Expanded bed affinity selection

Description

BACKGROUND

Separation of biomaterial has been applied in a variety of contexts. For example, separation techniques for separating proteins from other biomaterials are used in a number of analytical processes.

Acoustophoresis is a technique for separating particles and/or secondary fluids from a primary or host fluid using acoustics, such as acoustic standing waves. Acoustic standing waves can exert forces on particles in a fluid when there is a differential in density and/or compressibility, known as the acoustic contrast factor. The pressure profile in a standing wave contains areas of local minimum pressure amplitudes at standing wave nodes and local maxima at standing wave anti-nodes. Depending on their density and compressibility, the particles can be driven to and 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.

SUMMARY

This disclosure describes technologies relating to methods, systems, and apparatus for acoustic separation of materials. The materials being separated may be biomaterials. The separation may employ material support structures. The support structures may be beads. A functionalized material may be applied to the support structures that has an affinity for one or more materials to be separated. The support structures may be mixed in a fluid that contains the materials. The fluid mixture may be provided to a fluid column or flow chamber. The support structures can be retained in the column against a fluid or fluid mixture flow through the column by provision of an acoustic standing wave at one end of the column that can prevent the support structures from passing.

In accordance with some examples, an acoustic affinity system is implemented that can include the features of being closed, automated and/or single-use. The system can be considered closed if the components can be sealed from an open-air environment. An automated system is able to operate autonomously, with little or no operator intervention. The system is single use when components and materials employed for an affinity separation run, which may include multiple recirculations, are disconnected and discarded after an affinity separation run. A single use system can avoid the additional steps of cleaning and sterilizing the equipment components and materials for subsequent runs.

In some examples, methods, systems, and apparatuses are disclosed for separation of biomaterials accomplished by functionalized material distributed in a fluid chamber that bind the specific target materials. The specific target materials can be particles, including cells, recombinant proteins and/or monoclonal antibodies. The functionalized material, which may be beads and/or microcarrier structures are coated or otherwise provided with an affinity material for attracting and binding the specific target materials. The affinity material may be a protein, ligand or other material that can form a bond with the target material.

In some example implementations, the affinity material and the target material can form antigen-antibody interactions with binding sites on the functionalized material. In some instances, the target material become bound to the functionalized material when a ligand of the target material or the functionalized material is conjugated to a matrix on the complementary material. The functionalized material includes functionalized microbeads. The functionalized microbeads include a particular antigen ligand that has affinity for a corresponding antibody.

In some examples, material adhered to the support structures with the functionalized material remains in the column, while other free material in the fluid may pass through the acoustic standing wave to provide separation of materials. The support structures may be implemented to have a certain acoustic contrast factor based on their density, compressibility, size or other characteristics that permits the support structures to react more strongly to the acoustic standing wave than other materials in the fluid mixture.

The support structures may be agitated in the column to enhance the affinity process. In different modes, the column fluid mixture that passes through the acoustic standing wave can be recirculated to the column or not. The fluid flow in the column can be controlled to flow or not, and when flowing, the rate of flow can be controlled. The fluid may be stationary in the column and may have other processes applied thereto, such as temperature adjustment, agitation, incubation, and/or any other useful process. The volume of the column can be effectively modified, such as with the provision of a plunger or piston in the column. Heating or cooling can be applied to the column or the contents of the column, internally or externally to the column.

The particulates may include beads, and wherein at least one of the beads comprises a sphere with a diameter of about 20 to 300 μm and comprises at least one of DEAE (N, N-diethylaminoethyl)-dextran, glass, polystyrene plastic, acrylamide, collagen, or alginate. The cell-supporting material may include microbubbles that have a surface coating for growth of the cells. The cells may include, for example, T-cells, MRC-5 cells or stem cells.

An acoustic transducer can be used to generate the acoustic standing wave, which can generate pressure forces in one or multiple dimensions. In multiple dimensions, the acoustic standing wave forces can be of the same order of magnitude. For example, forces in the direction of wave propagation may be of the same order of magnitude as forces that are generated in a different direction. An interface region can be generated near a border of the acoustic standing wave that contributes to preventing support structures from passing. Multiple transducers may be used, some for generating an acoustic wave in one or modes, and others for generating an acoustic wave in another, different mode. For example, the acoustic wave can be a standing wave that can generate pressure forces in one dimension or in multiple dimensions. The acoustic wave can be generated in a mode to form an interface region to prevent passage of certain materials while permitting passage of other materials. The acoustic wave can be generated in a mode to trap and cluster certain materials that build in size until the gravity or buoyancy forces on the clusters surpass the other forces on the clusters, such as fluidic or acoustic forces, so that the clusters drop or rise out of the acoustic wave.

Collecting cells may be performed with or without turning off the acoustic transducer. An additive which enhances aggregation of the support structures into the flow chamber may be applied. The method may further include recirculating the support structures, such as beads, to a culturing chamber coupled to the flow chamber. The method may also include processing the collected cells for infusion into a subject patient. Subsequent to preferentially trapping, the method may include allowing the trapped cells and/or cell-supporting material to rise or settle out of the fluid due to a buoyance or gravity force. The rising or settling cells and/or support material may exit the flow chamber. The mode of trapping cells or support material for separation by rising or settling out of the fluid may be accompanied by a mode of preventing or permitting the cells and/or support material from passing through a fluid path. The mode of preventing or permitting passage may be implemented with an acoustic wave with an interface region across the fluid path.

In some example implementations, the material includes target compounds, such as recombinant proteins and monoclonal antibodies, viruses, and/or live cells (e.g., T cells). Beads or microcarriers with or without functionalized material on their surfaces may be the target compounds or components.

An example apparatus may include a flow chamber configured to receive fluid containing functionalized material. The flow chamber may be in the form of a column. An acoustic transducer is arranged in relation to the flow chamber, for example, acoustically coupled to the flow chamber, to provide an acoustic wave or signal into the flow chamber when excited. Excitation of the transducer can generate a multi-dimensional acoustic field inside the chamber that includes first spatial locales where acoustic pressure amplitude is elevated from a base level, such as, for example when the acoustic transducer is turned off, and second spatial locales where acoustic pressure amplitude has little or no elevation from the base level, for example the acoustic pressure amplitude may be equivalent to that when the acoustic transducer is turned off.

In some modes, the functional material may be driven to and retained at the first or second locales of the multidimensional acoustic field. In other modes, the functional material may be prevented from entering the multidimensional acoustic field in accordance with an edge effect at an interface region. Materials to be processed that include target materials for separation may be flowed into the flow chamber where functionalized material is retained such that a portion of the target materials with features complementary to the functionalized material become bound to the functionalized material while other portions of the materials pass through the flow chamber. The chamber may be configured for vertical flow which may be in an upward or downward direction. Fluid paths to the chamber may be provided at a top and/or bottom of the chamber. An acoustic transducer can be coupled to a top and/or bottom of the chamber to generate an acoustic field at that locale.

The functionalized microcarriers may also be circulated after the recombinant proteins or monoclonal antibody is eluted from the surface by a buffer or other process elution. This allows for greater surface area and affinity interaction of the functionalized microcarriers with the expressed proteins from the bioreactor, increasing the efficiency of the acoustic fluidized bed chromatography process.

In some example implementations, the apparatus provides functionalized particles, such as beads, in an arrangement that provides more space between particles, such as beads or cells, than packed columns. The lower density decreases the likelihood that non-target biomaterials will clog flow paths between the functionalized particles. In some example implementations, recirculating media containing the target biomaterials in effect increases the capture surface area of the apparatus by passing free target biomaterials past the functionalized particles multiple times. The reduced contact of non-target biomaterials such as cells can help preserve the viability of cells. The technology described here can be used in high- or low-density cell culture, new research applications, large production culture volumes, e.g., more than 1,000 liters, efficient monitoring and culture control, reduction of costs and contamination in cell culture applications.

The details of one or more implementations of the subject matter described in this specification are set forth in the accompanying drawings and the description below. Other features, aspects, and advantages of the subject matter will become apparent from the description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The disclosure is described in greater detail below, with reference to the accompanying drawings, in which:

FIG. 1 is a simplified diagram of an acoustic affinity process;

FIG. 2 is a side elevation view of an acoustic affinity system operated in an edge effect mode;

FIG. 3 is a side elevation view of an acoustic affinity system operated in a cluster mode;

FIG. 4 is a photograph of a front elevation view of a fluidized bed set up;

FIG. 5 is a diagram of an acoustic affinity system and process;

FIG. 6 is a diagram of an acoustic affinity system and process;

FIG. 7 is a diagram of an acoustic affinity system and process;

FIG. 8 is a diagram of an acoustic affinity system and process;

FIG. 9 is a diagram of an affinity positive selection in an acoustic affinity process;

FIG. 10 is a diagram of an affinity negative selection in an acoustic affinity process;

FIG. 11 is a graph showing the retention versus inflow fluid rate;

FIG. 12 is a graph showing the cell viability versus column volumes;

FIG. 13 is a graph showing a histogram of particle sizes;

FIG. 14 is a diagram of an acoustic affinity system and process with the recirculation;

FIG. 15 is a bar graph showing purity and recovery in a recirculation arrangement;

FIG. 16 is a diagram of a bead with functionalized material for targeting a CD3 marker;

FIG. 17 is a graph showing size distributions of different types of beads;

FIG. 18 is a graph showing binding ratios for different types of beads;

FIG. 19 is a diagram showing a comparative analysis between different affinity systems;

FIG. 20 shows four graphs illustrating cell population differences with changes in antibody titration ratios;

FIG. 21 is a chromatogram showing cell count per milliliter versus column volumes; and

FIG. 22 is a graph showing cell count in column outflow over time.

DETAILED DESCRIPTION

This disclosure describes methods, systems and apparatuses that employ an acoustic standing wave with nodes and antinodes to separate support structures such as beads or coated microbubbles from other materials in a chamber such as a column. The example implementations described herein may be operated in different modes. For example, in some modes, an acoustic wave is generated with certain characteristics across the chamber. The acoustic wave may be generated by an acoustic transducer, which may be located at one end of the column. The acoustic wave may cause an interface region to be generated that blocks certain materials from entering the acoustic wave, while permitting passage of other materials. The acoustic wave characteristics can be controlled to block or pass materials based on parameters such as compressibility, density, size, acoustic contrast factor, and any other parameter that is responsive to the acoustic waves. In other modes, an acoustic wave is generated with spatial locales that capture materials to form clusters that increase in size to a point where the gravity or buoyancy force on the cluster exceeds that of the acoustic or fluid drag force, causing the cluster to exit the acoustic wave.

The modes discussed herein may be employed together or separately or in combination. The modes may be employed or generated with one or more acoustic transducers. The acoustic field generated by the acoustic wave can be configured to block or permit passage of certain materials. For example, support structures for cells, which may be in the form of beads, bead/cell complexes or particles, may be blocked from passage through the acoustic field. Materials such as cells may be passed through the fluid chamber. The support structures include functionalized material that can bind with at least some of the material passed through the fluid chamber. The material that is bound to the support structures via the functionalized material is retained in the fluid chamber by the support structures being retained in the fluid chamber with the acoustic wave. Material that is not bound to the support structures may pass out of the fluid chamber through the acoustic wave. The technique of using acoustic waves to perform affinity separation obtains a number of advantages as described in more detail herein.

Referring to FIG. 1, a diagram illustrates an acoustic affinity process 100. Functionalized beads 102 are placed in a chamber 104 that contains targeted and non-targeted material. The target material corresponds to the functionalization provided to beads 102. Process 100 illustrates the target material being bound to beads 102 in an affinity binding process. Beads 102 are collected or influenced by an acoustic standing wave generated by transducer 106 between transducer 106 and reflector 108. The remaining material in chamber 104 can be removed by flowing fluid through chamber 104 while beads 102 are retained by the acoustic wave. Process 100 illustrated in FIG. 1 can be a positive or negative selection process, where the target material is desired to be itself collected or removed from the other materials, respectively.

Previous systems for affinity separation employed magnetically responsive beads. These beads may incur challenges during manufacturing processes as they do not dissolve or are not readily consumed in vivo and are preferentially completely removed from any treatment supplied to a patient. While such beads may be used in the present acoustic affinity separation system, the use of acoustics offers the possibility for the use of support structures, such as beads, that are tailored to be specifically acoustically responsive. For example, the beads can be nonmagnetic or non-magnetically responsive, and highly acoustically responsive. The acoustically responsive beads can be composed of a variety of materials, significantly increasing the flexibility of the processing system in which they are employed. These acoustic affinity beads can be composed of dissolvable material that is biocompatible, which can alleviate aggressive bead removal processes that are employed with magnetically responsive beads.

The acoustic affinity system can be configured to have increased throughput compared with current systems. For example, the fluid flow rate through the system can be increased over that typically used with conventional affinity systems. The system can be configured with larger channels that permit higher flow rates and volumes. The expansion of the cell population can be implemented within the presently disclosed systems or can be implemented externally and fed to the acoustic affinity system.

The configuration of the acoustic affinity system permits the use of multiple types of support structures or beads that may have different characteristics, such as different ranges of sizes or densities. The different groups of support structures or beads may be provided with different types of functionalized material such as proteins, antigens or antibodies to thereby enable multiplexing of affinity separation. This configuration permits complex, single-pass affinity selections to be realized.

In some example implementations, a column is provided with a volume of beads that have an affinity for a certain type of cell. Cells introduced into the column form a complex with the beads, which complexes can be separated from the column volume using acoustic techniques. The separation may be leveraged to harvest cultured cells of interest, and the extracted cells may be infused into a patient. Using acoustics with an affinity binding system to separate cultured cells of interest can be applicable to a variety of cell therapy applications, e.g., vaccine therapies, stem cell therapies, particularly allogenic and autologous therapies, or regenerative therapies.

An acoustic wave is generated in a flow chamber, such as a column, to effectuate separation of beads and bead complexes from unbound cells or materials in a fluid. The separation can be negative or positive, where the unwanted material to be excluded is bound to the beads, or where the material desired in the separation is bound to the beads, respectively. The material of interest, for either negative or positive selection, may be different types of cells, including adherent cells. Example adherent cells may include human multipotent stem cells (hMSC), human mesenchymal stem cells (also hMSC), human pluripotent stem cells (hPSC), human dermal fibroblasts (hDF), human chondrocytes, and some T lymphocytes. Adherent cells may differ in their antigen specificity (e.g. CD8 adherent cell). The lines used in cell therapy may be mono- or polyclonal (e.g. polyclonal CD8 adherent cell line), and CAR (chimeric antigen receptor) adherent cells (a.k.a. artificial adherent cell receptors, or chimeric adherent cell receptors, or chimeric immunoreceptors. These are T-cells modified to recognize a specific protein. The beads employed in the acoustic affinity separation system can be configured to bind or not bind to these cells or material of interest for negative or positive selection.

The bead technology described here can be used in high density cell culture, new research applications, large production culture volumes, e.g., more than 1,000 liters, efficient monitoring and culture control, reduction of costs and contamination in cell culture applications. The beads used may be commercially available, such as the MAGNE magnetic affinity beads or polystyrene beads supplied by Promega Corporation or MACS beads supplied by Miltenyi Biotec. The size of the beads, for example their diameter, may be in the nanometer or micrometer range. Cospheric beads may be used, which are beads with at least two layers. The layers may have different characteristics, such as differing contrast factors, structural rigidity, or any other characteristics that are desired to be combined in a single bead through the use of multiple layers.

