CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62/322,262, filed on April 14, 2016, and to U.S. Provisional Patent Application Serial No. 62/307,489, filed on March 12, 2016. The disclosures of these applications are hereby fully incorporated herein by reference in their entirety.

BACKGROUND

[0002] The ability to separate a particle/fluid mixture into its separate components is desirable in many applications. Physical size exclusion filters can be used for this purpose, where the particles are trapped on the filter and the fluid flows through the filter. Examples of physical filters include those that operate by tangential flow filtration, depth flow filtration, hollow fiber filtration, and centrifugation. However, physical filters can be complicated to work with. For instance, as the physical filter fills up, filtration capacity is reduced. Also, using such filters incurs periodic stopping to remove the filter and/or obtain or clear the particles trapped thereon.

[0003] Acoustophoresis is the separation of particles using high intensity sound waves, and without the use of membranes or physical size exclusion filters. It has been known that high intensity standing waves of sound can exert forces on particles. A standing wave has a pressure profile which appears to "stand" still in time. The pressure profile in a standing wave contains areas of net zero pressure at its nodes and anti- nodes. Depending on the density and compressibility of the particles, they will be trapped at the nodes or anti-nodes of the standing wave. However, conventional acoustophoresis devices have had limited efficacy due to several factors including heat generation, limits on fluid flow, and the inability to capture different types of materials. Improved acoustophoresis devices using improved fluid dynamics would be desirable.

BRIEF SUMMARY

[0004] The present disclosure relates to multi-stage acoustophoretic systems that can be used to achieve separation of particles from a particle/fluid mixture. In certain embodiments, the multi-stage acoustophoretic systems described herein can be used with bioreactors, such as in a perfusion process, to produce biomolecules such as recombinant proteins or monoclonal antibodies, and to separate these desirable products from a cell culture in the bioreactor A new mixture with an increased concentration of particles is obtained, or the particles themselves can be obtained or a clarified fluid containing biomolecules, such as recombinant proteins or monoclonal antibodies, may be produced. In more specific embodiments, the particles are biological cells, such as Chinese hamster ovary (CHO) cells, NSO hybridoma cells, baby hamster kidney (BHK) cells, or human cells; lymphocytes such as T cells (e.g., regulatory T-cells (Tregs), Jurkat T-cells), B cells, or NK cells; their precursors, such as peripheral blood mononuclear cells (PBMCs); algae or other plant cells, bacteria, viruses, or microcarriers. The acoustophoretic systems described herein are scalable and are generally useful for cell densities from about 20x 10 ⁶ cells/mL to about 50x 10 ⁶ cells/mL. Several different types of acoustophoretic systems are described herein.

[0005] In particular, the present disclosure provides a novel, scalable, potentially single-use technology for cell culture clarification based on acoustophoretic separation. Acoustic wave separation (AWS) technology involves the use of low-frequency acoustic forces to generate a multi-dimensional acoustic standing wave across a flow channel. Fluid media from a bioreactor enters the flow channel, and as the cells pass through the multi-dimensional acoustic standing wave, they are trapped or suspended by the acoustic forces. The trapped cells migrate to the pressure nodes of the standing wave and begin to clump together, eventually forming clusters that are large enough to settle out of the suspension by gravity. The permeate from the system shows a significant reduction in turbidity and reduces the area specification for secondary clarification using depth filtration and subsequent filtration for bioburden control.

[0006] Disclosed herein are multi-stage acoustophoretic systems, such as two-, three- and four-stage acoustophoretic systems. These multi-stage acoustophoretic systems can incorporate a filter "train" comprising depth filters, sterile filters, centrifuges, and affinity chromatography columns to purify a cell culture by separating recombinant proteins therefrom. In such systems, the frequency / power of the multi-dimensional acoustic standing wave(s) generated in the distinct stages of the system may be varied to send different concentrations or different size materials to subsequent filtration steps in the filter "train," thereby improving the efficiency of the clarification process by effectively managing the material that is processed in each step in the filter "train." In this way, the acoustic filters (i.e., the distinct stages of the system) can be used to manage the material that the next stage in the filter "train" receives for subsequent processing therein.

[0007] A multi-stage acoustophoretic system according to an example of the present disclosure includes three or more acoustophoretic devices fluidly connected to one another. The acoustophoretic devices may be connected in series, so that each acoustophoretic device shares a connection with at least another acoustophoretic device. In some examples, the acoustophoretic devices may or may not have a common connection. The acoustophoretic devices may commonly connect to a recovery pathway for recovering material following separation. Each acoustophoretic device may include a flow chamber having at least one inlet and at least one outlet; at least one ultrasonic transducer coupled to the flow chamber (e.g. located on a wall thereof), the transducer including a piezoelectric material that can be driven by a drive signal to create a multi-dimensional standing wave in the flow chamber; and a reflector opposite from the at least one ultrasonic transducer (e.g. located on the wall opposite the transducer). In particular embodiments, four acoustophoretic devices are present. In some embodiments, two or more acoustophoretic devices are used.

[0008] The acoustophoretic devices of the multi-stage acoustophoretic system can be fluidly connected to one another by tubing. The acoustophoretic devices of the multistage acoustophoretic system can be physically connected directly to one another, either one stage atop another, or side-by-side.

[0009] In particular embodiments, the acoustophoretic devices are configured to create multi-dimensional acoustic standing waves all having frequencies within one order of magnitude of each other. In particular embodiments, the acoustophoretic devices are configured to create multi-dimensional acoustic standing waves all having different frequencies than one another. In certain constructions, each multi-dimensional acoustic standing wave results in an acoustic radiation force having an axial force component and a lateral force component that are of the same order of magnitude. [0010] The multi-stage acoustophoretic system can further comprise a feed pump upstream of the furthest upstream of the at least three acoustophoretic devices and a pump (e.g., a peristaltic pump) downstream of each acoustophoretic device. That is, the multi-stage acoustophoretic system can comprise a feed pump upstream of a first acoustophoretic device, a first pump between the first acoustophoretic device and a second acoustophoretic device, a second pump between the second acoustophoretic device and a third acoustophoretic device, a third pump downstream of the third acoustophoretic device and upstream of a fourth acoustophoretic device (when present), and a fourth pump downstream of the fourth acoustophoretic device.

[0011] The multi-stage acoustophoretic system can also comprise a feed flowmeter upstream of the furthest upstream of the acoustophoretic devices, and a flowmeter downstream of each acoustophoretic device. That is, the multi-stage acoustophoretic system can comprise a feed flowmeter upstream of a first acoustophoretic device, a first flowmeter between the first acoustophoretic device and a second acoustophoretic device, a second flowmeter between the second acoustophoretic device and a third acoustophoretic device, a third flowmeter downstream of the third acoustophoretic device and upstream of a fourth acoustophoretic device, and a fourth flowmeter downstream of the fourth acoustophoretic device.

[0012] Each acoustophoretic device can have at least one dump diffuser at an inlet into the flow chamber. Each acoustophoretic devices can further comprise a port below the at least one ultrasonic transducer thereof. This port can be used to recover separated material from the acoustophoretic device.

