CN108136283B - Large-scale acoustic separation device - Google Patents

Abstract

An apparatus (100) for separating secondary fluids or particles from a primary fluid is disclosed. The device comprises an acoustic chamber (107), a fluid outlet (114) at a top end of the acoustic chamber, a concentrate outlet (116) at a bottom end of the acoustic chamber, and an inlet (112) at a first side end (122) of the acoustic chamber (107). An ultrasonic transducer (106) on a sidewall of the acoustic chamber and a reflector at an opposite sidewall create a multi-dimensional acoustic standing wave in the acoustic chamber (107) that captures and separates particles (e.g., cells) from the host fluid. The primary fluid is collected through a fluid outlet (114) and the particles are collected through a concentrate outlet (116). The device is a large device capable of handling several liters of liquid per hour and has a large internal space.

Description

Large-scale acoustic separation device

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application serial No. 62/211,142 filed on 28/8/2015; and claim priority from U.S. provisional patent application serial No. 62/252,068 filed on day 6 of 11/2015. The disclosures of these applications are incorporated herein by reference in their entirety.

Background

Acoustophoresis is the separation of particles and secondary fluids from a primary or primary fluid using a high intensity acoustic standing wave, and does not use membranes or physical size exclusion filters. It is well known that high intensity standing acoustic waves can exert forces on particles in a fluid when there are differences in density and/or compressibility, otherwise known as acoustic contrast factors. The pressure distribution in the standing wave includes regions of local minimum pressure amplitude at its nodes and local maxima at its anti-nodes. Depending on the density and compressibility of the particles, they will be trapped at the nodes or anti-nodes of the standing wave. Generally, the higher the frequency of the standing wave, the smaller the particles that can be trapped due to the pressure of the standing wave.

Material separation (e.g., acoustically separating a secondary fluid from a primary fluid or acoustically separating particles from a primary fluid flow) with different acoustic contrast factors (a combination of density and speed of sound through the material) has been demonstrated on a MEMS (micro-electro-mechanical systems) scale. On a MEMS scale, conventional acoustophoresis systems rely on the use of half-wave or quarter-wave acoustic chambers, which are typically less than one millimeter thick at frequencies of several megahertz and operate at very low flow rates (e.g., μ L/min). Such systems are not scalable as they benefit from extremely low reynolds number, laminar flow operation, and require minimal fluid dynamics optimization.

On a macroscopic scale, planar acoustic standing waves have been used to accomplish this separation process. However, a single plane wave typically traps particles or secondary fluids in such a way that they can only be separated from the primary fluid by closing the plane standing wave. This does not allow for continuous operation. Furthermore, the energy required to create the acoustic planar standing wave tends to heat the primary fluid through waste energy.

Thus, conventional acoustophoresis devices have limited efficacy due to a number of factors, including heating, the use of planar standing waves, restricting fluid flow, and the inability to capture different types of materials. It is therefore desirable to provide systems and methods for producing optimized particle clusters to improve gravitational separation and collection efficiency. There is a need for improved acoustophoretic devices that use improved fluid dynamics to make acoustophoresis a continuous process.

Disclosure of Invention

In various embodiments, the present disclosure relates to macro-scale acoustophoresis devices with improved fluid dynamics that can be used to improve the separation of particles (e.g., cells) from particle/fluid mixtures. More specifically, the device includes an acoustic chamber containing an ultrasonic transducer and a reflector that produce a multi-dimensional acoustic standing wave.

Disclosed herein are acoustophoretic devices for separating secondary fluids or particles from primary/primary fluids. For example, the particle may be a cell such as a Chinese Hamster Ovary (CHO) cell, NS0 hybridoma cell, Baby Hamster Kidney (BHK) cell, or a human cell; lymphocytes, such as T cells (e.g., regulatory T cells (tregs), Jurkat T cells), B cells, or NK cells; their precursors, such as Peripheral Blood Mononuclear Cells (PBMCs); algae or other plant cells, bacteria, viruses, or microcarriers.

Disclosed in various embodiments is an acoustophoretic device comprising: an acoustic chamber comprising at least one inlet at a first end thereof; at least one fluid outlet at a tip of the acoustophoretic device; at least one concentrate outlet at a bottom end of the acoustophoresis device; at least one ultrasonic transducer coupled to the acoustic chamber, the at least one ultrasonic transducer comprising a piezoelectric material configured to be driven by a voltage signal to generate a multi-dimensional acoustic standing wave in the acoustic chamber; and a reflector on the other side of the acoustic chamber relative to the at least one ultrasonic transducer; wherein the acoustic chamber comprises a planar cross-sectional area defined by a length and a width and a lateral cross-sectional area defined by the width and a height, wherein the length is greater than or equal to the width, and wherein the planar cross-sectional area is greater than the lateral cross-sectional area.

The at least one inlet may be part of a dump diffuser. The at least one inlet may include a height spanning approximately 60% of the height of the piezoelectric material. The base of the at least one inlet may be located along the base of the piezoelectric material. The dump diffuser may include at least one inlet flow port at an upper end of the plenum and a flow outlet at a lower end of the plenum having a shape that provides a flow direction perpendicular to an axial direction of the multi-dimensional acoustic standing wave generated by the at least one ultrasonic transducer.

Typically, dump diffusers are used to maximize the efficiency of acoustophoresis devices by reducing the non-uniformity in the acoustic chamber caused by gravity to make the incoming flow more uniform. The at least one inlet may be configured to allow fluid to enter the acoustic chamber at a flow rate of at least 800ml/min, and the fluid collector may be configured to allow fluid to exit the acoustic chamber at a flow rate of at least 25 ml/min.

In some embodiments, the at least one inlet comprises a first inlet at the first end of the acoustic chamber and a second inlet at a second end of the acoustic chamber opposite its first end, such that fluid flow into the acoustic chamber is uniform and symmetric.

Some embodiments of the acoustophoresis device further include a first sloped wall below the at least one inlet and leading to the at least one concentrate outlet, wherein the first sloped wall includes an angle of about 11 ° to about 60 ° relative to a first horizontal plane.

The at least one transducer may be a plurality of transducers spanning the length of the acoustic chamber. The plurality of transducers may be arranged in series in a single row. In some embodiments, the plurality of transducers includes a first row containing at least two transducers above a second row containing at least two transducers. The at least one concentrate outlet may comprise a plurality of concentrate outlets.

The acoustic chamber may comprise a volume of at least 40 cubic inches.

In various embodiments of the acoustophoretic device, a sloped top wall, a parabolically curved top wall, or a hypocycloidally curved top wall leads from the first and second ends of the acoustic chamber to at least one fluid outlet. In other embodiments, the at least one fluid outlet is connected to a central region of the acoustic chamber.

The multi-dimensional acoustic standing wave may include an axial force component and a lateral force component having the same order of magnitude.

The ultrasonic transducer may include: a housing having a top end, a bottom end, and an interior space; and a crystal at the bottom end of the housing, the crystal having an exposed outer surface and an inner surface, the crystal capable of generating an acoustic wave when driven by a voltage signal. In some embodiments, the backing layer contacts the inner surface of the crystal, the backing layer being made of a substantially acoustically transparent material. The substantially acoustically transparent material may be balsa wood, cork or foam. The substantially acoustically transparent material can have a thickness of up to 1 inch. The substantially acoustically transparent material may be in the form of a lattice. In other embodiments, the outer surface of the crystal is covered with a wear surface material having a thickness of a half wavelength or less, the wear surface material being a polyurethane, epoxy, or silicone coating. The outer surface of the crystal may also have a wear resistant surface formed by a matching layer or wear resistant plate of material adhered to the outer surface of the crystal. The matching layer or wear plate may be comprised of alumina. In other embodiments, the crystal does not have a backing layer or wear layer, i.e., the crystal does not have a backing layer or wear layer.

The multi-dimensional acoustic standing wave may be a three-dimensional standing wave.

Also disclosed in various embodiments is an acoustophoretic device comprising: an acoustic chamber comprising at least one inlet at a first end thereof; at least one fluid outlet at a tip of the acoustophoretic device; at least one concentrate outlet at a bottom end of the acoustophoresis device; at least one ultrasonic transducer coupled to the acoustic chamber, the at least one ultrasonic transducer comprising a piezoelectric material configured to be driven by a voltage signal to generate a multi-dimensional acoustic standing wave in the acoustic chamber; and a reflector on the other side of the acoustic chamber relative to the at least one ultrasonic transducer; wherein the at least one inlet is in the form of a dump diffuser comprising a flow outlet at a front lower end of a plenum, a first inlet flow port at an upper side end of the plenum and a second inlet flow port at an upper rear end of the plenum.

The flow rate through the acoustic chamber may be from about 1 milliliter per minute to about 800 milliliters per minute. The device of the present disclosure may have separation efficiencies above 90% for cell concentrations from as low as 50,000 cells per ml of fluid to 80,000,000 cells per ml of fluid.

In a particular embodiment, the multi-dimensional standing wave generates an acoustic radiation force having an axial force component and a lateral force component of the same order of magnitude. In particular embodiments, the acoustic standing wave may be a multi-dimensional acoustic standing wave (e.g., a three-dimensional acoustic standing wave). Examples of such multi-dimensional acoustic standing waves can be found in commonly owned U.S. patent No. 9,228,183, the entire contents of which are fully incorporated by reference.

These and other non-limiting features are described in more detail below.