Some implementations may use microbubbles as support structures to bind material of interest. The microbubbles can be composed by a shell of biocompatible materials and ligands capable of linking to the cells or material of interest, including proteins, lipids, or biopolymers, and by a filling gas. Low density fluids may be used for relative ease of manufacturing. The microbubble shell may be stiff (e.g., denaturated albumin) or flexible (phospholipids) and presents a thickness from 10 to 200 nm. The filling gas can be a high molecular weight and low-solubility filling gas or liquid (perfluorocarbon or sulfur hexafluoride), which can produce an elevated vapor concentration inside the microbubble relative to the surrounding fluid, such as blood, and increase the microbubble stability in the peripheral circulation. The microbubble shell can have a surface coating such as a lipid layer. The lipid layer may be utilized as scaffolds or substrates for material growth such as cells or biomolecules. Active groups may be easier to conjugate directly to the glass surface. The microbubbles may have a diameter in a range of 2 to 6 micrometers. The coated microbubbles may have a negative contrast factor.

Examples discussed above provide beads as support structures. Other support structures such as coated bubbles or microbubbles can be also used. For the sake of convenience, support structures may be referred to herein collectively as beads, which term is intended to encompass all types of support structures, including beads, bubbles, microcarriers and any other type of affinity material/support structure that can bind to or be bound to a target material of interest.

Cells are bound to beads, e.g., CD3/CD28 activated beads. As discussed in further detail below, the beads can be functionalized with surface chemistry such that the cells or material of interest can be attached to or adherent to the surface of the beads. The beads can include support matrices allowing for the growth of adherent cells in bioreactors or other cell culturing systems. In some cases, adherent cells will bind to the beads without the antigens on the surface and the beads can be functionalized or non-functionalized. Some examples of affinity applications include positive or negative selection of CD3+, CD3+CD4+ and/or CD3+CD8+ affinity selection for apheresis products. Other examples of affinity applications include positive or negative selection of TCR+ or TCR− cells.

Structurally, the beads include spheres with a diameter in a range of 1 to 300 μm, e.g., in the range of 125 to 250 μm. The spheres can have densities in a range of 1.02-1.10 g/cm³. In some instances, the beads can also include rod-like structures. The beads may be smooth or macroporous.

The core of the beads can be made from different materials, such as glass, polystyrene plastic, acrylamide, collagen, and alginate. The bead materials, along with different surface chemistries, can influence cellular behavior, including morphology and proliferation.

The beads can be coated with a variety of coatings such as glass, collagen (e.g., neutral or charged gelatin), recombinant proteins or chemical treatments to enhance cell attachment, which may lead to more desirable cell yields for a number of different cell lines.

Surface chemistries for the beads can include extracellular matrix proteins, recombinant proteins, peptides, and positively or negatively charged molecules. The surface charges of the micro carriers may be introduced from a number of different groups, including DEAE (N,N-diethylaminoethyl)-dextran, laminin or vitronectin coating (extra cellular matrix proteins). In the DEAE-dextran example, a mild positive charge can be added to the surface.

Other examples of bead coatings, for example with functionalized material for use in biological affinity processes, include streptavidin, monomeric avidin, protein A, anti-CD3, as well as other known functionalized material for binding biological material. Various combinations of antibodies, reagents and/or functionalized material can be used with the beads to bind to a cell of interest. A cell of interest may be identified with target proteins or markers, such as CD3, for example.

In some implementations, the beads are formed by substituting a cross-linked dextran matrix with positively charged DEAE groups distributed throughout the matrix. This type of bead can be used for established cell lines and for production of viruses or cell products from cultures of primary cells and normal diploid cell strains.

In some implementations, the beads are formed by chemically coupling a thin layer of denatured collagen to the cross-linked dextran matrix. Since the collagen surface layer can be digested by a variety of proteolytic enzymes, it provides opportunities for harvesting cells from the beads while maintaining increased or maximum cell viability and membrane integrity. The acoustic affinity system discussed herein can be operated with a number of types of beads, three general groupings of which are discussed below.

The beads may be constructed and configured according to cGMP (current good manufacturing practice) standards or regulations. One example group of beads that may be used in the acoustic affinity system are large, dense beads. These large beads may possess the following characteristics.

- Non-magnetic
- Average size of about 50 um
- Slower binding kinetics
- More easily separated using acoustic techniques
- Positive acoustic contrast factor
- Dissolvable and biocompatible
- poly(lactic-co-glycolic acid), PLGA
- Not internalized by cells

Another example group of beads are those referred to herein as medium sized beads. These medium sized beads may possess the following characteristics.

- Non-magnetic
- Average size in the range of about 1-10 um
- Dissolvable and biocompatible
- Binding kinetics faster than large beads
- Use large acoustic contrast
- Negative and positive contrast
- PLGA or proprietary lipid-based

Another example group of beads are those referred to herein as small beads. These small beads may possess the following characteristics.

- Non-magnetic
- Average size in the range of about 200 nm-2 um
- Dissolvable and biocompatible
- Very fast binding
- Separation through clustering
- Negative contrast factor, low speed of sound & high density
- Proprietary lipid-based

Different types of beads may be chosen for different types of applications. For example, larger beads may be used when the cells are cultured with the beads, or when the affinity binding takes place in a non-flowing mode.

The beads used for the affinity binding can be held back by or passed through an acoustic wave generated by an acoustic transducer. The acoustic transducer may generate a multi-dimensional acoustic standing wave in a flow chamber to create an acoustic field that includes locales of increased pressure radiation forces. The acoustic transducer can include a piezoelectric material that is excited to vibrate and generate an acoustic wave. The acoustic transducer can be configured to generate higher order vibration modes. For example, the vibrating material in the acoustic transducer can be excited to obtain a standing wave on the surface of the vibrating material. The frequency of vibration is directly related to the frequency of the excitation signal. In some implementations, the vibrating material is configured to have an outer surface directly exposed to a fluid layer, e.g., the fluid or mixture of beads and cultured cells in a fluid flowing through the flow chamber. In some implementations, the acoustic transducer includes a wear surface material covering an outer surface of the vibrating material, the wear surface material having a thickness of a half wavelength or less and/or being a urethane, epoxy, or silicone coating, polymer, or similar thin coating. In some implementations, the acoustic transducer includes a housing having a top end, a bottom end, and an interior volume. The vibrating material can be positioned at the bottom end of the housing and within the interior volume and has an interior surface facing to the top end of the housing. In some examples, the interior surface of the acoustic material is directly exposed to the top end housing. In some examples, the acoustic transducer includes a backing layer contacting the interior surface of the acoustic material, the backing layer being made of a substantially acoustically transparent material. One or more of the configurations can be combined in the acoustic transducer to be used for generation of a multi-dimensional acoustic standing wave.

The generated multi-dimensional acoustic standing wave can be characterized by strong gradients in the acoustic field in all directions, not only in the axial direction of the standing waves but also in lateral directions. In some instances, the strengths of such gradients are such that the acoustic radiation force is sufficient to overcome drag forces at linear velocities on the order of mm/s. Particularly, an acoustic radiation force can have an axial force component and a lateral force component that are of the same order of magnitude. As a consequence, the acoustic gradients result in strong trapping forces in the lateral direction.

The multi-dimensional acoustic standing wave can give rise to a spatial pattern of acoustic radiation force. The multidimensional acoustic standing wave may be generated from one transducer and reflector pair due to the multimode perturbations of the piezoelectric material in the transducer. The acoustic radiation force can have an axial force component and a lateral force component that are of the same order of magnitude. The spatial pattern may manifest as periodic variations of radiation force. More specifically, pressure node planes and pressure anti-node planes can be created in a fluid medium that respectively correspond to floor acoustic radiation force planes with maximum and minimum acoustic radiation force planes in between pressure nodal and anti-nodal planes. Pressure nodal planes are also acoustic displacement anti-nodal planes, and vice versa. The spatial pattern may function much like a comb filter in the fluid medium.

In some modes, discussed in greater detail below, the spatial pattern may create an interface region that blocks entry of particles with certain characteristics from entering or crossing the acoustic wave. In other modes of operation, discussed in greater detail below, the spatial pattern may be used to trap particles, for example, of a particular size or size range, while particles of a different size or size range may not be trapped. The modes may be employed separately or together in combination to provide both a barrier and trapping function, in the same or separate locale.

In a multidimensional acoustic standing wave, the acoustic radiation forces within a particular pressure nodal plane are such that particles are trapped at several distinct points within these planes. The trapping of particles leads to the formation of cluster of particles, which continuously grow in size, and, upon reaching a critical size, settle out or rise out of the primary fluid continuously because of enhanced gravitation or buoyancy settling. For example, the spatial pattern can be configured, for example, by adjusting the insonification frequency and/or phase, power, voltage and/or current supplied to the transducer, or fluid velocity or flow rate, to allow the cultured cells to freely flow through while trapping the support structures, such as beads or microbubbles, thereby separating at least the trapped support structures from cells or other materials in the fluid.

In some example implementations, one or more multi-dimensional acoustic standing waves are generated between an ultrasonic transducer and a reflector. An acoustic wave is continually launched from the acoustic transducer and reflected by the reflector to interfere with the launched acoustic wave to form an acoustic standing wave. The formation of the acoustic standing wave may depend on a number of factors, including frequency, power, medium, distance between the transducer and reflector, to name a few. The standing wave can be offset at the transducer or the reflector so that local minima or maxima are spaced from the transducer or from the reflector. The reflected wave (or wave generated by an opposing transducer) can be in or out of phase with the transducer generated wave. The characteristics of the standing wave can be modified and/or controlled by the drive signal applied to the transducer, such as by modifying and/or controlling the phase, amplitude or frequency of the drive signal. Acoustically transparent or responsive materials may also be used with the transducer or reflector to modify and/or control the standing wave.

As the fluid mixture flows between an ultrasonic transducer and reflector, or two facing ultrasonic transducers, between which one or more multi-dimensional acoustic standing waves are established, particles or secondary fluid cluster, collect, agglomerate, aggregate, clump, or coalesce. The clustering of material may take place at the nodes or anti-nodes of the multi-dimensional acoustic standing wave, depending on the particles' or secondary fluid's acoustic contrast factor relative to the host fluid. The particles form clusters that eventually exit the multi-dimensional acoustic standing wave nodes or anti-nodes when the clusters have grown to a size large enough to overcome the holding force of the multi-dimensional acoustic standing wave. For example, the clusters grow in size to a point where the gravity or buoyancy forces become dominant over the acoustic or fluid drag forces, causing the clusters to respectively sink or rise. For fluids/particles that are denser than the host fluid, such as is the case with most cells, the clusters sink and can be collected separately from the clarified host fluid. For fluids/particles that are less dense than the host fluid, the buoyant clusters float upwards and can be collected.

The scattering of the acoustic field off the particles creates secondary acoustic forces that contribute to driving particles or fluid droplets together. The multi-dimensional acoustic standing wave generates a three-dimensional acoustic radiation force, which acts as a three-dimensional trapping field. The acoustic radiation force is proportional to the particle volume (e.g. the cube of the radius) when the particle is small relative to the wavelength. The force is proportional to frequency and the acoustic contrast factor. The force scales with acoustic energy (e.g. the square of the acoustic pressure amplitude). When the acoustic radiation force exerted on the particles is stronger than the combined effect of fluid drag force and buoyancy and gravitational force, the particles are trapped within the acoustic standing wave. The particle trapping in a multi-dimensional acoustic standing wave results in clustering, concentration, agglomeration and/or coalescence of the trapped particles. Relatively large solids of one material can thus be separated from smaller particles of a different material, the same material, and/or the host fluid through enhanced gravitational/buoyancy separation.

The multi-dimensional standing wave generates acoustic radiation forces in a number of directions, including in the direction of acoustic wave propagation and in a direction that is the lateral to the acoustic wave propagation direction. As the mixture flows through the acoustic chamber, particles in suspension experience a strong axial force component in the direction of the standing wave. Since this acoustic force is across (e.g. perpendicular to) the flow direction, it is not aligned with the fluid drag force. The acoustic force can thus quickly move the particles to pressure nodal planes or anti-nodal planes, depending on the contrast factor of the particle. The lateral acoustic radiation force acts to move the concentrated particles towards the center of each planar node, resulting in clustering, agglomeration or clumping. The lateral acoustic radiation force component can overcome fluid drag for such clumps of particles, to continually grow the clusters, which can exit the mixture due to dominant gravity or buoyancy forces. The drop in drag per particle as the particle cluster increases in size, as well as the drop in acoustic radiation force per particle as the particle cluster grows in size, may separately or collectively influence operation of the acoustic separator device. In the present disclosure, the lateral force component and the axial force component of the multi-dimensional acoustic standing wave are of the same or different order of magnitude. In a multi-dimensional acoustic standing wave generated by a single transducer, the axial force can be comparable with the lateral force. The lateral force of such a multi-dimensional acoustic standing wave is much higher than the lateral force of a planar standing wave, usually by two orders of magnitude or more.

The multi-dimensional acoustic standing wave generated for various modes, including to form a barrier or for clustering, is obtained by exciting a piezoelectric material at a frequency that excites a fundamental 3D vibration mode of the transducer. The transducer may be composed of various materials that may be perturbed to generate an ultrasonic wave. For example, the transducer may be composed of a piezoelectric material, including a piezoelectric crystal or poly-crystal. Perturbation of the piezoelectric material, which may be a piezoelectric crystal or poly-crystal, in the ultrasonic transducer to achieve a multimode response allows for generation of a multidimensional acoustic standing wave. A piezoelectric material can be specifically designed to deform in a multimode response at designed frequencies, allowing for generation of a multi-dimensional acoustic standing wave. The multi-dimensional acoustic standing wave may be generated with distinct modes of the piezoelectric material such as a 3×3 mode that generates nine separate multidimensional acoustic standing waves. A multitude of multidimensional acoustic standing waves may also be generated by allowing the piezoelectric material to vibrate through many different mode shapes. Thus, the material can be selectively excited to operate in multiple modes such as a 0×0 mode (i.e. a piston mode), 1×1, 2×2, 1×3, 3×1, 3×3, and other higher order modes. The material can be operated to cycle through various modes, in a sequence or skipping past one or more modes, and not necessarily in a same order with each cycle. This switching or dithering of the material between modes allows for various multidimensional wave shapes, along with a single piston mode shape to be generated over a designated time. The transducers may be composed of a piezoelectric material, such as a piezoelectric crystal or poly-crystal, which may be made of PZT-8 (lead zirconate titanate). Such crystals may have a major dimension on the order of 1 inch and larger. The resonance frequency of the piezoelectric material may nominally be about 2 MHz and may be operated at one or more frequencies. Each ultrasonic transducer module may include single or multiple crystals. Multiple crystals can each act as a separate ultrasonic transducer and are can be controlled by one or multiple controllers, which controllers may include signal amplifiers. The control of the transducer can be provided by a computer control that can be programmed to provide control signals to a driver for the transducer. The control signals provided by the computer control can control driver parameters such as frequency, power, voltage, current, phase, or any other type of parameter used to excite the piezoelectric material. The piezoelectric material can be square, rectangular, irregular polygon, or generally of any arbitrary shape. The transducer(s) is/are used to create a pressure field that generates forces of the same order of magnitude in a lateral and an axial direction.