[0013] In certain embodiments, the inlet of the multi-stage acoustophoretic system can be fluidly connected to the outlet of a bioreactor. The multi-stage acoustophoretic system can further comprise at least one in-line filtration stage downstream of the furthest downstream of the three or more acoustophoretic devices. The in-line filtration stage can be selected from the group consisting of depth filters, sterile filters, centrifuges, affinity chromatography columns, or other filtration techniques known in the art for purification of proteins.

[0014] Methods for continuously separating a second fluid or a particulate from a host fluid using a multi-stage acoustophoretic system are also disclosed. All of the multi-dimensional standing wave(s) created in each acoustophoretic device of the multistage acoustophoretic system can have different frequencies, or the same frequency, or can have frequencies within the same order of magnitude.

[0015] In certain embodiments of the methods disclosed herein, a drive signal is sent to drive the ultrasonic transducer of the first acoustophoretic device to create a first multi-dimensional acoustic standing wave therein, such that at least a portion of the second fluid or particulate is continuously trapped in the first standing wave, with the residual mixture continuing into a second one of the acoustophoretic devices. Another drive signal is sent to drive the ultrasonic transducer of the second acoustophoretic device to create a second multi-dimensional acoustic standing wave therein, such that at least a portion of the second fluid or particulate is continuously trapped in the second standing wave, with the residual mixture continuing into a third one of the acoustophoretic devices. Another drive signal is sent to drive the ultrasonic transducer of the third acoustophoretic device to create a third multi-dimensional acoustic standing wave therein, such that at least a portion of the second fluid or particulate is continuously trapped in the third standing wave, with the residual mixture continuing into a fourth one of the acoustophoretic devices. Another drive signal is sent to drive the ultrasonic transducer of the fourth acoustophoretic device to create a fourth multidimensional acoustic standing wave therein, such that at least a portion of the second fluid or particulate is continuously trapped in the fourth standing wave.

[0016] The driving signals sent to each of the acoustophoretic devices can be different from each other and can be implemented for a specific range of particulate sizes to selectively filter particulates in the mixture. In certain embodiments, the voltage signal to at least one of the acoustophoretic devices is at least 50V. In certain embodiments, the voltage signal (which is AC) to each of the acoustophoretic devices is from 50V to about 60V (rms). In certain embodiments, the voltage signal to the furthest downstream of the acoustophoretic devices is from 40V to about 60V.

[0017] The second fluid or particulate can be Chinese hamster ovary (CHO) cells, NS0 hybridoma cells, baby hamster kidney (BHK) cells, or human cells; T cells, B cells, or NK cells; peripheral blood mononuclear cells (PBMCs); algae; plant cells, bacteria, viruses, or microcarriers. [0018] In certain embodiments of the methods disclosed herein, voltage signals are sent to drive the ultrasonic transducers of each of the acoustophoretic devices to create a multi-dimensional acoustic standing wave within each of the acoustophoretic devices, such that at least a portion of the second fluid or particulate is continuously trapped in each standing wave, and the frequency and power of each multi-dimensional acoustic standing wave is varied so as to selectively manage the size and/or concentration of the particulates in the mixture that pass from a selected one of the acoustophoretic devices to an immediately downstream one of the acoustophoretic devices.

[0019] These and other non-limiting characteristics are more particularly described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following is a brief description of the drawings, which are presented for the purposes of illustrating the exemplary embodiments disclosed herein and not for the purposes of limiting the same.

[0021] Figure 1 illustrates an example embodiment of a multi-stage acoustophoretic system according to the present disclosure. The acoustophoretic system includes four acoustophoretic stages fluidly connected to one another by tubing.

[0022] Figure 2A illustrates an example embodiment of four acoustophoretic devices / stages physically connected to one another for use in a multi-stage acoustophoretic system according to the present disclosure.

[0023] Figure 2B illustrates an isolated view of one of the acoustophoretic devices / stages of Figure 2A.

[0024] Figure 2C is a cross-sectional diagram of one of the acoustophoretic devices / stages of Figure 2A. The device includes opposing flow dump diffuser inlets generating flow symmetry and more uniform velocities.

[0025] Figure 3 illustrates three of the four acoustophoretic devices of the four-stage acoustophoretic system of Figure 1. The acoustophoretic devices are connected to one another and to pumps via tubing running therebetween. The figure also shows fluid entrained with cell culture media being continuously flowed into each device via an inlet thereof while sedimented / agglomerated cells fall / settle out of each device via a port thereof, and an outlet for the egress of residual fluid from the device to a subsequent device.

[0026] Figure 4 is a magnified side cross-sectional view illustrating cell culture media in a fluid flowing vertically downward through the flow chamber and passing into the acoustic wave zone of separation.

[0027] Figure 5 is a continuation of Figure 4, showing the cells in the fluid becoming trapped in the acoustic standing wave at the nodes of the pressure standing wave due to the lateral component of the acoustic radiation force.

[0028] Figure 6 is a continuation of Figure 5, showing the cells trapped in the acoustic standing wave agglomerating to form clusters of cells that settle out of suspension and fall to the bottom of the flow chamber due to decreased buoyancy / enhanced gravitational effects.

[0029] Figure 7 is a cross-sectional diagram of a conventional ultrasonic transducer.

[0030] Figure 8 is a cross-sectional diagram of an ultrasonic transducer of the present disclosure. An air gap is present within the transducer, and no backing layer or wear plate is present.

[0031] Figure 9 is a cross-sectional diagram of an ultrasonic transducer of the present disclosure. An air gap is present within the transducer, and a backing layer and wear plate are present.

[0032] Figure 10 is a graph of electrical impedance amplitude versus frequency for a square transducer driven at different frequencies.

[0033] Figure 11A illustrates the trapping line configurations for seven of the peak amplitudes of Figure 10 from the direction orthogonal to fluid flow.

[0034] Figure 11 B is a perspective view illustrating the separator. The fluid flow direction and the trapping lines are shown.

[0035] Figure 11C is a view from the fluid inlet along the fluid flow direction (arrow 251 ) of Figure 11 B, showing the trapping nodes of the standing wave where particles would be captured.

[0036] Figure 11 D is a view taken through the transducers face at the trapping line configurations, along arrow 253 as shown in Figure 11 B. [0037] Figure 12 is a graph showing the relationship of the acoustic radiation force, buoyancy force, and Stokes' drag force to particle size. The horizontal axis is in microns (pm) and the vertical axis is in Newtons (N).

[0038] Figure 13 is a performance chart showing the pressure drop for a system of the present disclosure having three acoustophoretic devices in series in a first experiment. The y-axis is pressure drop in psig, and runs from 0 to 30 in intervals of 5. The x-axis is volumetric throughput capacity in liters per square meter, and runs from 0 to 140 in intervals of 20.

[0039] Figure 14 is a set of two graphs showing the performance of a second experimental system. The bottom graph has a y-axis of percent reduction, and runs from 0% to 100% in intervals of 10%. The x-axis is test duration in minutes, and runs from 0 to 80 in intervals of 10. The top graph has a y-axis of percent reduction, and is logarithmic, with values of 0, 90%, and 99.0%.

