Patent Application
20220234012
Embodiments of the invention relate generally to bioprocessing systems and methods and, more particularly, to impeller and sparger assemblies for single-use bioreactor systems.
A variety of vessels, devices, components and unit operations are known for carrying out biochemical and/or biological processes and/or manipulating liquids and other products of such processes. In order to avoid the time, expense, and difficulties associated with sterilizing the vessels used in biopharmaceutical manufacturing processes, single-use or disposable bioreactor bags and single-use mixer bags are used as such vessels. For instance, biological materials (e.g., animal and plant cells) including, for example, mammalian, plant or insect cells and microbial cultures can be processed using disposable or single-use mixers and bioreactors.
Increasingly, in the biopharmaceutical industry, single use or disposable containers are used. Such containers can be flexible or collapsible plastic bags that are supported by an outer rigid structure such as a stainless steel shell or vessel. Use of sterilized disposable bags eliminates time-consuming step of cleaning of the vessel and reduces the chance of contamination. The bag may be positioned within the rigid vessel and filled with the desired fluid for mixing. An agitator assembly disposed within the bag is used to mix the fluid. Existing agitators are either top-driven (having a shaft that extends downwardly into the bag, on which one or more impellers are mounted) or bottom-driven (having an impeller disposed in the bottom of the bag that is driven by a magnetic drive system or motor positioned outside the bag and/or vessel). Most magnetic agitator systems include a rotating magnetic drive head outside of the bag and a rotating magnetic agitator (also referred to in this context as the “impeller”) within the bag. The movement of the magnetic drive head enables torque transfer and thus rotation of the magnetic agitator allowing the agitator to mix a fluid within the vessel. Magnetic coupling of the agitator inside the bag, to a drive system or motor external to the bag and/or bioreactor vessel, can eliminate contamination issues, allow for a completely enclosed system, and prevent leakage. Because there is no need to have a drive shaft penetrate the bioreactor vessel wall to mechanically spin the agitator, magnetically coupled systems can also eliminate the need for having seals between the drive shaft and the vessel.
Depending on the fluid being processed, the bioreactor system may include a number of fluid lines and different sensors, probes and ports coupled with the bag for monitoring, analytics, sampling, and liquid transfer. For example, a harvest port is typically located at the bottom of the disposable bag and the vessel, and allows for a harvest line to be connected to the bag for harvesting and draining of the bag. In addition, existing bioreactor systems typically utilize spargers for introducing a controlled amount of a specific gas or combination of gases into the bioreactor. A sparger outputs small gas bubbles into a liquid in order to agitate and/or dissolve the gas into the liquid. The delivery of gas via spargers helps in mixing a substance, maintaining a homogenous environment throughout the interior of the bag, and is sometimes essential for growing cells in a bioreactor. Ideally, the spargers and the agitator are in close proximity to ensure optimal distribution of the gases throughout the container.
One type of known sparger used in many cell culture processes is the drilled hole sparger. This type of sparger is well suited to deliver nominal gas flow through the bioreactor vessel, which is needed to control the partial pressure of carbon dioxide. One drawback, however, is that the size of the holes in the sparger is such that liquid from the vessel/processing environment can leak through the holes back into the gas supply line (particularly when the sparge gas is turned off or decreased). In some situations, the liquid can travel through the gas supply line and potentially reach upstream components such as the mass flow controller, affecting the operation thereof.
In addition to the above-noted drawbacks of existing sparger assemblies, many existing sparger assemblies have fixed pore/hole diameters, which produce a fixed distribution of bubble diameters in the bioreactor vessel. Due to this constraint, a compromise must often be made in the selection of the sparger for use in a given bioreactor, in order to select one sparger that produces a distribution of bubble diameters which is effective over a range of bioreactor operating conditions. Some bioreactor systems allow for the use of different (i.e., multiple) spargers in a single bioreactor in an effort to accommodate a wider range of operating conditions. Both of these options, however, require that sparger selection take place during the design process, prior to fabrication of the bioprocessing bag. This inability to produce different bubble diameter distributions under different sparge gas mass transfer requirements can cause a number of undesirable effects, such as excessive foaming on the surface of the bioreactor.