Drawings

The following is a brief description of the drawings that are presented for the purpose of illustrating the exemplary embodiments disclosed herein and not for the purpose of limiting the same.

Fig. 1 is an external perspective view of a first exemplary acoustophoretic device according to the present disclosure. The device has an acoustic chamber with a horizontal cross-sectional area greater than its vertical cross-sectional area.

Fig. 2 is a cross-sectional view of the acoustophoretic device of fig. 12.

Fig. 3A-3D illustrate four exemplary embodiments of top walls forming fluid paths leading from an acoustic chamber of an acoustophoretic device to a fluid outlet at the top of the device. Fig. 3A shows a top wall having a flat outer surface at a different angle than a flat inner surface. Fig. 3B shows a top wall whose flat outer surface has the same angle as the flat inner surface (i.e., a top wall having a constant thickness). Figure 3C shows a top wall with hypocycloidal curved outer and inner surfaces (i.e., the fluid path narrows very quickly). Fig. 3D shows the top wall forming a fluid path that connects only to the central region of the acoustic chamber.

Fig. 4A-4D illustrate exemplary arrangements of acoustophoresis devices having one or more concentrate outlets. In devices having multiple concentrate outlets, the outlets are evenly spaced from each other. Fig. 4A shows a device with a base having one concentrate outlet. Fig. 4B shows the device with a base having one concentrate outlet. Fig. 4C shows a device with a base having three concentrate outlets. Fig. 4D shows a device with a base with four concentrate outlets.

Fig. 5A-5C illustrate an exemplary embodiment of a transducer assembly of an acoustophoretic device according to the present disclosure. Fig. 5A shows a piezoelectric transducer assembly comprising a total of six rectangular transducers arranged in two rows of three transducers. Fig. 5B shows a piezoelectric transducer assembly comprising a total of six square transducers arranged side by side in a single row. Fig. 5C shows a piezoelectric transducer assembly comprising a total of five rectangular transducers arranged in two rows, with the upper row comprising two transducers and the lower row comprising three transducers.

FIG. 6 illustrates a simulation of the capture of clusters of particles by an acoustic standing wave generated by the transducer of the transducer assembly of FIG. 5C.

Fig. 7A and 7B illustrate further exemplary embodiments of piezoelectric transducer assemblies of an acoustophoretic device according to the present disclosure. Fig. 7A shows a piezoelectric transducer assembly including a total of three matrix transducers arranged side-by-side in a single row. Fig. 7B shows a piezoelectric transducer assembly including a total of eight square transducers arranged side-by-side in a single row.

FIG. 8 is a perspective view of an exemplary dump diffuser.

FIG. 9 is a side view of the example dump diffuser of FIG. 10.

Fig. 10 is a front cross-sectional view of a second exemplary acoustophoresis device according to the present disclosure. The device also has an acoustic chamber with a horizontal cross-sectional area greater than its vertical cross-sectional area.

Fig. 11 is a front external perspective view of the device of fig. 10.

Fig. 12 is a rear external perspective view of the device of fig. 10.

Fig. 13 is a graph showing the percent reduction/clarification over time (upper line) and phase contrast microscopy (lower line) for a 1.5% yeast mixture flowing at 810 milliliters per minute (mL/minute) through a 9 inch by 3 inch by 2 inch (length by width by height) acoustophoresis device according to the present disclosure without a dump diffuser and operating at 60 volts, 80 volts, and 100 volts. The lighter lines with dots indicate that the device uses a membrane, while the darker lines with squares indicate that the device does not use a membrane.

Fig. 14 is a graph showing the percent reduction/clarification over time (upper line) and phase contrast microscopy (lower line) for a 3% yeast mixture flowing at 810 milliliters/minute through a 9 inch by 3 inch by 2 inch (length by width by height) acoustophoresis device according to the present disclosure without a dump diffuser, with five Alternating Tangential Flow (ATF) membranes, and operating at 60 volts, 80 volts, and 100 volts.

Fig. 15 is a graph showing the percent reduction/clarification over time of a 3% yeast mixture flowing at 810 milliliters/minute through a 9 inch by 3 inch by 2 inch (length by width by height) acoustophoresis device according to the present disclosure having five Alternating Tangential Flow (ATF) membranes and operating at 80 volts and 100 volts. The shallower lines indicate devices using dump diffusers with a double row hole role according to the present disclosure, while the deeper lines indicate devices using half-plate dump diffusers according to the present disclosure.

Fig. 16 is a graph showing the percent reduction/clarification over time (upper line) and phase contrast microscopy (lower line) for a 3% yeast mixture flowing at 810 milliliters/minute through a 9 inch by 3 inch by 2 inch (length by width by height) acoustophoresis device using a half-plate dump diffuser, five Alternating Tangential Flow (ATF) membranes, and using a transducer assembly with two rows of transducers with the top row closed and the bottom row operating at 100 volts according to the present disclosure.

Fig. 17 is an external perspective view of a third exemplary acoustophoresis device according to the present disclosure. This embodiment specifically uses a dump diffuser in which the fluid enters the dump diffuser plenum along two different axes rather than just one (as in fig. 1).

Fig. 18 is a side cross-sectional perspective view of the device of fig. 17.

Fig. 19 is a side sectional view of the acoustophoresis device of fig. 19, illustrating additional aspects with fig. 18.

Fig. 20 is a front view of the acoustophoresis device of fig. 17, with transparent walls to show additional features.

Fig. 21 is an enlarged view of the flow chamber of the device of fig. 17.

FIG. 22 is an enlarged cross-sectional view of a transducer assembly of the acoustophoresis device of FIG. 17.

Fig. 23 is a cross-sectional view of a conventional ultrasonic transducer.

Fig. 24 is a cross-sectional view of an ultrasound transducer of the present disclosure. There is an air gap within the transducer and no backing layer or wear plate is present.

Fig. 25 is a cross-sectional view of an ultrasound transducer of the present disclosure. There is an air gap within the transducer and there is a backing layer and wear plate.

Fig. 26 is a graph showing the relationship between acoustic radiation force, gravity/buoyancy, and stokes' resistance and particle size. The horizontal axis is in micrometers (μm) and the vertical axis is in newtons (N).

FIG. 27 is a graph of electrical impedance magnitude versus frequency for a square transducer driven at different frequencies.

Fig. 28A illustrates a seven peak amplitude capture line configuration for the ultrasound transducer of the present disclosure. Fig. 28B is a perspective view illustrating a separator of the present disclosure. Showing the fluid flow direction and the capture line. Fig. 28C is a view of the fluid inlet looking in the direction of fluid flow (arrow 814) of fig. 28B, showing a capture node that captures a standing wave of particles. FIG. 28D is a view taken through the face of the transducer along the capture line structure along arrow 816 as shown in FIG. 28B.

Detailed Description

The present disclosure may be understood more readily by reference to the following detailed description of the desired embodiments and the examples included therein. The following specification and claims will refer to a number of terms which shall be defined to have the following meanings.

Although specific terms are used in the following description for the sake of clarity, these terms are intended to refer only to the particular structure selected for the illustrated embodiments in the drawings, and are not intended to define or limit the disclosure. In the following drawings and description, it should be noted that like reference numerals indicate components having similar functions.

The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The term "comprising" is used herein to require the presence of the recited component and to allow the presence of other components. The term "comprising" should be interpreted as including the term "consisting of," which only allows for the presence of the component and any impurities that may result from the manufacture of the component.

Numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement techniques used in the present application to determine the value.

All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (e.g., a range of "2 grams to 10 grams" is inclusive of the endpoints, 2 grams and 10 grams, and all intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they may be imprecise enough to include values near these ranges and/or values.

The modifier "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. The modifier "about" when used in connection with a range is also to be construed as disclosing the range defined by the absolute values of the two endpoints. For example, a range of "from about 2 to about 10" also discloses a range of "from 2 to 10". The term "about" may mean plus or minus 10% of the number referred to. For example, "about 10%" may mean a range of 9% to 11%, and "about 1" may mean 0.9 to 1.1.

It should be noted that many of the terms used herein are relative terms. For example, the terms "upper" and "lower" are positionally opposite to each other, i.e., an upper component is located higher than a lower component in a given direction, but these terms may be changed if the device is turned upside down. For a given structure, the terms "inlet" and "outlet" are relative to the fluid flowing through them, e.g., fluid flows into the structure through the inlet and out of the structure through the outlet. The terms "upstream" and "downstream" are relative to the direction of fluid flow through the various components, i.e., the flowing fluid flows through the upstream component before flowing through the downstream component. It should be noted that in a circuit, a first component may be described as being upstream and downstream of a second component.

The terms "horizontal" and "vertical" are used to indicate a direction relative to an absolute reference, i.e. the ground plane. However, these terms should not be construed as requiring structures to be absolutely parallel or absolutely perpendicular to each other. For example, the first and second vertical structures need not be parallel to each other. The terms "top" and "bottom" are used to refer to surfaces that are always higher at the top than at the bottom, relative to an absolute reference (i.e., the surface of the earth). The terms "upward" and "downward" are also relative to absolute reference; and upward tends to oppose the earth's gravity.

The term "parallel" should be interpreted as meaning that the distance between two surfaces remains approximately constant in the sense of hierarchy, rather than the strict mathematical sense that the surfaces will not intersect when extended to infinity.