In some examples, the size, shape, and thickness of the piezoelectric material can determine the transducer displacement at different frequencies of excitation. Transducer displacement with different frequencies can be used to target certain material in an ensonified fluid. For example, higher frequencies with shorter wavelengths can target smaller sized material. Lower frequencies with longer wavelengths can target smaller sized material. In these cases of higher and lower frequencies, material that is not influenced by the acoustic wave may pass through without significant change. Higher order modal displacements can generate three-dimensional acoustic standing waves with strong gradients in the acoustic field in all directions, thereby creating strong acoustic radiation forces in all directions, which forces may, for example be equal in magnitude, leading to multiple trapping lines, where the number of trapping lines correlate with the particular mode shape of the transducer.

The piezoelectric crystals of the transducers described herein can be operated at various modes of response by changing the drive parameters, including frequency, for exciting the crystal. Each operation point has a theoretically infinite number of vibration modes superimposed, where one or more modes are dominant. In practice, multiple vibration modes are present at arbitrary operating points of the transducer, with some modes dominating at a given operating point.

Referring to FIG. 2, a system 200 operating in interface barrier mode is illustrated. An acoustic interface region 202 is employed to block beads 204 from passing through acoustic wave 206. Acoustic wave 206 is generated by an acoustic transducer 208 continually launching an acoustic wave that is reflected by a reflector 210 to generate a standing wave with localized minima (nodes) and maxima (anti-nodes). A pressure rise may be generated on the upstream side of acoustic wave 206 at interface region 202, along with an acoustic radiation force acting on the incoming suspended particles. Interface region 202, also referred to as providing an edge, boundary or barrier effect, can act as a barrier to certain materials or particles. In system 200, a majority, or substantially all, of beads 204 are prevented from entering acoustic wave 206. Other materials can pass through interface region 202. Acoustic wave 206 is configured to influence beads 204, while other material experiences a lower influence to permit them to pass through acoustic wave 206.

Interface region 202 is located at an upstream bounding surface or region of the volume of fluid that is ensonified by acoustic transducer 208. For example, the fluid may flow across interface region 202 to enter the ensonified volume of fluid and continue in a downstream direction. The frequency of acoustic standing wave 206 may be controlled to have desired characteristics, such that, for example, different contrast factor materials may be held back by or allowed through acoustic standing wave 206. Interface region 202 can be generated and controlled to influence, for example, particles of a first size range to be retained. Acoustic standing wave 206 can be generated and controlled to permit, for example, particles of a second size range that is different from the first to pass through. Acoustic standing wave 206 that forms interface region 202 may be modulated so as to block or pass selective materials. The modulation can be employed to block or pass selective materials at different times while fluid flows through the acoustic field generated by acoustic standing wave 206.

In some example implementations, acoustic standing wave 206 produces a three-dimensional acoustic field, which, in the case of excitation by transducer 208 implemented as a rectangular transducer, can be described as occupying a roughly rectangular prism volume of fluid across the direction of fluid flow. Acoustic wave 206 can be generated as a standing wave. The generation of acoustic wave 206 can be achieved with two transducers facing each other across the fluid flow. A single transducer, e.g., transducer 208, may be used to launch acoustic wave 206 through the fluid toward an interface boundary region that provides a change in acoustic properties, such as may be implemented with a chamber wall or reflector 210. The acoustic wave reflected from the interface boundary can contribute to forming a standing acoustic wave with the acoustic wave launched from transducer 208. During operation at different or changing flow rates, the location of interface region 202 may move upstream or downstream.

The acoustic field generated by acoustic standing wave 206 exerts an acoustic radiation pressure (e.g., a pressure rise) and an acoustic radiation force on the fluid and materials at interface region 202. The radiation pressure influences material in the fluid to block upstream materials with certain characteristics from entering the acoustic field. Other materials with different characteristics than the blocked materials are permitted to pass through the acoustic field with the fluid flow. The characteristics that affect whether the materials or particles are blocked or passed by the acoustic field include material compressibility, density, size and acoustic contrast factor. The parameters that can influence the generation or modulation of the acoustic wave include frequency, power, current, voltage, phase or any other drive parameters for operating transducer 208. Other parameters impacting acoustic wave 206 include transducer size, shape, thickness, as well as chamber size and fluid parameters such as density, viscosity and flow rate.

Referring to FIG. 3, a system 300 operating in clustering mode is illustrated. One or more multi-dimensional acoustic standing waves 306 are created between an ultrasonic transducer 308 and a reflector 310. An acoustic wave is continually launched from acoustic transducer 308 and reflected by reflector 310 to interfere with the launched wave, thereby forming a standing wave 306 that has local minima and maxima, or nodes and anti-nodes, respectively. The reflected wave (or wave generated by an opposing transducer) can be in or out of phase with the transducer-generated wave. The characteristics of the standing wave can be modified and/or controlled by the drive signal applied to transducer 308, such as by modifying and/or controlling the phase, amplitude or frequency of the drive signal. Acoustically transparent or responsive materials may also be used with transducer 308 or reflector 310 to modify and/or control standing wave 306.

In a clustering mode, beads 304, bead complexes 314 and/or particles such as cells cluster, collect, agglomerate, aggregate, clump, or coalesce within multi-dimensional standing wave 306. The clustering may occur at the nodes or anti-nodes of multi-dimensional acoustic standing wave 306, depending on the acoustic contrast factor of beads 304 or the particles relative to the host fluid. For example, beads 304, bead complexes 314 or particles that have a positive acoustic contrast factor are driven to the nodes of multi-dimensional acoustic standing wave 306, while beads 304, bead complexes 314 or particles that have a negative acoustic contrast factor are driven to the anti-nodes. The clustered beads 304, bead complexes 314 or particles form clusters 312 that eventually exit the nodes or anti-nodes of multi-dimensional acoustic standing wave 306 when clusters 312 have grown to a size large enough to overcome the holding force of multi-dimensional acoustic standing wave 306. For example, as clusters 312 grow in size in multi-dimensional acoustic standing wave 306, gravity or buoyancy forces begin to dominate over acoustic and/or fluid drag forces. Once the size of a cluster 312 is large enough to cause the gravity or buoyancy forces on cluster 312 to exceed the acoustic and/or fluid drag forces, cluster 312 exits multi-dimensional acoustic standing wave 306.

For beads 304, bead complexes 314 or particles that, for example, have a positive acoustic contrast factor, clusters 312 typically sink with gravity forces. For beads 304, bead complexes 314 or particles that, for example, have a negative acoustic contrast factor, clusters 312 typically rise with buoyancy forces. Gravity is not depicted in FIG. 3, and the orientation of system 300 can be with gravity aligned with or against the fluid flow direction. With gravity against the direction of fluid flow, clusters 312 are depicted as sinking due to gravity forces. With gravity aligned with the direction of fluid flow, clusters 312 are depicted as rising due to buoyancy forces.

In this mode of operation, beads 304, and bead complexes 314, are retained in the chamber by sinking or rising out of the acoustic wave. The beads tend to be lightly clustered in this mode and tend to be redistributed in the chamber to permit additional interaction with target material or cells. In addition, an agitator can be provided to the chamber to promote movement and redistribution of the clustered beads.

Particles such as cell Type A are not captured in multi-dimensional acoustic standing wave 306. The characteristics of the Type A cells and multi-dimensional acoustic standing wave 306 permit the Type A cells to pass without being captured and/or clustering. The Type B cells are bound to beads 304 to form bead complexes 314. Accordingly, Type B cells may themselves pass through multi-dimensional acoustic standing wave 306 but may be driven into a cluster 312 if bound to beads 304.

Referring to FIG. 4, a set up for a fluidized bed system 400 is illustrated. The fluidized bed is composed of cospheric beads with a range of about 10% to about 30% packing. The acoustic transducer is attached to a top of the column housing the fluidized bed. Connections are provided at a base of the column for introducing or removing fluid that may entrain beads, cells or other materials. The configuration and operation of system 400 can be controlled with a controller that provide signals to operate a driver for the transducer, as well as fluid control devices, such as pumps, valves or switches. The controller receives feedback from sensors, which can include turbidity sensors, fluid flow sensors and/or valve sensors. The controller also receives feedback from the acoustic transducer to contribute to providing a close loop transducer control. The different modes of operation of the transducer(s) can be implemented by the controller. The controller can be employed to provide automated operation for system 400 in accordance with the examples discussed herein. For example, the controller can be provided with a number of automation profiles from which an operator can select to implement an automated acoustic affinity cell selection process. As illustrated in FIG. 4, the acoustic transducer is employed in a mode to generate an edge effect or interface region as discussed above. Testing on the throughput of the column with the transducer operated in this mode has established some guidelines for flow velocities or flow rates that can be employed in the column while the beads are maintained in the column by the acoustic standing wave and edge effect.

Referring to FIG. 5, a fluidized bed system 500 is illustrated. In this example implementation, column 502 is packed with affinity beads 504, which may be in the range of about 10% to about 30% packing where % packing indicates the percentage of bead volume versus volume of the entire column. Beads 504 are provided with affinity structures to bind to target cells 506. An acoustic transducer 512 capable of generating an acoustic field is coupled to a top of column 502. In operation, a mix of target cells 506 and nontargeted cells 508 is input into column 502 via an inlet 510. As the mix of cells flows through column 502, target cells 506 bind with beads 504. Nontargeted cells 508 tend not to bind with beads 504 for lack of a complementary affinity structure. As the mixture flows through column 502 towards transducer 512, beads 504 are free to move within the fluidized bed of column 502. As beads 504 approach the acoustic field generated by transducer 512, they are blocked by the acoustic edge effect and/or being trapped in the acoustic field. In any case, beads 504 are prevented from passing to the output of column 502. As target cells 506 bind to beads 504, target cells 506 are prevented from exiting column 502 along with the beads 504 to which they are bound. Nontargeted cells 508 are not influenced as strongly by the acoustic field as are beads 504 and can pass through the acoustic field and exit column 502.

This affinity technique employed with fluidized bed system 500 can be implemented on a single-pass basis. System 500 can be configured with the choice of beads to select for material that passes through and exits column 502, or to select for material that is bound to the beads and retained in column 502. The passed or retained material can be positively or negatively selected.

Referring to FIG. 6, an affinity separation process 600 is illustrated. Process 600 includes an external incubation step where affinity beads and cells are combined together to obtain bead complexes. The mix of bead complexes and uncombined material in a fluid is fed into a column 602. As the fluid mix travels along column 602, the bead complexes are directed into column 602 by an acoustic field generated by transducer 604. The uncombined material exits column 602 by passing through the acoustic field. This separation step retains the bead complexes while removing a majority of the uncombined material. Once the bead complexes are loaded into column 602, a flush process can be implemented with the introduction of a buffer fluid at the base of column 602. The remaining uncombined material moves with the buffer fluid through the acoustic field generated by transducer 604. The bead complexes also move with the buffer fluid along column 602 but are blocked from exiting by the acoustic field.

Process 600 offers a number of features that are advantageous for affinity separation of materials. For example, binding of target material to the beads can take place externally, which also permits flexible incubation steps. The acoustic separation provides a gentle and high throughput separation process that quickly reduces the amount of uncombined material in mix with bead complexes. For example, the separation process can be completed in less than one hour. Process 600 is also flexibly scalable and can handle processing volumes in the range of about 10 mL to about 1 L. In addition, all types of beads may be used in process 600, providing significant flexibility for unique or custom affinity separation processes.

Referring to FIG. 7, a fluidized bed system 700 is illustrated. A column 702 is provided, which can be implemented as any of the columns illustrated in FIGS. 4-6. In a first wash process, column 702 is loaded with affinity beads. A wash solution is passed through column 702 while acoustic transducer 704 is on to generate an acoustic field near a top of column 702. The acoustic field retains the affinity beads in column 702 while the wash solution passes through to wash the affinity beads. A capture process is implemented in which cellular material is introduced into column 702. Target cellular material binds to the affinity beads to form bead complexes and is blocked from exiting column 702 by the acoustic field generated by the acoustic transducer 704. Nontargeted material can pass through the acoustic field and can exit column 702. After the capture process, a flush process is provided where fluid is introduced to column 702 to flow the nontargeted material out of column 702. The bead complexes are retained in column 702 against the fluid flow by the acoustic field generated by the acoustic transducer 704.

System 700 offers a number of advantageous features for affinity separation processes, including internal bead binding and low shear forces imposed on the material in column 702. The internal bead binding with low shear forces can be important when larger beads are used due to potentially greater binding energy that is associated with larger beads. For example, it may take longer, or a greater amount of energy, for targeted cellular material to be captured by the larger beads. Lower shear forces can thus help to avoid impeding binding with larger beads. System 700 can employ acoustic transducer 704 to create an acoustic edge effect, which can lead to improved throughput. For example, the processes of binding and separation can be completed in under 2 hours. System 700 is scalable and can handle processing volumes in the range of about 10 mL to about 1 L. the fluidized bed employed in system 700 can be used with beads or with cells for the purposes of affinity separation and/or separation alone.

Referring to FIG. 8, a cell selection system 800 is illustrated. System 800 includes a column 802 that is provided with a stirring mechanism 804. Stirring mechanism 804 can be implemented as a stir bar near a base of column 802. An affinity separation process can be implemented in system 800 using column 802 as a fluidized bed. Column 802 is loaded with affinity beads, for example in a range of about 10% to about 30% packing. The affinity beads are washed with the introduction of a fluid into column 802 while the acoustic field is generated by acoustic transducer 806. The fluid exits column 802 while the affinity beads are blocked from exiting by the acoustic field. A mix of cellular material is introduced into column 802 while acoustic transducer 806 generates an acoustic field near a top of column 802. All of the cellular material is retained in column 802, along with the affinity beads, with the implementation of the acoustic field. Excess fluid may pass the acoustic field and exit column 802 while the cells and affinity beads are blocked from exiting.

During the wash process and the introduction of the cellular material, transducer 806 may be operated in different modes or with different characteristics to, in one case, block the affinity beads from exiting during the wash process, and in another case, block both of the affinity beads and the cellular material from exiting. For example, the frequency used to drive transducer 806 may be different to retain the affinity beads than the frequency when both the cells and affinity beads are retained.

Once column 802 is loaded with affinity beads and cellular material, stirring mechanism 804 can be employed to agitate column 802. The agitation contributes to moving the affinity beads and the cellular material within column 802. As the affinity beads and cellular material move within column 802 the affinity binding process for targeted material can be enhanced. This incubation step can be implemented with no fluid flow and with transducer 806 being unenergized.

Once the incubation/binding process is completed, the affinity bead/targeted material complexes can be washed, and nontargeted material can be removed from column 802. The targeted material may be separated from the affinity beads with a solution provided to column 802 that promotes detachment of the targeted material from the affinity beads. For example, the solution can include enzymes (e.g., trypsin) in a buffer. For example, The targeted material may then be removed from column 802, while the affinity beads are retained with the acoustic field generated by the acoustic transducer 806.