In connection with the above, high performance bioreactor systems must provide good bulk mixing in combination with efficient gas dispersion in order to achieve a high gas surface area and bubble size distribution, and thus provide high oxygen transfer rates and kLa (the volumetric mass-transfer coefficient that describes the efficiency with which oxygen can be delivered to a bioreactor for a given set of operating conditions) values desired in intensified cell culture and/or microbial applications. Traditional solutions for achieving high kLa values employ multiple impellers mounted on a single shaft. With single-use bioreactors, however, the use of multiple impellers results in a bulky format of the disposable bag, which cannot be collapsed efficiently. Moreover, longer shafts with multiple impellers requires stabilization, which increases the complexity and cost of the vessel and bag design, and renders bag installation more cumbersome and less user friendly.
In view of the above, there is a need for impeller and/or sparger assemblies that provide for increased oxygen transfer rates and kLa values in a bioreactor system to support increased cell culture cell densities. In addition, there is a need for sparger assemblies that prevent or inhibit backflow of liquid from the bioreactor vessel into the sparge gas supply lines, and which enable sparge gas bubble diameter and/or distribution to be selectively adjusted during the cell culturing process.
In an embodiment, a sparger assembly for a bioprocessing system is provided. The sparger assembly includes a first layer having a plurality of pores of a first size, and a second layer disposed above the first layer and having a plurality of holes of a second size, the second size being greater than the first size. The pores of the first layer and the holes of the second layer allow for the passage of a sparge gas through the first layer and the second layer.
In another embodiment of the invention, a bioprocessing system is provided. The bioprocessing system includes a vessel, a flexible bioprocessing bag positionable within the vessel, and a sparger assembly positioned at a bottom of the flexible bioprocessing bag. The sparger assembly includes a first layer having a plurality of pores of a first size, and a second layer disposed above the first layer and having a plurality of holes of a second size, the second size being greater than the first size, wherein the pores of the first layer and the holes of the second layer allow for the passage of a sparge gas through the first layer and the second layer.
In yet another embodiment of the invention, a sparger assembly is provided. The sparger assembly includes a base layer, a dielectric layer disposed above the base layer, a top layer disposed above the dielectric layer, the top layer having an upper surface, at least one electrode in contact with the dielectric layer, and at least one sparge gas opening in at least the hydrophobic layer for facilitating the formation of a bubble of sparge gas on the upper surface of the top layer for introduction of the sparge gas into a bioreactor vessel.
In yet another embodiment, a bioprocessing system is provided. The system includes a vessel, a flexible bioprocessing bag positionable within the vessel, and a sparger assembly positioned at a bottom of the flexible bioprocessing bag. The sparger assembly incudes a base layer, a dielectric layer disposed above the base layer, a top layer disposed above the dielectric layer, the top layer having an upper surface, at least one electrode in contact with the dielectric layer, and at least one sparge gas opening in at least the hydrophobic layer for facilitating the formation of a bubble of sparge gas on the upper surface of the top layer for introduction of the sparge gas into a bioreactor vessel.
In yet another embodiment, a method for bioprocessing is provided. The method includes the steps of positioning a sparger assembly in a bioreactor vessel, the sparger assembly having a base layer, a dielectric layer disposed above the base layer, a top layer disposed above the dielectric layer, the top layer having an upper surface, at least one electrode in contact with the dielectric layer, and at least one sparge gas opening in at least the hydrophobic layer for facilitating the formation of a bubble of sparge gas on the upper surface of the top layer for introduction of the sparge gas into the bioreactor vessel, electrically connecting the at least one electrode to a voltage source, and adjusting a voltage supplied to the at least one electrode to adjust a diameter of the bubble formed on the upper surface of the top layer.
The present invention will be better understood from reading the following description of non-limiting embodiments, with reference to the attached drawings, wherein below:
Reference will be made below in detail to exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference characters used throughout the drawings refer to the same or like parts.
As used herein, the term “flexible” or “collapsible” refers to a structure or material that is pliable, or capable of being bent without breaking, and may also refer to a material that is compressible or expandable. An example of a flexible structure is a bag formed of polyethylene film. The terms “rigid” and “semi-rigid” are used herein interchangeably to describe structures that are “non-collapsible,” that is to say structures that do not fold, collapse, or otherwise deform under normal forces to substantially reduce their elongate dimension. Depending on the context, “semi-rigid” can also denote a structure that is more flexible than a “rigid” element, e.g., a bendable tube or conduit, but still one that does not collapse longitudinally under normal conditions and forces.
A “vessel,” as the term is used herein, means a flexible bag, a flexible container, a semi-rigid container, a rigid container, or a flexible or semi-rigid tubing, as the case may be. The term “vessel” as used herein is intended to encompass bioreactor vessels having a wall or a portion of a wall that is flexible or semi-rigid, single use flexible bags, as well as other containers or conduits commonly used in biological or biochemical processing, including, for example, cell culture/purification systems, mixing systems, media/buffer preparation systems, and filtration/purification systems, e.g., chromatography and tangential flow filter systems, and their associated flow paths. As used herein, the term “bag” means a flexible or semi-rigid container or vessel used, for example, as a bioreactor or mixer for the contents within.
