rotary element that has or can be provided with (a passage for) a rotational axis in a way such that the magnet(s) are located symmetrically inside the mold versus the axis heating the mold to melt the polymer part(s) or powder in order to obtain a single piece rotary magnetic element,

Advantageously, an exemplary process according to an aspect of the invention further comprises a packaging step and a sterilization step, to provide sterile liquid substance circulation (mixing) devices to be used, for example in clinical batch production process. There exist several applications where it can be appropriate to provide a container provided with a liquid substance circulation (mixing) system. A first exemplary application is the use of graduated container for the preparation, complementation, dilution or adjustment of liquid substance. Generally, in laboratories, the procedure is to add into the container a stirring bar, preparing, complementing, diluting or adjusting the liquid substance, removing the stirring bar and adjusting the volume of the liquid substance to the expected final volume. This leads to inaccurate value, to contamination risk, to a waste of time, and so on. For the production of clinical batches or sterile solutions, all steps are earned out in so called "white rooms" and liquid substances to be used should be sterile by

1851-09 PCT - 38 - autoclaving and/or filtration according to their content, which increases the working costs and contamination risk by the technician. For this reason, the use of disposable pouches containing ready-to-use liquid substance has really been increasing.

Unfortunately, it is generally needed to adjust the liquid substance by making, for example, concentration, salinity, pH, osmolality, dissolved gas concentration and/or complementation adjustments. Similarly, it is more and more frequent to harvest or collect media containing molecules of interest (production of proteins, viruses, DNA, antibiotics, metabolites, and so on) in such pouches or bags. The harvested or collected media should generally be subjected to further steps requiring homogenization of the content of the pouch or bag.

To solve this problem, some embodiments of the invention encompass provision of a liquid substance container comprising the liquid circulation device. For example, the liquid substance container can be a graduated container wherein the graduation takes into account the volume of the liquid substance circulation (mixing) device, In another example, the liquid substance container contains the liquid substance circulation (mixing) device and can be an empty pouch or bag designed to harvest a liquid substance from a chemical, biochemical or biotechnological process or can be a pouch or bag containing a basic liquid substance to be adjusted, complemented, diluted, concentrated or even ready- to-use or containing a suspension which should be maintained in movement to prevent sedimentation. This type of container prevents contamination, ensures a sterile container comprising a sterile circulation device, both being provided sterile and ready-to-use, depyrogenated, endotoxin free, and so on.

In a particularly preferred embodiment, the container comprises an amount of solute in the form of powder, aggregates, pellets, granules and the like or in the form of liquid concentrate, or a number of particles to be suspended, and at least one solvent or diluent inlet. Indeed, it is well known that storage of ready-to-use solutions in a flexible bag or in a rigid container is very expensive in terms of required space. Moreover, the storage period of ready-to-use liquid substance are generally short, resulting in an over- consumption of this latter. Some embodiments of the invention provide a sterile container containing the predetermined amount of solute to which only water has to be added. Due

1851-09 PCT - 39 - to the presence of the circulation device within the container, the preparation step of the liquid substance based upon the solute is made considerably easier.

In a particularly useful application, the liquid substance circulation (mixing) device is used in a culture medium flexible bag or in a medical mixing flexible bag retained in a rigid container.

In the field of cell culture, it is also known to procure sterile pouches containing liquid culture medium often needing complementation (addition of serum, antibiotics, particular nutriments, metabolic markers, and the like) resulting in homogenization and mixing problems, in particular for large scale pouches or bags. An exemplary system is the WAVE Bioreactor system (mark of Invitrogen Corporation, Carlsbad, California, USA, now merged with Applied Biosystems as Life Technologies) which uses a disposable culture device in pouches or bags which presents a problem of circulation of the medium since the pouch or bag undergoes a rocking movement on an agitating plate, which makes it possible to make turbulences in the medium without homogenizing it or making it circulate. Consequently many zones in the pouch are not homogeneous and the cells and/or the cells on carriers sediment and aggregate, which they do not withstand. In addition, for pouches of 500 liters, the agitating plate must be oversized, which means that the mechanical elements of the circulation device of the WAVE Bioreactor are subjected to high wearing forces and the enormous agitating plate is difficult to design, and is mechanically fragile and very bulky.

Consequently, aspects of the invention, as mentioned above, set out to resolve this problem by procuring a liquid substance container comprising a circulation device which can be used for homogenizing culture media in pouches to be complemented without risk of contaminating the culture medium, including for pouches ranging up to 200 or 1,000 liters without involving any additional investment cost, nor any considerable additional cost in use.

In the field of cell culture, some cell culture methods can be carried out within pouches or bags directly. The use of the device in culture pouches or bags affords good circulation of the medium and optimum homogenization. Consequently it is possible to cultivate cells in suspension (on carriers or not) in the pouches or bags, since good

1851-09 PCT - 40 - circulation is achieved, in particular with a filtration membrane at the liquid substance inlet, to avoid cell damages or crushing of cell carriers by the rotary magnetic element.

Finally, aspects of the invention also relate to a mixing device suitable for being used in a flexible bag as described above or in any other type of container, and which is based on the finding that (as already mentioned above) it is advantageous to guide the liquid flow (deviate it from its natural path) when or after it exits the mixing device. When passing through an opening in the wall of a mixing device with rotary magnetic element, since the wall has a given thickness, the outlet flow undergoes a small local deviation from its natural rotating path and tangential velocity. However, it immediately tends to return to the rotational pattern generated by the centrifugal forces, i.e., it becomes again circular (in the case of a cylindrical device) or helical (in the case of a cone shaped device) which limits the mixing efficiency. One aspect of the invention aims at solving that problem by providing a mixing device comprising a rotary magnetic element located in a compartment delimited by a wall, the mixing device further comprising at least one liquid inlet opening located in a central area of the wall; at least one liquid outlet opening located in a peripheral area of the wall; and deflector(s) that substantially alter the natural rotational direction of the liquid exiting the outlet opening. The details given previously as to the rotary magnetic element, the materials constituting the wall of the mixing device and the relative dimensions and/or shape of both do apply to this aspect of the invention. Pressure build up is also preferably promoted and a shape of the wall such that its inner sections are circular (hence: a cylindrical or a helical shape) are also preferred.

The parameters which are specific to this aspect of the invention are mainly related to the nature, shape and dimension of the deflector(s). Normally (without deflector(s)), when the liquid exits the compartment of the mixing device, its flow path is tangential to the wall of the compartment at the outlet point. The deflector(s) of this aspect of the invention act to shift this liquid outflow path from a given angle after the liquid has impacted on the deflector(s) so that the flow path tends to become more radial (i.e. the flow direction when the liquid leaves the deflector(s) tends to become more parallel to the radial direction at the outlet point). This angle shift is preferably of at least 5°, more preferably of at least 10° and even more preferably of at least 20°. It preferably

1851-09 PCT - 41 - allows directing the liquid outflow in any preferred direction, for instance the corners of the parallelepiped-shaped containers in general and flexible bags in particular. These corners are well known to persons of ordinary skill in the art as being dead zones in a mixing process. By this angle shift at each of the deflector(s), the global flow pattern created by the mixer within its action area is changed from a circular path to several divergent jets, the number of jets being equal to the number of outlets,

This directional change of the velocity vector occurs directly when the flow leaves (is no longer in contact with) the deflector(s). This permits mixing of the substance away from the mixing device and throughout the bag in a more efficient manner. It allows avoiding dead zones for instances in the corners of the container (bag) to be mixed.