Referring to FIG. 9, an affinity selection process 900 for positive selection in a straight column with a single pass is illustrated. Process 900 begins with the loading of column 902 with affinity beads and washing the beads. Acoustic transducer 904 generates an acoustic field near a top of column 902 during the loading and washing processes. A mix of cellular material is then fed into column 902. Target material is bound to the affinity beads to form bead complexes. The nontargeted material exits column 902 through the acoustic field. The targeted material is retained with affinity beads in column 902, while the nontargeted material exits column 902. The bead complexes are washed with the introduction of a buffer into column 902. A detachment buffer is introduced to column 902 to cause the targeted material to detach from the affinity beads. With the acoustic field in place, the detached targeted material exits column 902 and is collected, while the affinity beads are retained.

Referring to FIG. 10, an affinity selection process 1000 for negative cell selection in a straight column with a single pass is illustrated. Process 1000 begins with the loading of column 1002 with affinity beads to a desired void fraction. The loading process can be implemented while acoustic transducer 1004 is removed from column 1002. With acoustic transducer 1004 connected to a top of column 1002, the affinity beads are washed with the introduction of a buffer. This washing process also serves to expand the bead volume to form a fluidized bed. With acoustic transducer 1004 generating an acoustic field, a mix of cellular material is fed into column 1002. Target material is bound to the affinity beads to form bead complexes. The nontargeted material exits column 1002 through the acoustic field. The targeted material is retained with the affinity beads in column 1002, while the nontargeted material exits column 1002 and is collected as the desired product. This negative cell selection removes the targeted material from the mix of cellular material in a single pass. The affinity beads can be multiplexed or configured to bind with more than one type of targeted material, which permits multiplexed negative selection in a single pass.

Referring to FIG. 11, a graph 1100 illustrates bead retention with an acoustic field versus fluid inflow rate for an acoustic fluidized bed column. As shown in graph 1100, 100% of the beads are retained in the column as the fluid inflow rate increases from 0 to about 10 mL per minute. As the fluid inflow rate increases beyond 10 mL per minute, more and more beads pass through the acoustic field. The data presented in graph 1100 is useful to understand the breakthrough fluid inflow rate that causes beads to pass through the acoustic field. This test used SP Sepharose “Fast Flow” beads with an average diameter of 90 um and an average density of 1.033 g/cc. The terminal velocity was 52.2 cm/hr. The column parameters were: volume −40 ml; height −20 cm; and diameter −1.6 cm. The expanded void fraction was 70% with a starting bead concentration of 7.86E+05 cells/ml. Operating parameters were: frequency −1 MHz and power −3 W.

Referring to FIG. 12, a graph 1200 illustrates total viable cells recovered in an acoustic affinity system versus column volumes where column volumes indicates the amount of input to the system normalized by the volume of the column. As shown in graph 1200, the total viable cells, in millions of cells per milliliter, increases significantly after about a half a column volume. This data shows the efficiency of binding in the acoustic affinity system. For example, almost no unbound cells are observed during the initial half a column volume of supplying a cellular material feed to the fluidized bed column.

Referring to FIG. 13, a graph 1300 illustrates a histogram of beads exiting a fluidized bed column in accordance with particle diameter. Graph 1300 shows that at lower flowrates, small particles escape the column while larger particles are retained. In addition, the average size of an escaping particle increases with flow rate.

Referring to FIG. 14, a fluidized bed system 1400 for implementing acoustic affinity cell selection with the recirculation is illustrated. System 1400 includes a column 1402 and an acoustic transducer 1404. Column 1402 includes annular ribs 1406 that can impede the flow of fluid and force fluid flow toward the center of column 1402. Ribs 1406 can help prevent undesired effects such as channeling within column 1402.

System 1400 is operated similarly to those discussed above. For example, system 1400 may be used for positive or negative selection and can employ different modes of operation with the acoustic transducer 1404. System 1400 illustrates the use of recirculation to improve target cell recovery, by providing more opportunities for target cells to bind with beads in column 1402. After the beads are loaded into column 1402 and washed, a pass 1 feed is supplied to column 1402. The outflow of column 1402 resulting from the pass 1 feed is collected for use as a pass 2 feed. The pass 2 feed is used as the input for a feed supply in a follow-on recirculation pass. Although not shown, the pass 2 feed can generate an outflow that can be collected for another follow-on recirculation pass. Any number of recirculations can be employed. Each of the example systems and fluidized beds discussed herein can be configured to have multiple recirculation passes.

Referring to FIG. 15, a graph 1500 illustrates the purity (P) and percentage recovery (R) in a fluidized bed system with a number of recirculated feed passes. Graph 1500 shows that purity is maintained at a high level, greater than 90% for recirculation passes 1 and 2, and greater than 80% for recirculation pass 3. The recovery of cells increases with each recirculation pass, nearing 100% with the third recirculation.

Referring to FIG. 16, an example implementation of a bead with the functionalized material is illustrated. The bead is configured to have an affinity for CD3 receptors on a cell. The bead may be coated with streptavidin, monomeric avidin, protein A, and/or anti-CD3. A biotin-anti-CD3 complex may be used to provide the affinity target for the CD3 receptor on the cell. The anti-CD3-biotin antibody may be replaced or substituted with an anti-TCR-biotin antibody. The streptavidin coated beads can provide a greater binding surface area than other types of coatings. For example, the streptavidin coated beads can have a greater cell binding/cm²ratio than other coatings. The term coating is used to refer to functionalized material on a surface of a bead, and may cover portions or all of a bead surface. Alternatively, or in addition, a portion of a bead may be coated with streptavidin and another portion may be coated with another functionalized material to implement multiplexed affinity processes.

Referring to FIG. 17, a graph showing size distributions of different types of beads is illustrated. The y-axis is graduated in terms of percentage, while the x-axis is graduated by size in micrometers. The graph illustrates the different size distributions of the GE Sepharose beads and the ABT beads. The beads are distributed over a size that is greater than the size of the cells. The size differential between the beads on the cells can be used as an acoustic contrast factor to distinguish between and separate the beads and the cells.

Referring to FIG. 18, a graph illustrates binding ratios for different types of beads. The binding ratios are for an initial cell population of 100,000 cells. The y-axis represents cells/ cm²and the x-axis represents the different types of beads. As illustrated, the Sepharose beads, with an average size of 34 μm in diameter, attained a binding ratio of greater than 30,000 cells/ cm². The Promega beads, with an average size of 65 μm in diameter, attained a binding ratio of about 65,000 cells/ cm². The Pluribeads, with an average size of 70 μm in diameter, attained a binding ratio greater than the Sepharose beads, and less than 40,000 cells/ cm².

Referring to FIG. 19, a diagram illustrating a comparative analysis between different affinity systems is shown. One system uses APC and anti-CD3, while the other system uses APC, anti-biotin, biotin, and anti-CD3. As shown in the diagram, the system including the biotin resulted in about 65% binding, while the system without biotin resulted in about 55% binding. The comparative implementation includes antiCD3-fluorophore vs. antiCD3-biotin+antibiotin-fluorophore.

Referring to FIG. 20, several graphs are illustrated representing cell count for different antibody titration ratios. As the graphs illustrate, the cell populations have greater separation as the titration ratio increases.

Referring to FIG. 21, a chromatogram illustrating cell count per milliliter versus column volumes is shown. Column volumes referred to the amount of fluid entrained with material that is presented to or recirculated in a fluidized bed. It is desirable in the fluid flow through the column to achieve plug flow.

Referring to FIG. 22, a graph illustrating cell count in column outflow over time is shown. Turbidity of the column outflow correlates well with cell concentration.

Several experimental tests for acoustic affinity cell separation were conducted. The results of the tests are tabulated as examples below.

EXAMPLE 1

Four fluidized bed platform tests were performed with different cell concentrations (100 e6/mL and 10 e6/mL) and different capturing antibody combinations (Anti-TCR a/b only vs Anti-TCR a/b and Anti-CD52) on the first day of testing. The initial feed concentration (100 e6/mL and TCR a/b-population was 74-78%).

All samples were incubated with the corresponding capturing antibody combinations listed in Table 1 in 2% BSA in PBS for 20 minutes on the IKA roller (30 rpm). Cells were washed twice with 2% BSA in PBS and finally re-suspended in 10 mL 2% BSA in PBS. A sample was removed for flow cytometry and used as the initial population for tests A through D. Next, 4 ml of a 50% solid Promega bead slurry were loaded into the fluidized bed column and washed with 30 ml of a 2% BSA in PBS solution to remove residual ethanol and particulates. This initial washing step was performed at a flow rate and power of 1 ml/min and 0.75 W.

The feed cell population was then separated using the fluidized bed unit packed with avidin-conjugated methacrylate beads (Promega) which operated at the following conditions; flow rate −1 mL/min and power −0.75 W. The first fraction, denoted as the outflow, was collected after the entire sample was loaded into the fluidized bed. A second fraction, denoted as the flush, was collected after flushing the fluidized bed with 30 ml of a 2% BSA in PBS solution at 1 ml/min and 0.75 W. This flushing step is implemented to ensure all uncaptured cells are recovered. Once this process was completed, the remaining contents of the column were retrieved and collected as the third fraction, denoted as the holdup. Samples from all three fractions were collected for flow cytometry. For the purposes of conducting a mass balance, the mass and cell count for each fraction was recorded.

TABLE 1

Fluidized Bed (FB) platform test parameters

Day 1, Fluidized Bed (FB) test parameters

Cell conc.

[×10{circumflex over ( )}6/
Sample volume
Total cell #
Bead volume
TCR a/b
CD52

Label
Antibody
Bead
mL]
[mL]
[×10{circumflex over ( )}6]
[mL]
[mL]
[mL]
Analytics

FB_A
TCR a/b
Promega
100
10
1000
2
1.5
—
Counting & Flow

FB_B
TCR a/b and CD52
Promega
100
10
1000
2
1.5
0.56
Counting & Flow

FB_C
TCR a/b
Promega
10
10
100
2
0.15
—
Counting & Flow

FB_D
TCR a/b and CD52
Promega
10
10
100
2
0.15
0.056
Counting & Flow

The purity increased by approximately 15% for all samples after separation by the fluidized bed unit, where the initial cell population consisted of 76% TCR knockout cells. In tests conducted at a higher cell concentration (100E6 cells/mL), samples A and B yielded a purity of 13% and 10% in the outflow and 11% and 8% in the flush respectively showing a slight decrease in purity in the second fraction. The purity in the holdup fraction was 73.2% and 68.1% for samples A and B indicating that 100% purity was not achieved. Tests conducted at lower cell concentrations (10E6 cells/mL) yielded a higher purity of 90.5% and 92.4% in the outflow fraction and even higher purity in the flush fraction of 94.8% and 93.2% in samples C and D respectively. This result showed that lower cell concentrations were better with current conditions used with the fluidized bed unit. Overall employing the combination of anti-TCR and anti-CD52 as capturing antibodies did not yield significantly different purity compared to using anti-TCR as the sole capturing antibody.

TABLE 2

TCR a/b− purity and recovery from Fluidized Bed test

Day 1, Fluidized Bed (FB) test results

TCR a/b− purity [%]
Recovery

Label
Control
Outflow
Flush
Hold-up
[%]

FB_A
74.50%
84.10%
83.10%
73.00%
6.70%

FB_B
75.40%
83.10%
81.90%
67.50%
4.80%

FB_C
78.10%
90.50%
94.80%
78.40%
33.50%

FB_D
76.30%
92.30%
93.20%
81.30%
32.90%

The total TCR− recovery for each test is equal to the sum of TCR− cells in the flow-through and flush fractions divided by the starting TCR− cell count (See Eq. 6 in Appendix). There are two mechanisms by which TCR-cells could be retained in the fluidized bed system: acoustic retention and inefficient flushing. Acoustic retention occurs when a free cell experiences a greater force from the acoustic field compared to the drag force exerted by the fluid flow. This happens at high power to flow rate ratios and can be prevented by optimizing operating conditions. Cells also tend to disperse into the volume of the system, making a flush step necessary to improve recovery. The flushing step should have a uniform velocity distribution, otherwise a large volume of buffer is needed to recover TCR− cells as the incoming wash buffer mixes with the fluidized bed. This type of cell retention can be reduced by increasing flush velocity and volume and improving the fluidized bed inlet design.

For each fluidized bed test the total TCR− cell recovery can be seen in Table 2.

The lowest recoveries were seen while testing high cell densities (100 e6/ml). Tests A and B had TCR− recoveries of 7% and 5% respectively. A “clogging” effect in the column was observed during these tests where beads and cells agglomerated together in very large clumps. Rather than acting as a fluid these solid clumps caused channeling in the column and prevented cells from escaping. It is also possible that non-specific binding occurred as the column fouled.

Tests C and D had similar recoveries, 34% and 33%. The two tests with 10 e6/ml behaved as expected but still had relatively low TCR− cell recoveries. This is due to the low fluid velocity and inefficient flush step described previously and can be improved by optimizing operating conditions and improving the fluidized bed inlet design. Changing the antibody had a minimal effect on cell recovery.

EXAMPLE 2

Four Acoustic Separator unit tests were performed with different affinity bead types (Promega, Dynabead, PolyStyrene 6 um and 14 um). On Day 2, fixed antibody combination (Anti-TCR and Anti-CD52) and antibody volume of 0.15 mL and 0.052 mL, respectively) were used. The initial TCR a/b-population was 77%.

All samples were incubated with anti-TCR and anti-CD52 in 2% BSA in PBS for 20 minutes on the IKA roller (30 rpm). Cells were washed twice with 2% BSA in PBS. A sample was removed for flow cytometry and used as the initial population for tests L through Q. Samples were incubated with the corresponding bead candidate listed in Table 3. for 30 minutes on the IKA roller (30 rpm) in 10 mL of 2% BSA in PBS and then separated using the Acoustic Separator unit operated at the following conditions; flow rate −1 mL/min and power −0.75W. The first fraction, denoted as the outflow was collected after the entire sample passed through the acoustic field. A second fraction denoted as the flush was collected after flushing the fluidized bed with 30 ml of a 2% BSA in PBS solution. Once this process was completed, the remaining contents of the column were retrieved and collected as the third fraction, denoted as the holdup. Samples from all three fractions were collected for flow cytometry. For the purposes of conducting a mass balance, the mass and cell count for each fraction was recorded.

TABLE 3

Acoustic Separator(AC) platform test parameters

Day 2, Acoustic Separator (AC) parameters

Cell conc.