As used herein, the term “removably connected” or “removably coupled” means that the aeration manifolds/sparger elements and base plate are connected in such a way as to be easily connected and/or removed to allow for easy user customization of a sparger assembly without special tools. In other words, “removably connected” is an opposite of “permanently connected”.
Embodiments of the invention provide bioreactor systems and sparger assemblies for a bioreactor system. In an embodiment, a sparger assembly for a bioprocessing system includes a base plate and at least one aeration manifold connected to the base plate in spaced vertical relation to the base plate. Each aeration manifold includes at least one inlet for receiving a gas and a plurality of gas outlet openings for delivering the gas to a fluid within the bioprocessing system.
With reference to
The vessel 12 may include one or more sight windows 22, which allows one to view a fluid level within the flexible bag 20, as well as a window 24 positioned at a lower area of the vessel 12. The window 24 allows access to the interior of the vessel 12 for insertion and positioning of various sensors and probes (not shown) within the flexible bag 20, and for connecting one or more fluid lines to the flexible bag 20 for fluids, gases, and the like, to be added or withdrawn from the flexible bag 20. Sensors/probes and controls for monitoring and controlling important process parameters include any one or more, and combinations of: temperature, pressure, pH, dissolved oxygen (DO), dissolved carbon dioxide (pCO2), mixing rate, and gas flow rate, for example.
With specific reference to
The flexible bag 20 contains an impeller 28 attached to a magnetic hub 30 at the bottom center of the inside of the bag, which rotates on an impeller plate 32 also positioned on the inside bottom of the bag 20. Together, the impeller 28 and hub 30 (and in some embodiments, the impeller plate 32) form an impeller assembly. A magnetic drive 34 external to the vessel 12 provides the motive force for rotating the magnetic hub 30 and impeller 28 to mix the contents of the flexible bag 20. While
In an embodiment, the impeller plate 32 may be configured as a sparger assembly that is used to introduce a specific gas or air into the fluid within the bag 20 in order to agitate and/or dissolve the air or gas into the fluid. Accordingly, in some embodiments, the impeller and sparger, and the components thereof, form a combined impeller/sparger assembly. In other embodiments, the sparger assembly and the impeller assembly may be separate and/or discrete components. In either implementation, the sparger assembly and the impeller assembly are in close proximity to ensure optimal distribution of gases throughout the bag 20, as discussed in detail hereinafter. As discussed below, it is envisioned that the sparger assembly (which may also serve as an impeller plate supporting the impeller) may take one of various configurations.
For example,
In an embodiment, the gas outlet openings 120 may all be the same size. In other embodiments, the gas outlet openings 120 of the first aeration manifold 112 may be a different size than the has outlet openings 120 of the second aeration manifold 114. For example, the gas outlet openings 120 of the first aeration manifold 112 may be smaller than the gas outlet openings 120 of the second aeration manifold 114. In such an implementation, therefore, the first aeration manifold 112, with its comparatively small gas outlet openings 120 that produce relatively small gas bubbles, may be utilized to supply oxygen, while the second aeration manifold 114, with its comparatively large gas outlet openings 120 that produce relatively large gas bubbles, is particularly suited for stripping or sweeping out CO2 with air, for example. Where a porous frit is utilized, the openings/pores will not have the same size, however, the various aeration manifolds may have openings with the same or different average size.
With further reference to
In an embodiment, the base plate 110 may further include an aperture 126 or fitting for fluid coupling with drain tubing for draining or harvesting of the contents of the flexible bag 20. Incorporating the impeller mounting shaft 124 and the drain aperture 126 into the base plate 110 facilitates positioning of the flexible bag 20 within the bioreactor vessel 10, as well as facilitates alignment of the magnetic hub 30 with the magnetic drive system and drain port in the flexible bag 20 with the drain tubing connected to the bottom of the bioreactor vessel 20.