The present application relates to "same order of magnitude". Two numbers are of the same order of magnitude if the quotient of the larger number divided by the smaller number is a value of at least 1 and less than 10.

The term "virus" refers to an infectious agent that is capable of replicating only within another living cell, and additionally exists in the form of a viral particle formed from a capsid that surrounds and contains DNA or RNA, and in some cases, a lipid envelope that surrounds the capsid.

The term "crystal" refers to a single crystal or polycrystalline material used as a piezoelectric material.

Acoustophoresis is a low power, pressure drop free, non-blocking solid state method for removing particles from a fluid dispersion: i.e. it is used to achieve separation more commonly with porous filters, but it does not have the disadvantages of filters. In particular, the acoustophoresis device of the present disclosure is suitable for use with a bioreactor and operates on a macro-scale for separation in high flow rate flow systems. The acoustophoretic device is designed to produce a high intensity multi-dimensional ultrasonic standing wave that results in acoustic radiation forces greater than the combined effects of fluid drag and buoyancy or gravity, and is therefore able to capture (i.e., remain stationary) the suspended phase (i.e., cells) allowing more time for the acoustic waves to increase particle concentration, agglomeration, and/or coalescence. This is a significant difference from previous methods, in which the particle trajectory is only changed by the action of acoustic radiation forces. Thus, in the present device, the radiation force is used as a filter to prevent target particles (e.g. biological cells) from traversing through the plane of the standing wave. The trapping capacity of the standing wave can be varied as desired, for example by varying the flow rate of the fluid, the acoustic radiation force and the shape of the acoustophoretic device, to maximize cell entrapment through trapping and sedimentation. The technology provides a green and sustainable alternative for separating the secondary phase, and energy cost is obviously reduced. Excellent particle separation efficiency has been demonstrated for particle sizes as small as 1 micron. The acoustophoresis device of the present invention has the ability to produce an ultrasonic standing wave field capable of trapping particles in a flow field having a flow rate greater than 1 milliliter per minute (mL/minute).

The sound field scattered from the particles forms a three-dimensional acoustic radiation force, which acts as a three-dimensional trapping field. When the particles are small relative to the wavelength, the acoustic radiation force is proportional to the particle volume (e.g., the cube of the radius). It is proportional to the frequency and the acoustic contrast factor. It also varies with acoustic energy (e.g., the square of the sound pressure amplitude). For harmonic excitation, the sinusoidal spatial variation of the force is responsible for driving the particles to stable positions within the standing wave. Particles are trapped within the acoustic standing wave field when the acoustic radiation force exerted on the particles is stronger than the combined effect of the fluid drag and buoyancy/gravity forces. The effect of the acoustic forces (i.e., transverse and axial acoustic forces) on the trapped particles forms tightly packed clusters by concentration, agglomeration, aggregation, agglomeration and/or coalescence of the particles, which continue to settle by enhanced gravity for particles heavier than the primary fluid or rise by enhanced buoyancy for particles lighter than the primary fluid when a critical size is reached. In addition, secondary interparticle forces, such as the Bjerkness forces, contribute to particle agglomeration.

Most biological cell types exhibit higher density and lower compressibility than the media in which they are suspended, such that the acoustic contrast factor between the cells and the media has a positive value. Thus, axial Acoustic Radiation Force (ARF) drives the cells towards the standing wave pressure node. The axial component of the acoustic radiation force drives cells with positive contrast factors to the pressure node plane, while cells or other particles with negative contrast factors are driven to the pressure anti-node plane. The radial or lateral component of the acoustic radiation force is the force that captures the cell. The radial or lateral component of the ARF is greater than the combined effect of fluid resistance and gravity. For minicells or emulsions, the resistance FD can be expressed as:

wherein U is_fAnd U_pIs the fluid and cell velocity, R_pIs the particle radius, μ_fAnd mu_pIs the dynamic viscosity of the fluid and the cells, and

is the ratio of the dynamic viscosity. Buoyancy force F_BExpressed as:

for cells to be captured in a multi-dimensional ultrasonic standing wave, the force balance or sum of force vectors on the cell can be assumed to be zero, so an expression for the transverse acoustic radiation force FLRF can be found, given by:

F_LRF＝F_D+F_B。

this equation can be used to estimate the magnitude of the lateral acoustic radiation force for a cell of known size and material properties and for a given flow rate.

One theoretical model for calculating acoustic radiation force is based on the formula developed by Gor' kov. The primary acoustic radiation force FA is defined as a function of the field potential U, F_A＝-▽(U)，

Wherein the field potential U is defined as

And f₁And f₂Are unipolar and bipolar contributions defined by

Where p is the acoustic pressure, u is the fluid particle velocity, and Λ is the cell density ρ_pAnd fluid density ρ_fA is the cell sound velocity c_pSpeed of sound c of fluid_fRatio of (A) to (B), V_oIs the cell volume, and<>indicating the time of averaging over the period of the wave.

The model of Gork' ov is for a single particle in a standing wave and is limited to particle sizes that are small relative to the wavelength of the acoustic field in the fluid and particle. It also does not take into account the effect of the viscosity of the fluid and particles on the radiation force. Thus, this model cannot be used in the macroscopic ultrasonic separator discussed herein because the particle clusters can grow considerably. A more complex and complete model of the acoustic radiation force, not limited by the particle size, is therefore used. The model implemented was based on the theoretical work of Yurii Ilinskii and Evgenia Zabolotskaya, as described in the AIP Conference records (Conference Proceedings), volume 1474-1, page 255-258 (2012). These models also include the effect of fluid and particle viscosity and therefore more accurately calculate acoustic radiation force. Additional internal models for calculating the acoustic trapping force of cylindrical objects have been developed, such as a "hockey puck" of trapped particles in a standing wave, which closely resembles a cylinder.

The lateral force of the total Acoustic Radiation Force (ARF) produced by the ultrasound transducer of the present disclosure is significant and sufficient to overcome the fluidic resistance. Thus, such a transverse ARF can be used to retain cells within the acoustic standing wave as the fluid flows through the standing wave. In addition, as described above, this effect of acoustic forces (i.e., lateral and axial acoustic forces) on the trapped particles results in the formation of tightly packed clusters through concentration, agglomeration, and/or coalescence of the particles, which settle due to enhanced gravitational (particles heavier than the primary fluid) or buoyancy (particles lighter than the primary fluid). Thus, relatively large solids of one material can be separated from smaller particles of a different material, the same material, and/or the host fluid by enhanced gravity separation.

The multi-dimensional standing wave generates acoustic radiation forces in an axial direction (i.e., in the direction of the standing wave, between the transducer and the reflector, perpendicular to the flow direction) and a lateral direction (i.e., in the flow direction). As the mixture flows through the acoustic chamber, the suspended particles experience a strong axial force component in the direction of the standing wave. Since this acoustic force is perpendicular to the flow direction and resistance, it will quickly move the particle to the pressure node plane or anti-node plane depending on the particle's contrast factor. The transverse acoustic radiation force then acts to move the concentrated particles towards the center of each planar node, causing coalescence or aggregation. The transverse acoustic radiation force component overcomes the fluid resistance, which allows the particle mass to grow continuously and then settle out of the mixture due to gravity. The resistance of each particle decreases as the size of the particle cluster increases and the acoustic radiation force of each particle decreases as the size of the particle cluster increases, either considered together or separately in the operation of the acoustic separator device. In at least some examples of the present disclosure, the lateral force component and the axial force component of the multi-dimensional acoustic standing wave are of the same order of magnitude. In this regard, it is noted that in a multi-dimensional acoustic standing wave, the axial force may have a different value than the lateral force, e.g., weaker or stronger, or may be equal or equivalent, but the lateral force of the multi-dimensional acoustic standing wave is greater than the lateral force of a planar standing wave, sometimes by two orders of magnitude or more.

The acoustophoretic filtration device can be used in at least two different ways. First, the standing wave can be used to capture specific biomolecules (e.g., phytochemicals, recombinant proteins, or monoclonal antibodies) and separate these desired products from the cells, cell debris, and culture medium. The specific biomolecules can then be transferred and collected for further processing. Second, the standing wave can be used to capture cells and cell debris present in the cell culture medium. Cells and cell debris with positive contrast factors move to the nodes of the standing wave (rather than the anti-nodes). Physical scrubbing of the cell culture medium also occurs as cells and cell debris condense at the nodes of the standing wave, whereby more cells are captured by contact with cells already held within the standing wave. This generally separates the cells and cell debris from the cell culture medium. When the cells in the standing wave agglomerate to the point where mass can no longer be maintained by the acoustic wave, the aggregated cells and cell debris that have been captured can settle out of the fluid stream by gravity and can be collected separately. To assist this gravitational settling of cells and cell debris, the standing wave may be interrupted to allow all cells to settle out of the filtered fluid stream. This process is useful for dewatering. The particular biomolecule may have been previously removed or retained in the fluid stream (i.e., cell culture medium).