[×10{circumflex over ( )}6/
Sample volume
Total cell #
Bead volume
TCR a/b
CD52

Label
Antibody
Bead
mL]
[mL]
[×10{circumflex over ( )}6]
[mL]
[mL]
[mL]
Analytics

AC_A
TCR a/b and CD52
Promega
10
10
100
2
0.15
0.056
Counting & Flow

AC_B
TCR a/b and CD52
Dynabead
10
10
100
0.015
0.15
0.056
Counting & Flow

AC_C
TCR a/b and CD52
PS (6 um)
10
10
100
0.015
0.15
0.056
Counting & Flow

AC_D
TCR a/b and CD52
PS (14 um)
10
10
100
0.015
0.15
0.056
Counting & Flow

The purity increased by approximately 13% for all samples after separation by the Acoustic Separator unit, where the initial cell population consisted of 77% TCR knockout cells. The sample incubated with Dyna beads resulted in the highest purity of 89.4% in the outflow fraction while the sample incubated with Polystyrene (10-14 μm) beads resulted in the lowest purity of 84.3%. This trend was also observed in the flush fraction where samples incubated with Dyna beads yielded 91.1% purity and Polystyrene beads yielded 84.5% purity. The purity in all samples increased slightly from the outflow (84.3%-89.8%) to the flush fraction (84.6%-91.1%).

TABLE 4

TCR a/b− purity and recovery from Fluidized Bed test

Day 2, Acoustic Separator (AC) test results

TCR a/b− purity [%]
Recovery

Label
Control
Outflow
Flush
Hold-up
[%]

AC_A
76.20%
85.70%
86.20%
79.20%
79.90%

AC_B
77.60%
89.80%
91.10%
88.90%
17.00%

AC_C
77.70%
88.30%
89.40%
84.70%
26.20%

AC_D
77.40%
84.30%
84.60%
79.70%
27.10%

The total TCR− cell recoveries for each acoustic separator system test can be seen in Table 4. In this figure it appears that the recovery is affected by the bead type, with 50 um Promega beads having an 80% TCR− cell recovery and 4.5 um Dyna-beads having just 17% recovery. Both polystyrene particles had similar recoveries, 26% and 27% for 6 um and 14 um beads respectively. Since every test was performed with the same operating conditions, similar recoveries were expected so this relationship should be confirmed in future work. Like in the fluidized bed, TCR− recovery in the Acoustic Separator system can be increased by increasing flow velocity and by improving the inlet and collector designs.

EXAMPLE 3

Two Fluidized Bed (FB) processes and two Acoustic Separator(AS) processes were performed. Different pump systems (Syringe pump and peristaltic pump) were tested on the Fluidized Bed unit and two new bead candidates were tested on the Acoustic Separator unit on the first day of testing. The initial feed concentration was 10⁷cells/mL and TCR a/b-population was about 80%.

Sample preparation and acoustic unit operating procedures were the same as previous examples. Briefly, feed samples for the Fluidized Bed were incubated with biotinylated anti-TCR a/b antibody (Table 5) in 2% BSA in PBS for 20 minutes on the IKA roller (30 rpm). Cells were washed twice with 2% BSA in PBS and finally re-suspended in 10 mL 2% BSA in PBS. For the feed samples for the Acoustic Separator unit, bead incubation was followed by antibody-cell incubation. 1×10⁶cells from each feed sample were collected separately for flow cytometry and used as the initial population for each test.

The fluidized bed column was loaded with 2 ml Promega bead slurry (avidin-conjugated methacrylate beads) and then washed with 30 ml of a 2% BSA in PBS solution to remove residual ethanol and particulates. This initial washing step was performed at 3 mL/min and 2.25 W. Two different pumps (Syringe pump—FB_A and Peristaltic pump—FB_B) were evaluated on day 1. The feed cell population was then separated using the fluidized bed unit packed with Promega beads which operated at 3 mL/min and 4 mL column volume. For the Acoustic Separator unit operation, bead labeled feed were separated at the following conditions; flow rate −1mL/min and power −0.75W.

For the performance evaluation, processed samples from both units were collected and analyzed from three different fractions—outflow, flush and holdup (Table 6). The first fraction of the processed sample, denoted as the outflow, was collected after the entire sample was loaded into the fluidized bed. A second fraction, denoted as the flush, was collected after flushing the fluidized bed with 30 ml of 2% BSA in PBS solution. This flushing step is necessary to ensure all uncaptured cells are recovered. Once this process was completed the remaining contents of the column were retrieved and collected as the third fraction, denoted as the holdup. Samples from all three fractions were collected for flow cytometry. The mass and cell concentration count for each fraction was recorded for the cell recovery evaluation.

TABLE 5

Fluidized Bed(FB) and Acoustic Separator (AS) unit test parameters (bead volume = 1 cc, slurry volume = 4 ml.

Day 1, Fluidized Bed (FB) and Acoustic Separator (AS) test, parameters

Cell conc.

[×10{circumflex over ( )}6/
Feed volume
Bead volume
TCR a/b
Power
Flow rate

Label
Antibody
Bead
mL]
[mL]
[mL]
[mL]
[W]
[mL/min]
Comments

FB_A
Anti-TCR
Promega
10
10
2
0.15
2.25
3
Syringe pump

FB_B
Anti-TCR
Promega
10
10
2
0.15
2.25
3
Peristaltic pump

AS_C
Anti-TCR
PLGA
10
10
0.15
0.15
0.75
1
Syringe pump

AS_D
Anti-TCR
WAX
10
10
0.15
0.15
0.75
1
Syringe pump

The purity increased by approximately 11˜12% for all samples after separation by the Fluidized Bed unit and almost no change after separation by the Acoustic Separator unit, where the initial cell population consisted of 80% TCR knockout cells (Table 6). For the Fluidized Bed tests, both peristaltic pump(FB_A) and syringe pump(FB_B) resulted in similar level of purity (90˜92%) in the flow through and flush fraction. In addition to fluidized bed testing, two different micron sized bio-degradable particle candidates (AS_A—PLGA and AS_B—Wax) were tested in Acoustic Separator unit.

Table 6 also shows recovery results. Fluidized Bed with peristaltic pump (FB_A) and syringe pump (FB_B) showed 78% and 61% of TCR− recovery, respectively. Based on the results, FDS decided to use peristaltic pump for upcoming platform validation. Peristaltic pump enables flexibility of further process optimization and closed system development. Acoustic Separator for PLGA and Wax resulted low recovery (50% and 38%, respectively).

TABLE 6

Fluidized Bed(FB) and Acoustic Separator (AS) unit test parameters

Day 1, Fluidized Bed (FB) and Acoustic Separator (AS) test, results

Processed TCR a/b− [%]
Recovery

Label
Control
Flow through
Flush
Hold-up
[%]

FB_A
80.20%
92.24%
91.44%
86.90%
78.04%

FB_B
79.10%
90.43%
91.26%
73.20%
61.37%

AS_A
80.00%
80.40%
80.10%
79.50%
49.54%

AS_B
80.60%
81.00%
83.70%
79.70%
38.49%

EXAMPLE 4

Four fluidized bed unit tests were performed with different operation procedures. The residence time of feed cells in the column was increased by re-circulation of the processed sample or by holding samples in the column for a longer time period. The initial feed concentration was 10⁷cells/mL and TCR a/b-population was about 80%.

The same procedure was performed for feed and initial bead loading of the Fluidized Bed unit as in day 1. Table 7 shows four different operation procedures, no recirculation (FB_E, no recirc.), one recirculation (FB_F, 1 recirc.), 4 recirculations (FB_G, 4 recirc.) and stop and flow (FB_H, Stop and Flow). Specifically, in the stop and flow condition, 2.5 mL of feed samples were loaded with (3 mL/min) and flow stopped for 3 min 20 sec. This procedure was repeated until all the feed volume was loaded into the column. All the feed cells were held in the column by higher power condition (4.5 W) for a total of 13 min 20 sec. Once the recirculation steps and stop and flow steps were finished, the column was flushed with 30 mL of 2% BSA solution. The processed samples were collected and analyzed as in day 1.

TABLE 7

Fluidized Bed (FB) platform and Acoustic Separator (AS) unit test parameters (bead volume = 1 ml)

Day 2, Fluidized Bed (FB) test, parameters

Cell conc.

[×10{circumflex over ( )}6/
Feed volume
Bead volume
TCR a/b
Power
Flow rate

Label
Antibody
Bead
mL]
[mL]
[mL]
[mL]
[W]
[mL/min]
Comments

FB_E
TCR
Promega
10
10
2
0.15
2.25
3
No recirc.

FB_F
TCR
Promega
10
10
2
0.15
2.25
3
1 recirc.

FB_G
TCR
Promega
10
10
2
0.15
2.25
3
4 recirc.

FB_H
TCR
Promega
10
10
2
0.15
2.25
3
Stop and Flow

The purity increased by approximately 9-18% for all samples after separation, where the initial cell population consisted of 80% TCR knockout cells (Table 8). One recirculation (FB_F) resulted 95.6% and 97.7% of purity in Flow through and Flush portion, respectively. Notably, 4 recirculations (FB_G) showed low purity and we observed some temperature increase due to the 4 times of recirculation. The temperature rising also happened in stop and flow condition (FB_H).

TABLE 8

TCR a/b− purity and recovery from

Fluidized Bed and Acoustic Separator test

Day 2, Fluidized Bed (FB) test, results

Processed TCR a/b− [%]
Recovery

Label
Control
Flow through
Flush
Hold-up
[%]

FB_E
80.50%
93.16%
96.37%
73.50%
51.86%

FB_F
79.80%
95.64%
97.69%
59.20%
82.64%

FB_G
80.50%
89.50%
84.80%
56.10%
98.89%

FB_H
80.10%
95.11%
91.51%
73.30%
85.75%

For the TCR a/b− recovery, Recirculation and Stop and Flow condition showed good results. Adding more recirculation steps showed better recovery (one recirc. −82.64% and 4 recirc. 98.89%) and stop and flow condition also resulted high recovery (85.75%).

In accordance with the present disclosure, an acoustic affinity system is discussed that provides a number of advantageous features. For example, the systems and methods discussed herein can provide increased recovery and purity for target cellular material. The systems and methods are scalable, capable of handling a relatively wide range of material volumes. Positive and negative selection can be implemented in accordance with the present disclosure. Positive selection can include implementations with apheresis products. Negative and positive selection can be implemented on a multiplexed basis, where multiple types of cellular material can be selected in one pass. The systems and processes discussed herein can be fully automated and can be figured to be used with consumable components. The acoustic affinity cell selection system can be integrated with a cellular concentrate-wash device and/or system for downstream applications.

The methods, systems, and devices discussed above are examples. Various configurations may omit, substitute, or add various procedures or components as appropriate. For instance, in alternative configurations, the methods may be performed in an order different from that described, and that various steps may be added, omitted, or combined. Also, features described with respect to certain configurations may be combined in various other configurations. Different aspects and elements of the configurations may be combined in a similar manner. Also, technology evolves and, thus, many of the elements are examples and do not limit the scope of the disclosure or claims.

Specific details are given in the description to provide a thorough understanding of example configurations (including implementations). However, configurations may be practiced without these specific details. For example, well-known processes, structures, and techniques have been shown without unnecessary detail to avoid obscuring the configurations. This description provides example configurations only, and does not limit the scope, applicability, or configurations of the claims. Rather, the preceding description of the configurations provides a description for implementing described techniques. Various changes may be made in the function and arrangement of elements without departing from the spirit or scope of the disclosure.

Also, configurations may be described as a process that is depicted as a flow diagram or block diagram. Although each may describe the operations as a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations may be rearranged. A process may have additional stages or functions not included in the figure.

Having described several example configurations, various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the disclosure. For example, the above elements may be components of a larger system, wherein other structures or processes may take precedence over or otherwise modify the application of the invention. Also, a number of operations may be undertaken before, during, or after the above elements are considered. Accordingly, the above description does not bound the scope of the claims.

A statement that a value exceeds (or is more than) a first threshold value is equivalent to a statement that the value meets or exceeds a second threshold value that is slightly greater than the first threshold value, e.g., the second threshold value being one value higher than the first threshold value in the resolution of a relevant system. A statement that a value is less than (or is within) a first threshold value is equivalent to a statement that the value is less than or equal to a second threshold value that is slightly lower than the first threshold value, e.g., the second threshold value being one value lower than the first threshold value in the resolution of the relevant system.

Claims

1. A separation system, comprising: a vertically oriented column oriented column that includes an upper end and a lower end with an opening on each of the upper end and the lower end, each opening being operable as an inlet or an outlet and configured to permit a fluid flow through the column, the column including an acoustic section being defined by an ensonified volume on one of the upper end or the lower end and a fluidized bed section that is separate from the acoustic section, the fluidized bed section including support structures dispersed in a fluid forming a fluidized bed in the fluidized bed section;an acoustic transducer at the acoustic section of the column and that generates an acoustic wave in the column in a direction transverse to a longitudinal axis of the column to form the ensonified volume; andthe acoustic transducer being excited to generate the acoustic wave to form an edge effect across the column to block the support structures from passing through the acoustic wave and retain the support structures in the fluidized bed section of the column against fluid flow.
2. The system of claim 1, further comprising a mechanical agitator coupled to the column for agitating the fluid.
3. The system of claim 1, wherein the acoustic transducer is operated in one or more of a plurality of modes.
4. The system of claim 3, wherein the plurality of modes comprise a clustering mode and an edge effect mode.
5. The system of claim 1, wherein the acoustic transducer is configured to generate a multi-dimensional acoustic wave in the acoustic section of the column.
6. The system of claim 1, wherein the support structures further comprise mobile support structures bound to cellular material in the fluidized bed section of the column that are responsive to the acoustic wave.
7. The system of claim 6, wherein the mobile support structures comprise affinity beads with an affinity for the cellular material.
8. The system of claim 7, wherein the affinity beads comprise column packing in a range of from about 10% to about 30% of an entire column volume.
9. The system of claim 7, wherein the affinity beads comprise one or more of anti-TCR or anti-CD52 capturing antibodies.
10. The system of claim 7, wherein the affinity beads are configured as avidin-conjugated methacrylate beads.
11. A method for separating materials, comprising: providing support structures in a vertically oriented column for binding with a first material, wherein the column comprises: an upper end and a lower end with an opening on each of the upper end and the lower end, each opening being operable as an inlet or an outlet and configured to permit a fluid flow through the column;an acoustic section being defined by an ensonified volume on one of the upper end or the lower end and a fluidized bed section that is separate from the acoustic section, the fluidized bed section including support structures dispersed in a fluid forming a fluidized bed in the fluidized bed section; andan acoustic transducer at the acoustic section of the column and arranged to generate an acoustic wave in the column in a direction transverse to a longitudinal axis of the column to form the ensonified volume; andflowing a fluid mixture that includes the first material into the column; andgenerating an acoustic wave with the acoustic transducer in the acoustic section of the column to block the support structures from leaving the fluidized bed section of the column with the fluid flow.
12. The method of claim 11, further comprising agitating the fluid mixture in the column with a mechanical agitator.
13. The method of claim 11, further comprising generating the acoustic wave in one or more of a plurality of modes.
14. The method of claim 13, wherein the plurality of modes comprise a clustering mode and an edge effect mode.
15. The method of claim 11, further comprising generating a multi-dimensional acoustic wave in the acoustic section of the column.
16. The method of claim 11, wherein the support structures comprise affinity beads with an affinity for cellular material.
17. The method of claim 16, further comprising packing the column in a range of from about 10% to about 30% with the affinity beads of an entire column volume.
18. The method of claim 16, wherein the affinity beads comprise one or more of anti-TCR or anti-CD52 capturing antibodies.
19. The method of claim 16, wherein the affinity beads are configured as avidin-conjugated methacrylate beads.
20. An acoustic affinity separation method, comprising: providing affinity beads in a fluidized bed section of a vertically oriented column, wherein the column comprises: an upper end and a lower end with an opening on each of the upper end and the lower end, each opening being operable as an inlet or an outlet and configured to permit a fluid flow through the column;an acoustic section being defined by an ensonified volume on one of the upper end or the lower end and a fluidized bed section that is separate from the acoustic section, the fluidized bed section including support structures dispersed in a fluid forming a fluidized bed in the fluidized bed section; andan acoustic transducer at the acoustic section of the column and arranged to generate an coustic wave in the column in a direction transverse to a longitudinal axis of the column to form the ensonified volume;generating an acoustic wave with the acoustic transducer in the acoustic section of the column;flowing a cellular material fluid mixture in the column and through the acoustic wave;configuring the acoustic wave to maintain the affinity beads in the fluidized bed section of the column and from passing through the acoustic wave.