In the mixing device, there are preferably several outlet openings, the number of which depending in fact on the size and shape of the container it is designed for, These outlets are preferably located on a side portion of the wall of the mixer. They are preferably all identical. Advantageously, they are all equipped with deflector(s) and even more preferably, with identical deflector(s) so that the flow pattern is symmetrical. The terms "equipped with" mean in fact that there are some deflector(s) somewhere in the flow leaving the outlet opening but not necessarily directly at the opening.

In the case where the wall of the mixing device is conical, the inlet opening is generally unique and located at the top of the cone while the outlet openings are spread all over it. In the case the wall of the mixing device comprises a top portion (roof) and a side portion (side wall), the latter is preferably vertical and comprises the outlet openings. If the container has a high H/D ratio of at least 2, the mixing device may comprise at least one outlet opening at the top of its wall (roof) but in a peripheral region thereof (and not in the centre). Deflector(s)may also be provided at that opening. According to a first embodiment, the wall of the mixing device has an external surface which, when viewed in radial sections through the outlet opening(s), is not circular and/or comprises portions in relief - a "radial section" being a section through a plane which is perpendicular to the axis of the device and which passes through the outlet oρening(s)). This is, for instance, the case when the mixing device comprises several side wall portions separated by outlet slots and having a convex external surface as described earlier, or even a concave

1851-09 PCT - 42 - external surface. Other geometries of the portions may also give a deflecting effect; for example, if the portions each comprise at least one excrescence.

According to a second embodiment, the wall of the mixing device has an external surface which, when viewed in radial sections through the outlet opening^), is circular but the wall has a high thickness when compared to the dimensions of the openings so that these act as tubes that guide and/or deflect the liquid flow. Typically, this is the case when the ratio e/d of the thickness (e) to the diameter (d) of the opening (when viewed in a radial cut, i.e., it may be the diameter if the opening is circular or it may be the width in the case of a slot) is at least equal to one preferably at least equal to two and even to four. In this embodiment, the higher the ratio, the more the outlet flow pattern will be altered. Generally speaking, the alteration becomes really substantial when the ratio is at least equal to 0.5.

In these two first embodiments, the deflector(s) are hence formed by the wall of the device itself. In a third embodiment, the deflector(s) are part(s) affixed to or integrated to the external surface of the wall and which may be tubes prolonging the outlet opening(s) or baffles or the like either integrally molded with the wall or being fixed to it. In a fourth embodiment, the deflector(s) are parts separate from the wall of the mixing device but which are intended to be located somewhere into the outlet flow to deviate its rotational flow pattern. Their size, shape and location are preferably adapted to the size and shape of the container intended to be mixed.

The above described mixing device is particularly useful when used in a flexible bag or rigid container for mixing pharmaceutical solutions and suspensions preferably in a sterile environment. Some aspects of the invention also concern a mixing system comprising a flexible mixing bag as described above; a rigid support for the bag; and a magnetic driver adapted to drive the device in the bag. Other characteristics and advantages of one or more embodiments of the invention will appear more clearly in the light of the following description of particular non-limiting exemplary embodiment(s) of the invention, while referring to the figures.

1851-09 PCT - 43 - As it can be seen in FIGS. Ia and Ib, the liquid substance circulation (mixing) device 1 comprises an upper portion 2a comprising an upper plate 3 and an inlet 4 in the central area. According to the illustrated preferred embodiment, the upper portion 2a of the device 1 further comprises a guide 5 upstream of the inlet 4 in view of the liquid substance circulation (mixing) flow and an anti-vortex element 6.

In FlG. Ib, it can be seen that the upper portion 2a of the circulation device comprises retention member(s) 7 for maintaining the rotary magnetic element in place (during transport for example), These member(s) are baffles arranged to form a cross.

FIG. 2 illustrates the rotary magnetic element 8 comprising a central hole 9 provided for receiving a bearing element 10 (see FIG. 3). and two permanent magnets 1 1 "opposite one to each other" or symmetrically at each side, and connected to a medium portion 12. The medium portion 12 has the central hole 9 formed therein. In some embodiments, the medium portion 12 comprises on its upper surface a recess 27 provided to accommodate the retention member(s) 7 illustrated in FIG, Ib. Indeed, the rotary magnetic element 8 is provided to rotate while the upper portion 2a of the device 1, comprising the retention member(s) 7, is provided to be static. It is therefore advantageous to provide a recess 27 avoiding friction strength between the retention member(s) 7 and the rotary magnetic element 8 but maintaining the blocking effect of the retention member(s) 7. As it can be seen in FIG. 3, the bearing element 10 is provided for receiving a protrusion 13 (see FIG. 4) extending perpendicularly from the bottom plate 14 within a central cavity 15. The bearing element 10 comprises a bottom surface 16 being not symmetric, for example with a truncated and/or beveled region 17, i.e., having an irregular cross section, for preventing the rotation of the bearing element 10 when the rotary magnetic element 8 is rotating.

FlG. 4 illustrates the bottom portion 2b of the circulation device. The liquid substance circulation (mixing) device comprises a bottom plate 14 (being optionally the same as the bottom surface of the container into which the circulation device according to the invention is intended to be inserted). The liquid substance circulation (mixing) device further comprises a compartment 18 delimited by the upper plate 3 and the inlet in the central area 4 shown in FIG. 1, the bottom plate 14 and a plurality of peripheral side wall

1851-09 PCT - 44 - portions 19a, 19b, 19c, 19d, and so on extending vertically from the bottom plate 14. Each side wall portion 19a, 19b, 19c, 19d is separated from the other by an outlet slot 20. Each outlet slot 20 and each side walϊ portion 19a, 19b, 19c, 19d is respectively disposed symmetrically to the others. Preferably, the compartment 18 presents a substantially circular internal cross section and each side wall portion 19a, 19b, 19c, 19d presents a convex external surface 21a, 21b, 2Ic₅ 2 Id.

In the illustrated embodiment, there are four (4) side wall portions together defining a quatrefoil circumferential shape.

The bottom portion further comprises connector(s) 22, of the "quick connect" type provided to fix the upper portion 2a of the liquid substance circulation (mixing) device 1 to the bottom portion 2b. The upper portion 2a comprises reciprocal connector 23 provided to accommodate the connector 22 for fixing the upper 2a and the bottom 2b portions of the liquid circulation device according to the invention. The bottom plate further comprises a cavity 24 with a truncated cross section 26 adapted to receive the bearing element 10 and to prevent the rotation of this latter. Moreover, for a perfect fitting, the protrusion 13 comprises some embossings 25.

Preferably, the rotary magnetic element 8 comprises an outer surface of polyetheretherketone and the bearing element 10 is made of ultra high molecular weight polyethylene, or the opposite. The latter is more preferred (see above). FIG. 5 is an exploded view of the illustrated device. As can be seen, it is particularly easy to assemble. The bottom portion 2b comprises the bottom plate 14 and the plurality of side wall portions 19a, 19b, 19c, 19d, and so on are each separated from the other by outlet slots 20 delimited a compartment 18, being further delimited by the upper plate 3 of the upper portion. The upper plate comprises a ring 26 provided to be accommodated in the compartment created when the device is assembled.