In the present disclosure, perfusion bioreactors may also be used to produce cells that may then be used for various applications, including cell therapy. In this type of method, biological cells used in cell therapy are cultured and expanded in a bioreactor (i.e., the number of cells in the bioreactor is increased by cell propagation). These cells may be lymphocytes, such as T cells (e.g., regulatory T cells (Tregs), Jurkat T cells), B cells, or NK cells; their precursors, such as Peripheral Blood Mononuclear Cells (PBMCs); and so on. In a perfusion bioreactor, a cell culture medium (also called the main fluid) containing some cells is sent from the bioreactor to a filtration device that generates an acoustic standing wave. Most of the cells are captured and held in the acoustic standing wave, while the remaining host fluid and other cells in the host fluid are returned to the bioreactor. As the number of captured cells increases, they form larger clusters that settle out of the acoustic standing wave at critical dimensions due to gravity. The clusters may fall outside the acoustic standing wave region, e.g., below the acoustic standing wave, to a concentrate outlet from which cells may be recovered for cell therapy. Only a small fraction of the cells are captured and removed from the bioreactor through the concentrate outlet, and the remainder continues to multiply in the bioreactor, allowing for continuous production and recovery of the desired cells.

In these applications, the acoustophoresis device of the present disclosure may act as a cell retention device. The systems described herein operate at a range of cell recirculation rates, effectively maintaining cells within a range of perfusion (or media removal) rates, and can be tuned to either fully retain or selectively pass a percentage of cells through fluid flow rate, transducer power or frequency manipulation. Both power and flow rate can be monitored and used as feedback for an automated control system.

The target cells may also be held in the flow chamber of the external filtering device by using standing acoustic waves so that other parts can be brought into close proximity and introduced for the purpose of altering the target cells. Such a procedure would include the capture of T cells and the subsequent introduction of modified lentiviral material with specific gene splicing, such that a lentivirus with specific gene splicing would transfect the T cells and generate chimeric antigen receptor T cells also known as CAR-T cells.

The acoustic filtering device of the present disclosure is designed to maintain a high intensity three-dimensional acoustic standing wave. The device is driven by a function generator and an amplifier (not shown). Device performance is monitored and controlled by a computer. It may sometimes be desirable to modulate the frequency or voltage amplitude of the standing wave due to acoustic streaming effects. The modulation may be done by amplitude modulation and/or by frequency modulation. The duty cycle of the standing wave propagation can also be used to achieve some result of material trapping. In other words, the beams may be turned on and off at different frequencies to achieve the desired results.

The acoustophoresis device of the present disclosure can handle higher flow rates and greater flow rates than conventional devices. A first exemplary embodiment of an acoustophoretic device 100 for separating secondary fluid or particles from primary/primary fluid is shown in fig. 1 and 2. Fig. 1 is an external perspective view, and fig. 2 is a front cross-sectional view of the device. The design of the device provides a vertical plane or line of flow symmetry so that a more uniform flow of fluid occurs through the device.

Referring first to fig. 1, an acoustophoretic device 100 is formed from a sidewall 110. As shown here, the sidewall 110 has a rectangular shape such that the device has a first side end 122; a second side end 124 spaced apart from the first side end 122 and opposite the first side end 122; a front side 126; a rear side 128 spaced apart from the front side 126 and opposite the front side 126; a tip 130; and a bottom end 132 spaced apart from the top end 130 and opposite the top end 130. A support frame 160 for the device is also shown here. The legs 162 are shown extending from the support frame. The support frame may be integral with the apparatus 100 or a structure separate from the apparatus 100.

Referring now to fig. 2, the top wall 140 is located on top of the side wall 110 and the base 150 is located below the side wall 110. The side walls 110, top wall 140, and base 150 together enclose the interior space 107. At least one concentrate outlet 116 is located at the bottom end of the apparatus 100. As will be explained further herein, the concentrated particles will exit the interior space 107 through the concentrate outlet. The base is shown with two inclined walls 152, the walls 152 sloping downwardly to the concentrate outlet 116. Note that due to the cross-sectional view, these walls 152 appear as straight lines, whereas in three dimensions the walls are conical.

At the first lateral end 122 there is at least one fluid inlet 112 allowing fluid to enter the inner space 107 from outside the device 100. As shown here, there are two fluid inlets 112, one on each of the lateral ends 122, 124. At least one fluid outlet 114 is present at the top end of the device 100. As will be explained further herein, the fluid will exit the interior space 107 through the fluid outlet. The fluid inlet 112 and the fluid outlet 114 are also visible in fig. 1.

Referring now to fig. 1 and 2 together, at least one ultrasonic transducer 106 and at least one reflector 108 are located on opposite sides of the interior space, and an acoustic chamber 120 is present therebetween. As shown here, three ultrasound transducers 106 are located on the back side 128 of the device and the reflector 108 is located on the front side 126 of the device.

It should be noted that the volume of the acoustic chamber 120 and the interior space 107 are not coextensive. The volume of the acoustic chamber is defined by the side walls 110. Conversely, the interior space 107 also includes space from the top wall 140 and the base 150. It should also be noted that the sloped wall 152 has an interior angle a measured relative to a horizontal plane (defined herein by the acoustic bottom 121), where the angle a is about 10 ° to about 60 °, including about 30 ° to about 45 °, in embodiments.

Still referring to fig. 1 and 2 together, the acoustic chamber 120 has a length 101 between a first lateral end and a second lateral end; a width 103 between the front side and the back side; and a height 105 defined by the height of the ultrasonic transducer. Thus, length 101 and width 103 define a planar cross-sectional area (i.e., a horizontal cross-sectional area), while width 103 and height 105 define a lateral cross-sectional area (i.e., a vertical cross-sectional area). As seen here, the planar cross-sectional area is greater than the lateral cross-sectional area.

In particular embodiments, the acoustic chamber 120 may have a volume of at least 40 cubic inches such that a large volume of fluid may be processed within the acoustic chamber. In this regard, the fluid inlet 112 of the device may be configured to allow fluid to enter the acoustic chamber at a flow rate of at least 800 milliliters per minute (mL/min).

Fig. 3A-3D are front views of four different top walls 140 that may be used in an acoustophoresis device. The top wall forms a fluid path from the acoustic chamber 120 of the acoustophoretic device to the fluid outlet 114 at the top of the device. In these figures, each top wall 140 has an inner surface 142 and an outer surface 144. It should be noted that other top wall shapes and configurations may also be used, as will be seen later herein. In a particular embodiment, the fluid outlet may be configured to allow fluid to flow out of the acoustic chamber at a flow rate of at least 25 mL/min.

Fig. 3A shows the top wall 140 having a flat outer surface 144 that has a different angle than the flat inner surface 142, such that the top wall is thicker near the fluid outlet 114. The inner surface 142 extends from the fluid outlet 114 to a length 141 that is approximately the same as the length 101 of the acoustic chamber 107 of fig. 2.

Fig. 3B shows the top wall 140 with the flat outer surface 144 having the same angle as the flat inner surface 142, i.e. the top wall has a constant thickness. Likewise, the inner surface 142 extends from the fluid outlet 114 to a length 141 that is substantially the same as the length 101 of the acoustic chamber 107 of fig. 2.

Figure 3C shows the top wall 140 having an inner surface 142 and an outer surface 144 with hypocycloidal curves. Likewise, the inner surface 142 extends from the fluid outlet 114 to a length 141 that is substantially the same as the length 101 of the acoustic chamber 107 of fig. 2. The hypocycloidal shape of the inner surface causes the fluid path to narrow up to the fluid outlet 114 very quickly.

Fig. 3D shows the top wall 140 having an inner surface 142, the inner surface 142 extending only to a short length 141. In this embodiment, the length 141 is much shorter than the length 101 of the acoustic chamber, so that fluid is only expelled from the central region of the acoustic chamber to the fluid outlet 114.

Fig. 4A-4D are front views of four different bases 150 that may be used in an acoustophoresis device. The base forms a fluid path from the acoustic chamber 120 of the acoustophoretic device to the concentrate outlet 116 at the bottom of the device. It should be noted that other shapes and configurations may also be used for the base, as will be seen later herein. The legs 162 are also visible here, although they need not be integral with the base.

Fig. 4A shows a base with one concentrate outlet 116. Two inclined walls 152 lead from the sides of the acoustic chamber to the concentrate outlet 116.

Fig. 4B also shows the base having one concentrate outlet 116. Here the sloped wall 152 is shallower than in fig. 4A.

Fig. 4C shows a base with three concentrate outlets 116. The outlets 116 are evenly spaced from one another. The inclined wall 152 leads to each concentrate outlet.

Fig. 4D shows a base with four concentrate outlets. The outlets 116 are evenly spaced from one another. The inclined wall 152 leads to each concentrate outlet.

Fig. 5A-5C illustrate three different embodiments of a transducer assembly formed from a plurality of ultrasonic transducers, which can be used in the acoustophoretic devices of the present disclosure. Multiple transducers allow for greater particle capture efficiency, especially when the transducers have different resonant frequencies, thereby capturing a greater range of particle (e.g., cell) sizes. The transducer assemblies 170 are oriented along the length of the acoustic chamber and the side ends 122,124 and the tip 130 are labeled in each figure for orientation of the assemblies.

Fig. 5A shows a piezoelectric transducer assembly 170 that includes a total of six rectangular transducers 106 arranged in two rows 172,174 of three transducers each. It is envisaged that in such an arrangement the transducers together span the entire width and height of the acoustic chamber.

Fig. 5B shows a piezoelectric transducer assembly 170, which piezoelectric transducer assembly 170 includes a total of six square transducers 106 arranged side-by-side in a single row 172. The transducers collectively span the entire width of the transducer assembly, but not the entire height of the transducer assembly.