[0040] Figure 15 is a set of graphs showing the performance of a third experimental system. The bottom graph has a y-axis of percent reduction, and runs from 0% to 100% in intervals of 10%. The x-axis is test duration in minutes, and runs from 0 to 80 in intervals of 10. The top graph has a y-axis of percent reduction, and is logarithmic, with values of 0, 90%, and 99.0%.

[0041] Figure 16 is a graph showing the performance or volumetric throughput (VT) per stage of a fifth experimental four-stage acoustophoretic system. The top line along the right side of the graph represents the DOHC filter pressure drop. The dotted line extending from the DOHC pressure drop line represents the DOHC filter turbidity. The bottom line along the right side of the graph represents the XOHC filter pressure drop. The dotted line extending from the XOHC pressure drop line represents the XOHC filter turbidity.

[0042] Figure 17 is another graph showing the performance or volumetric throughput (VT) across all four stages of the fifth four-stage acoustophoretic experimental system. The top line along the right side of the graph represents the total pressure drop. The second line from the top along the right side of the graph represents the DOHC filter pressure drop. The dotted line extending from the DOHC pressure drop line represents the DOHC filter turbidity. The bottom line along the right side of the graph represents the XOHC filter pressure drop. The dotted line extending from the XOHC pressure drop line represents the XOHC filter turbidity.

[0043] Figure 18 illustrates the performance of a sixth experimental system compared to depth flow filtration (DFF). The DFF performance is shown on the top line and used filters having areas of 35 m ² and 1 1 .6 m ² to achieve the desired performance. Along the bottom line, the use of a three-stage AWS system in conjunction with a depth filter reduced the filter area used to 10.2 m ² , and the use of a four-stage AWS system in conjunction with a depth filter further reduced the filter area used to just 4.5 m ²

[0044] Figure 19 illustrates an exemplary setup of a three-stage acoustophoretic system used as the experimental system for some of the performance testing of the present disclosure.

[0045] Figure 20 illustrates an exemplary setup of a four-stage acoustophoretic system used as the experimental system for much of the performance testing of the present disclosure.

[0046] Figure 21 is a graph showing turbidity reduction vs feed PCM for the system of Figure 20. The y-axis is turbidity reduction by percentage, and runs from 0% to 100% in intervals of 10%. The x-axis is feed PCM by percentage, and runs from 0% to 15% in intervals of 5%. The double-compound line with large circular data points represents an experimental run at [QF / (QS/QF)] = [0.6x / 10%]. The dashed line with large triangular data points represents an experimental run at [QF / (QS/QF)] = [1 .0x / 10%]. The line with box-X data points represents a first experimental run at [QF / (QS/QF)] = [1 .0x / 20%]. The triple-compound line with large diamond-shaped data points represents a second experimental run at [QF / (QS/QF)] = [1 .Ox / 20%]. The dotted line with large square data points represents a third experimental run at [QF / (QS/QF)] = [1 .Ox / 20%]. The optimal (i.e., greatest) turbidity reduction occurred at a feed PCM of ~ 5-6%.

[0047] Figure 22 is a graph showing percent reduction vs feed flow rate (QF) for three different factors (TCD, turbidity, PCM) for the system of Figure 20 at QS/QF = 20%. The y-axis is reduction by percentage, and runs from 0% to 100% in intervals of 10%. The x-axis is feed flow rate (x flow), and runs from 0 to 3.5 in intervals of 0.5. [0048] Figure 23 is a graph showing turbidity reduction vs flow ratio for the system of Figure 20 at QS/QF = 20%. The y-axis is turbidity reduction by percentage, and runs from 0% to 100% in intervals of 10%. The x-axis is flow ratio (Qsi/Qs2), and runs from 0 to 5 in intervals of 1 . The top line represents 0.6x flow, and the bottom line represents 1 .3x flow.

[0049] Figure 24 is a graph showing PCM vs solids flow ratio for the system of Figure 20. The y-axis is packed cell mass (PCM) by percentage, and runs from 0% to 50% in intervals of 5%. The x-axis is flow ratio (Qsi/Qs2), and runs from 0 to 5 in intervals of 1 . The top line along the right side of the graph represents solids stage 2. The middle line along the right side of the graph represents solids stage 1 . The bottom line along the right side of the graph represents permeate.

[0050] Figure 25 is a graph showing turbidity reduction vs flow ratio for the system of Figure 20. The y-axis is turbidity reduction by percentage, and runs from 0% to 100% in intervals of 10%. The x-axis is flow ratio (QS/QF), and runs from 0% to 25% in intervals of 5%.

[0051] Figure 26 is a graph showing % reduction for three different factors (turbidity, TCD, PCM) vs feed PCM for the system of Figure 20. The y-axis is reduction by percentage, and runs from 0% to 100% in intervals of 10%. The x-axis is feed PCM by percentage, and runs from 0% to 12% in intervals of 2%. The diamond-shaped data points represent the normalized turbidity reduction. The square data points represent the TCD reduction. The triangular data points represent the PCM reduction.

[0052] Figure 27 is a graph showing % reduction vs feed cell viability for the system of Figure 20. The y-axis is reduction by percentage, and runs from 0% to 100% in intervals of 10%. The x-axis is feed cell viability by percentage, and runs from 0% to 100% in intervals of 20%. Again, the diamond-shaped data points represent the normalized turbidity reduction, the square data points represent the TCD reduction, and the triangular data points represent the PCM reduction.

[0053] Figure 28 is another graph showing the performance of the seventh experimental system. The performance of each stage is shown over time, and shows that the performance does not deteriorate. The y-axis is TCD reduction by percentage, and runs from 0% to 100% in intervals of 10%. Along the x-axis, the leftmost set of bars represents data taken after 2 hours, the set of bars second from the left represents data taken after 4 hours, the middle set of bars represents data taken after 6 hours, the set of bars second from the right represents data taken after 9 hours, and the rightmost set of bars represents data taken after 12.5 hours. Within each set of bars, the leftmost bar represents stage 1 , the second bar from the left represents stage 2, the second bar from the right represents stage 3, and the rightmost bar represents stage 4.

[0054] Figure 29 is another graph showing the performance of the seventh experimental system. The graph shows comparative results for the depth filter area used for a depth filter (DFF) alone (left-side image) versus the equivalent area for a DFF used with an acoustophoretic filter to obtain the same capacity (right-side image). In both images, the y-axis is the specified area of the depth filter in m ² , and runs from 0 to 60 in intervals of 1 0. In both images, along the x-axis, the leftmost set of bars represents a first customer, the middle set of bars represents a second customer, and the rightmost set of bars represents a third customer. Within each set of bars, the leftmost bar represents stage 1 , the second bar from the left represents stage 2, the second bar from the right represents stage 3, and the rightmost bar (when present) represents stage 4.