The bottom plate 16 of the bearing element 10 and the cavity 24 of the bottom plate 14 of the bottom portion 2b present both an irregular cross section (truncated and/or beveled region 17 and 26) being the same in order to insert the bottom plate 16 of the bearing element 10 in the cavity 24 of the bottom plate of the bottom portion 2b to prevent the rotation of this latter.

1851-09 PCT - 45 - When mounting the illustrated device, the protrusion 13 is inserted into the bearing element 10. The embossings 25 of the protrusion 13 perfectly fit the internal surface of the bearing element 10 to prevent the rotation of this latter.

The rotary magnetic element 8 is then placed around the bearing element 10, this latter being placed in the central hole 9 of the medium portion 12.

After having placed the rotary magnetic element 8 on the bearing element 10, being on its turn, placed on the protrusion 13 of the bottom portion 2b of the device I₅ the upper portion 2a has just to be connected to the bottom portion 2b by connecting the connector(s) 22 to the reciprocal connector(s) 23. As can be seen, the embodiment illustrated in FIG. 5 comprises a tubular guide 5 upstream of the inlet 4 with respect to a liquid substance circulation flow.

FIG. 6 illustrates an alternative embodiment of the rotary magnetic element 8 having a cross shaped cross section. According to the invention, the rotary magnetic element 8 can present optionally an aerodynamic (i.e., hydrodynamic) geometry and could create a liquid substance flow rate within the range from 0.6 to 40 liters/min for small scale process and within the range from 10 and 300 liters/min, in particular from 20 to 250 liters/min and preferably from 25 to 200 liters/min, for large scale application.

FIGS. 7 and 8 illustrate preferred applications of the liquid circulating device according to aspects of the invention. According to some aspects of the invention, provision is made for packaging liquid substance circulation (mixing) device 1 in a sterile individual package or supplying it in a non-sterile and packaged form or even non-sterile and non-packaged form. The non-sterile circulation device 1 remains a device for single usage if required, but it can also be reusable, if the users wish to wash and reuse it. In a particular embodiment of the invention, provision is also made for the liquid substance circulation (mixing) device according to the invention to be autoclaved with a view to its reuse. Consequently, the circulation device 1 according to the invention is designed to be used in normally used containers 28 such as solution storage CARBOYS or parallelepiped bags 28 (see FIG. 8), or in solution or suspension preparation receptacles 28 (see FIG. 7).

1851-09 PCT - 46 - It suffices, if it is packaged individually and sterile, to immerse the liquid substance circulation (mixing) device 1 in the receptacle 28 containing the solution or suspension to be prepared and then to actuate the stator part (driver) in order to set the rotative magnetic element 8 in rotation. According to aspects of the invention, the container 28 may be a container 28 supplied with the liquid substance circulation (mixing) device which is directly welded in the bottom thereof. In the embodiment illustrated, bottom portion 2b comprises a bottom plate 14 but, when the container 28 is directly supplied with the liquid substance circulation (mixing) device, the bottom plate 14 of the liquid substance circulation (mixing) device 1 can be the same as the bottom surface of the container 28, In addition, when the container 28 is either designed to be used conjointly with the circulation device with a particular fluid, or directly supplied with the circulation device, provision is made for the graduation 29 to directly take account of the volume of the circulation device according to the invention. In the first case, this makes it possible to use the disposable circulation device 1 and to use a new one at each use without having to remove it before the solution is made up to the mark. Of course, due to the increased sweeping operation of the circulation device according to the invention, when such a container, for example a graduated container is used to prepare a solution, the preparation time is considerably preserved and the quality of the solution is improved. Indeed, when the device is used to prepare a buffer, the acid or base concentration is important with respect to the buffer capacity of the solution and to the concentration to reach (saline conditions, and so on).

When preparing a buffer solution with a device which brings the solution in rotation, i.e. with just a common stirred bar, pH adjustment is really time consuming and it very often occurs that too much acid or base is added because the measured value of the pH is not the exact one that the solution presents. This is because the solution is not immediately homogenous and this drawback generally leads to a varying concentration due to acidic and base addition, reacting together to create a salt modifying the osmolarity and osmolality and even the concentration of the buffer solution. This is also prejudicial to the reproduction of experiments. The rotary magnetic element in rotation creates a liquid substance suction siphon in the central area and expels the liquid

1851-09 PCT - 47 - substance sucked radially and tangentially along the external surface of the guide, which leads to a nearly perfect instant homogeneity.

FIG. 9 is a schematic view of the liquid substance circulation (mixing) device 1 used in pouch or bag 28, for example to cultivate cells or to produce a solution or a suspension in a closed system. In the field of cell culture, the cells can be cultivated on (miciO)caniers, in suspension or in a fixed bed. The cells on (micro)carriers or not, both in suspension can be circulated by the device. Preferably, in those kinds of applications, a filter or a membrane 299 is present upstream the liquid substance inlet or upstream the tubular guide 5 if present with respect to a liquid circulation flow (see arrows) for confining the cells in the pouch or bag being a culture vessel 28. The confinement of the cells is advantageous to prevent them to enter into the liquid substance circulation (mixing) device 1 in order to avoid imposing excessively high stresses on them or preventing blockage of the rotary magnetic element 8. In the case of a reactor or a container 28 for producing a suspension of particles, aggregates, powder, granules, and the like, it is advantageous to place this type of filter or membrane 29 in order to prevent the particles in suspension in the liquid substance entering the liquid substance circulation (mixing) device,

In the illustrated embodiment, the pouch or bag 28 may be used as a culture device or as a medium reservoir for another device. In the case of a culture device of the culture pouch type, the liquid substance circulation (mixing) device 1 is used for stirring the culture medium. In the case of a medium reservoir, since generally the packaged medium commercially available in pouches or bags does not comprise the required additives, the device 1 may be used for homogenizing the medium contained in the pouch or bag 28 and the additives added via a feeding connection 30. It is advantageous for the circulation device 1 to be directly integrated in the pouch or bag 28 at the time of manufacture. The presence of this liquid substance circulation (mixing) device ensures stirring of the medium and cells by the circulation of the medium within the culture pouch with a perfect sweeping of this latter.

The liquid substance circulation (mixing) device 1 makes it possible to use an appreciably smaller driving system requiring less mechanism than the devices generally provided for mobilizing the culture medium in this type of culture devices, such as the

1851-09 PCT - 48 - tilting stirring plate of the aforementioned WAVE system. Consequently the liquid substance circulation (mixing) device 1 is, in at least some cases, much less expensive than those generally used for culture pouches or bags 28.