Fig. 5C shows a piezoelectric transducer assembly 170, which piezoelectric transducer assembly 170 includes a total of five rectangular transducers 106 arranged in two rows, with an upper row 174 including two transducers and a lower row 172 including three transducers. It should be noted that the transducers in the upper row 174 are staggered/offset relative to the transducers in the lower row 172. One benefit of this arrangement is shown in fig. 6, which indicates the location of the multi-dimensional acoustic standing wave 176 to be generated by the transducer. As can be seen here, the staggering of the transducers causes the acoustic standing waves 176 to also be staggered so that the standing waves generated by the upper row 174 are staggered with the standing waves in the lower row 172. As previously described, particles/cells trapped in the multi-dimensional acoustic standing wave will agglomerate and form clusters that eventually settle out of the standing wave and flow down to the concentrate outlet. This staggering allows the downward falling clusters from the upper row 174 to avoid passing through the standing acoustic waves created in the lower row 172 so that the clusters formed in the lower row 172 are not disturbed or disrupted.

It is also contemplated that multiple transducers may be arranged in series in a single row, such as in fig. 7A and 7B. FIG. 7A shows a transducer assembly 170 that includes a total of three rectangular transducers 106 arranged side-by-side in a single row. FIG. 7B shows a transducer assembly 170 having a total of eight square transducers 106 arranged side-by-side in a single row.

Returning now to fig. 1 and 2, the device 100 has symmetrical fluid inlets 112 arranged on opposite sides of the acoustic chamber. In a particular embodiment, these inlets are in the form of dump diffusers (dump diffusers) that provide a more uniform flow of the mixture of primary fluid and particles into the acoustic chamber.

Briefly, each dump diffuser includes an inlet through which the primary fluid/secondary fluid or particulate mixture flows into the hollow chamber. This mixture fills the chamber in the dump diffuser, which reduces/eliminates flow pulsations and flow non-uniformities caused by the pump, hose and horizontal inlet flow in situations where the effects of gravity dominate. The mixture then flows horizontally out of the dump diffuser and into the acoustic chamber 107. The acoustic chamber (black arrows) into which the dump diffuser brings the heavier mixture is located above the bottom of the chamber and below the ultrasonic transducer 106 and the clusters of nodes formed in the acoustic standing wave. This minimizes any interference of the influent material with the clusters.

The structure and operation of the dump diffuser is shown in fig. 8 and 9. FIG. 8 is a perspective view of the dump diffuser 530 with the front panel removed, showing the interior and exterior of the dump diffuser. FIG. 9 is a perspective view of a front plate of the dump diffuser.

Beginning with fig. 9, dump diffuser 530 includes a housing 531 having an upper end 532, an opposite lower end 534, two sides 538, a front surface 536, and a rear surface 539. A hollow chamber 540 exists within the housing 531. The dump diffuser also includes an inlet port 542 that receives the mixture and leads to the chamber 540. Inlet ports 542 are present on the upper end and side 538 of the housing; here two inlet ports are visible. Fig. 11 is a photograph of the front plate 546 mounted on the front surface 536 of the housing. As shown here, the diffuser outlets 544 are located on the lower end 534 and are in the form of two rows of holes, although these could also be in the form of slots.

Referring now to fig. 2 and 8, in use, a primary fluid/secondary fluid or particulate mixture enters dump diffuser 530 through inlet port 542 and fills chamber 540. The pressure then pushes the mixture out evenly through the diffuser outlet 544. These diffuser outlets 544 also pass through the sidewall 110 of the apparatus 100 and may also be considered fluid inlets 112 into the interior space 107. The diffuser outlet is placed above the bottom 121 of the acoustic chamber. In an embodiment, the diffuser outlet is located at a height 515 above the chamber bottom 121 that is between 0% and 100% of the height 105 of the acoustic chamber, and more specifically between 5% and 25% of the height of the acoustic chamber. The diffuser outlet 544 provides a flow direction parallel to the axial direction of the acoustic standing wave generated by the ultrasonic transducer. The plurality of diffuser outlets are also arranged such that they are in opposite positions such that the horizontal velocity of the fluid will be reduced to zero at the centre of the acoustic chamber.

The flow streamlines through the acoustic chamber are ideally symmetrical as this minimizes non-uniformity, turbulence, circulation and turbulence of the clusters falling to the concentrate outlet 116 to be collected. Symmetry may also maximize inlet flow distribution and gravity during particle collection. Since it is heavier than the permeate exiting at the top of the device, the (relatively) heavy entering mixture enters near the bottom of the acoustic chamber. The symmetrical inlet also ensures that the incoming mixture spreads at the bottom of the chamber due to gravity and provides a near uniform velocity profile from bottom to top. Due to the two opposing inlet flows, the horizontal velocity of the mixture decreases towards zero as it approaches the center of the acoustic chamber and may be equal to zero. In this example, uniform velocity facilitates separation and collection of results. The uniform velocity avoids peak velocities that may prevent the acoustic standing wave from overcoming particle drag that may prevent the cluster from growing and continuously leaving the acoustic standing wave by gravity or buoyancy.

As the particle clusters settle, the axial acoustic forces associated with the standing wave keep the clusters intact. This effect ensures a fast fall of the clusters with high terminal velocity, of the order of about 1 cm/sec. This rate is very fast compared to the chamber flow rate, which is on the order of 0.1 cm/sec to 0.3 cm/sec. The shallow wall angle of the base means that the distance that the cylindrical particle clusters descend before leaving the acoustic chamber can be very short, so that little dispersion of the clusters occurs. Ideally, the system operates with 3 to 12 capture lines per square inch of transducer. The symmetry, minimal flow disturbance of the central collection area and shallow collector walls provide good particle collection.

A second exemplary embodiment of an acoustophoretic device 600 is shown in fig. 10-12. Fig. 10 is a front cross-sectional view. Fig. 11 is an external perspective view of the front of the device. Fig. 12 is an external perspective view of the rear of the device. In this arrangement, the planar cross-sectional area of the acoustic chamber is also greater than the lateral cross-sectional area of the acoustic chamber.

Beginning with fig. 10, an acoustophoretic device 600 shares many similarities with the device 100 of fig. 1. The device 600 has a first lateral end 122 and an opposite second lateral end 124. There are side walls 110, a top wall 140 and a base 150 to define an interior space 107. A dump diffuser 530 is present on each side end 122,124, which serves as a fluid inlet 112 to the interior space 107 of the device. Here, the top wall 140 includes a parabolic inner surface 142 that leads to the fluid outlet 114 at the top end 130. Two concentrate outlets 116 exist in the base 152, with the sloped wall 152 leading to each outlet at the bottom end 132. Five ultrasonic transducers 106 are shown, the rectangles indicating piezoelectric material for generating multi-dimensional acoustic standing waves.

One notable aspect of the device (more evident in fig. 10) is the placement of dump diffuser 530/fluid inlet 112 relative to ultrasound transducer 106. As seen here, the fluid inlet 112 has a height 113 that is about 60% of the height 176 of the piezoelectric material. Moreover, the base 111 of the fluid inlet 112 is positioned along, i.e., aligned with, the base 177 of piezoelectric material. In an embodiment, the height of the fluid inlet may be about 5% to about 75% of the height of the piezoelectric material.

Referring now to fig. 11, the reflector 108 is visible on the front side 126 of the device 600. Further, it can be seen that the fluid outlet 114 and concentrate outlet 116 lead from the top/bottom end of the interior space to the rear side 128 of the device.

Also visible in FIG. 11 is an alternative configuration of the dump diffuser 530. The dump diffuser in fig. 8 has two inlet flow ports 542 located on the side 538. By contrast, the dump diffuser shown in fig. 8 has three inlet flow ports 542. Two inlet flow ports 542 are located on the side 538. The third inlet flow port 542 is located on an aft face 539 on the upper end 532 of the diffuser.

Referring now to fig. 12, an ultrasound transducer assembly 170 is seen. Five connectors 171 are visible, one connector 171 corresponding to one transducer 106 visible in fig. 10.

Experiments were performed using the acoustophoresis device of fig. 1. The dimensions of the acoustic chamber are 9 inches by 3 inches by 2 inches (length by width by height). As shown in fig. 5A, the device has six transducers arranged in two rows. The experiment measures the percent reduction/clarification and Packed Cell Mass (PCM) of the influent/yeast mixture over time.

In the graph in fig. 13, the yeast mixture is 1.5% yeast and flows through the device at a flow rate of 810 milliliters per minute (mL/minute). The inlet of the device is not part of the dump diffuser (i.e., there is no front plate as shown in fig. 9). The ultrasonic transducers of the device were operated at 60 volts, 80 volts and 100 volts. The apparatus operates using an Acoustically Transparent Film (ATF), and also operates without using any such ATF. As can be seen in fig. 28, the PCM (lower line) measured at 60 volts with and without ATF is about 20% -28%, about 28% -35% at 80 volts and about 35% -38% at 100 volts. The percent reduction/clarification of the mixture with and without ATF (upper line) was about 75% -80% at 60 volts, about 80% -90% at 80 volts and about 85% -90% at 100 volts, although these devices appear to have slightly better separation/clarification efficiency without ATF. Both the percent reduction and the PCM value indicate that operating at higher voltages results in better separation of water and yeast.