[0055] Figure 30 is another graph showing the performance of the seventh experimental system. The y-axis is the cell density reduction by percentage, and runs from 0% to 100% in intervals of 1 0%. The x-axis is TCD reduction by percentage for various cell viabilities. Along the x-axis, the leftmost bar represents a cell density of 22x 1 0 ⁶ cells/mL at a cell viability of 82%, the second bar from the left represents a cell density of 22x 1 0 ⁶ cells/mL at a cell viability of 80%, the third bar from the left represents a cell density of 22x 10 ⁶ cells/mL at a cell viability of 64%, the third bar from the right represents a cell density of 40x 1 0 ⁶ cells/mL at a cell viability of 62%, the second bar from the right represents a cell density of 70x 1 0 ⁶ cells/mL at a cell viability of 60%, and the rightmost bar represents a cell density of 1 00χ 1 0 ⁶ cells/mL at a cell viability of 70%.

DETAILED DESCRIPTION

[0056] The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein. In the following specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings.

[0057] Although specific terms are used in the following description for the sake of clarity, these terms are intended to refer only to the particular structure of the embodiments selected for illustration in the drawings, and are not intended to define or limit the scope of the disclosure. In the drawings and the following description below, it is to be understood that like numeric designations refer to components of like function. Furthermore, it should be understood that the drawings are not to scale.

[0058] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0059] As used in the specification and in the claims, the term "comprising" may include the embodiments "consisting of" and "consisting essentially of." The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named components/steps and permit the presence of other components/steps. However, such description should be construed as also describing compositions or processes as "consisting of" and "consisting essentially of" the enumerated components/steps, which allows the presence of only the named components/steps, along with any impurities that might result therefrom, and excludes other components/steps.

[0060] Numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0061] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of "from 2 grams to 10 grams" is inclusive of the endpoints, 2 grams and 10 grams, and all the intermediate values).

[0062] A value modified by a term or terms, such as "about" and "substantially," may not be limited to the precise value specified. The approximating language may correspond to the precision of an instrument for measuring the value. The modifier "about" should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression "from about 2 to about 4" also discloses the range "from 2 to 4."

[0063] It should be noted that many of the terms used herein are relative terms. For example, the terms "upper" and "lower" are relative to each other in location, i.e. an upper component is located at a higher elevation than a lower component in a given orientation, but these terms can change if the device is flipped. The terms "inlet" and "outlet" are relative to a fluid flowing through them with respect to a given structure, e.g. a fluid flows through the inlet into the structure and flows through the outlet out of the structure. The terms "upstream" and "downstream" are relative to the direction in which a fluid flows through various components, i.e. the flow fluids through an upstream component prior to flowing through the downstream component. It should be noted that in a loop, a first component can be described as being both upstream of and downstream of a second component.

[0064] The terms "horizontal" and "vertical" are used to indicate direction relative to an absolute reference, i.e. ground level. The terms "upwards" and "downwards" are also relative to an absolute reference; an upwards flow is always against the gravity of the earth.

[0065] The present application refers to "the same order of magnitude." Two numbers are of the same order of magnitude if the quotient of the larger number divided by the smaller number is a value of at least 1 and less than 10.

[0066] The present application also refers to an "acute" angle. For purposes of the present disclosure, the term "acute" refers to an angle between 0° and 90°, exclusive of 0° and 90°.

[0067] Advances in fed batch cell culture have led to higher cell densities of up to 50x10 ⁶ cells/mL and product titers of > 5g/L. Accompanied by a shift towards the use single-use technology in cell culture, improvements in efficiency of the cell harvest and clarification stages are sought to generate harvested cell culture fluid (HCCF) for capture chromatography and subsequent downstream processing. The evolution of continuous processes where there is a preference for a continuous feed of HCCF available for direct load to the continuous multicolumn capture chromatography step may also factor into efficiency considerations. Existing cell culture clarification using either centrifugation or depth filtration are typically operated in batch mode and use bulk storage of feed or HCCF during the process.

[0068] The acoustophoretic separation technology of the present disclosure employs ultrasonic standing waves to trap, i.e., hold stationary, secondary phase materials, including fluids and/or particles, in a host fluid stream. The trapping of secondary phase materials is an important distinction from previous approaches where particle trajectories were merely altered by the effect of the acoustic radiation force. The scattering of the acoustic field off the particles results in a three-dimensional acoustic radiation force, which acts as a three-dimensional trapping field. The three-dimensional acoustic radiation force generated in conjunction with an ultrasonic standing wave is referred to in the present disclosure as a three-dimensional or multi-dimensional standing wave. 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. It is proportional to frequency and the acoustic contrast factor. It also scales with acoustic energy (e.g. the square of the acoustic pressure amplitude). For harmonic excitation, the sinusoidal spatial variation of the force is what drives the particles to the stable positions within the standing waves. 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 particle can be trapped within the acoustic standing wave field. This trapping results in concentration, agglomeration and/or coalescence of the trapped particles. Additionally, secondary inter-particle forces, such as Bjerkness forces, aid in particle agglomeration. Heavier-than-the-host-fluid (i.e. denser than the host fluid) particles are separated through enhanced gravitational settling.

[0069] One specific application for the acoustophoresis device is in the processing of bioreactor materials. It is desirable to filter as much as possible or all of the cells and cell debris from the expressed materials that are in the fluid stream. The expressed materials are composed of biomolecules such as recombinant proteins or monoclonal antibodies, and are the desired product to be recovered. Through the use of acoustophoresis, the separation of the cells and cell debris is very efficient and leads to very little loss of the expressed materials. This separation technique is an improvement over current filtration processes (depth filtration, tangential flow filtration, centrifugation), which show limited efficiencies at high cell densities, where the loss of the expressed materials in the filter beds themselves can be up to 5% of the materials produced by the bioreactor. The use of mammalian cell cultures including Chinese hamster ovary (CHO), NSO hybridoma cells, baby hamster kidney (BHK) cells, and human cells has proven to be a very efficacious way of producing/expressing the recombinant proteins and monoclonal antibodies used in today's pharmaceuticals. The filtration of the mammalian cells and the mammalian cell debris through acoustophoresis aids in greatly increasing the yield of the bioreactor. The acoustophoresis techniques discussed herein permit the cells and/or their expressed materials, to be recovered.

[0070] In the acoustophoresis techniques discussed herein, the contrast factor is the difference between the compressibility and density of the particles and the fluid itself. These properties are characteristic of the particles and the fluid themselves. Most cell types present a higher density and lower compressibility than the medium in which they are suspended, so that the acoustic contrast factor between the cells and the medium has a positive value. The axial acoustic radiation force (ARF) drives the cells, with a positive contrast factor, to the pressure nodal planes, whereas cells or other particles with a negative contrast factor are driven to the pressure anti-nodal planes. The radial or lateral component of the acoustic radiation force helps trap the cells. In some examples, radial or lateral components of the ARF are larger than the combined effect of fluid drag force and gravitational force.

[0071] As the cells agglomerate at the nodes of the standing wave, there is also a physical scrubbing of the cell culture media that occurs whereby more cells are trapped as they come in contact with the cells that are already held within the standing wave. This phenomenon, or combination of phenomena, contributes to separating the cells from the cell culture media. The expressed biomolecules remain in the nutrient fluid stream (i.e. cell culture medium).