Turning now to
As shown in
Similar to the embodiment of
In yet another embodiment, an immediately adjacent pair of aeration manifolds, e.g., aeration manifolds 212, 216 may have gas outlet openings 224 of a first size, while another immediately adjacent pair of aeration manifolds, e.g., aeration manifolds 214, 218 may have gas outlet openings of a second size, wherein the second size is different from the first size. The configuration of the base plate 210 and aeration manifolds 212, 214, 216, 218, and the selectively removable nature of the aeration manifolds, allows the configuration of the sparger assembly 200 to be easily adjusted according to user preferences. In particular, this design allows for plug-and-play like functionality, enabling a user to mount various combinations of aeration manifolds to the base plate 210 to provide a sparger assembly of various configurations. For example, a user can easily mount three aeration manifolds with smaller gas outlet openings 224 in combination with a single aeration manifold with larger gas outlet openings 224 to increase oxygen delivery to the system, where desired, or mount three aeration manifolds with larger gas outlet openings 224 in combination with a single aeration manifold with smaller gas outlet openings 224 to enhance CO2 removal, without having to adjust rate of gas delivery to the sparger assembly 200.
As discussed above in connection with
Referring now to
With reference to
While
Turning now to
With reference to
As shown in
In an embodiment, the inner aeration manifolds 714 and outer aeration manifolds are raised above the support plate 710 substantially the same distance. In another embodiment, as best shown in
While the sparger assemblies of the invention have hereinbefore been described as having sparger elements/aeration manifolds that are arc or arch shaped, and arranged in a manner so as to form a circle or portion of an arc, the invention is not so limited in this regard. In particular, the aeration manifolds, themselves, may have any shape desired (e.g., rectangular, triangular, ovular, etc.) and may be arranged in an annular, circular, rectangular or any polygon shape. Other arrangements of the aeration manifolds on the base plate are also possible. For example,
Turning to
In connection with the embodiments described above, by providing a sparger assembly that includes aeration manifolds for gas distribution that are raised from the base plate (or at least above a bottom surface of the vessel), sparge gas can be input into the bioreactor in close association with the impeller, which provides for more efficient gas dispersion in order to achieve a high gas surface area and bubble size distribution. Moreover, because the aeration manifolds are removably connected to the base plate, the sparger assembly may be universally configurable and adaptable to provide almost any gas distribution profile desired. In particular, the modular nature of the sparger assemblies described herein (i.e., base plate and removable aeration manifolds) allows for easy customization and creation of a sparger assembly, including customization of gas outlet height, gas outlet opening location, sparging ‘density’, etc.
In any of the embodiments described above, the interior of the aeration manifolds may be designed for optimized flow distribution such as, for example, by using a manifold groove system that promotes reduced pressure losses. In some embodiments, various components of the sparger assemblies, including the aeration manifolds, may be manufactured through additive manufacturing, which can be used to provide transitions from solid to porous materials with incorporated fluid channels to reduce the number of parts and ease of assembly. While the embodiments described above disclose hollow aeration manifolds having gas outlet openings, the manifolds may also be comprised of a porous frit wherein the openings for gas release are the pores in the porous frit.
In an embodiment, the pattern of apertures, holes or pores in the aeration manifolds of the spargers described herein can be any regular geometric pattern or a random pattern. In an embodiment, the apertures of one or more of the aeration manifolds may be arranged in a pattern which is configured such that the spacing between the apertures, holes or pores, s, is greater than the diameter of the gas bubbles that are produced by an aperture, hole or pore of diameter, d. Having a spacing between the apertures, holes or pores which is greater than the gas bubbles diameter assists in preventing adjacent gas bubbles from coalescing, as it keeps the bubbles from contacting each other at the surface of the sparger element/aeration manifold. The diameter of a gas bubble produced by an aperture, hole or pore of a specific diameter is dependent not only on the diameter of the hole or pore but is also greatly affected by factors such as the surface energy of the material from which the sparger is constructed and also on the physical and chemical properties of the liquid in which the bubbles are being created since that affects the surface tension of the air/liquid interface of the gas bubble surface.
With reference to
Referring now to
While the impeller assembly 800 is shown in
With specific reference to
As alluded to above, the impeller assembly 800 may be seated on the bottom of the flexible bag 20 in close association with a sparger assembly. For example, the impeller assembly 800 may be connected to a base plate of one of the sparger assemblies disclosed herein, such that the impeller blades 812 are in close association with the gas outlet openings of the sparger assembly. Through testing, it has been shown that the vertically straight portion 816 of the blades 812 of the impeller assembly 800 is particularly efficient in breaking the bubbles input into the flexible bag 20 by the sparger assembly, and delivers high power to the bioreactor system 10. In addition, testing has demonstrated that the angled portion 818 of the blades 812 facilitates mixing of the contents of the flexible bag 20. Accordingly, this combination of straight and angled blade portions yields improved bubble break-up and efficient gas distribution (kLa) with optimum power consumption (i.e., without requiring greater power input or agitation at very high speeds, which can cause shear damage and produce eddies that are harmful to the cells).