CLAIM OF PRIORITY

This application is a continuation-in-part application of and claims priority to U.S. patent application Ser. No. 15/963,809, filed on Apr. 26, 2018, which claims the benefit of U.S. Patent Application Ser. No. 62/490,574, filed on Apr. 26, 2017. This application is also a continuation-in-part application of and claims priority to U.S. patent application Ser. No. 15/222,800, filed on Jul. 28, 2016, which claims the benefit of U.S. Patent Application Ser. No. 62/197,801, filed on Jul. 28, 2015. The entire contents of each of the above applications is hereby incorporated herein by reference.

US Referenced Citations (499)

Number	Name	Date	Kind
2473971	Ross	Jun 1949	A
2667944	Crites	Feb 1954	A
3372370	Cyr	Mar 1968	A
3555311	Weber	Jan 1971	A
4055491	Porath-Furedi	Oct 1977	A
4065875	Srna	Jan 1978	A
4118649	Schwartzman et al.	Oct 1978	A
4125789	Van Schoiack	Nov 1978	A
4158629	Sawyer	Jun 1979	A
4165273	Azarov et al.	Aug 1979	A
4173725	Asai et al.	Nov 1979	A
4204096	Barcus et al.	May 1980	A
4254661	Kossoff et al.	Mar 1981	A
4320659	Lynnworth et al.	Mar 1982	A
4344448	Potts	Aug 1982	A
4398325	Piaget et al.	Aug 1983	A
4484907	Sheeran, Jr.	Nov 1984	A
4552669	Sekellick	Nov 1985	A
4666595	Graham	May 1987	A
4673512	Schram	Jun 1987	A
4699588	Zinn et al.	Oct 1987	A
4743361	Schram	May 1988	A
4759775	Peterson et al.	Jul 1988	A
4800316	Wang	Jan 1989	A
4821838	Chen	Apr 1989	A
4836684	Javorik et al.	Jun 1989	A
4860993	Goode	Aug 1989	A
4878210	Mitome	Oct 1989	A
4983189	Peterson et al.	Jan 1991	A
5059404	Mansour	Oct 1991	A
5059811	King et al.	Oct 1991	A
5062965	Bernou et al.	Nov 1991	A
5085783	Feke et al.	Feb 1992	A
5164094	Stuckart	Nov 1992	A
5225089	Benes et al.	Jul 1993	A
5371429	Manna	Dec 1994	A
5395592	Bolleman et al.	Mar 1995	A
5431817	Braatz et al.	Jul 1995	A
5443985	Lu et al.	Aug 1995	A
5452267	Spevak	Sep 1995	A
5475486	Paoli	Dec 1995	A
5484537	Whitworth	Jan 1996	A
5527460	Trampler et al.	Jun 1996	A
5560362	Sliwa, Jr. et al.	Oct 1996	A
5562823	Reeves	Oct 1996	A
5594165	Madanshetty	Jan 1997	A
5604301	Mountford et al.	Feb 1997	A
5626767	Trampler et al.	May 1997	A
5688405	Dickinson et al.	Nov 1997	A
5711888	Trampler et al.	Jan 1998	A
5766947	Rittershaus	Jun 1998	A
5831166	Kozuka et al.	Nov 1998	A
5834871	Puskas	Nov 1998	A
5902489	Yasuda et al.	May 1999	A
5912182	Coakley et al.	Jun 1999	A
5947299	Vazquez et al.	Sep 1999	A
5951456	Scott	Sep 1999	A
6090295	Raghavarao et al.	Jun 2000	A
6166231	Hoeksema	Dec 2000	A
6216538	Yasuda et al.	Apr 2001	B1
6221258	Feke et al.	Apr 2001	B1
6205848	Faber et al.	Jun 2001	B1
6273262	Yasuda et al.	Aug 2001	B1
6332541	Coakley et al.	Dec 2001	B1
6391653	Letcher et al.	May 2002	B1
6475151	Koger et al.	Nov 2002	B2
6482327	Mori et al.	Nov 2002	B1
6487095	Malik et al.	Nov 2002	B1
6592821	Wada et al.	Jul 2003	B1
6641708	Becker et al.	Nov 2003	B1
6649069	DeAngelis	Nov 2003	B2
6699711	Hahn et al.	Mar 2004	B1
6727451	Fuhr et al.	Apr 2004	B1
6763722	Fjield et al.	Jul 2004	B2
6881314	Wang et al.	Apr 2005	B1
6929750	Laurell et al.	Aug 2005	B2
6936151	Lock et al.	Aug 2005	B1
7008540	Weavers et al.	Mar 2006	B1
7010979	Scott	Mar 2006	B2
7061163	Nagahara et al.	Jun 2006	B2
7081192	Wang et al.	Jul 2006	B1
7093482	Berndt	Aug 2006	B2
7108137	Lal et al.	Sep 2006	B2
7150779	Meegan, Jr.	Dec 2006	B2
7186502	Vesey	Mar 2007	B2
7191787	Redeker et al.	Mar 2007	B1
7322431	Ratcliff	Jan 2008	B2
7331233	Scott	Feb 2008	B2
7340957	Kaduchak et al.	Mar 2008	B2
7373805	Hawkes et al.	May 2008	B2
7541166	Belgrader et al.	Jun 2009	B2
7601267	Haake et al.	Oct 2009	B2
7673516	Janssen et al.	Mar 2010	B2
7674630	Siversson	Mar 2010	B2
7837040	Ward et al.	Nov 2010	B2
7846382	Strand et al.	Dec 2010	B2
7968049	Takahashi et al.	Jun 2011	B2
8075786	Bagajewicz	Dec 2011	B2
8080202	Takahashi et al.	Dec 2011	B2
8134705	Kaduchak et al.	Mar 2012	B2
8256076	Feller	Sep 2012	B1
8266950	Kaduchak et al.	Sep 2012	B2
8273253	Curran	Sep 2012	B2
8273302	Takahashi et al.	Sep 2012	B2
8309408	Ward et al.	Nov 2012	B2
8319398	Vivek et al.	Nov 2012	B2
8334133	Fedorov et al.	Dec 2012	B2
8387803	Thorslund et al.	Mar 2013	B2
8592204	Lipkens et al.	Nov 2013	B2
8679338	Rietman et al.	Mar 2014	B2
8691145	Dionne et al.	Apr 2014	B2
8873051	Kaduchak et al.	Oct 2014	B2
8889388	Wang et al.	Nov 2014	B2
9272234	Lipkens et al.	Mar 2016	B2
9357293	Claussen	May 2016	B2
9365815	Miyazaki et al.	Jun 2016	B2
9368110	Hershey et al.	Jun 2016	B1
9388363	Goodson et al.	Jul 2016	B2
9391542	Wischnewskiy	Jul 2016	B2
9403114	Kusuura	Aug 2016	B2
9410256	Dionne et al.	Aug 2016	B2
9416344	Lipkens et al.	Aug 2016	B2
9421553	Dionne et al.	Aug 2016	B2
9422328	Kennedy, III et al.	Aug 2016	B2
9457139	Ward et al.	Oct 2016	B2
9457302	Lipkens et al.	Oct 2016	B2
9458450	Lipkens et al.	Oct 2016	B2
9464303	Burke	Oct 2016	B2
9476855	Ward et al.	Oct 2016	B2
9480375	Marshall et al.	Nov 2016	B2
9480935	Mariella, Jr. et al.	Nov 2016	B2
9488621	Kaduchak et al.	Nov 2016	B2
9504780	Spain et al.	Nov 2016	B2
9512395	Lipkens et al.	Dec 2016	B2
9513205	Yu et al.	Dec 2016	B2
9514924	Morris et al.	Dec 2016	B2
9517474	Mao et al.	Dec 2016	B2
9532769	Dayton et al.	Jan 2017	B2
9533241	Presz, Jr. et al.	Jan 2017	B2
9550134	Lipkens et al.	Jan 2017	B2
9550998	Williams	Jan 2017	B2
9556271	Blumberg et al.	Jan 2017	B2
9556411	Lipkens et al.	Jan 2017	B2
9566352	Holmes et al.	Feb 2017	B2
9567559	Lipkens et al.	Feb 2017	B2
9567609	Paschon et al.	Feb 2017	B2
9572897	Bancel et al.	Feb 2017	B2
9573995	Schurpf et al.	Feb 2017	B2
9574014	Williams et al.	Feb 2017	B2
9580500	Schurpf et al.	Feb 2017	B2
9587003	Cancel et al.	Mar 2017	B2
9597357	Gregory et al.	Mar 2017	B2
9597380	Chakraborty et al.	Mar 2017	B2
9605074	Shah	Mar 2017	B2
9605266	Rossi et al.	Mar 2017	B2
9606086	Ding et al.	Mar 2017	B2
9608547	Ding et al.	Mar 2017	B2
9611465	Handa et al.	Apr 2017	B2
9616090	Conway et al.	Apr 2017	B2
9623348	McCarthy et al.	Apr 2017	B2
9629877	Cooper et al.	Apr 2017	B2
D787630	Lipkens et al.	May 2017	S
9644180	Kahvejian et al.	May 2017	B2
9645060	Fiering	May 2017	B2
9656263	Laurell et al.	May 2017	B2
9657290	Dimov et al.	May 2017	B2
9662375	Jensen et al.	May 2017	B2
9663756	Lipkens et al.	May 2017	B1
9670477	Lipkens et al.	Jun 2017	B2
9670938	Beliavsky	Jun 2017	B2
9675668	Bancel et al.	Jun 2017	B2
9675902	Lipkens et al.	Jun 2017	B2
9675906	Lipkens et al.	Jun 2017	B2
9677055	Jones et al.	Jun 2017	B2
9685155	Hershey et al.	Jun 2017	B2
9686096	Lipkens et al.	Jun 2017	B2
9688958	Kennedy, III et al.	Jun 2017	B2
9689234	Gregory et al.	Jun 2017	B2
9689802	Caseres et al.	Jun 2017	B2
9695063	Rietman et al.	Jul 2017	B2
9695442	Guschin et al.	Jul 2017	B2
9810665	Fernald et al.	Nov 2017	B2
9833763	Fernald et al.	Dec 2017	B2
9869659	Buckland et al.	Jan 2018	B2
9872900	Ciaramella et al.	Jan 2018	B2
9873126	Mao et al.	Jan 2018	B2
9873894	Conway et al.	Jan 2018	B2
9878056	Bancel et al.	Jan 2018	B2
9878536	Foresti et al.	Jan 2018	B2
9879087	DeSander et al.	Jan 2018	B2
9990297	Conway et al.	Jan 2018	B2
9907846	Morein et al.	Mar 2018	B2
9909117	Kaduchak	Mar 2018	B2
9909313	Grubbs	Mar 2018	B1
9913656	Stulen	Mar 2018	B2
9913866	O'Shea et al.	Mar 2018	B2
9925277	Almarsson et al.	Mar 2018	B2
9926382	Fischer et al.	Mar 2018	B2
9937207	Gregory et al.	Apr 2018	B2
9938390	Storti et al.	Apr 2018	B2
9943599	Gehlt et al.	Apr 2018	B2
9944702	Galetto	Apr 2018	B2
9944709	Galetto	Apr 2018	B2
9994743	El-Zahab	Apr 2018	B2
9974898	Spain et al.	May 2018	B2
9983459	Arnold	May 2018	B2
10006052	Jarjour	Jun 2018	B2
20020038662	Schuler et al.	Apr 2002	A1
20020134734	Campbell et al.	Sep 2002	A1
20030015035	Kaduchak et al.	Jan 2003	A1
20030028108	Miller et al.	Feb 2003	A1
20030195496	Maguire	Oct 2003	A1
20030209500	Kock et al.	Nov 2003	A1
20030230535	Affeld et al.	Dec 2003	A1
20040016699	Bayevsky	Jan 2004	A1
20040035208	Diaz et al.	Feb 2004	A1
20040057886	Zumeris et al.	Mar 2004	A1
20040112841	Scott	Jun 2004	A1
20040124155	Meegan, Jr.	Jul 2004	A1
20040149039	Cardelius	Aug 2004	A1
20050031499	Meier	Feb 2005	A1
20050055136	Hofmann et al.	Mar 2005	A1
20050121269	Namduri	Jun 2005	A1
20050145567	Quintel et al.	Jul 2005	A1
20050196725	Fu	Sep 2005	A1
20050239198	Kunas	Oct 2005	A1
20060037915	Strand et al.	Feb 2006	A1
20060037916	Trampler	Feb 2006	A1
20060050615	Swisher	Mar 2006	A1
20070053795	Laugharn, Jr. et al.	Mar 2007	A1
20070138108	Hadfield et al.	Jun 2007	A1
20070224676	Haq	Sep 2007	A1
20070267351	Roach et al.	Nov 2007	A1
20070272618	Gou et al.	Nov 2007	A1
20070284299	Xu et al.	Dec 2007	A1
20080011693	Li et al.	Jan 2008	A1
20080067128	Hoyos et al.	Mar 2008	A1
20080105625	Rosenberg et al.	May 2008	A1
20080181838	Kluck	Jul 2008	A1
20080217259	Siversson	Sep 2008	A1
20080245709	Kaduchak et al.	Oct 2008	A1
20080245745	Ward et al.	Oct 2008	A1
20080264716	Kuiper et al.	Oct 2008	A1
20080272034	Ferren et al.	Nov 2008	A1
20080272065	Johnson	Nov 2008	A1
20080316866	Goodemote et al.	Dec 2008	A1
20090029870	Ward et al.	Jan 2009	A1
20090042253	Hiller et al.	Feb 2009	A1
20090048805	Kaduchak et al.	Feb 2009	A1
20090053686	Ward et al.	Feb 2009	A1
20090087492	Johnson et al.	Apr 2009	A1
20090098027	Tabata et al.	Apr 2009	A1
20090104594	Webb	Apr 2009	A1
20090126481	Burris	May 2009	A1
20090178716	Kaduchak et al.	Jul 2009	A1
20090194420	Mariella, Jr. et al.	Aug 2009	A1