[0072] Desirably, the ultrasonic transducer(s) generate a three-dimensional or multidimensional acoustic standing wave in the fluid that exerts a lateral force on the suspended particles to accompany the axial force so as to increase the particle trapping capabilities of the standing wave. Ultrasonic transducer publications in acoustic related literature provide typical results that indicate that the lateral force in planar or one- dimensional acoustic wave generation is two orders of magnitude smaller than the axial force. In contrast, the technology disclosed in this application provides for a lateral force to be of the same order of magnitude as the axial force.

[0073] It is also possible to drive multiple ultrasonic transducers with arbitrary phasing and/or different or variable frequencies. Multiple transducers may work to separate materials in a fluid stream while being out of phase with each other and/or while operating at different or variable frequencies. Alternatively, or in addition, a single ultrasonic transducer that has been divided into an ordered array may also be operated such that some components of the array will be out of phase with other components of the array.

[0074] Three-dimensional (3-D) or multi-dimensional acoustic standing waves are generated from one or more piezoelectric transducers, where the transducers are electrically or mechanically excited such that they move in a multi-excitation mode. The types of waves thus generated can be characterized as composite waves, with displacement profiles that are similar to leaky symmetric (also referred to as compressional or extensional) Lamb waves. The waves are leaky because they radiate into the water layer, which result in the generation of the acoustic standing waves in the water layer. Symmetric Lamb waves have displacement profiles that are symmetric with respect to the neutral axis of the piezoelectric element, which causes multiple standing waves to be generated in a 3-D space. Through this manner of wave generation, a higher lateral trapping force is generated than if the piezoelectric transducer is excited in a "piston" mode where only a single, planar standing wave is generated. Thus, with the same input power to a piezoelectric transducer, the 3-D or multi-dimensional acoustic standing waves can have a higher lateral trapping force which may be up to and beyond 10 times stronger than a single acoustic standing wave generated in piston mode.

[0075] It may be desirable, at times, due to acoustic streaming, to modulate the frequency or voltage amplitude of the standing wave. Such modulation may be done by amplitude modulation and/or by frequency modulation. The duty cycle of the propagation of the standing wave may also be utilized to achieve certain results for trapping of materials. In other words, the acoustic beam may be turned on and shut off at different frequencies to achieve desired results.

[0076] In certain applications, multiple acoustophoretic cell filtration devices may be implemented for clarification of a bioreactor cell culture and separation of the biomolecules / proteins from the cells that express them. The present disclosure relates to acoustophoretic systems that are made of modular components, and to kits of such modules. Each module cooperatively engages other modules, and can then be reversibly separated. The kits and modules permit the user to make different configurations of acoustophoretic systems as desired to provide for improved settling and improved separation of particles from fluid. Briefly, particles that are suspended in a host fluid can be subjected to multiple transducers generating multiple standing waves to induce separation from the fluid itself. Improved fluid dynamics can also be provided using the modular components, increasing separation of particles from fluid.

[0077] The use of multiple standing waves from multiple ultrasonic transducers allows for multiple separation stages. For example, in a flow path that runs past four ultrasonic stage-reflector pairs (i.e., four acoustophoretic stages), the first stage (and its standing wave) collects a certain amount of the particles, the second stage (and its standing wave) collects particles that passed through the first stage, the third stage (and its standing wave) collects particles that passed through the first and second stages, and the fourth stage (and its standing wave) collects particles that passed through the first, second, and third stages. This construction can be useful where the particle/fluid ratio is high (i.e. large volume of particles), and the separation capacity of any upstream transducers is reached. This construction can also be useful for particles that have a bimodal or greater size distribution, where each transducer can be implemented to capture particles within a certain size range.

[0078] Figure 1 illustrates a first example embodiment of a multi-stage acoustophoretic system 2000. System 2000 includes a first acoustophoretic device 2010, a second acoustophoretic device 2020, a third acoustophoretic device 2030, and a fourth acoustophoretic device 2040. Each device can be considered a different acoustophoretic or filtration stage. In this regard, system 2000 is a four-stage acoustophoretic system because each acoustophoretic device can be constructed as described herein including a transducer-reflector pair(s) to create at least one multidimensional acoustic standing wave within each device. The acoustic chambers of each device can, in certain embodiments, have an area of 1 inch x 2 inches. The nominal flow rate through the system can be about 4 L/hour, typically about 3.64 L/hour (i.e., about 60 mL/min).

[0079] In the system 2000 depicted in Figure 1 , the devices 2010, 2020, 2030, 2040 are connected to each other in series, with each device / stage being connected to adjacent stages by tubing 2060 running therebetween. Attaching adjacent stages to one another using tubing (as opposed to directly connecting the device of each stage to adjacent devices) provides for better separation of fluid and particulate.

[0080] Pumps (e.g., peristaltic pumps) may be provided between each device, and additional pumps can be provided upstream of the first device and downstream of the last device. In this regard, it is noted that in the four-stage system 2000 depicted in Figure 1 , five pumps are present for the four devices / stages: (1 ) a feed pump 2008 upstream of the first acoustophoretic device 2010; (2) a first pump 2018 downstream of the first acoustophoretic device 2010 and upstream of the second acoustophoretic device 2020; (3) a second pump 2028 downstream of the second acoustophoretic device 2020 and upstream of the third acoustophoretic device 2030; (4) a third pump 2038 downstream of the third acoustophoretic device 2030 and upstream of the fourth acoustophoretic device 2040; and (5) a fourth pump 2048 downstream of the fourth acoustophoretic device 2040. Put more simply, the embodiment of system 2000 depicted in Figure 1 includes a feed pump 2008 upstream of the first acoustophoretic device 2010 and a pump 2018, 2028, 2038, 2039 downstream of each acoustophoretic device 2010, 2020, 2030, 2040, respectively. The pumps 2008, 2018, 2028, 2038, 2048 are fluidly connected between respective adjacent devices / stages by tubing 2060

[0081] In addition to pumps, the embodiment of system 2000 depicted in Figure 1 includes flowmeters adjacent each pump. As illustrated here, five flowmeters are present: (1 ) a first flowmeter 2009 upstream of the first acoustophoretic device 2010; (2) a second flowmeter 2019 downstream of the first acoustophoretic device 2010 and upstream of the second acoustophoretic device 2020; (3) a third flowmeter 2029 downstream of the second acoustophoretic device 2020 and upstream of the third acoustophoretic device 20430; (4) a fourth flowmeter 2039 downstream of the third acoustophoretic device 2030 and upstream of the fourth acoustophoretic device 2040; and (5) a fifth flowmeter 2049 downstream of the fourth acoustophoretic device 2040. Put more simply, the embodiment of system 2000 depicted in Figure 1 includes a feed flowmeter 2009 upstream of the first acoustophoretic device 2010 and flowmeters 2019, 2029, 2039, 2049 downstream of each acoustophoretic device 2010, 2020, 2030, 2040, respectively. As with the pumps, the flowmeters 2009, 2019, 2029, 2039, 2049 are fluidly connected between respective adjacent devices / stages by tubing 2060. Thus, fluid flow is through a feed pump, then a flowmeter, then the first acoustophoretic / filtration stage, then a pump, then a flowmeter, etc., ending with a final pump and a flowmeter.