In this respect, the impeller assembly 800 optimizes bulk mixing and efficient gas distribution at the gas sparger to provide high oxygen transfer rates and kLa values, which is desirable in intensified cell culture and/or microbial applications. In contrast to existing systems and devices, the impeller assembly 800 achieves this performance while maintaining a relatively low profile (i.e., it remains bottom driven and sits closely to the bottom of the bag 20, allowing for the bag to still be easily collapsed for storage and transport). This simple design also allows for easy user installation and configuration. In particular, in some embodiments, the impeller assembly 800 may be quickly and easily positioned on the mounting shaft of the base plate of a sparger assembly, in the manner hereinbefore described.
Referring now to
The blades 854 are substantially similar to the blades 812 of the impeller assembly 800 of
Turning now to
While the impeller assemblies 850, 870 of
Turning now to
In an embodiment, the hub 910 is a magnetic hub configured to be driven by an external magnetic drive system or motor, as discussed above. While
In an embodiment, one or more the blades 912, 914 are connected to the hub 910 at angles offset from a radial line extending from the impeller axis. For example, blades 912 may be angled forward of a radial line extending from the impeller axis with respect to a direction of rotation 916 of the impeller assembly 900, while blades 914 may be angled rearward of a radial line extending from the impeller axis with respect to the direction of rotation 916 of the impeller assembly 900. As shown in
This canted configuration of the blades 912, 914 of the impeller assembly is more clearly shown in
In operation, the blades 912 oriented at leading angles with respect to a true radial line 918 extending from the central axis 920 function to pull liquid inwardly towards the hub 910, in the direction of arrows B, as shown in
With reference to
In use, the impeller assembly 1000 may be mounted to the mounting shaft of a sparger assembly, as discussed above. As indicated above, the slots 1014 are positioned so that when the blades 1012 rotate, the slots 1014 pass closely over the gas outlet openings in the sparger assembly.
Referring to
It is contemplated that slots or apertures may be integrated with any existing impeller designs or configurations for a bioreactor system, as well as the impeller assembly configurations described herein. By utilizing an impeller with slots or apertures in the area of the blade that passes closely over the gas outlet openings of the sparger assembly, the interfacial contact between the blades of the impeller and the fluid within the flexible bag 20 may be increased. Accordingly, the impeller assemblies 1000, 1100 provide for more efficient gas distribution at the gas sparger to provide high oxygen transfer rates and kLa values desired for enhanced cell culturing, without increasing the power requirements on the impeller drive system.
Embodiments of the impeller assemblies and sparger assemblies disclosed herein provide various means of increasing kLa of a bioreactor system (i.e., achieving more efficient gas distribution) to support intensified cell culture and/or microbial applications. It is contemplated that the impeller assemblies disclosed herein may be utilized in conjunction with any existing sparger assembly. Similarly, the sparger assemblies disclosed herein may be utilized in connection with a number of existing impeller assemblies. Still further, it is envisioned that any of the impeller assemblies disclosed herein may be utilized in conjunction with any of the sparger assemblies also disclosed herein, to provide both improved bulk mixing and efficient gas dispersion. In this respect, the configuration of both the impeller assemblies and sparger assemblies of the invention facilitates simple user manipulation or configuration of a combined impeller and sparger assembly. In particular, the impeller and/or sparger assemblies of the invention can be easily manipulated (e.g., by interchanging the aeration manifolds on the sparger and/or connecting different impellers to the sparger base plate) to achieve almost any level of bulk mixing or gas dispersion desired, depending on the particular cell culturing or bioprocessing operations being carried out within the bioprocessing system 10.
In addition to the impeller and sparger assemblies described above, the present invention further provides various sparger assembly constructions that provide for additional operational advantages over existing sparer devices. For example,
As shown in
In an embodiment, the first layer or section 806 is formed from a hydrophobic material and may be, for example, a sintered part such as a porous frit. In an embodiment, the first layer 806 may be formed from a variety of hydrophobic materials such as a polymer material including, for example, polyethylene, polytetrafluoroethylene (PTFE), polypropylene, fluorocarbons, etc., and the pores may be formed therein via any means known in the art. Regardless of the specific material and manufacturing method utilized, the size of the pores 808 of the first layer 806 is such that the first layer 806 is water impermeable and gas permeable, meaning that water is prevented from passing through the first layer 806, but gas (e.g., oxygen or carbon dioxide) is permitted to pass through the first layer 806 via the pores 808. In an embodiment, the size of the pores 808 in the first layer 806 is between about 2 and about 20 micrometers. In an embodiment, the first layer 806 is formed from a hydrophobic material and has a three-dimensionally interconnected pore structure such that gas is permitted to pass through the interconnected pores linearly and/or in a zig-zag or traversing manner so as to enter from the bottom of the first layer 806 and exit the top of the first layer 806.