20090227042	Gauer et al.	Sep 2009	A1
20090045107	Ward et al.	Dec 2009	A1
20090295505	Mohammadi et al.	Dec 2009	A1
20100000945	Gavalas	Jan 2010	A1
20100078323	Takahashi et al.	Apr 2010	A1
20100078384	Yang	Apr 2010	A1
20100124142	Laugharn et al.	May 2010	A1
20100139377	Huang et al.	Jun 2010	A1
20100192693	Mudge et al.	Aug 2010	A1
20100193407	Steinberg et al.	Aug 2010	A1
20100206818	Leong et al.	Aug 2010	A1
20100255573	Bond et al.	Oct 2010	A1
20100261918	Chianelli et al.	Oct 2010	A1
20100317088	Radaelli et al.	Dec 2010	A1
20100323342	Gomez et al.	Dec 2010	A1
20100330633	Walther et al.	Dec 2010	A1
20110003350	Schafran et al.	Jan 2011	A1
20110024335	Ward et al.	Feb 2011	A1
20110092726	Clarke	Apr 2011	A1
20110095225	Eckelberry et al.	Apr 2011	A1
20110123392	Dionne et al.	May 2011	A1
20110125024	Mueller	May 2011	A1
20110146678	Ruecroft et al.	Jun 2011	A1
20110154890	Holm et al.	Jun 2011	A1
20110166551	Schafer	Jul 2011	A1
20110189732	Weinand et al.	Aug 2011	A1
20110207225	Mehta et al.	Aug 2011	A1
20110245750	Lynch et al.	Oct 2011	A1
20110262990	Wang et al.	Oct 2011	A1
20110278218	Dionne et al.	Nov 2011	A1
20110281319	Swayze et al.	Nov 2011	A1
20110309020	Rietman et al.	Dec 2011	A1
20120088295	Yasuda et al.	Apr 2012	A1
20120145633	Polizzotti et al.	Jun 2012	A1
20120161903	Thomas et al.	Jun 2012	A1
20120163126	Campbell et al.	Jun 2012	A1
20120175012	Goodwin et al.	Jul 2012	A1
20120231504	Niazi	Sep 2012	A1
20120267288	Chen et al.	Oct 2012	A1
20120325727	Dionne et al.	Dec 2012	A1
20120325747	Reitman et al.	Dec 2012	A1
20120328477	Dionne et al.	Dec 2012	A1
20120329122	Lipkens et al.	Dec 2012	A1
20130017577	Arunakumari et al.	Jan 2013	A1
20130115664	Khanna et al.	May 2013	A1
20130175226	Coussios et al.	Jul 2013	A1
20130206688	El-Naas	Aug 2013	A1
20130217113	Srinivasan et al.	Aug 2013	A1
20130277316	Dutra et al.	Oct 2013	A1
20130277317	LoRicco et al.	Oct 2013	A1
20130284271	Lipkens et al.	Oct 2013	A1
20133146412	Schultz	Nov 2013
20140011240	Lipkens et al.	Jan 2014	A1
20140017758	Kniep et al.	Jan 2014	A1
20140102947	Baym et al.	Apr 2014	A1
20140141413	Laugharn, Jr. et al.	May 2014	A1
20140154795	Lipkens et al.	Jun 2014	A1
20140319077	Lipkens et al.	Oct 2014	A1
20140329997	Kennedy, III et al.	Nov 2014	A1
20140377834	Presz, Jr. et al.	Dec 2014	A1
20150053561	Ward et al.	Feb 2015	A1
20150060581	Santos et al.	Mar 2015	A1
20150252317	Lipkens et al.	Sep 2015	A1
20150274550	Lipkens et al.	Oct 2015	A1
20150321129	Lipkens et al.	Nov 2015	A1
20160060615	Walther et al.	Mar 2016	A1
20160089620	Lipkens et al.	Mar 2016	A1
20160102284	Lipkens et al.	Apr 2016	A1
20160121331	Kapur et al.	May 2016	A1
20160123858	Kapur et al.	May 2016	A1
20160145563	Berteau et al.	May 2016	A1
20160153249	Mitri	Jun 2016	A1
20160175198	Warner et al.	Jun 2016	A1
20160184790	Sinha et al.	Jun 2016	A1
20160202237	Zeng et al.	Jul 2016	A1
20160208213	Doyle et al.	Jul 2016	A1
20160230168	Kaduchak et al.	Aug 2016	A1
20160237110	Gilmanshin et al.	Aug 2016	A1
20160237394	Lipkens et al.	Aug 2016	A1
20160237395	Lipkens et al.	Aug 2016	A1
20160252445	Yu et al.	Sep 2016	A1
20160279540	Presz, Jr. et al.	Sep 2016	A1
20160279551	Foucault	Sep 2016	A1
20160312168	Pizzi	Oct 2016	A1
20160314868	El-Zahab et al.	Oct 2016	A1
20160319270	Lipkens et al.	Nov 2016	A1
20160325039	Leach et al.	Nov 2016	A1
20160325206	Presz, Jr. et al.	Nov 2016	A1
20160332159	Dual et al.	Nov 2016	A1
20160339360	Lipkens et al.	Nov 2016	A1
20160347628	Dionne et al.	Dec 2016	A1
20160355776	Lipkens et al.	Dec 2016	A1
20160361670	Lipkens et al.	Dec 2016	A1
20160363579	Lipkens et al.	Dec 2016	A1
20160368000	Dionne et al.	Dec 2016	A1
20160369236	Kennedy, III et al.	Dec 2016	A1
20160370326	Kaduchak et al.	Dec 2016	A9
20170000413	Clymer et al.	Jan 2017	A1
20170002060	Bolen et al.	Jan 2017	A1
20170002839	Burkland et al.	Jan 2017	A1
20170007679	Maeder et al.	Jan 2017	A1
20170008029	Lipkens et al.	Jan 2017	A1
20170016025	Poirot et al.	Jan 2017	A1
20170016027	Lee et al.	Jan 2017	A1
20170020926	Mata-Fink et al.	Jan 2017	A1
20170029802	Lipkens et al.	Feb 2017	A1
20170035866	Poirot et al.	Feb 2017	A1
20170037386	Jones et al.	Feb 2017	A1
20170038288	Ward et al.	Feb 2017	A1
20170042770	Warner et al.	Feb 2017	A1
20170044517	Lipkens et al.	Feb 2017	A1
20170049949	Gilmanshin et al.	Feb 2017	A1
20170056448	Glick et al.	Mar 2017	A1
20170058036	Ruiz-Opazo et al.	Mar 2017	A1
20170065636	Moriarty et al.	Mar 2017	A1
20170066015	Lipkens et al.	Mar 2017	A1
20170067021	Moriarty et al.	Mar 2017	A1
20170067022	Poirot et al.	Mar 2017	A1
20170072405	Mao	Mar 2017	A1
20170073406	Schurpf et al.	Mar 2017	A1
20170073423	Juillerat et al.	Mar 2017	A1
20170073638	Campana et al.	Mar 2017	A1
20170073684	Rossi et al.	Mar 2017	A1
20170073685	Maeder et al.	Mar 2017	A1
20170080070	Weinschenk et al.	Mar 2017	A1
20170080423	Dauson et al.	Mar 2017	A1
20170081629	Lipkens et al.	Mar 2017	A1
20170088809	Lipkens et al.	Mar 2017	A1
20170088844	Williams	Mar 2017	A1
20170089826	Lin	Mar 2017	A1
20170096455	Baric et al.	Apr 2017	A1
20170107536	Zhang et al.	Apr 2017	A1
20170107539	Yu et al.	Apr 2017	A1
20170119820	Moriarty et al.	May 2017	A1
20170128523	Ghatnekar et al.	May 2017	A1
20170128857	Lipkens et al.	May 2017	A1
20170130200	Moriarty et al.	May 2017	A1
20170136168	Spain et al.	May 2017	A1
20170137491	Matheson et al.	May 2017	A1
20170137774	Lipkens et al.	May 2017	A1
20170137775	Lipkens et al.	May 2017	A1
20170137802	Lipkens et al.	May 2017	A1
20170145094	Gaietto	May 2017	A1
20170151345	Shah	Jun 2017	A1
20170152502	Scharenberg et al.	Jun 2017	A1
20170152503	Scharenberg et al.	Jun 2017	A1
20170152504	Scharenberg et al.	Jun 2017	A1
20170152505	Scharenberg et al.	Jun 2017	A1
20170152527	Paschon et al.	Jun 2017	A1
20170152528	Zhang et al.	Jun 2017	A1
20170158749	Cooper et al.	Jun 2017	A1
20170159005	Lipkens et al.	Jun 2017	A1
20170159007	Lipkens et al.	Jun 2017	A1
20170166860	Presz, Jr. et al.	Jun 2017	A1
20170166877	Bayle et al.	Jun 2017	A1
20170166878	Thanos et al.	Jun 2017	A9
20170166903	Zhang et al.	Jun 2017	A1
20170173080	Lee et al.	Jun 2017	A1
20170173128	Hoge et al.	Jun 2017	A1
20170173498	Lipkens et al.	Jun 2017	A9
20170175073	Lipkens et al.	Jun 2017	A1
20170175125	Welstead et al.	Jun 2017	A1
20170175139	Wu et al.	Jun 2017	A1
20170175144	Zhang et al.	Jun 2017	A1
20170175509	Abdel-Fattah et al.	Jun 2017	A1
20170175720	Tang et al.	Jun 2017	A1
20170183390	Springer et al.	Jun 2017	A1
20170183413	Galetto	Jun 2017	A1
20170183418	Galetto	Jun 2017	A1
20170183420	Gregory et al.	Jun 2017	A1
20170184486	Mach et al.	Jun 2017	A1
20170189450	Conway et al.	Jul 2017	A1
20170190767	Schurpf et al.	Jul 2017	A1
20170191022	Lipkens et al.	Jul 2017	A1
20170232439	Suresh et al.	Aug 2017	A1
20170374730	Flores	Dec 2017	A1
20180000311	Lipkens et al.	Jan 2018	A1
20180000870	Britt	Jan 2018	A1
20180000910	Chakraborty et al.	Jan 2018	A1
20180001011	Paschon et al.	Jan 2018	A1
20180008707	Bussmer et al.	Jan 2018	A1
20180009158	Harkness et al.	Jan 2018	A1
20180009888	Baumeister et al.	Jan 2018	A9
20180009895	Smith et al.	Jan 2018	A1
20180010085	Lipkens et al.	Jan 2018	A1
20180014846	Rhee	Jan 2018	A1
20180015128	Britt	Jan 2018	A1
20180015392	Lipkens et al.	Jan 2018	A1
20180016570	Lipkens et al.	Jan 2018	A1
20180016572	Tang	Jan 2018	A1
20180020295	Pander et al.	Jan 2018	A1
20180021379	Gaietto et al.	Jan 2018	A1
20180022798	Shurpf et al.	Jan 2018	A1
20180028683	Wong et al.	Feb 2018	A1
20180043473	Helvajian et al.	Feb 2018	A1
20180049767	Gee et al.	Feb 2018	A1
20180051089	Galettto et al.	Feb 2018	A1
20180051265	Cooper	Feb 2018	A1
20180052095	Cumbo et al.	Feb 2018	A1
20180052147	Zeng	Feb 2018	A1
20180055529	Messerly et al.	Mar 2018	A1
20180055530	Messerly et al.	Mar 2018	A1
20180055531	Messerly et al.	Mar 2018	A1
20180055532	Messerly et al.	Mar 2018	A1
20180055997	Cabrera et al.	Mar 2018	A1
20180056095	Messerly et al.	Mar 2018	A1
20180057810	Zhang et al.	Mar 2018	A1
20180058439	Locke et al.	Mar 2018	A1
20180066223	Lim	Mar 2018	A1
20180066224	Lipkens et al.	Mar 2018	A1
20180066242	Zhang	Mar 2018	A1
20180067044	Kaduchak et al.	Mar 2018	A1
20180071363	Ghatnekar et al.	Mar 2018	A1
20180071981	Collino et al.	Mar 2018	A1
20180078268	Messerly	Mar 2018	A1
20180080026	Rossi et al.	Mar 2018	A1
20180085743	Yavorsky et al.	Mar 2018	A1
20180087044	Lipkens et al.	Mar 2018	A1
20180088083	Sinha	Mar 2018	A1
20180092338	Hering et al.	Apr 2018	A1
20180092660	Ethicon	Apr 2018	A1
20180094022	Bracewell et al.	Apr 2018	A1
20180095067	Huff et al.	Apr 2018	A1
20180098785	Price et al.	Apr 2018	A1
20180100134	Lim	Apr 2018	A1
20180100204	O'Shea	Apr 2018	A1
20180010758	Vincent et al.	May 2018	A1
20180119174	Scharenberg et al.	May 2018	A1
20180130491	Mathur	May 2018	A1
20180136167	Smith et al.	May 2018	A1
20180143138	Shreve et al.	May 2018	A1
20180143167	Mziray et al.	May 2018	A1
20180147245	O'Shea et al.	May 2018	A1
20180147576	Lavieu et al.	May 2018	A1
20180148740	Conway et al.	May 2018	A1
20180148763	Shimada et al.	May 2018	A1
20180153946	Alemany et al.	Jun 2018	A1
20180155716	Zhang et al.	Jun 2018	A1
20180157107	Koyama	Jun 2018	A1
20180161775	Kapur et al.	Jun 2018	A1
20180177490	Shiraishi	Jun 2018	A1
20180178184	Holland	Jun 2018	A1
20180180610	Taha	Jun 2018	A1