In addition, since the driving system is practically identical for all the liquid substance circulation (mixing) devices 1, it is also advantageous at this level since only the investment of a single driving system and several liquid substance circulation (mixing) devices 1 is to be made, whatever the diverse applications required. The liquid substance circulation (mixing) device 1 is intended, in at least some cases, to be produced as a disposable device, and therefore at low cost for enabling its single use. When the liquid substance circulation (mixing) device 1 is integrated in the container 28, its use make the preparation of solution, suspension, culture and the like easier because it is provided within the container. The container 28 and the circulation device 1 are both sterile and intended for a single-use. Moreover, for container 28 provided with a solute amount which can be any one of the solutes, the user just has to place the container 28 (being rigid, a pouch, a flexible bag or the like) upon the driving system, to connect a feeding tube on the one side to the water reservoir or feeding reservoir and on the other side to a connection 30 provided on the container.

As can be seen in FIG. 10, it can be advantageous that the flexible bag or pouch 28 comprises a reservoir 32. In other applications, for example dialysis of a protein in a buffer A against a buffer B, it is advantageous for the container 28 to comprise an internal reservoir 32 comprising one part consisting of a dialysis membrane having a suitable pore size according to the size of the protein to be dialyzed. The internal reservoir 32 of the container 28 should retain the protein to be dialyzed but not the buffer A. Consequently, in this type of application, it is advantageous to procure, at the internal reservoir 32, a supplementary addition tube 31 in order to be able to place the sample to be dialyzed in the internal reservoir. Since the homogenization of the liquid substance is maximum and the liquid substance circulation (mixing) device 1 according to the invention affords excellent circulation, this particular type of container 28 in this particular application substantially reduces the dialysis time. In another field, the reservoir 32 is provided for containing carriers and microcarriers. For example, in the field of cell culture, the cells on (micro)carriers can be

1851-09 PCT - 49 - confined in a culture zone 32 which will be fed by the swept medium brought into circulation by the liquid substance circulation (mixing) device 1 according to the invention. A grille, filter or membrane (not shown) can be provided for retaining these within the internal reservoir 32. In this case, the addition tube 31 allows, for example, the inoculation of carriers or microcarriers with cells. Advantageously, the grille, filter or membrane also prevents the cells from circulating in the space internal to the container 18 and external to the reservoir 32 and prevents the cells from entering the circulation device.

In this type of application, provision is preferably made for the medium to be able to circulate through the internal reservoir 32 in order to nourish the cells. Since homogenization is maximum and circulation is optimum, the medium is perfectly stirred and the cells are well supplied with nutriments. The tube 30 can also serve to enrich the medium with nutriment, and to add regulation substances. As mentioned before, it may be advantageous to provide for the presence of sensors in order to monitor the culture parameters such as the nutriment concentration, the pH, the dissolved oxygen, and so on. In addition, provision can also be made to add a fluid inlet in the bottom portion which allows an entry of a liquid or a gas into the compartment 18. In the case when oxygen is fed, the oxygen will be stirred with the medium and the dissolution of the oxygen in the culture medium will be increased. In addition, this stirring significantly reduces the size of any bubbles present, which reduces any damage to the cells through the stresses normally generated by the bubbles.

FIG. 1 1 illustrates the upper portion 2a of an alternative embodiment of the liquid substance circulation (mixing) device wherein the liquid substance inlet 4 is bored through the upper plate 3 under the guide 5. FlG. 12 illustrates the upper portion 2a of another alternative embodiment of the liquid substance circulation (mixing) device comprising the anti-vortex element 6 without a guide.

FIG. 13 includes two schematic views (one from above (a) and one axial cut (b) through a plane comprising the axis of the device) of a mixing device adapted for the "free positioning" embodiment. In this embodiment, the mixing device I is not fitted or otherwise welded to the bag. Rather, the device comprises a positioning mechanism as

1851-09 PCT - 50 - described earlier in the specification. The advantage of this version of the mixing device is the limited welding to the bag, thus limiting risks of leakages for poor welds.

This positioning mechanism includes extension arms 33 equipped with magnets

34 which are adapted to interact with corresponding magnets 35 located outside of the bag 28, optionally in the rigid container used to handle the flexible bag 28. When the magnets are engaged, the mixing device 1 is positioned correctly versus the magnetic driver 36 also located outside the bag 28 and optionally in the rigid container.

The views are namely schematic due to the fact that the magnets 34 should not be visible in FIG. 13A (since they are located under the extension arms 33) and also because the magnetic impeller is not shown in FIG. 13B - while it should be since it is inside the device 1.

Before engagement with the driver, the mixing device may be held in place inside the bag both through the fact that the bag has been put under vacuum and through the use of a protective cover as described earlier in the specification. An alternative form of positioning mechanism is illustrated in FIG. 14 which also comprises two schematic views (as defined above) of an embodiment of the invention. This mixing device is also adapted for the "free positioning" embodiment of the present invention. This positioning mechanism comprises three (3) tabs 37 which are fixed (e.g., welded) to the flexible bag 28. The mechanism can include two or more tabs. The geometry of the tabs 37 and the manner in which the mixing device 1 is inserted in them allows the mixing device 1 to move to some extent horizontally relative to the bag side wall 28 before engagement with the driver 36. The limited freedom to move permits the proper positioning of the device for coupling to the driver 36, which coupling occurs through the magnetic forces exerted by the driver on the impeller and vice versa. FIGS. 15 and 16 (both also comprising two schematic views as defined above, but where the axial plane for the cut passes through the magnets) illustrate a rotary magnetic element 8 intended to be retained in a device according to another embodiment of the invention, In this embodiment, already described above, the element 8 is in the form of a disc (alternative to the shape of an impeller) having embedded therein and disposed symmetrically about its axis of rotation, four (4) magnets 11. Again, magnets 11 should not appear on figures "a" because they are embedded in the disc; they are only shown to

1851-09 PCT - 51 - show their location. Alternatively, one or more such magnets can be used. This disc has portions in relief in the shape of four (4) blades 38. Alternatively, one or more such blades can be used.

In FIG. 15, the blades 38 are curved. In FIG. 16, the blades 38 are straight. This latter embodiment allows rotation of the element in both clockwise and counterclockwise directions with identical performance.

FIGS. 17a to d relate to two different embodiments of the mixing device where the compartment has no complete bottom surface (bell embodiment). FIGS. 17a and b (again comprising two schematic views as described above) illustrate a mixing device without bottom plate suitable for the "free positioning" version. The device simply comprises a top wall 2a and side wall portions 19 with a convex external surface 21. The side wall portions 19 are merely lying on the bottom of the flexible bag (not shown). Although the device shows four side wall portions, one or more could be utilized,

The portions define slots 20 between them. In the case of one portion, the slot would be located on one position on the portion. In this mixing device, the medium to be agitated is sucked in through inlet openings 4 and is propelled outside the device through the slots 20 all through the centrifugal effect generated by rotary magnetic element 8 rotating within the compartment of the device. In this embodiment, the element rotates about a protrusion 13 extending from the top wall 2a of the mixer. The element 8 is retained on the protrusion 13 by connector 22. Alternatively, the element can be a magnetic stir bar or other magnetic agitator that sits on the surface of the flexible bag or other container. The device illustrated is equipped with positioning member(s) including both extension arms 33 and bridges 37.