The second layer or section 810, as described above, has openings 812 of a size that permit the passage of gas (and which may, in some embodiments, likewise permit the passage water therethrough). In an embodiment, the second section 810 may take the form of a drilled hole, marco sparger, having a plurality of discrete holes through the second section 810 that are monodisperse in size/diameter. In an embodiment, the size of the openings 812 in the second layer is between about 100 and about 500 micrometers in diameter.
In operation, the sparger assembly 800 is placed inside a bioreactor vessel, for example, inside flexible bag 20 of the bioreactor/bioprocessing system 10 in a manner heretofore known in the art. Various bioprocessing or cell culturing operations may then be carried out within the flexible bag 20, as known in the art. Sparge gas 814 is supplied to the housing 802 through connector 804, and passes through the porous first layer 806, and subsequently through the openings 812 in the second layer 810, where bubbles 816 of a desired size are formed and dispersed into the liquid within the interior 820 of the flexible bag 20. The size of the openings 812 in the second layer 810, and the flow rate of the gas, are chosen so as to produce bubbles 816 of a desired size (such as, for example, to provide a nominal gas flow through the liquid in the interior 820 of the bag to control the partial pressure of carbon dioxide). Typically, the size of the openings in such drilled hole, macro spargers is such that liquid from the interior of the bag may leak beyond the sparger and into the gas supply line, particularly at low gas flow rates or when sparging is ceased. The presence of the hydrophobic first layer 806 beneath the second layer or section 810, however, prevents the liquid from leaking past the first layer 806 (while simultaneously permitting the passage of spage gas from the housing 802 into the interior 820 of the bag during sparging).
The sparger 800 of the invention therefore allows for the formation of bubbles of a desired size typical of existing drill hole, macro spargers, but which also inhibits or prevents liquid backflow from the processing volume into the sparge gas supply or delivery line. It is contemplated that such sandwiched-layer sparger construction may be incorporated into any of the sparger configurations described herein (and as shown in, for example,
Turning now to
In an embodiment, the layers 852, 854, 856 may be sandwiched together and mounted or received within a sparger housing (not shown) having a tube connector for selective connection to the supply of sparge gas. For example, the housing and tube connector may be constructed similar to those shown in the sparger assembly of
As further shown in
It is envisioned that the electrodes 860 could take several forms and employ different shapes. For example, in one embodiment, the electrodes 860 may be arrayed in two or more concentric rings located around the openings 858. In an embodiment, there may be a concentric ring closely surrounding each opening with a larger common plane surrounding all of the concentric rings on the surface of the sparger. The rings which surround the openings could have a more complex shape, such as concentric rings with interdigitated fingers extending between the rings. In an embodiment, the electrode to electrode spacing could be as small as, for example, 50 um.
In some embodiments, the electrode 860 closest to the opening 858 may be in contact with the opening or within 50um of the opening. The electrode closest to the opening could be in contact with the opening such that it is part of the opening and take the form of a plated through-hole.
In an embodiment, the base layer 852 may be formed from, for example, a polyimide (Kapton), glass epoxy and/or ceramic materials commonly used in the electronics industry such as silicone dioxide (SiO2). Any base layer material with questionable/unverified biocompatibility could be encapsulated/coated in other biocompatible materials such as polydimethylsiloxane (PDMS), Ethylene tetrafluoroethylene (ETFE), Polyvinylidene chloride (PVDC) in any area where product contact could occur. The electrodes in contact with the fluid inside the bioreactor bag could be gold plated which would make them biocompatible. In embodiments where the base layer is polyimide or glass epoxy, then printed circuit board fabrication techniques could be used to construct the sparger assembly 850 (e.g., spin coating followed by oven drying could be used to overlay the hydrophobic layer over the other two layers).