In the graph of fig. 14, the yeast mixture was 3.0% yeast and flowed through the device at a flow rate of 810 ml/min. Also, the inlet of the device is not part of the dump diffuser. The ultrasonic transducers of the device were operated at 60 volts, 80 volts and 100 volts. Five acoustically transparent membranes (ATFs) were used to operate these devices. Here, the measured PCM (lower line) is about 18% -25% at 60 volts, about 25% -29% at 80 volts and about 29% -30% at 100 volts. The percent reduction/clarification of the mixture (upper line) is about 55% to 75% at 60 volts, about 75% to 82% at 80 volts, and about 75% to 82% at 100 volts.

In the graph of fig. 15, the yeast mixture was 3.0% yeast and flowed through the device at a flow rate of 810 ml/min. The inlet of the device is part of a dump diffuser with a front plate. The darker line represents the dump diffuser, where the front plate is configured as a half plate (i.e., one large slot at the bottom of the front plate), and the lighter line represents the dump diffuser where the front plate has two rows of holes. The ultrasonic transducers of the device were operated at 80 volts and 100 volts. These devices operate using five ATFs. For the two rows of well front plates, the PCM (lower line) measured at 80 volts was about 20% -30%, and measured at 100 volts was about 30% -35%. The PCM for the half-panel front panel is about 20% -30% at 80 volts and about 30% -35% at 100 volts. Notably, once the top row of ultrasonic sensors is closed, there is no significant change in the PCM for the front half-panel. The percent reduction/clarification of the mixture for the two rows of wells (upper line) was about 68% -80% at 80 volts and about 85% -90% at 100 volts when the top row was open. The percent reduction/clarification of the mixture for the half-panel front panel is about 68% -80% at 80 volts, about 85% -90% at 100 volts with the top row open, and then about 65% -75% at 100 volts with the top row closed.

In the graph of fig. 16, the yeast mixture was 3.0% yeast and flowed through the device at a flow rate of 810 ml/min. The inlet of the device is part of the dump diffuser, using a half-plate front plate. Only the bottom row of transducers is used and the ultrasound transducers of the device operate at 100 volts. The PCM measured (lower line) was about 18% -32% and the percent reduction/clarification of the mixture (upper line) was about 60% -78%.

A third exemplary embodiment of an acoustophoretic device 700 is shown in fig. 17-22. Fig. 17 is an external perspective view. Fig. 18 is a perspective side sectional view of the device. Fig. 19 is a side sectional view of the device. Fig. 20 is a front cross-sectional view of the device. Figure 21 is an enlarged perspective side cross-sectional view of the acoustic chamber. Fig. 22 is an enlarged perspective side cross-sectional view of an ultrasound transducer. This particular embodiment is also constructed in a modular fashion from multiple components.

Beginning with fig. 17, an acoustophoresis device 700 bears some similarities to the device shown in fig. 1 and 10. The device 700 has a first lateral end 122 and an opposite second lateral end 124. There are side walls 110, a top wall 140 and a base 150 to define an interior space 107. A dump diffuser 530 is present on each side end 122,124, which serves as a fluid inlet 112 to the interior space 107 of the device. The dump diffuser here has three inlet flow ports 542, similar to that described in the apparatus of fig. 10. The top wall 140 includes a conical inner surface 142 that leads to the fluid outlet 114 at the tip 130. The concentrate outlet 116 is present in the base 150, with the tapered surface leading to the concentrate outlet at the bottom end 132. An ultrasound transducer 106 is present on the back side and a reflector 108 is present on the front side opposite the transducer.

Referring now to fig. 18, it can be seen that the fluid outlet 114 and the concentrate outlet 116 lead from the top/bottom end of the interior space 107 to one side end 124 of the device, i.e. there is a common side of the fluid inlet 112. As described above, the fluid outlet 112 generally allows for the recovery of clarified fluid from the interior space 107. The concentrate outlet 116 generally allows for the recovery or collection of particles, cells.

Referring now to fig. 19, the inner surface 142 of the top wall 140 leading to the fluid outlet 114 can be seen, as can the inclined wall 152 leading to the concentrate outlet 116. The bottom of the acoustic chamber is indicated by dashed line 121 and the top of the acoustic chamber is indicated by dashed line 119. The inner surface 142 has an inner angle B measured relative to the imaginary line 119, wherein the angle B is in embodiments from about 11 ° to about 60 °, including from about 30 ° to about 45 °. Similarly, the sloped wall 152 has an angle a relative to the imaginary line 121, where the angle a is about 11 ° to about 60 °, including about 30 ° to about 45 °, in embodiments. An O-ring 180 may be disposed between the top wall/base and the acoustic chamber to provide a fluid seal therebetween.

Referring now to fig. 20, it is again seen that the fluid outlet 114 and the concentrate outlet 116 lead from the top/bottom end of the inner space 107 to one of the lateral ends 124 of the device, i.e. the common side where the fluid inlet 112 is present.

The piezoelectric material 178 of the ultrasonic transducer can be seen, as well as the fluid inlet 112 from dump diffuser 530 into the acoustic chamber 107. A hollow chamber 540 is also visible. The piezoelectric material 178 has a height 176. Fluid inlet 112 also has a height 113. The height 113 of the fluid inlet 112 is about 60% of the height 176 of the piezoelectric material 178. In an embodiment, the height of the fluid inlet may be about 5% to about 75% of the height of the piezoelectric material. Again, the bottom edge 111 of the fluid inlet 112 is aligned with the bottom edge 177 of the piezoelectric material.

Turning now to fig. 21, an enlarged view of the acoustic chamber 107 of the device 700 can be seen, the acoustic chamber 107 being sandwiched between the ultrasound transducer 106 and the reflector 108. There is a very small gap (e.g., 0.010 inches) between fluid inlet 112 and transducer 106. The gap may be filled with, for example, an O-ring, as shown in fig. 19. There is also a very short gap (e.g., <0.025 inches) between the bottom edge 177 of the piezoelectric material of the transducer and the sloped wall 152 of the base.

Fig. 22 provides an enlarged cross-sectional view of the ultrasound transducer 106. As shown in fig. 18 and 19, the ultrasound transducer 106 is typically located in a sidewall of the device. As shown here, the ultrasound transducer includes a housing 190. There is an air gap 194 within the housing of the transducer. The connector 191 is present and spaced from the piezoelectric material 178 in crystal form. The piezoelectric material 178 is attached to the housing using a potting material 192 such as epoxy. An adhesive backing film 193, for example made of Polyetheretherketone (PEEK), is then attached to the piezoelectric material 178 and the outer surface of the housing. The film may act as an abrasion resistant layer. The wear layer typically has a thickness of a half wavelength or less (e.g., 0.050 inches). Additional features of the ultrasound transducer used in the present device will be explained in more detail herein.

One particular application disclosed herein for an acoustophoretic device is the treatment of bioreactor materials. The fluid flow entering these devices is a mixture of primary/primary fluid (e.g., water, cell culture medium) and secondary particles. The secondary particles may comprise cells and specific materials, such as biomolecules (e.g. recombinant proteins or monoclonal antibodies or viruses). These devices can be used to aggregate larger particles, such as cells, in a mixture so that there are two different streams exiting the device. First, a flow of aggregated cells and some fluid may be discharged through the concentrate outlet. Second, a clarified fluid stream containing a particular substance (e.g., a biomolecule) may be discharged through the fluid outlet. Depending on the material that it is desired to recover, either of these two streams exiting the apparatus may be recycled to the bioreactor.

The acoustophoresis device of the present disclosure using three-dimensional acoustic standing waves may also be coupled with standard filtration processes upstream or downstream, such as depth filtration using diatomaceous earth, Tangential Flow Filtration (TFF), or other physical filtration processes, as desired.

Desirably, the flow rate through the device of the present disclosure can be a minimum of 1 milliliter per minute (mL/min), or a minimum of about 800mL/min, and more desirably, higher flow rates can also be achieved. In alternative units, these flow rates may be about 0.005mL/min, or about 4.5mL/min/cm2 per square centimeter of the cross-sectional area of the acoustic chamber. This is true for batch, fed-batch and perfusion bioreactors.

It may be helpful to explain now how to generate a multi-dimensional acoustic standing wave, in particular a three-dimensional acoustic standing wave. The multi-dimensional acoustic standing waves required for particle collection are obtained by driving the ultrasonic transducer at a frequency that creates the acoustic standing waves and excites the fundamental 3D vibration mode of the transducer crystal. Perturbing the piezoelectric crystal in an ultrasound transducer in a multimode manner allows for the generation of a multi-dimensional acoustic standing wave. Piezoelectric crystals can be specifically designed to deform in a multimode manner at a design frequency, allowing a multi-dimensional acoustic standing wave to be generated. The multi-dimensional acoustic standing waves may be generated by different modes of the piezoelectric crystal, such as a 3x3 mode that would generate a multi-dimensional acoustic standing wave. Multiple multi-dimensional acoustic standing waves may also be generated by allowing the piezoelectric crystal to vibrate in many different modes (modes flaps). Thus, the crystal may excite multiple modes, such as a 0x0 mode (i.e., piston mode) to 1x1, 2x2, 1x3, 3x1, 3x3, and other higher order modes, and then cycle back through the lower modes of the crystal (not necessarily in direct order). This switching or dithering of the crystal between modes allows for the generation of various multi-dimensional waveforms and a single piston mode shape in a given time.