An alternate embodiment of the device illustrated in FIGS.17c and 17d is quite similar to the device of FIGS.17a and 17b, However, in this embodiment, a foundation disc 1' is provided below the side wall portions 19 of the mixing device 1. This foundation disc 1' is preferably shaped in a circle and its surface extends from about the inside wall of the side wall portions to the outside wall of the side wall portions, It can have a rim that extends a few centimeters inside or outside of this region to assist in creating a coupling seal to the surface of the bag. The foundation disc can be shaped in a variety of ways such as circular, square or otherwise, but always with a "hole" in the

1851-09 PCT - 52 - middle thereof below the area of rotation of the element 8. If positioning member(s) are not used with this device, the foundation disc can be used as a surface to weld the mixing device 1 to a container surface. Hence this particular embodiment is adapted for both the "precise positioning" and the "free aligning" versions. The "precise location" embodiment is illustrated in FIG. 17e, where the mixing device 1 is shown as being welded to a flexible bag 28 retained in a rigid container 28'. The weld is made between the foundation disc 1 ' and the bag 28.

FIG. 18 illustrates various alternative embodiments of the side wall portions 19 of a mixing device suitable for some embodiments of the invention. Each side wall portion 19 includes a section (when viewed in a plane parallel to the bottom of the mixing device) with a shape that includes deflector(s) to alter the natural flow path of liquid as it exits slots 20. The shapes include triangles 18a and half moons 18b (as described above) but can include many other shapes that provide for the deflector(s). Various other side wall portion designs are illustrated in FIGS, 18d to 18i. These geometries all have in common the fact that they include deflectors either by the shape and/or thickness of the wall portions itself (FIGS. 18a to 18d) and/or by the fact that they have an excrescence (like a baffle) integrally molded with the wall portions (FIGS. 18e to 18i). FIG. 18c shows an embodiment where the deflectors are formed from the wall of the slots 20 which are formed as channels in an annular side wall. In this embodiment, liquid is forced to deflect away from the normal rotational flow path due to the channel-like slots 20.

FIGS. 18j to 18m show still further embodiments of deflectors, namely:

- FIGS. 18 j and 18k: external baffles (i.e., not integrated to the wall portions of the mixing device but to the flexible bag, which is not shown) located in the outlet flow and which are curved

- FIG. 18£: half moons but which are offset so that the inner section of the mixing device is no longer circular

- FIG. 18m: external baffles located in the outlet flow but which are straight and distributed evenly approximately at the middle of each side wall portion.

1851-09 PCT - 53 - While the former drawings are related to embodiments of mixing devices with radial outflow directions (where the liquid flows out of the device through its side walls), the embodiments of FIG. 19 (which includes schematic axial cuts) show axial outlet directions (the liquid flowing out through peripheral points on the top surface of the device). FIG, 19a shows an embodiment of the mixing device with an axial central inlet and axial peripheral outlets (see arrows) and having an internal baffle 39. FIG. 19b shows the same embodiment without an internal baffle. FIG. 19c shows the same embodiment as FIG. 19a but with guide 5 upstream of the inlet to increase the vertical action of the device. FIG. 19d shows the same embodiment but with guiding tubes 40 at the outlets, the tubes prolonging the outlets and transforming the axial outflow directions into radial outflow directions.

FIG. 20 shows a trolley that can be used in a "precise location" embodiment in cooperation with an intermediate frame as the one pictured in FIGS. 21 to 23 and comprising:

- a body 40 supporting a driver 36;

- at one end of the body, two wheels 41 intended to roll on the floor;

- at least one foldable foot 42 bearing a wheel 43 which rolls on the floor when the trolley is disengaged from a container (like the one shown in FIGS. 22 and 23) and which folds back when the trolley is engaged with the container; and at the other end of the body, two wheels 44 which are never in contact with the floor but are able to roll on the rail of a frame 45 like the one of FIG. 21 when the trolley is engaged with the container.

At the end close to wheels 41 rolling on the floor, the trolley comprises a handle

46 in order to be able to move it easily. At the same end, it also comprises a support 57 for the controller of the driver and hanging member(s) for hanging the end of the trolley to the container in order to put and keep the driver at its right location against the bottom of the container. These hanging member(s) comprise hooks 47 collaborating with springs 48.

1851-09 PCT - 54 - The intermediate frame 45 shown in FIGS. 21 to 23 comprises a guiding rail comprising a bottom plate 49 and two side plates 49'. In FIG. 21, the rail is shown straight (because this figure is schematic) but in fact, it is inclined as shown in FIGS. 22 and 23. This guiding rail is equipped with two locking castors 50 collaborating with matching holes on the trolley (not shown). The intermediate frame 45 also comprises a compartment 51 for tubing security which can be closed by folding back movable plate 52 and four hollow upward extensions 53 designed to receive the feet 54 of the rigid container 55 pictured in FIGS. 22 and 23.

Compartment 51 and movable plate 52 are not present in the frame shown in FIGS. 22 and 23. These figures show the trolley of FIG. 20 and another intermediate frame 45 mounted on the rigid container 55 respectively in a schematic view and in a view from above, with the container in phantom (i.e. shown as a transparent item, which is generally not the case). As can be seen, frame 45 is inserted between the container 55 - which rests with its feet 54 in the hollow extensions 53 - and a roller plate 56 which is a standard one, into which the container 55 directly fits as well. In these figures, the trolley is shown in a position such that the driver 36 is at a precise location relative to the container, which is underneath its bottom and approximately in the center of it. It is kept there thanks to the locking castors 50 and the hanger(s) including the hooks 47 and springs 48. As can be seen on these figures, the foldable foot 42 has come down after it has passed the beam 55' of the container but since the trolley is hanging up, it does not touch the floor. The same applies to wheels 41.

FIG. 24 shows schematic views of a rigid container, with (FIGS. 24a and b) or without (FIGS. 24c and d) wheels 56 and respectively with a square or rectangular bottom frame (FIGS. 24a and c) or with a frame in "U" shape (FIGS. 24b and d) allowing insertion of the driver 36 by the open end of the "U," and hence avoiding to pass underneath the beam 55 ' formed by the frame.

In another aspect, an alternative embodiment of a liquid substance circulation device is depicted in FIG. 25. Elements in this figure similar to those in the earlier figures have received the same reference character, incremented by twenty-five hundred; for example, element 6 in FIG. Ia has an analog in element 2506 in FIG. 25. The device shown in FIG. 25, or aspects thereof, can be used in many different applications; in one

1851-09 PCT - 55 - non-limiting example, it can be combined with a bag 28 having a relatively large volume; for example, on the order of 500 liters.

In the embodiment of FlG. 25, anti-vortex element 2506 has six instead of four arms (element 6 in FIG, Ia has four arms, i.e., is cross-shaped). Furthermore, guide 2505 can have a height similar to that of guide 5; however, the diameter of the device as a whole, and that of the guide 2505, can, in at least some embodiments, be substantially larger than that of the embodiments shown in the earlier figures, such that the aspect ratio (height to diameter) of the guide is much lower and the guide functions more as a structural stiffener for upper plate 2503. The diameter of guide 2505 can be larger than that of inlet 2504, in at least some instances.