[0082] In some embodiments, such as that depicted in Figure 2A, the individual device stages 2010, 2020, 2030, 2040 are physically located side-by-side, and are physically connected to each other. However, physical proximity is not required - the stages can be separated from each other, and be fluidly connected by tubing, such as depicted in Figure 1 and Figure 3. Figure 2B is a rear view of the first acoustophoretic stage 2010.

[0083] Figure 2C shows a cross-sectional diagram of an exemplary acoustophoretic device / stage 2100, which can be used as any of the devices / stages of the multi-stage acoustophoretic systems described herein. This device can be used to ameliorate some of the problems with a fluid at low particle Reynolds numbers, and create a more uniform flow through the device. The device 2100 has upward, vertical flow through the acoustic chamber 2111. The acoustic chamber also has two opposing dump diffusers 2112 and a collector design which provides a vertical plane or line of flow symmetry. Generally, the cross-section of the device in the flow direction is circular or rectangular. In this example, the acoustic chamber is empty (in the absence of fluid), e.g., there are no other structures in the chamber between the transducer and the reflector, and fluid is permitted to flow through the acoustic chamber. At least one permeate outlet 2114 is present at the upper end of the acoustic chamber. At least one concentrate outlet 2116 is present at the lower end of the acoustic chamber. A shallow wall 2118 is present at the lower end of the acoustic chamber, and leads to the concentrate outlet 2116. The shallow wall is angled relative to a horizontal plane, which may be described by the bottom of the acoustic chamber. At least one ultrasonic transducer (not shown) is coupled to the acoustic chamber, and may be located, for example, on a sidewall of the acoustic chamber. At least one reflector (not shown), or another ultrasonic transducer, is positioned opposite the ultrasonic transducer, and may be located, for example, on the sidewall opposite the ultrasonic transducer. The multidimensional standing wave may be generated using a transducer and an opposing reflector, or may be generated using two opposing transducers.

[0084] This device 2110 includes a symmetrical, dual dump diffuser, plenum inlet configuration. Here, two dump diffusers 2112 are placed on opposite sides of the device. Each dump diffuser has a plenum /chamber with an upper end 2120 and a lower end 2122. The plenum volume provides flow diffusion and dramatically reduces incoming flow non-uniformities. An inlet flow port 2124 is located above the lower end 2122, and at least one flow outlet 2126 is located at the lower end of the plenum. A solid wall 2128 is present at the upper end of the plenum. These dump diffuser flow outlets can be in the form of slots or a line of holes, and they are placed above the bottom of the acoustic chamber. The diffusers 2112 provide a flow direction through the acoustic chamber that is at an angle, such as normal, to the axial direction of the acoustic standing waves generated by the ultrasonic transducer. The acoustic chamber inlets are also arranged so that they are in opposing locations, so that the horizontal velocity decreases toward zero in the center of the acoustic chamber.

[0085] The dump diffusers contribute to reducing or eliminating downward flow of the fluid mixture in the acoustic chamber. The fluid mixture fills up the plenum in the dump diffuser and then flows horizontally into the acoustic chamber, where the mixture flows vertically upwards past the acoustic standing waves. The dump diffuser reduces / eliminates flow pulsations and flow non-uniformities that result from pumps, hosing and/or horizontal inlet flow where gravity effects dominate. The dump diffuser brings the mixture into the acoustic chamber below the ultrasonic transducer, and thus below the nodal clusters or lines that form in the ultrasonic standing waves. This arrangement helps to reduce or minimize any disturbances of the clusters that might otherwise be caused by inflowing material.

[0086] The vertical plane or line of symmetry is aligned with gravity forces. Also shown are flow streamlines which are desirably symmetrical to reduce or minimize non- uniformities, eddy disturbances, circulation, and disturbance of clusters falling through the concentrate outlet 2116 to be collected. Symmetry also contributes to even distribution of gravity forces in the inlet flow distribution and particle collection process. Because it is heavier than the permeate exiting at the top of the device, the (relatively) heavy incoming mixture comes in near the bottom of the acoustic chamber, spreads out across the bottom of the chamber due to gravity forces, and provides near uniform velocity profiles from bottom to top. The horizontal velocity of the mixture may decrease to approach or equal zero as the mixture nears the center of the acoustic chamber due to the dual opposing inlet flows. This reduction in horizontal velocity contributes to reduced or minimized interference between the chamber flow and dropping particle clusters. A uniform velocity enhances and may maximize separation and collection results. The lateral acoustic forces of the acoustic standing wave can overcome particle drag and permit the particles to be trapped to form clusters and to grow and be continuously removed from the acoustic standing wave. The uniform velocity can aid in avoiding uneven disturbances or interference with the lateral acoustic forces. The uniform velocity permits an inlet flow distributor to be optional.

[0087] As the particle clusters drop out, the axial acoustic forces associated with the standing wave contribute to keeping the clusters intact. Keeping the clusters intact can help to maintain rapid dropping of the clusters with high terminal velocities, on the order of 1 cm/sec. The dropping velocity of the clusters can be extremely fast compared to the chamber flow velocities, which may be on the order of 0.1 cm/sec to 0.3 cm/sec in some examples. The shallow wall angle means the particle clusters, which may have a cylindrical shape, can travel a relatively short distance before they exit the acoustic chamber, so that little dispersion of the clusters occurs. Preferably, the system operates with 3 to 12 crystal vibration nodes per square inch of transducer. The symmetry, reduced flow disturbance in the central collection region, and shallow collector walls obtain improved collection, and may permit baffles / laminar plates to be optional.

[0088] Figure 3 is a diagram that generally illustrates the fluid flow path through an acoustophoretic stage 2010. Fresh fluid/cell culture media mixture is continuously introduced to the stage via an inlet 2012 at a top end thereof, and flows through the flow chamber 2050. Within the flow chamber, cells are agglomerated and fall / settle out of the acoustic standing waves. These sedimented cell agglomerates / aggregates then fall to the bottom of the flow chamber 2050 and can be recovered via port 2016. The residual mixture flows out of the flow chamber through an outlet 2014 at a top end of the device and continues on to downstream devices, as described herein.

[0089] Figures 4-6 illustrate the functioning of an acoustophoretic stage. As shown in Figure 4, a fluid/cell culture media mixture enters the acoustophoretic stage via tubing and passes into an acoustic wave zone of separation, i.e. the area in which the at least one multi-dimensional acoustic standing wave is created. This acoustic wave zone is defined between the ultrasonic transducer and the reflector. As depicted in Figure 5, due to the effect of the lateral component of the acoustic radiation force, the cells become trapped in the acoustic standing wave at the nodes thereof. As depicted in Figure 6, as the cells agglomerate at the nodes, a reduction in acoustic radiation force and/or decreased buoyancy / enhanced gravitational effects cause the agglomerated cells to settle out of suspension and fall to the bottom of the flow chamber.