Some further explanation of the ultrasound transducers used in the devices, systems, and methods of the present disclosure may also be helpful. In this regard, the transducer uses a piezoelectric crystal, typically made of PZT-8 (lead zirconate titanate). Such crystals may have a diameter of 1 inch and a nominal resonant frequency of 2MHz, and may also have larger dimensions. Each ultrasound transducer module may have only one crystal, or may have multiple crystals, each serving as an independent ultrasound transducer and controllable by one or more amplifiers. The crystals may be square, rectangular, irregular polygonal, or generally any shape. The transducer is used to generate a pressure field that generates forces of the same order of magnitude in a direction orthogonal (transverse) to the direction of the standing wave as well as in the direction of the standing wave (axial).

Fig. 23 is a cross-sectional view of a conventional ultrasonic transducer. The transducer has a wear plate 50 at the bottom end, an epoxy layer 52, a ceramic crystal 54 (made of, for example, PZT), an epoxy layer 56, and a backing layer 58. On either side of the ceramic crystal there are electrodes: a positive electrode 61 and a negative electrode 63. An epoxy layer 56 attaches a backing layer 58 to the crystal 54. The entire assembly is housed in a housing 60, which housing 60 may be made of, for example, aluminum. An electrical adapter 62 provides a connection for wires to pass through the housing and connect to leads (not shown) attached to the crystal 54. Typically, the backing layer is designed to increase damping and produce a broadband transducer that is uniformly displaced over a wide frequency range, and is designed to suppress excitation at specific eigenmodes of vibration. The wear plates are typically designed as impedance transformers to better match the characteristic impedance of the medium into which the transducers radiate.

Fig. 24 is a cross-sectional view of an ultrasound transducer 81 of the present disclosure. The transducer 81 is shaped as a disk or plate and has an aluminum housing 82. Piezoelectric crystals are blocks of perovskite ceramic crystals, each of which is composed of a larger divalent metal ion (typically lead or barium) and a small tetravalent metal ion (typically titanium or zirconium) in the lattice of the O2-ion. By way of example, a PZT (lead zirconate titanate) crystal 86 defines the bottom end of the transducer and is exposed from the exterior of the housing. The crystal is supported at its periphery by a small resilient layer 98 (e.g., silicone or similar material) located between the crystal and the housing. In other words, there is no wear layer. In a particular embodiment, the crystal is an irregular polygon, and in a further embodiment is an asymmetric irregular polygon.

The screw 88 threadably attaches the aluminum top plate 82a of the housing to the main body 82b of the housing. The top plate includes a connector 84 for powering the transducer. The top surface of PZT crystal 86 is connected to positive and negative electrodes 90, 92 separated by insulating material 94. The electrodes may be made of any conductive material, such as silver or nickel. Power is supplied to the PZT crystal 86 through electrodes on the crystal. Note that the crystal 86 has no backing layer or epoxy layer. In other words, there is an air gap 87 in the transducer between the aluminum top plate 82a and the crystal 86 (i.e., the air gap is completely empty). As shown in fig. 5, a minimal backing 58 and/or wear plate 50 may be provided in some embodiments.

The transducer design can affect the performance of the system. A typical transducer is a layered structure in which a ceramic crystal is bonded to a backing layer and a wear plate. Because transducers have a high mechanical impedance presented by standing waves, conventional wear plate design criteria (e.g., half-wavelength thickness for standing wave applications, or quarter-wavelength thickness for radiation applications) and fabrication methods may not be suitable. In contrast, in one transducer embodiment of the present disclosure, the absence of a wear plate or backing allows the crystal to vibrate at one of its eigenmodes (i.e., near the eigenfrequency) with a high Q factor. The vibrating ceramic crystal/disk is directly exposed to the fluid flowing through the flow chamber.

Removing the backing (e.g., venting the back of the crystal) also allows the ceramic crystal to vibrate in higher order vibrational modes with little damping (e.g., higher order modal displacement). In a transducer containing a crystal with a backing, the crystal vibrates like a piston with a more uniform displacement. Removing the backing can cause the crystal to vibrate in a non-uniform displacement mode. The higher the order number of the mode shape of the crystal, the more nodal lines the crystal may have. Higher order modal displacements of the crystal will produce more trapping lines, and while the dependence of the trapping lines on the nodes is not necessarily one-to-one, driving the crystal at higher frequencies will not necessarily produce more trapping lines.

In some embodiments, the crystal may have a backing that minimally affects the Q factor of the crystal (e.g., less than 5%). The backing may be made of a substantially acoustically transparent material, such as balsa wood, foam, or cork, which allows the crystal to vibrate at higher order modes and maintain a high Q factor while still providing some mechanical support for the crystal. The backing layer may be solid or may be a lattice with holes through the layer such that the lattice follows the nodes of the vibrating crystal in a particular higher order vibration mode, providing support at the node locations while allowing the rest of the crystal to vibrate freely. The goal of lattice work or acoustically transparent materials is to provide support without lowering the Q factor of the crystal or interfering with the excitation of a particular mode shape.

Direct crystal contact with the fluid also contributes to the high Q factor by avoiding the damping and energy absorbing effects of the epoxy and wear plates. Other embodiments may have wear plates or wear surfaces to prevent PZT containing lead from contacting primary fluid. This may be desirable, for example, in biological applications such as the separation of blood. Such applications may use wear resistant layers such as chromium, electrolytic nickel or electroless nickel. Chemical vapor deposition may also be used to apply layers of poly (p-xylylene) (e.g., parylene) or other polymers or polymer films. Organic and biocompatible coatings such as silicone or polyurethane may also be used as wear resistant surfaces.

Fig. 26 is a log-log graph (log y-axis, log x-axis) showing changes in acoustic radiation force, fluid resistance, and buoyancy with particle radius, and provides an explanation for separating particles using acoustic radiation force. Buoyancy is a force that is dependent on particle volume and therefore is negligible for micron-sized particle sizes, but grows and becomes significant for particle sizes on the order of hundreds of microns. The fluid resistance (stokes' resistance) varies linearly with the fluid velocity and therefore typically exceeds the buoyancy of micron-sized particles, but is negligible for larger sized particles of hundreds of microns. The acoustic radiation force scaling is different. When the particle size is small, the Gor' kov equation is accurate and the acoustic trapping force is proportional to the volume of the particle. Eventually, as the particle size increases, the acoustic radiation force no longer increases as the cube of the particle radius and will quickly disappear at some critical particle size. For further increasing the particle size, the magnitude of the radiation force is again increased, but in phase opposition (not shown). This pattern repeats for increasing particle size.

First, as the suspension flows through the system primarily with small micron-sized particles, the acoustic radiation force needs to balance the combined effects of fluid drag and buoyancy to cause the particles to be trapped in the standing wave. In fig. 26, this occurs at the particle size labeled Rc 1. The figure shows that all larger particles will also be captured. Thus, when small particles are trapped in the standing wave, particle coalescence/agglomeration/aggregation/agglomeration occurs, resulting in a continuous increase in effective particle size. When the particles are clustered, the total resistance to clustering is much lower than the sum of the resistances on the individual particles. In essence, as the particles agglomerate into clusters, they are shielded from fluid flow between each other and reduce the overall resistance of the clusters. As the particle cluster size increases, acoustic radiation forces reflect off the cluster such that the net acoustic radiation force per unit volume decreases. The acoustic lateral force on the particles may be different from the drag force so that the clusters remain stationary and increase in size. For example, the acoustic transverse force may be greater than the resistive force to allow particles to be captured, clustered, and increased in size.

The particle size increase continues until buoyancy becomes dominant, as represented by the second critical particle size Rc 2. The buoyancy per unit volume of the cluster remains constant with cluster size as it is a function of particle density, cluster concentration and the gravitational constant. Thus, as the cluster size increases, buoyancy on the cluster increases faster than the acoustic radiation force. At size Rc2, the particles will rise or fall depending on their relative density with respect to the primary fluid. At this size, the acoustic forces are secondary, gravity/buoyancy becomes dominant, and the particles naturally settle or rise from the primary fluid. Not all particles will escape and the remaining particles and new particles entering the acoustic chamber will continue to move to the three-dimensional nodal position, repeating the build-up and settling process. This phenomenon explains the rapid fall and rise of acoustic radiation force after exceeding the dimension Rc 2. Thus, fig. 6 explains how small particles can be continuously captured in a standing wave, grow into larger particles or clumps, and then eventually rise or settle due to increased buoyancy.

The size, shape and thickness of the transducer determine the displacement of the transducer at different excitation frequencies, which in turn affects the particle separation efficiency. Higher order modal displacements produce three dimensional acoustic standing waves with strong gradients in all directions in the acoustic field, thereby producing equally strong acoustic radiation forces in all directions, resulting in a plurality of trapping lines, where the number of trapping lines is related to the particular mode shape of the transducer.

FIG. 27 shows the measured electrical impedance magnitude of the transducer as a function of frequency near the 2.2MHz transducer resonance. The minimum in the transducer electrical impedance corresponds to the acoustic resonance of the water column and represents the potential operating frequency. Numerical modeling has shown that at these acoustic resonance frequencies, the transducer displacement curve changes significantly, directly affecting the acoustic standing wave and the resulting trapping force. Since the transducer operates near its thickness resonance, the displacements of the electrode surfaces are substantially out of phase. The typical displacement of the transducer electrodes is not uniform and varies based on the excitation frequency. Higher order transducer displacement modes result in higher trapping forces and multiple stable trapping lines for trapped particles.