In the embodiment of FlG. 25, bottom portion 2502b can be molded or otherwise formed with a substantially hexagonal shape with rounded apexes, for ease of injection molding, including reduced chances of air entrapment. In particular, the substantially hexagonal shape with rounded apexes can be formed from six side wall portions 2519 each with an outlet opening 2520. The rounding of the apexes can be at the bases of deflector baffles 7000. Baffles 7000 are similar to those shown in FIG. 18m except that they are coupled to wall portions 2519, and are also somewhat similar to those shown in FIG. 18e except that they are straight.

In a non-limiting exemplary embodiment, outlet slots 2520 can be about 6 cm high and about 4.2 cm wide, for an area of about 25.2 square cm, and other dimensions can be approximated by scaling the drawings; although it is again to be emphasized that the dimensions and other features are by way of example and not limitation.

In still another aspect, a variety of different types of upper portions ("caps") can be provided. While not intended to be limiting, certain types of upper portions are particularly advantageous for bioreactor applications where cell cultivation is at a small scale (e.g., about 30 mL or less) where the liquid substance circulation device diameter is approximately equivalent in diameter to the bottom of the container in which the mixing takes place. By providing a suitably sloped "cap" to the liquid substance circulation device, "dead zones" with low fluid velocity can be reduced or minimized. Such dead zones are undesirable in that they allow cells to settle and adhere to the top surface, which is harmful in terms of cell density and viability. While the different types of

1851-09 PCT - 56 - upper portions ("caps") to be discussed immediately below are not limited to such small scale applications, it is believed that they are most beneficial in such applications, inasmuch as at higher scales, the relative size of the liquid substance circulation device to the container is small (e.g., about 12 cm diameter of liquid substance circulation device in a container with a diameter of about 60 cm). More generally, this aspect of the invention applies to mixing of Liquid-Solid applications for which the solid phase could settle on the cap of the mixing system.

FIGS, 26 and 34 show a so-called "volcano" configuration of upper portion or cap 3202. This includes a straight inlet portion 3204 and a conical portion 3206. The total height Z can be any desired value; in a non-limiting example, it is about 20 mm. Furthermore the diameter of the portion 3204 can be any desired value; but in a non- limiting example, it could have an internal diameter of about 8 mm. Yet further, the base diameter of the conical poition 3206 can be any desired value; but in a non-limiting example, it could have an external diameter of about 26 mm. The value of Theta can be any value which provides adequate prevention of dead zones; in a non-limiting example, Theta ranges from 15 degrees to 75 degrees, most preferably from about 60 degrees to about 70 degrees. Portion 3202 may have outlets 3208 which allow the material being mixed to exit. In other words, material to be mixed is inlet through straight inlet portion 3204, mixed in the lower portion 3256 by impeller 3258, and outlet through outlets 3208. Any number of outlets 3208 can be included; for example, four spaced at 90 degrees about the large diameter of conical poition 3206. Outlets 3208 could be located in recesses 3210 in conical portion 3206. For purposes of attachment to a lower portion, upper portion 3202 may have fasteners (not shown) which may be configured to mate with portions such as portions 22 described above, in a manner similar to portions 23 as described above (e.g., via a "snap-in" action). Region 3250 between straight inlet portion 3204 and conical portion 3206 does not contain material being mixed, thereby reducing the chance for dead zones. It could contain air (if portion 3202 was hollow) or could be solid plastic or the like. For the avoidance of doubt, the material to be mixed is inlet through portion 3204, which is the same diameter from top to bottom, mixed in a lower portion similar to that seen in the earlier figures, then exhausted in a direction 180

1851-09 PCT - 57 - degrees opposite to the inlet through outlet(s) 3208. Region 3250 may be solid or hollow but does not contain material to be mixed,

FIG. 34 shows the upper portion 3202 on lower portion 3256 in a closely fit mixing vessel 3254. FIGS. 27 and 35 show a so-called "crater" configuration of upper portion or cap

3302. This includes a conical inlet portion 3306 which funnels down to a smaller end 3304, The total height H can be any desired value; in a non-limiting example, it is about 20 mm. Furthermore the diameter of the portion 3304 can be any desired value; but in a non-limiting example, it could have an internal diameter of about 8 mm. Yet further, the large diameter of the conical portion 3306 can be any desired value; but in a non-limiting example, it could have an external diameter of about 26 mm. The value of Alpha can be any value which provides adequate prevention of dead zones; in a non-limiting example, Alpha ranges from 15 degrees to 75 degrees, most preferably from about 60 degrees to about 70 degrees. A base plate 3308 may be provided. Fluid outlets 3310 are provided; any desired number can be included. Material to be mixed is inlet through conical area "crater" 3306 and enters lower portion (not shown in FIG. 27) where it is mixed, and it is then outlet through outlets 3310. Portion 3302 could be attached to a lower portion 3356 including impeller 3358, for example, as described for the example in FIG. 26. Region 3350 between "crater" 3306 and cylindrical side wall 3352 does not contain materia! being mixed, thereby reducing the chance for dead zones. It could contain air (if portion 3302 was hollow) or could be solid plastic or the like.

FIG. 35 shows the upper portion 3302 on lower portion 3356 in a closely fit mixing vessel 3354.

FIGS. 28 and 29 show an ellipsoidal or spherical cap configuration of upper portion or cap 3502 located on a lower portion 3504 within a mixing container 3506. It will be appreciated that this is, in essence, a form of the "volcano" in which the top is in the form of an ellipsoidal or spherical cap rather than a cone. In this regard, portion 3502 is a spherical cap, i.e., the region of a sphere which lies above (or below) a given plane. If the plane passes through the center of the sphere, the cap is a called a hemisphere. As best seen in FIG. 29, in an example, the portion 3502 is less than hemispherical; however, the example in the figures is not intended to be limiting. Portion 3502 includes inlets

1851-09 PCT - 58 - 3508. Lower portion 3504 can include outlet openings 3520 and deflector baffles 3522. In a non-limiting exemplary embodiment, holes 3508 may have a diameter of about 8 mm, lower portion 3504 may be about 80 mm in diameter and 20 mm in height, and upper portion 3502 may also be about 80 mm in diameter and 20 mm in height. FIG, 36 shows an alternative embodiment of the so-called "crater" configuration of upper portion or cap 3602. This includes a conical inlet portion 3606 which funnels down to a smaller end 3604. The dimensions could be, for example, similar to the embodiment described above, or could_} for example, be larger for use with a larger mixing vessel 3654. A base plate 3608 may be provided. Material to be mixed is inlet through conical area "crater" 3606 and enters lower portion 3656 where it is mixed by impeller 3658, and it is then outlet through radial outlets 3620. Portion 3602 could be attached to lower portion 3656, for example, as described for the example in FIG. 26. Region 3650 between "crater" 3606 and cylindrical side wall 3652 does not contain material being mixed, thereby reducing the chance for dead zones. It could contain air (if portion 3602 was hollow) or could be solid plastic or the like. FIG. 36 shows the upper portion 3602 on lower portion 3656 in a mixing vessel 3654 which is not a close fit.

For FIGS. 26-29 and 34-36, the rotary magnetic element and its associated mounting (if any) can be as described with regard to the other embodiments above.

