WO2018200830A1 - Expanded bed affinity cell selection - Google Patents
Expanded bed affinity cell selection Download PDFInfo
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- WO2018200830A1 WO2018200830A1 PCT/US2018/029594 US2018029594W WO2018200830A1 WO 2018200830 A1 WO2018200830 A1 WO 2018200830A1 US 2018029594 W US2018029594 W US 2018029594W WO 2018200830 A1 WO2018200830 A1 WO 2018200830A1
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- WIPO (PCT)
- Prior art keywords
- acoustic
- column
- beads
- standing wave
- affinity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
- B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3823—Affinity chromatography of other types, e.g. avidin, streptavidin, biotin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
Definitions
- 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.
- the particles can be driven to and trapped at the nodes or anti-nodes of the standing wave.
- the higher the frequency of the standing wave the smaller the particles that can be trapped.
- 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.
- 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.
- the affinity material and the target material can form antigen-antibody interactions with binding sites on the functionalized material.
- 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.
- 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.
- 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 ⁇ 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.
- the acoustic standing wave forces can be of the same order of magnitude.
- 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.
- 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.
- 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.
- 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.
- 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 microcarners with the expressed proteins from the bioreactor, increasing the efficiency of the acoustic fluidized bed chromatography process.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- an acoustic affinity system is implemented that can include in 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 the 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.
- 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.
- 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.
- 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.
- 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 MAG E 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.
- 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.
- support structures examples discussed above provide beads as support structures.
- Other support structures such as coated bubbles or microbubbles can be also used.
- 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 that can bind to or be bound to a target material of interest.
- Cells are bound to beads, e.g., CD3/CD28 activated beads.
- 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.
- the beads include spheres with a diameter in a range of 1 to 300 ⁇ , e.g., in the range of 125 to 250 ⁇ .
- the spheres can have densities in a range of 1.02-1.10 g/cm 3 .
- 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.
- 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).
- DEAE N, N-diethylaminoethyl
- laminin or vitronectin coating extra cellular matrix proteins.
- a mild positive charge can be added to the surface.
- 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.
- 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.
- cGMP current good manufacturing practice
- 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.
- medium sized beads are those referred to herein as medium sized beads. These medium sized beads may possess the following characteristics.
- small beads are those referred to herein as small beads. These small beads may possess the following characteristics.
- 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.
- 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.
- 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.
- 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.
- the interior surface of the acoustic material is directly exposed to the top end housing.
- 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.
- 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.
- an acoustic radiation force can have an axial force component and a lateral force component that are of the same order of magnitude.
- 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.
- the spatial pattern may create an interface region that blocks entry of particles with certain characteristics from entering or crossing the acoustic wave.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the clusters sink and can be collected separately from the clarified 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).
- the particles 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.
- 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.
- 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.
- 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.
- 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.
- 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 3x3 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.
- the material can be selectively excited to operate in multiple modes such as a 0x0 mode (i.e. a piston mode), lxl, 2x2, 1x3, 3x1, 3x3, 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.
- 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.
- multiple vibration modes are present at arbitrary operating points of the transducer, with some modes dominating at a given operating point.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 multidimensional acoustic standing wave 306, depending on the acoustic contrast factor of beads 304 or the particles relative to the host fluid.
- 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 multidimensional 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.
- clusters 312 typically sink with gravity forces.
- clusters 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.
- 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.
- an agitator can be provided to the chamber to promote movement and redistribution of the clustered beads.
- Type A 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.
- 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.
- 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.
- 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.
- a fluidized bed system 500 is illustrated.
- 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.
- 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.
- beads 504 are free to move within the fluidized bed of column 502.
- 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.
- 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.
- 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.
- 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.
- 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 lOmL to about 1L.
- all types of beads may be used in process 600, providing significant flexibility for unique or custom affinity separation processes.
- 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.
- 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 lOmL to about 1L. 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.
- 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.
- 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.
- 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.
- 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.
- This incubation step can be implemented with no fluid flow and with transducer 806 being unenergized.
- 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.
- the solution can include enzymes (e.g., trypsin) in a buffer.
- 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.
- 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.
- 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.
- 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.
- a graph 1100 illustrates bead retention with an acoustic field versus fluid inflow rate for an acoustic fluidized bed column.
- 100% of the beads are retained in the column as the fluid inflow rate increases from 0 to about 10 mL per minute.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Example 1 Several experimental tests for acoustic affinity cell separation were conducted. The results of the tests are tabulated as examples below.
- the feed cell population was then separated using the fluidized bed unit packed with avi din-conjugated methacrylate beads (Promega) which operated at the following conditions; flow rate - lmL/min and power - 0.75W.
- 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 30ml of a 2% BSA in PBS solution at lml/min and 0.75W. This flushing step is implemented to ensure all uncaptured cells are recovered.
- 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.
- 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).
- 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.
- 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.
- a second fraction denoted as the flush was collected after flushing the fluidized bed with 30ml 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.
- 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 ⁇ ) beads resulted in the lowest purity of 84.3%.
- TCR- cell recoveries for each acoustic separator system test can be seen in Table 4.
- Table 4 The total TCR- cell recoveries for each acoustic separator system test can be seen in Table 4.
- the recovery is affected by the bead type, with 50um Promega beads having an 80% TCR- cell recovery and 4.5um Dyna-beads having just 17%) recovery.
- Both polystyrene particles had similar recoveries, 26% and 27% for 6um and 14um 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.