[0090] As briefly explained above and illustrated in Figure 3, it should be noted that, in many embodiments of the multi-stage acoustophoretic system described herein, the ultrasonic transducer(s) is directly adjacent to the flow chamber, and is directly exposed to any fluid passing through the flow chamber. The transducer can be separated from the flow chamber by a thin film of, for example, polyether etherketone (PEEK) or any suitable material that is acoustically transparent, or other suitable material that can act as a barrier between the transducer and the fluid in the flow chamber. The reflector is solid or flexible, and can be made of a high acoustic impedance material such as steel or tungsten, or any other suitable material providing good acoustic reflection. One specifically contemplated material for the reflector of the multi-stage acoustophoretic systems described herein is borosilicate glass. Another transducer may also be used as a reflector.

[0091] The various parts of the multi-stage acoustophoretic system described herein, such as the flow chamber, can be made from any suitable material that can house a fluid mixture. Such suitable materials for the flow chamber and associated parts include medical grade plastics, such as polycarbonates or polymethyl methacrylates, or other acrylates. One specifically contemplated material for the flow chamber / housing of the multi-stage acoustophoretic systems described herein is polyphenylsulfone (PPS). The material may be configured to be at least somewhat transparent as a clear window to permit the internal flow channels and flow paths to be seen during operation of the acoustophoresis device / system.

[0092] The multi-dimensional acoustic standing wave used for particle collection is obtained by driving an ultrasonic transducer at a frequency that both generates the acoustic standing wave and excites a fundamental 3D vibration mode of the piezoelectric material of the transducer. Perturbation of the piezoelectric material in an ultrasonic transducer in a multimode fashion allows for generation of a multidimensional acoustic standing wave. A piezoelectric material can be specifically designed to deform in a multimode fashion at designated frequencies, allowing for generation of a multidimensional acoustic standing wave. The multi-dimensional acoustic standing wave may be generated by distinct modes of the piezoelectric material such as a 3x3 mode that would generate 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 piezoelectric material can be excited to generate multiple modes such as a 0x0 mode (i.e. a piston mode) to a 1x1 , 2x2, 1x3, 3x1 , 3x3, and other higher order modes and then cycle back through the lower modes of the piezoelectric material (not necessarily in straight order). This switching or dithering of the piezoelectric material between modes allows for various multidimensional wave shapes, along with a single piston mode shape to be generated over a designated time.

[0093] Some further explanation of the ultrasonic transducers used in the devices, systems, and methods of the present disclosure may be useful as well. In this regard, the transducers use a piezoelectric material, which can be a ceramic material, a crystal or a polycrystal, such as PZT-8 (lead zirconate titanate). Such crystals may have a 1 inch diameter and a nominal 2 MHz resonance frequency. Each ultrasonic transducer module can have only one piezoelectric material, or can have multiple piezoelectric materials. The multiple piezoelectric materials can each act as a separate ultrasonic transducer and may be controlled by one or multiple controllers, drivers or amplifiers.

[0094] Figure 7 is a cross-sectional diagram of a conventional ultrasonic transducer. This transducer has a wear plate 50 at a bottom end, epoxy layer 52, piezoelectric crystal 54 (made of, e.g. PZT), an epoxy layer 56, and a backing layer 58. On either side of the piezoelectric crystal, there is an electrode: a positive electrode 61 and a negative electrode 63. The epoxy layer 56 attaches backing layer 58 to the crystal 54. The entire assembly is contained in a housing 60 which may be made out of, for example, aluminum. An electrical adapter 62 provides a connection for wires to pass through the housing and connect to leads (not shown) which attach to the crystal 54. Typically, backing layers are designed to add damping and to create a broadband transducer with uniform displacement across a wide range of frequency and are designed to suppress excitation at particular vibrational eigen-modes. Wear plates are usually designed as impedance transformers to better match the characteristic impedance of the medium into which the transducer radiates.

[0095] Figure 8 is a cross-sectional view of an ultrasonic transducer 81 of the present disclosure. Transducer 81 is shaped as a disc or a plate, and has an aluminum housing 82. The aluminum housing has a top end and a bottom end. The transducer housing may also be composed of plastics, such as medical grade HDPE or other metals. The piezoelectric element is a mass of perovskite ceramic, each consisting of a small, tetravalent metal ion, usually titanium or zirconium, in a lattice of larger, divalent metal ions, usually lead or barium, and O ^2" ions. As an example, a PZT (lead zirconate titanate) piezoelectric element 86 defines the bottom end of the transducer, and is exposed at the exterior of the bottom end of the housing. The piezoelectric element is supported on its perimeter by a small elastic layer 98, e.g. epoxy, silicone or similar material, located between the piezoelectric element and the housing. Transducer 81 does not include a wear plate or backing material. In some embodiments, a layer of plastic or other material (not shown) is provided over the exterior surface of piezoelectric element 86. The material has a feature of separating piezoelectric element 86 from the fluid in which the acoustic standing wave is being generated. The material may be relatively thin, e.g. in the range of 10 pm - 1 mm, and may be fastened to piezoelectric element 86 with adhesive, for example. The material may be substantially acoustically transparent, as may be the adhesive. Piezoelectric element 86 has an exterior surface (which is exposed) and an interior surface.

[0096] Screws attach an aluminum top plate 82a of the housing to the body 82b of the housing via threads 88. The top plate includes a connector 84 for powering the transducer. The top surface of the PZT piezoelectric element 86 is connected to a positive electrode 90 and a negative electrode 92, which are separated by an insulating material 94. The polarities of electrodes 90, 92 can be reversed, and electrodes 90, 92 can be located on opposing sides or the same side of piezoelectric element 86, where the same side can be an interior surface or an exterior surface. The electrodes can be made from any conductive material, such as silver or nickel. Electrical power is provided to the PZT piezoelectric element 86 through the electrodes on the piezoelectric element. Note that the piezoelectric element 86 has no backing layer or epoxy layer. Transducer 81 has an interior volume or an air gap 87 in the transducer between aluminum top plate 82a and the piezoelectric element 86 (e.g., the housing is empty or contains atmospheric air). A minimal backing 58 (on the interior surface) and/or wear plate 50 (on the exterior surface) may be provided in some embodiments, as seen in Figure 9.

[0097] The transducer design can affect performance of the system. A typical transducer is a layered structure with the ceramic piezoelectric element bonded to a backing layer and a wear plate. Because the transducer is loaded with the high mechanical impedance presented by the standing wave, the traditional design guidelines for wear plates, e.g., half wavelength thickness for standing wave applications or quarter wavelength thickness for radiation applications, and manufacturing methods, may not be appropriate. In the example of transducer 81 , illustrated in Figure 8, there is no wear plate or backing, allowing piezoelectric element 86 to vibrate in one of its eigenmodes with a high Q-factor, or in a combination of several eigenmodes. Piezoelectric element 86 has an exterior surface that is directly exposed to the fluid flowing through a flow chamber to which transducer 81 is mounted.