It will be appreciated that the exemplary embodiments of FIGS. 26, 34 and 27, 35 include axial outlets 3208, 3310, and are thus particularly suited to small-scale applications where there is little or no radial clearance between the mixing device and the vessel. On the other hand, the exemplary embodiments of FIGS. 28, 29 and 36 are shown within somewhat larger vessel 3506, 3654, respectively, where there is sufficient radial clearance to employ radial outlets 3520, 3620 in the lower portion (indeed, once there is significant space between the mixing device and mixing vessel, use of radial outlets is preferred to reduce dead zones, as discussed elsewhere herein). Of course, "conical volcano" embodiments with radial outlets are possible, as are ellipsoidal or spherical cap volcano configurations with axial outlets.

FIG. 30 shows raw data for cell density (recombinant CHO cell line in suspension) versus time for a flat design corresponding roughly to FIG. 25; a "chimney" design corresponding roughly to FIG, 5; a "volcano" design corresponding roughly to

1851-09 PCT - 59 - FIG. 26; and a "crater" design corresponding roughly to FIG. 27. FIG. 32 is a corresponding plot. The superior performance of the "volcano" and "crater" designs in terms of cell density after elapsed time is quite clear.

FIG. 31 shows raw data for cell viability versus time for a flat design corresponding roughly to FIG. 25; a "chimney" design corresponding roughly to FIG. 5; a "volcano" design corresponding roughly to FIG. 26; and a "crater" design corresponding roughly to FIG. 27. FIG. 33 is a corresponding plot. The superior performance of the "volcano" and "crater" designs in terms of ceil viability after elapsed time is apparent. Given the discussion of FIGS. 26-36, it will be appreciated that, in general terms_} a liquid substance circulation device, according to an aspect of the invention includes a lower portion 3504, 3256, 3356, 3656 with an upstanding side wall. Also included is an upper portion 3202, 3302, 3502, 3602 associated with the lower portion. As used herein, including the claims, "associated with" includes lower and upper portions that are formed separately and fastened together with snapping portions or other mechanical fasteners, gluing, welding, or the like, as well as upper and lower portions that are formed together as one piece; for example, by injection molding or the like. The upper portion and the upstanding side wall of the lower portion cooperatively delimit at least a portion of a compartment, in which an impeller such as 3258, 3358, 3658 is located. The impeller can rotate on a fixed axis or be loose but captured within the compartment. As used herein, including the claims, "cooperatively delimiting at least a portion of the compartment" includes the case where there is no "bottom" to the lower portion and the "bottom" of the compartment is defined by the bottom of the mixing vessel 3254, 3354, 3654, 3506, as well as the case where there is a bottom to the lower portion. As used herein, an "upstanding" side wall of the lower portion is either vertical, or if not vertical, is sloped more closely to the vertical than the upper portion; furthermore, both the case of a discrete transition in slope or a smooth (continuous) transition in slope between the upper and lower portions is intended to be encompassed by this terminology.

The upper portion has at least one axial inlet 3204, 3304, 3508, 3604 to the impeller. The upper portion has a slope sufficient to reduce likelihood of solid-phase settling on the upper portion. At least one outlet 3208, 3310, 3520, 3620 from the

1851-09 PCT - 60 - compartment is formed in at least one of the upper portion and the upstanding side wall of the lower portion.

As used herein, including the claims, "axial" means coincident with or parallel to the axis of rotation of the impeller; "radial" means perpendicular to such axis. Accordingly, flow out of a radial outlet is at an angle Lambda = 90 degrees, as best seen in FIG. 36, while the corresponding value in the case of an axial outlet is 180 degrees.

The radial outflow is beneficial to reduce or eliminate dead zones by "scouring" the floor of the mixing vessel 3506, 3654. "Un-scouied" flat or near-flat surfaces may result in the undesirable "dead zones" where cells may settle. Both axial and radial outlets are preferably peripheral as defined above.

With reference to FIGS. 26, 28, 29, and 34 in some instances, the lower portion has an outside diameter and the upper portion has a central apex (e.g., apex of cone or top of hemisphere) and is sloped from the central apex to the outside diameter of the lower portion to reduce the likelihood of solid-phase settling. By way of example and not limitation, the upper portion can be conical in shape tapered at an angle Theta of from 15 degrees to 75 degrees, most preferably from 60 degrees to 70 degrees.

In some instances, such as FIGS. 26 and 34, the at least one axial input to the impeller is a central inlet, the at least one outlet from the compartment is an axial outlet formed in the upper portion, and the central inlet 3204 is smaller in diameter than the outside diameter of the lower portion.

As many axial outlets as are desired can be used. In at least some instances, the axial outlet comprises a first axial outlet, and the upper portion 3202 is formed with three additional axial outlets 3208 for a total of four axial outlets 3208. The four axial outlets can be spaced ninety degrees apart about the upper portion. Other numbers of outlets could be used, preferably spaced equi-angularly.

With reference to FIGS. 27, 35 and 36, in some instances, the upper portion 3302,

3602 has a sloped interior surface with a larger diameter 3306, 3606 spaced away from the lower portion and a smaller diameter 3304, 3604 spaced adjacent the lower portion. The smaller diameter forms the at least one axial inlet to the impeller. The upper portion further has a cylindrical outer surface 3352, 3652 defining an interior region 3350, 3650

1851-09 PCT - 61 - between the cylindrical outer surface and the sloped interior surface, with material to be mixed being excluded from the interior region. The sloped interior surface is sloped sufficiently to reduce the likelihood of the solid-phase settling.

By way of example and not limitation, the sloped interior surface can be tapered at an angle, Alpha, of from 15 degrees to 75 degrees, most preferably from 60 degrees to 70 degrees.

In some cases, such as FIGS. 27 and 35, the at least one outlet from the compartment is an axial outlet formed in the upper portion. In some cases, the axial outlet 3310 comprises a first axial oxitlet, the upper portion is formed with three additional axial outlets for a total of four axial outlets 3310, and the four axial outlets are spaced ninety degrees apart about the upper portion. Note that the fourth outlet is not visible in FIGS. 27 and 35 as it is hidden behind the conical surface.

In some instances, such as FIG. 36, the at least one outlet from the compartment comprises a first radial outlet 3620 formed in the lower portion, and the lower portion is formed with sufficient additional radial outlets to reduce dead regions adjacent the lower portion 3620 (for example, as in any of the other radial embodiments).

With reference to FIGS. 28 and 29, in some instances, the upper portion 3502 has a convex curved profile to reduce the likelihood of the solid-phase settling. The convex curved profile can be, for example, a spherical cap or an ellipsoidal cap. In a non-limiting example, the radial outlet 3520 comprises a first radial outlet formed in the lower portion, and the lower portion is formed with sufficient additional radial outlets to reduce dead regions adjacent the lower portion (e.g., in the adjacent portions of the vessel). In some instances, there may be, for example, three additional radial outlets for a total of four radial outlets 3520 (only two are visible in FIG. 28 as the others are hidden behind the spherical cap), and the four radial outlets are spaced ninety degrees apart about the lower portion 3504. A plurality of deflectors 3522 can optionally be located between the radial outlets.

Still with reference to FIGS. 28 and 29, in some cases, the at least one axial inlet

3508 comprises a first axial inlet, the upper portion 3502 is formed with two additional axial inlets for a total of three axial inlets 3508, and the three axial inlets are spaced one hundred twenty degrees apart about an apex of the upper portion 3502. Other numbers of

1851-09 PCT - 62 - inlets could be used, preferably spaced equi-angularly. The axial inlets shown in FIGS. 28 and 29 could, for example, be otherwise similar to inlet 3204 described elsewhere.