- TCR- recovery in the Acoustic Separator system can be increased by increasing flow velocity and by improving the inlet and collector designs.
- FB Fluidized Bed
- AS Acoustic Separator
- 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 (30rpm). Cells were washed twice with 2% BSA in PBS and finally re- suspended in lOmL 2% BSA in PBS. For the feed samples for the Acoustic Separator unit, bead incubation was followed by antibody-cell incubation. 1 x 10 6 cells 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 2ml Promega bead slurry (avidin- conjugated methacrylate beads) and then washed with 30ml of a 2% BSA in PBS solution to remove residual ethanol and particulates. This initial washing step was performed at 3mL/min and 2.25W.
- 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 3mL/min and 4mL column volume.
- bead labeled feed were separated at the following conditions; flow rate - lmL/min and power - 0.75W.
- 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
- 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 10 7 cells/mL and TCR a/b- population was about 80 %.
- 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, 2.5mL of feed samples were loaded with (3mL/min) and flow stopped for 3min 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 (4.5W) for a total of 13min 20sec. Once the recirculation steps and stop and flow steps were finished, the column was flushed with 30mL of 2% BSA solution. The processed samples were collected and analyzed as in day 1.
- an acoustic affinity system is discussed that provides a number of advantageous features.
- 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.
- 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.
- 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.
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Abstract
Description
Claims
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CA3082883A CA3082883C (en) | 2017-04-26 | 2018-04-26 | Expanded bed affinity cell selection |
AU2018256852A AU2018256852A1 (en) | 2017-04-26 | 2018-04-26 | Expanded bed affinity cell selection |
EP18724088.2A EP3642621A1 (en) | 2017-04-26 | 2018-04-26 | Expanded bed affinity cell selection |
SG11202003720YA SG11202003720YA (en) | 2017-04-26 | 2018-04-26 | Expanded bed affinity cell selection |
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US201762490574P | 2017-04-26 | 2017-04-26 | |
US62/490,574 | 2017-04-26 |
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WO2018200830A1 true WO2018200830A1 (en) | 2018-11-01 |
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PCT/US2018/029594 WO2018200830A1 (en) | 2017-04-26 | 2018-04-26 | Expanded bed affinity cell selection |
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EP (1) | EP3642621A1 (en) |
AU (1) | AU2018256852A1 (en) |
CA (2) | CA3082883C (en) |
SG (1) | SG11202003720YA (en) |
WO (1) | WO2018200830A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114502717A (en) * | 2019-08-30 | 2022-05-13 | 弗洛设计声能学公司 | Acoustic affinity cell selection for multi-target receptors |
WO2023017126A1 (en) * | 2021-08-13 | 2023-02-16 | Bluetech Aps | Industrial method for isolating an analyte from a liquid mixture |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080272034A1 (en) * | 2004-08-16 | 2008-11-06 | Searete Llc, | Separation of particles from a fluid by wave action |
WO2013148376A1 (en) * | 2012-03-26 | 2013-10-03 | Duke University | Acoustically responsive particles |
US20150321129A1 (en) * | 2014-05-08 | 2015-11-12 | Flodesign Sonics, Inc. | Acoustophoretic device with piezoelectric transducer array |
WO2017019916A1 (en) * | 2015-07-28 | 2017-02-02 | Flodesign Sonics | Acoustic affinity separation |
-
2018
- 2018-04-26 EP EP18724088.2A patent/EP3642621A1/en not_active Withdrawn
- 2018-04-26 SG SG11202003720YA patent/SG11202003720YA/en unknown
- 2018-04-26 CA CA3082883A patent/CA3082883C/en active Active
- 2018-04-26 WO PCT/US2018/029594 patent/WO2018200830A1/en unknown
- 2018-04-26 AU AU2018256852A patent/AU2018256852A1/en not_active Abandoned
- 2018-04-26 CA CA3165215A patent/CA3165215A1/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080272034A1 (en) * | 2004-08-16 | 2008-11-06 | Searete Llc, | Separation of particles from a fluid by wave action |
WO2013148376A1 (en) * | 2012-03-26 | 2013-10-03 | Duke University | Acoustically responsive particles |
US20150321129A1 (en) * | 2014-05-08 | 2015-11-12 | Flodesign Sonics, Inc. | Acoustophoretic device with piezoelectric transducer array |
WO2017019916A1 (en) * | 2015-07-28 | 2017-02-02 | Flodesign Sonics | Acoustic affinity separation |
Non-Patent Citations (1)
Title |
---|
PER AUGUSTSSON ET AL: "Acoustofluidics 11: Affinity specific extraction and sample decomplexing using continuous flow acoustophoresis", LAB ON A CHIP, vol. 12, no. 10, 1 January 2012 (2012-01-01), pages 1742, XP055183547, ISSN: 1473-0197, DOI: 10.1039/c2lc40200a * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114502717A (en) * | 2019-08-30 | 2022-05-13 | 弗洛设计声能学公司 | Acoustic affinity cell selection for multi-target receptors |
WO2023017126A1 (en) * | 2021-08-13 | 2023-02-16 | Bluetech Aps | Industrial method for isolating an analyte from a liquid mixture |
Also Published As
Publication number | Publication date |
---|---|
AU2018256852A1 (en) | 2020-10-22 |
SG11202003720YA (en) | 2020-05-28 |
CA3165215A1 (en) | 2018-11-01 |
CA3082883C (en) | 2022-08-30 |
CA3082883A1 (en) | 2018-11-01 |
EP3642621A1 (en) | 2020-04-29 |
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