In operation, the sparger assembly 850 is placed inside a bioreactor vessel, for example, inside flexible bag 20 of the bioreactor/bioprocessing system 10 in a manner heretofore known in the art. Various bioprocessing or cell culturing operations may then be carried out within the flexible bag 20, as known in the art. Sparge gas, when supplied to the housing (not shown), passes through the openings 858 in the sparger assembly 850, initially forming bubbles, e.g. bubble 862, on the exposed upper surface 861 of the top layer 856. The bubbles 858 are subsequently released form the upper surface 861 of the top layer 856 and enter the liquid within the bioreactor vessel.
The sparger assembly 850 of the invention therefore allows for bubble diameter to be selectively varied by altering the voltage applied to the electrodes, irrespective of the diameter of the openings(s) 858 (i.e., even with a fixed opening diameter). This allows the sparge gas bubble diameter distribution produced by the sparger assembly 850 to be continuously adjusted, as needed, to match, for example, the oxygen mass transfer requirements and/or the carbon dioxide clearance requirements during a bioreactor run.
The principle of operation of the sparger assembly 850 is similar to electrowetting on dielectric (EWOD) techniques currently used to manipulate fluids in microfluidic devices. Currently, spargers used in many bioprocessing fields, including in the pharmaceutical industry use fixed diameter, circular, pores/holes in the sparger element. It is contemplated, however, that the EWOD techniques described herein may be utilized in conjunction with different pore/opening geometries (such as a hole with a lobed cross section) and complementary electrode geometries/patterns to enable even greater or more precise control of bubble diameter. [000110] In an embodiment, the sparger assembly 850 may include various arrays of electrodes associated with a discrete set of (or single) sparge gas pore/opening. The arrays of electrodes may each be individually controllable by a controller 900 or microprocessor, i.e., each array of electrodes may be dynamically addressed electronically. This allows individual pores/openings or groups of pores/openings to be differentially activated, allowing for sections or patterns of individual pores/openings on a single sparger element or assembly to be adjusted to produce different distributions of bubble diameters, as desired.
The ability to control bubble diameter across the entire sparger assembly, and even vary bubble diameter at certain discrete areas across the sparger assembly relative to other discrete areas of the sparger assembly, provides a wider range of operational effectiveness than has heretofore been possible with existing devices and, particularly, sparger devices having fixed hole/opening sizes. The ability to selectively change the bubble diameter of the sparge gas allows for the generation of bubbles having a diameter that is, for example, efficient at the beginning of a cell culture process and does not cause excessive foaming when the cell density is low, and for selective adjustment mid-process, in real time, to produce bubbles having a different diameter that are more efficient when the cell density is higher. In some embodiment, the bubble diameter may be adjusted in the manner described herein in conjunction with varying the impeller speed to provide better and more efficient mass transfer control during the bioprocessing operation.
It is contemplated that the sparger construction shown in
In an embodiment, a sparger assembly for a bioprocessing system is provided. The sparger assembly includes a first layer having a plurality of pores of a first size, and a second layer disposed above the first layer and having a plurality of holes of a second size, the second size being greater than the first size. The pores of the first layer and the holes of the second layer allow for the passage of a sparge gas through the first layer and the second layer. In an embodiment, the first size is sufficient to allow for passage of gas through the first layer, and to inhibit the passage of water through the first layer. In an embodiment, the first layer is formed form a sintered hydrophobic material. In an embodiment, the second layer is configured as a drilled hole sparging element. In an embodiment, the first size is between about 2 and about 20 micrometers. In an embodiment, the second size is between about 100 and about 500 micrometers. In an embodiment, the sparger assembly also includes a housing receiving the first layer and the second layer, the housing being configured to receive a sparge gas from a supply, wherein the sparge gas supplied to the housing is permitted to pass through the first layer and the second layer into a bioprocessing vessel, and wherein the first pore size of the first layer is such that water is not permitted to pass from the bioprocessing vessel past the first layer. In an embodiment, the first layer is gas permeable and water impermeable. In an embodiment, the second layer is gas and water permeable.
In another embodiment of the invention, a bioprocessing system is provided. The bioprocessing system includes a vessel, a flexible bioprocessing bag positionable within the vessel, and a sparger assembly positioned at a bottom of the flexible bioprocessing bag. The sparger assembly includes a first layer having a plurality of pores of a first size, and a second layer disposed above the first layer and having a plurality of holes of a second size, the second size being greater than the first size, wherein the pores of the first layer and the holes of the second layer allow for the passage of a sparge gas through the first layer and the second layer. In an embodiment, the first layer is gas permeable and water impermeable. In an embodiment,the second layer is gas and water permeable. In an embodiment,the first layer is formed form a sintered hydrophobic material. In an embodiment,the second layer is configured as a drilled hole sparging element.