In another aspect, the liquid substance circulation devices just described can be located within appropriate liquid containers, such as 3506 (or in a bag as described above). In some instances, the liquid substance circulation device is a "close fit," in which case axial outlets as in FIGS. 26 and 27 are advantageous. In other cases, where sufficient radial clearance exists, radial outlets, as in FIGS. 28 and 29, are appropriate. A "close fit" as used herein means that the liquid substance circulation device has an outside diameter equal to the inside diameter of the liquid container or with just enough clearance between the two to allow for easy insertion; the whole bottom surface of the bioreactor is intended to accommodate the circulation device. Radial outlets are preferred when the radial clearance between the outside diameter of the liquid substance circulation device and the inside diameter of the liquid container is large enough such that stagnant or dead zones (where cells would collect) would be formed on the floor of the liquid container if radial outlets were not used.

Exemplary presently preferred embodiments include a configuration similar to that in FIGS. 28 and 29 in a mixing vessel 3506 with a volume of about one liter, as well as a configuration similar to that shown in FiGS. 26 and 34 with a mixing vessel 3254 having a volume of about 20 to 30 milliliters and a lower portion with about a 2 cm diameter.

Although the preferred embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions or substitutions are possible, without departing from the scope and spirit of the invention as defined in the accompanying claims.

1851-09 PCT - 63 -

Claims

CLAIMSWhat is claimed is:

1. A liquid substance circulation device, comprising: a lower portion with an upstanding side wall; an upper portion associated with said lower portion, said upper portion and said upstanding side wall of said lower portion cooperatively delimiting at least a portion of a compartment; and an impeller located within said compartment; wherein: said upper portion has at least one axial inlet to said impeller; said upper portion has a slope sufficient to reduce likelihood of solid-phase settling on said upper portion; and at least one outlet from said compartment is formed in. at least one of said upper portion and said upstanding side wall of said lower portion.

2. The device of Claim 1, wherein said upstanding side wall is vertical.

3. The device of Claim 2, wherein: said lower portion has an outside diameter; and said upper portion has a central apex and is sloped from said central apex to said outside diameter of said lower portion to reduce said likelihood of said solid-phase settling.

4. The device of Claim 3, wherein said upper portion is conical in shape and is tapered at an angle of from 15 degrees to 75 degrees.

5. The device of Claim 4, wherein: said at least one axial inlet to said impeller comprises a central inlet; said at least one outlet from said compartment comprises an axial outlet formed in said upper portion; and

1851-09 PCT - 64 - said central inlet is smaller in diameter than said outside diameter of said lower portion.

6. The device of Claim 3, wherein said upper portion has a convex curved profile to reduce said likelihood of said solid-phase settling.

7. The device of Claim 6, wherein said convex curved profile comprises one of a spherical cap and an ellipsoidal cap.

8. The device of Claim 6, wherein: said at least one outlet from said compartment comprises a first radial outlet formed in said lower portion; and said lower portion is formed with sufficient additional radial outlets to reduce dead regions adjacent said lower portion.

9. The device of Claim 8, further comprising a plurality of deflectors located between said radial outlets.

10. The device of Claim 2, wherein: said upper portion has a sloped interior surface with a larger diameter spaced away from said lower portion and a smaller diameter spaced adjacent said lower portion, said smaller diameter forming said at least one axial inlet to said impeller; said upper portion further has a cylindrical outer surface defining an interior region between said cylindrical outer surface and said sloped interior surface, with material to be mixed being excluded from said interior region; and said sloped interior surface is sloped sufficiently to reduce said likelihood of said solid-phase settling.

11. The device of Claim 10, wherein said sloped interior surface is conical and is tapered at an angle of from 15 degrees to 75 degrees.

1851-09 PCT - 65 -

12. The device of Claim 11, wherein said at least one outlet from said compartment comprises an axial outlet formed in said upper portion.

13. The device of Claim 11, wherein: said at least one outlet from said compartment comprises a first radial outlet formed in said lower portion; and said lower portion is formed with sufficient additional radial outlets to reduce dead regions adjacent said lower portion.

14. An apparatus comprising: a liquid container; and a liquid substance circulation device, located within said liquid container, said liquid substance circulation device in turn comprising: a lower portion with an upstanding side wall; a upper portion associated with said lower portion, said upper portion and said upstanding side wall of said lower portion cooperatively delimiting at least a portion of a compartment; and an impeller located within said compartment; wherein: said upper portion has at least one axial inlet to said impeller; said upper portion has a slope sufficient to reduce likelihood of solid-phase settling on said upper portion; and at least one outlet from said compartment is formed in at least one of said upper portion and said upstanding side wall of said lower portion.

15. The apparatus of Claim 14, wherein said upstanding side wall is vertical.

16. The apparatus of Claim 14, wherein: said lower portion has an outside diameter; and

1851-09 PCT - 66 - said upper portion has a central apex and is sloped from said central apex to said outside diameter of said lower portion to reduce said likelihood of said solid-phase settling.

17. The apparatus of Claim 14, wherein said upper portion is conical in shape and is tapered at an angle of from 15 degrees to 75 degrees.

18. The apparatus of Claim 14, wherein: said lower portion is a close fit to said liquid container; and said at least one outlet comprises an axial outlet formed in said upper portion.

19. The apparatus of Claim 14, wherein said upper portion has a convex curved profile to reduce said likelihood of said solid-phase settling.

20. The apparatus of Claim 19, wherein said convex curved profile comprises one of a spherical cap and an ellipsoidal cap.

21. The apparatus of Claim 19, wherein: said lower portion is not a close fit to said liquid container; said at least one outlet from said compartment comprises a first radial outlet formed in said lower portion; and said lower portion is formed with sufficient additional radial outlets to reduce dead regions in said liquid container adjacent said lower portion.

22. The apparatus of Claim 21, further comprising a plurality of deflectors located between said radial outlets.

23. The apparatus of Claim 14, wherein: said upper portion has a sloped interior surface with a larger diameter spaced away from said lower portion and a smaller diameter spaced adjacent said lower portion, said smaller diameter forming said at least one axial inlet to said impeller;

1851-09 PCT - 67 - said upper portion further has a cylindrical outer surface defining an interior region between said cylindrical outer surface and said sloped interior surface, with material to be mixed being excluded from said interior region; and said sloped interior surface is sloped sufficiently to reduce said likelihood of said solid-phase settling.

24. The apparatus of Claim 23, wherein said sloped interior surface is conical and is tapered at an angle of from 15 degrees to 75 degrees.

25. The apparatus of Claim 23, wherein: said lower portion is a close fit to said liquid container; and said at least one outlet from said compartment comprises an axial outlet formed in said upper portion.

26. The apparatus of Claim 21, wherein: said lower portion is not a close fit to said liquid container; said at least one outlet from said compartment comprises a first radial outlet formed in said lower portion; and said lower portion is formed with sufficient additional radial outlets to reduce dead regions in said liquid container adjacent said lower portion.

1851-09 PCT - 68 -