Foreign Referenced Citations (168)

Number	Date	Country
2002236405	Sep 2002	AU
105 087 788	Nov 2015	CN
104722106	Apr 2016	CN
30 27 433	Feb 1982	DE
32 18 488	Nov 1983	DE
196 48 519	Jun 1998	DE
103 19 467	Jul 2004	DE
10 2008 006 501	Sep 2008	DE
10 2014 206 823	Oct 2015	DE
0 292 470	Nov 1988	EP
0 167 406	Jul 1991	EP
0 641 606	Mar 1995	EP
1 175 931	Jan 2002	EP
1 254 669	Nov 2002	EP
1 308 724	May 2003	EP
2 209 545	Jul 2010	EP
270152	Jan 2018	EP
2419511	Jan 2018	EP
3068888	Jan 2018	EP
3257600	Jan 2018	EP
3274453	Jan 2018	EP
3274454	Jan 2018	EP
3275894	Jan 2018	EP
278108	Feb 2018	EP
3279315	Feb 2018	EP
3286214	Feb 2018	EP
2289535	Mar 2018	EP
2545068	Mar 2018	EP
2675540	Mar 2018	EP
2750683	Mar 2018	EP
2796102	Mar 2018	EP
3066201	Mar 2018	EP
3066998	Mar 2018	EP
3107552	Mar 2018	EP
3288660	Mar 2018	EP
3288683	Mar 2018	EP
3289362	Mar 2018	EP
3291842	Mar 2018	EP
3291852	Mar 2018	EP
3292142	Mar 2018	EP
3292195	Mar 2018	EP
3292515	Mar 2018	EP
3294343	Mar 2018	EP
3294764	Mar 2018	EP
3294857	Mar 2018	EP
3294871	Mar 2018	EP
3294888	Mar 2018	EP
3294896	Mar 2018	EP
3296302	Mar 2018	EP
3297740	Mar 2018	EP
3298046	Mar 2018	EP
3164488	Apr 2018	EP
3301115	Apr 2018	EP
3302783	Apr 2018	EP
3302789	Apr 2018	EP
3303558	Apr 2018	EP
3306310	Apr 2018	EP
2675901	May 2018	EP
2956772	May 2018	EP
3323444	May 2018	EP
3324996	May 2018	EP
3327127	May 2018	EP
3337819	Jun 2018	EP
2 420 510	May 2006	GB
9-136090	May 1997	JP
1442486	Sep 2014	KR
2037327	Jun 1995	RU
2085933	Jul 1997	RU
629496	Oct 1978	SU
WO 198707178	Dec 1987	WO
WO 8911899	Dec 1989	WO
WO 9005008	Mar 1990	WO
WO 9501214	Jan 1995	WO
WO 9734643	Sep 1997	WO
WO 1998017373	Apr 1998	WO
WO 9850133	Nov 1998	WO
WO 0041794	Jul 2000	WO
WO 02072234	Sep 2002	WO
WO 02072236	Sep 2002	WO
WO 03089567	Oct 2003	WO
WO 2004079716	Sep 2004	WO
WO 2009063198	May 2009	WO
WO 2009111276	Sep 2009	WO
WO 2009144709	Dec 2009	WO
WO 2010024753	Apr 2010	WO
WO 2010040394	Apr 2010	WO
WO 2011023949	Mar 2011	WO
WO 2011025890	Mar 2011	WO
WO 2011027146	Mar 2011	WO
WO 2011130321	Oct 2011	WO
WO 2011131947	Oct 2011	WO
WO 2011161463	Dec 2011	WO
WO 2013043044	Mar 2013	WO
WO 2013043297	Mar 2013	WO
WO 2013049623	Apr 2013	WO
WO 2013055517	Apr 2013	WO
WO 2013138797	Sep 2013	WO
WO 2013148376	Oct 2013	WO
WO 2013159014	Oct 2013	WO
WO 2014014941	Jan 2014	WO
WO 2014029505	Feb 2014	WO
WO 2014035457	Mar 2014	WO
WO 2014046605	Mar 2014	WO
WO 2014055219	Apr 2014	WO
WO 2014124306	Aug 2014	WO
WO 2014153651	Oct 2014	WO
WO 2014165177	Oct 2014	WO
WO 2015006730	Jan 2015	WO
WO 2015102528	Jul 2015	WO
WO 2016004398	Jan 2016	WO
WO 2016124542	Aug 2016	WO
WO 2016176663	Nov 2016	WO
WO 2016209082	Dec 2016	WO
WO 2017011519	Jan 2017	WO
WO 2017021543	Feb 2017	WO
WO 2017041102	Mar 2017	WO
2017132694	Aug 2017	WO
WO 20174201349	Nov 2017	WO
WO 2017218714	Dec 2017	WO
WO 2018009894	Jan 2018	WO
WO 2018002036	Jan 2018	WO
WO 2018005873	Jan 2018	WO
WO 2018013558	Jan 2018	WO
WO 2018013629	Jan 2018	WO
WO 2018013840	Jan 2018	WO
WO2018014174	Jan 2018	WO
WO2018015561	Jan 2018	WO
WO 20180011600	Jan 2018	WO
WO2018018958	Feb 2018	WO
WO2018021920	Feb 2018	WO
WO2018022158	Feb 2018	WO
WO 2018022513	Feb 2018	WO
WO2018022619	Feb 2018	WO
WO2018022651	Feb 2018	WO
WO2018022930	Feb 2018	WO
WO2018023114	Feb 2018	WO
WO2018024639	Feb 2018	WO
WO2018026644	Feb 2018	WO
WO2018026941	Feb 2018	WO
WO2018028647	Feb 2018	WO
WO 2018034343	Feb 2018	WO
WO2018034885	Feb 2018	WO
WO 2018035141	Feb 2018	WO
WO 2018035423	Feb 2018	WO
WO20180202691	Feb 2018	WO
WO2018034655	Mar 2018	WO
WO 2018038711	Mar 2018	WO
WO 2018039119	Mar 2018	WO
WO 2018039407	Mar 2018	WO
WO 2018039408	Mar 2018	WO
WO 2018039410	Mar 2018	WO
WO 2018039412	Mar 2018	WO
WO 2018039515	Mar 2018	WO
WO 2018045284	Mar 2018	WO
WO 2018049226	Mar 2018	WO
WO 2018050738	Mar 2018	WO
WO 2018057825	Mar 2018	WO
WO 2018063291	Apr 2018	WO
WO 2018058275	May 2018	WO
WO 2018081476	May 2018	WO
WO 2018091879	May 2018	WO
WO2018094244	May 2018	WO
WO 20180814701	May 2018	WO
WO 2018098671	Jun 2018	WO
WO 2018102752	Jun 2018	WO
WO 2018106163	Jun 2018	WO
WO 2018112145	Jun 2018	WO
WO 2018112335	Jun 2018	WO

Non-Patent Literature Citations (86)

Entry
Hammarstrom et al. (Lab on Chip, 2012, 12, 4296-4304). (Year: 2012).
Chromatogrpahy forum re: column volume discussion (Year: 2013).
Hoke (Infection and Immunity, May 2008, p. 2063-2069 (Year: 2008).
U.S. Appl. No. 99/908,288, filed Mar. 2018, Harkness.
Alvarez et al.; ShockWaves, vol. 17, No. 6, pp. 441-447, 2008.
Augustsson et al., Acoustophoretic microfluidic chip for sequential elution of surface bound molecules from beads or cells, Biomicrofluidics, Sep. 2012, 6(3):34115.
Benes et al.; Ultrasonic Separation of Suspended Particles, 2001 IEEE Ultrasonics Symposium; Oct. 7-10, 2001; pp. 649-659; Atlanta, Georgia.
Castilho et al.; Animal Cell Technology: From Biopharmaceuticals to Gene Therapy; 11—Animal Cell Separation; 2008.
Castro; Tunable gap and quantum quench dynamics in bilayer graphene; Jul. 13, 2010; Mathematica Summer School.
Chitale et al.; Understanding the Fluid Dynamics Associated with Macro Scale Ultrasonic Separators; Proceedings of Meetings on Acoustics, May 2015.
Cravotto et al.; Ultrasonics Sonochemistry, vol. 15, No. 5, pp. 898-902, 2008.
Garcia-Lopez, et al.; Enhanced Acoustic Separation of Oil-Water Emulsion in Resonant Cavities. The Open Acoustics Journal. 2008, vol. 1, pp. 66-71.
Grenvall et al.; Concurrent Isolation of Lymphocytes and Granulocytes Using Prefocused Free Flow Acoustophoresis; Analytical Chemistry; vol. 87; pp. 5596-5604; 2015.
Higginson et al.; Tunable optics derived from nonlinear acoustic effects; Journal of Applied Physics; vol. 95; No. 10; pp. 5896-5904; 2004.
Hill et al.; Ultrasonic Particle Manipulation; Microfluidic Technologies for Miniaturized Analysis Systems, Jan. 2007, pp. 359-378.
Ilinskii et al.; Acoustic Radiation Force on a Sphere in Tissue; AIP Conference Proceedings; 2012.
Kuznetsova et al.; Microparticle concentration in short path length ultrasonic resonators: Roles of radiation pressure and acoustic streaming; Journal of the Acoustical Society of America, American Institute of Physics for the Acoustical Society of America, vol. 116, No. 4, Oct. 1, 2004, pp. 1956-1966, DOI: 1.1121/1.1785831.
Latt et al.; Ultrasound-membrane hybrid processes for enhancement of filtration properties; Ultrasonics sonochemistry 13.4 (2006): 321-328.
Li et al.; Electromechanical behavior of PZT-brass unimorphs; J. Am. Ceram. Soc. vol. 82; No. 7; pp. 1733-1740, 1999.
Lipkens et al.; The effect of frequency sweeping and fluid flow on particle trajectories in ultrasonic standing waves; IEEE Sensors Journal, vol. 8, No. 6, pp. 667-677, 2008.
Lipkens et al.; Frequency sweeping and fluid flow effects on particle trajectories in ultrasonic standing waves; Acoustics 08, Paris, Jun. 29-Jul. 4, 2008.
Lipkens et al.; Prediction and measurement of particle velocities in ultrasonic standing waves; J. Acoust. Soc. Am., 124 No. 4, pp. 2492 (A) 2008.
Lipkens et al.; Separation of micron-sized particles in macro-scale cavities by ultrasonic standing waves; Presented at the International Congress on Ultrasonics, Santiago; Jan. 11-17, 2009.
Lipkens et al.; Separation of bacterial spores from flowering water in macro-scale cavities by ultrasonic standing waves; submitted/uploaded to http://arxiv.org/abs/1006.5467 on Jun. 28, 2010.
Lipkens et al., Macro-scale acoustophoretic separation of lipid particles from red blood cells, The Journal of the Acoustical Society of America, vol. 133, Jun. 2, 2013, p. 045017, XP055162509, New York, NY.
Meribout et al.; An Industrial-Prototype Acoustic Array for Real-Time Emulsion Layer Detection in Oil Storage Tanks; IEEE Sensors Journal, vol. 9, No. 12, Dec. 2009.
Musiak et al.; Design of a Control System for Acoustophoretic Separation, 2013 IEEE 56th International Midwest Symposium on Circuits and Systems (MWSCAS), Aug. 2013, pp. 1120-1123.
Nilsson et al.; Review of cell and particle trapping in microfluidic systems; Department of Measurement Technology and Industrial Electrical Engineering, Div. of Nanobiotechnology, Lund University, P.O. Box 118. Lund, Sweden, Analytica Chimica Acta 649, Jul. 14, 2009, pp. 141-157.
Pangu et al.; Droplet transport and coalescence kinetics in emulsions subjected to acoustic fields; Ultrasonics 46, pp. 289-302 (2007).
Phys. Org. “Engineers develop revolutionary nanotech water desalination membrane.” Nov. 6, 2006. http://phys.org/news82047372.html.
Ponomarenko et al.; Density of states and zero Landau level probed through capacitance of graphene; Nature Nanotechnology Letters, Jul. 5, 2009; DOI: 10.1038/NNANO.2009.177.
“Proceedings of the Acoustics 2012 Nantes Conference,” Apr. 23-27, 2012, Nantes, France, pp. 278-282.
Ryll et al.; Performance of Small-Scale CHO Perfusion Cultures Using an Acoustic Cell Filtration Device for Cell Retention: Characterization of Separation Efficiency and Impact of Perfusion on Product Quality; Biotechnology and Bioengineering; vol. 69; Iss. 4; pp. 440-449; Aug. 2000.
Seymour et al., J. Chem. Edu., 1990, 67(9), p. 763, published Sep. 1990.
Volpin et al.; Mesh simplification with smooth surface reconstruction; Computer-Aided Design; vol. 30; No. 11; 1998.
Wang et al.; Retention and Viability Characteristics of Mammalian Cells in an Acoustically Driven Polymer Mesh; Biotechnol. Prog. 2004, pp. 384-387 (2004).
Wicklund et al.; Ultrasonic Manipulation of Single Cells; Methods in Molecular Biology; vol. 853; pp. 1777-196; 2012.
Annex to Form PCT/ISA/206—Communication Relating to the Results of the Partial International Search Report dated Jul. 18, 2013.
European Search Report of European Application No. 11769474.5 dated Sep. 5, 2013.
European Search Report of European Application No. 11796470.0 dated Jan. 5, 2016.
European Search Report of European Application No. 13760840.2, dated Feb. 4, 2016.
European Search Report of European Application No. 13721179.3 dated Mar. 23, 2016.
European Search Report for European Application No. 14749278.9 dated Jan. 13, 2017.
Extended European Search Report for European Application No. EP 12833859.7 dated Mar. 20, 2015.
Extended European Search Report for European Application No. EP 14787587.6 dated Jan. 2, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2011/032181 dated Dec. 20, 2011.
International Search Report and Written Opinion for International Application No. PCT/US2011/040787 dated Feb. 27, 2012.
International Search Report and Written Opinion for International Application No. PCT/US2012/051804 dated Nov. 16, 2012.
International Search Report and Written Opinion for International Application No. PCT/US2013/037404 dated Jun. 21, 2013.
International Search Report and Written Opinion for International Application No. PCT/US2013/032705 dated Jul. 26, 2013.
International Search Report and Written Opinion for International Application No. PCT/US2013/050729 dated Sep. 25, 2013.
International Search Report and Written Opinion for International Application No. PCT/US2013/059640 dated Feb. 18, 2014.
International Search Report and Written Opinion for International Application No. PCT/US2014/015382 dated May 6, 2014.
International Search Report and Written Opinion for International Application No. PCT/US2014/035557 dated Aug. 27, 2014.
International Search Report and Written Opinion for International Application No. PCT/US2014/043930 dated Oct. 22, 2014.
International Search Report and Written Opinion for International Application No. PCT/US2014/046412 dated Oct. 27, 2014.
International Search Report and Written Opinion for International Application No. PCT/US2014/064088 dated Jan. 30, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/010595 dated Apr. 15, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/019755 dated May 4, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/030009 dated Jul. 30, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/039125 dated Sep. 30, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/053200 dated Dec. 28, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/066884, dated Mar. 22, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/024082 dated Jun. 27, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/031357 dated Jul. 26, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/038233 dated Sep. 26, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2015/024365 dated Oct. 13, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/041664 dated Oct. 18, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/044586 dated Oct. 21, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/049088 dated Nov. 28, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/050415 dated Nov. 28, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/037104 dated Dec. 16, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2017/015197 dated Apr. 3, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2017/015450 dated Apr. 10, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2016/047217 dated Apr. 11, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2016/048243 dated Apr. 20, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2017/017788 dated May 8, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2017/030903 dated Jul. 19, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2017/025108 dated Jul. 20, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2017/031425 dated Aug. 30, 2017.
Sony New Release: <http://www.sony.net/SonyInfo/News/Press/201010/10-137E/index.html>.
International Search Report and Written Opinion for International Application No. PCT/US2017/031425 dated Oct. 23, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2018/026617, dated Jul. 4, 2018.
International Search Report and Written Opinion for International Application No. PCT/US2018/31267, dated Aug. 1, 2018.
Jin Zuwei, “Expanded Bed Absorption-Challenges and Advances in Column and Process Design”, Pharmaceutical Engineering, vol. 35 No. 1, Jan./Feb. 2015, 12 pages.
Office Action received for European Patent Application No. 18724088.2 dated Feb. 22, 2021, 5 pages.

Related Publications (1)

	Number	Date	Country
	20190062690 A1	Feb 2019	US

Provisional Applications (3)

Number	Date	Country
62508385	May 2017	US
62490574	Apr 2017	US
62197801	Jul 2015	US

Continuation in Parts (2)

	Number	Date	Country
Parent	15963809	Apr 2018	US
Child	15983823		US
Parent	15522800	Jul 2016	US
Child	15963809		US

Expanded bed affinity selection

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

Field of Search

CPC

International Classifications

Term Extension

Abstract