In yet another embodiment of the invention, a sparger assembly is provided. The sparger assembly includes a base layer, a dielectric layer disposed above the base layer, a top layer disposed above the dielectric layer, the top layer having an upper surface, at least one electrode in contact with the dielectric layer, and at least one sparge gas opening in at least the hydrophobic layer for facilitating the formation of a bubble of sparge gas on the upper surface of the top layer for introduction of the sparge gas into a bioreactor vessel. In an embodiment, the top layer is formed from a hydrophobic material. In an embodiment, the at least one electrode is electrically coupled to a voltage source, wherein the voltage source is controllable to supply a voltage to the at least one electrode, and wherein adjusting the voltage supplied to the at least one electrode varies a diameter of the bubble formed on the upper surface of the top layer. In an embodiment, the at least one opening is a plurality of openings including at least a first array of openings and a second array of openings, and the at least one electrode is a plurality of electrodes including at least a first array of electrodes associated with the first array of openings and a second array of electrodes associated with the second array of openings. In an embodiment, the at least one electrode is sandwiched between the base layer and the dielectric layer. [000116] In yet another embodiment, a bioprocessing system is provided. The system includes a vessel, a flexible bioprocessing bag positionable within the vessel, and a sparger assembly positioned at a bottom of the flexible bioprocessing bag. The sparger assembly incudes a base layer, a dielectric layer disposed above the base layer, a top layer disposed above the dielectric layer, the top layer having an upper surface, at least one electrode in contact with the dielectric layer, and at least one sparge gas opening in at least the hydrophobic layer for facilitating the formation of a bubble of sparge gas on the upper surface of the top layer for introduction of the sparge gas into a bioreactor vessel. In an embodiment, the top layer is formed from a hydrophobic material. In an embodiment, the at least one electrode is electrically coupled to a voltage source, wherein the voltage source is controllable to supply a voltage to the at least one electrode, and wherein adjusting the voltage supplied to the at least one electrode varies a diameter of the bubble formed on the upper surface of the top layer. In an embodiment, the at least one electrode is sandwiched between the base layer and the dielectric layer.
In yet another embodiment, a method for bioprocessing is provided. The method includes the steps of positioning a sparger assembly in a bioreactor vessel, the sparger assembly having a base layer, a dielectric layer disposed above the base layer, a top layer disposed above the dielectric layer, the top layer having an upper surface, at least one electrode in contact with the dielectric layer, and at least one sparge gas opening in at least the hydrophobic layer for facilitating the formation of a bubble of sparge gas on the upper surface of the top layer for introduction of the sparge gas into the bioreactor vessel, electrically connecting the at least one electrode to a voltage source, and adjusting a voltage supplied to the at least one electrode to adjust a diameter of the bubble formed on the upper surface of the top layer. In an embodiment, the method further includes fluidly connecting the sparger assembly to a supply of sparge gas. In an embodiment, the method further includes supplying the sparge gas to the sparger assembly to form a first bubble having a first diameter on the upper surface of the top layer, and adjusting a voltage supplied to the at least one electrode to produce a second bubble having a second diameter on the upper surface of the top layer, wherein the first diameter is different from the second diameter. In an embodiment, the at least one opening is a plurality of openings including at least a first array of openings and a second array of openings, and the at least one electrode is a plurality of electrodes including at least a first array of electrodes associated with the first array of openings and a second array of electrodes associated with the second array of openings. The method may further includes supplying a first voltage to the first array of electrodes to produce a plurality of bubbles having a first diameter, and supplying a second voltage that is different from the first voltage to the second array of electrodes to produce a plurality of bubbles having a second diameter that is different from the first diameter.
As used herein, an element or step recited in the singular and proceeded with the word “a” or “an” should be understood as not excluding plural of said elements or steps, unless such exclusion is explicitly stated. Furthermore, references to “one embodiment” of the present invention are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the recited features. Moreover, unless explicitly stated to the contrary, embodiments “comprising,” “including,” or “having” an element or a plurality of elements having a particular property may include additional such elements not having that property. [000119] This written description uses examples to disclose several embodiments of the invention, including the best mode, and also to enable one of ordinary skill in the art to practice the embodiments of invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to one of ordinary skill in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
This application is a national stage of International Application No. PCT/EP2020/064216 filed on May 21, 2020, which claims priority to U.S. Provisional Patent Application No. 62/850,725 filed on May 21, 2019, all of which are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/064216 | 5/21/2020 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62850725 | May 2019 | US |