To investigate the effect of the transducer displacement profile on acoustic trapping force and particle separation efficiency, the experiment was repeated ten times, all conditions being identical except for the excitation frequency. Ten consecutive acoustic resonance frequencies, represented by circled numbers 1-9 and the letter a in fig. 27, are used as excitation frequencies. The conditions were 30 minutes duration of the experiment, a 1000ppm oil concentration of about 5 microns SAE-30 oil droplets, a flow rate of 500ml/min and 20W applied power.

The capture line of the oil droplets was observed and characterized as the emulsion passed through the transducer. Characterization involves observing and patterning the number of capture lines that pass through the fluidic channel for seven of the ten resonant frequencies identified in fig. 27, as shown in fig. 28A.

Fig. 28B shows an isometric view of the system with the capture line position being determined. Fig. 28C is a view of the system as it would appear looking down at the portal along arrow 814. Fig. 28D is a view of the system as it would appear when looking directly at the transducer face along arrow 816.

The effect of the excitation frequency clearly determines the number of capture lines, which varies from a single capture line at the excitation frequency of the acoustic resonances 5 and 9 to nine capture lines for the acoustic resonance frequency 4. Four or five capture lines were observed at other excitation frequencies. Different displacement profiles of the transducer may produce different (more) capture lines in the standing wave, with more gradients in the displacement profile generally producing higher capture forces and more capture lines. It is to be noted that although the different capture line profiles shown in fig. 28A are obtained at the frequencies shown in fig. 27, these capture line profiles may also be obtained at different frequencies.

FIG. 28A shows different crystal vibration modes possible by driving the crystal to vibrate at different fundamental frequencies of vibration. The 3D mode of crystal vibration is propagated by the acoustic standing wave all the way through the fluid in the chamber to the reflector and back. The resulting multi-dimensional standing wave can be considered to comprise two components. The first component is a flat out-of-plane motion component (uniform displacement on the crystal surface) of the crystal that generates the standing wave, and the second component is a displacement amplitude variation in which peaks and valleys occur in both lateral directions of the crystal surface. Three-dimensional force gradients are created by standing waves. These three-dimensional force gradients cause lateral radiation forces that stop and trap particles against flow by overcoming viscous drag. In addition, the transverse radiation force is responsible for creating tightly packed clusters of particles. Thus, particle separation and gravity-driven collection depend on the creation of a multi-dimensional standing wave that can overcome particle resistance as the mixture flows through the acoustic standing wave. As schematically shown in fig. 28A, a plurality of particle clusters are formed along the capture line in the axial direction of the standing wave.

The present disclosure has been described with reference to exemplary embodiments. Modifications and alterations will occur to others upon reading and understanding the preceding detailed description. The disclosure is susceptible to all modifications and variations coming within the scope of the appended claims or the equivalents thereof.

Claims (20)

1. An acoustophoretic device, comprising:

an acoustic chamber comprising a first end and a second end opposite the first end;

an inlet at the first end and the second end, respectively;

at least one fluid outlet at the apex of the acoustic chamber;

at least one concentrate outlet at the bottom end of the acoustic chamber;

at least two ultrasonic transducers arranged side-by-side and coupled to the acoustic chamber and each configured to generate acoustic waves in the acoustic chamber between the inlets of the first and second ends; and

a reflector on the other side of the acoustic chamber relative to the at least two ultrasonic transducers;

when the at least two ultrasonic transducers are excited to a higher order mode of vibration, each of the at least two ultrasonic transducers cooperates with the reflector to generate acoustic waves in the acoustic chamber that can capture secondary fluids or particles in a mixture comprising a primary fluid such that the secondary fluids or particles agglomerate, aggregate, cluster, or coalesce together to be concentrated and exit the acoustic waves due to enhanced gravity or buoyancy; and is

The at least one concentrate outlet is configured to receive a concentrated secondary fluid or particulate and the at least one fluid outlet is configured to receive a clarified primary fluid.

2. The acoustophoretic device of claim 1, further comprising a fluid path through the acoustic chamber for receiving the mixture, the fluid path passing between the at least two ultrasonic transducers and the reflector.

3. The acoustophoretic device of claim 1, wherein at least one of the inlets to the acoustic chamber is part of a dump diffuser.

4. The acoustophoretic device of claim 3, wherein each of the at least two ultrasonic transducers comprises a piezoelectric material, and the at least one inlet comprises a height that spans 60% of a height of the piezoelectric material.

5. The acoustophoretic device of claim 4, wherein a base of the at least one inlet is positioned along a base of the piezoelectric material.

6. The acoustophoretic device of claim 1, wherein the inlets at the first and second ends of the acoustic chamber, respectively, allow the mixture to enter the acoustic chamber simultaneously via the first and second ends.

7. The acoustophoretic device of claim 3, further comprising a first sloped wall located below the at least one inlet and leading to the at least one concentrate outlet, wherein the first sloped wall includes an angle of 11 ° to 60 ° relative to a first horizontal plane.

8. The acoustophoretic device of claim 1, wherein the at least two ultrasonic transducers span at least a portion of the acoustic chamber.

9. The acoustophoretic device of claim 1, wherein the acoustic wave is a multi-dimensional acoustic standing wave comprising an axial force component and a lateral force component having the same order of magnitude.

10. The acoustophoretic device of claim 1, further comprising an acoustically transparent membrane on a face of the at least two ultrasonic transducers.

11. A method of separating a secondary fluid or particulate from a mixture comprising a primary fluid, comprising:

placing the mixture in an acoustophoresis device, the acoustophoresis device comprising:

an acoustic chamber comprising a first end and a second end opposite the first end;

an inlet at the first end and the second end, respectively;

at least one fluid outlet at the apex of the acoustic chamber;

at least one concentrate outlet at the bottom end of the acoustic chamber;

at least two ultrasonic transducers arranged side-by-side and coupled to the acoustic chamber and each configured to generate acoustic waves in the acoustic chamber between the inlets of the first and second ends;

a reflector on the other side of the acoustic chamber relative to the at least two ultrasonic transducers;

the at least two ultrasonic transducers and the reflector cooperate to generate sound waves in the acoustic chamber when the at least two ultrasonic transducers are excited to a higher order mode;

exciting each of the at least two transducers to a higher order mode of vibration to produce the acoustic wave in the acoustic chamber; and

capturing the secondary fluid or particles via the acoustic waves such that the secondary fluid or particles agglomerate, aggregate, cluster or coalesce together to be concentrated and separated from the primary fluid;

receiving a concentrated secondary fluid or particulate at the at least one concentrate outlet;

receiving clarified primary fluid at the at least one fluid outlet.

12. The method of claim 11, further comprising flowing the mixture through a dump diffuser into the acoustic chamber.

13. The method of claim 11, further comprising flowing the mixture into the acoustic chamber via each inlet located at the first end and the second end, respectively.

14. The method of claim 11, wherein the at least two ultrasonic transducers span at least a portion of the acoustic chamber.

15. The method of claim 11, wherein the acoustic wave is a multi-dimensional acoustic standing wave comprising an axial force component and a lateral force component having the same order of magnitude.

16. The method of claim 11, further comprising generating the acoustic wave in the acoustic chamber through an acoustically transparent membrane.

17. A method of separating a secondary fluid or particle from a mixture comprising a primary fluid, comprising:

flowing a mixture of the primary fluid and the secondary fluid or particles through an acoustophoresis device at a rate of at least 25mL/min, the acoustophoresis device comprising:

an acoustic chamber having a volume of at least 40 cubic inches and comprising a first end and a second end opposite the first end;

an inlet at the first end and the second end, respectively;

at least two ultrasonic transducers arranged side-by-side and coupled to the acoustic chamber and each configured to generate acoustic waves in the acoustic chamber; and

a reflector on the other side of the acoustic chamber relative to the at least two ultrasonic transducers;

the at least two ultrasonic transducers and the reflector cooperate to generate sound waves in the acoustic chamber when the at least two ultrasonic transducers are excited to a higher order mode;

exciting each of the at least two transducers to a higher order mode of vibration to produce the acoustic wave in the acoustic chamber; and

capturing the secondary fluid or particles via the acoustic waves such that the secondary fluid or particles agglomerate, aggregate, cluster, or coalesce together and are separated from the primary fluid.

18. The method of claim 17, further comprising flowing the mixture into the acoustic chamber at a rate of at least 800 mL/min.

19. The method of claim 17, further comprising subjecting the mixture to a pressure of from 0.005mL/min/cm²To 4.5mL/min/cm²Into the acoustic chamber at a rate within a range of (a).

20. An acoustophoretic device, comprising:

an acoustic chamber having a volume of at least 40 cubic inches and comprising inlets at a first end and a second end opposite the first end thereof, respectively;

at least one fluid outlet at the top end of the acoustic chamber;

at least one concentrate outlet at a bottom end of the acoustic chamber;

at least two ultrasonic transducers arranged side-by-side and coupled to the acoustic chamber and each configured to generate a multi-dimensional acoustic wave in the acoustic chamber; and

a reflector on the other side of the acoustic chamber relative to the at least two ultrasonic transducers;

each of the at least two ultrasonic transducers cooperates with the reflector to create a multi-dimensional acoustic standing wave in the chamber when each of the at least two ultrasonic transducers is excited;

wherein the acoustic chamber comprises a planar cross-sectional area defined by a length and a width and a lateral cross-sectional area defined by the width and a height, wherein the length is greater than or equal to the width, and wherein the planar cross-sectional area is greater than the lateral cross-sectional area.

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