[0098] Removing the backing (e.g. making the piezoelectric element air backed) also permits the ceramic piezoelectric element to vibrate at higher order modes of vibration with little damping (e.g. higher order modal displacement). In a transducer having a piezoelectric element with a backing as previously implemented, the piezoelectric element vibrates with a more uniform displacement, like a piston. Removing the backing allows the piezoelectric element to more readily vibrate in a non-uniform displacement mode. The higher order the mode shape of the piezoelectric element, the more nodal lines the piezoelectric element can generate. The higher order modal displacement of the piezoelectric element creates more trapping lines, although the correlation of trapping line to node may not necessarily be one to one, and driving the piezoelectric element at a higher or lower frequency may not necessarily produce more or less trapping lines for a given frequency of operation.

[0099] In some embodiments of the acoustic filtering device of the present disclosure, the piezoelectric element may have a backing that has a relatively small effect on the Q-factor of the piezoelectric element (e.g. less than 5%). The backing may be made of a substantially acoustically transparent material such as balsa wood, foam, or cork which allows the piezoelectric element to vibrate in a higher order mode shape and maintains a high Q-factor while still providing some mechanical support for the piezoelectric element. The backing layer may be a solid, or may be a lattice having holes through the layer, such that the lattice follows the nodes of the vibrating piezoelectric element in a particular higher order vibration mode, providing support at node locations while allowing the rest of the piezoelectric element to vibrate freely. The goal of the lattice work or acoustically transparent material is to provide support without lowering the Q-factor of the piezoelectric element or interfering with the excitation of a particular mode shape.

[0100] Placing the piezoelectric element in direct contact with the fluid also contributes to the high Q-factor by avoiding the dampening and energy absorption effects of the epoxy layer and the wear plate. Other embodiments of the transducer(s) may have wear plates or a wear surface to prevent the PZT, which contains lead, from contacting the host fluid. These layers between the transducer and the host fluid may be desirable in, for example, biological applications such as separating blood, biopharmaceutical perfusion, or fed-batch filtration of mammalian cells. Such applications might use a wear layer such as chrome, electrolytic nickel, or electroless nickel. Chemical vapor deposition could also be used to apply a layer of poly(p-xylylene) (e.g. Parylene) or another polymer. Organic and biocompatible coatings such as silicone or polyurethane are also usable as a wear surface. Thin films, such as a PEEK film, can also be used as a cover of the exterior surface of the piezoelectric material, with the advantage of being a biocompatible material. In one embodiment, the PEEK film is adhered to the face of the piezo-material using pressure sensitive adhesive (PSA). Other films that may possess low acoustic impedance and/or that are biocompatible can be used as well.

[0101] In the implementations discussed herein, particles are trapped in the ultrasonic standing wave, i.e., remain in a stationary position. The particles are collected along well defined trapping lines, separated by half a wavelength. Within each nodal plane, the particles are trapped in the minima of the acoustic radiation potential. The axial component of the acoustic radiation force drives the particles, with a positive contrast factor, to the pressure nodal planes, whereas particles with a negative contrast factor are driven to the pressure anti-nodal planes. The radial or lateral component of the acoustic radiation force contributes to trapping the particle in a lateral direction. The radial or lateral component of the acoustic radiation force is on the same order of magnitude as the axial component of the acoustic radiation force. As discussed above, the lateral force can be increased by driving the transducer in higher order mode shapes, as opposed to a form of vibration where the crystal effectively moves as a piston having a uniform displacement. The acoustic pressure is proportional to the driving voltage of the transducer. The electrical power is proportional to the square of the voltage.

[0102] The transducer can be driven by a drive signal, such as a voltage signal (AC or DC), a current signal, a magnetic signal, an electromagnetic signal, a capacitive signal, or any other type of signal to which the transducer is responsive to create a multi-dimensional acoustic standing wave. In embodiments, the voltage signal driving the transducer can have a pulsed, sinusoidal, square, sawtooth, or triangle waveform; and have a frequency of 500 kHz to 10 MHz. The voltage signal can be driven with pulse width modulation, which can be used to produce any desired waveform. The voltage signal can be amplitude or frequency modulated. The drive signal may be turned on or off and/or configured with start/stop capability to, for example, eliminate streaming.

[0103] In some examples, the size, shape, and thickness of the transducer can determine the transducer displacement at different frequencies of excitation. Transducer displacement with different frequencies may affect separation efficiency. In some examples, the transducer is operated at frequencies near the thickness resonance frequency (half wavelength). The presence of gradients in transducer displacement can result in more places for particles to be trapped. Higher order modal displacements can generate three-dimensional acoustic standing waves with strong gradients in the acoustic field in all directions, thereby creating similarly strong acoustic radiation forces in all directions, which forces may, for example, be on the same order of magnitude. The higher order modal displacements can lead to multiple trapping lines. The number of trapping lines correlates with the particular mode shape of the transducer.

[0104] To investigate the effect of the transducer displacement profile on acoustic trapping force and separation efficiencies, an experiment was repeated ten times using a 1 "x1 " square transducer, with all conditions identical except for the excitation frequency. Ten consecutive acoustic resonance frequencies, indicated by circled numbers 1 -9 and letter A on Figure 10, were used as excitation frequencies. The conditions were experiment duration of 30 min, a 1000 ppm oil concentration of approximately 5-micron SAE-30 oil droplets, a flow rate of 500 ml/min, and an applied power of 20W. Oil droplets were used because oil is less dense than water, and can be separated from water using acoustophoresis.

[0105] Figure 10 shows the measured electrical impedance amplitude of the transducer as a function of frequency in the vicinity of the 2.2 MHz transducer resonance when operated in a water column containing oil droplets. The minima in the transducer electrical impedance correspond to acoustic resonances of the water column and represent potential frequencies for operation. Additional resonances exist at other frequencies where multi-dimensional standing waves are excited. Numerical modeling has indicated that the transducer displacement profile varies significantly at these acoustic resonance frequencies, and thereby directly affects the acoustic standing wave and resulting trapping force. Since the transducer may be operated near its thickness resonance, the displacements of the electrode surfaces are essentially out of phase. The displacement of the transducer electrodes may not be uniform and varies depending on frequency of excitation. As an example, at one frequency of excitation with a single line of trapped oil droplets, the displacement has a single maximum in the middle of the electrode and minima near the transducer edges. At another excitation frequency, the transducer profile has multiple maxima leading to multiple trapped lines of oil droplets. Higher order transducer displacement patterns can result in higher trapping forces and multiple stable trapping lines for the captured oil droplets.

[0106] To investigate the effect of the transducer displacement profile on acoustic trapping force and oil separation efficiencies, an experiment was repeated ten times, with all conditions identical except for the excitation frequency. Ten consecutive acoustic resonance frequencies, indicated by circled numbers 1 -9 and letter A on Figure 10, were used as excitation frequencies. The conditions were experiment duration of 30 min, a 1000 ppm oil concentration of approximately 5-micron SAE-30 oil droplets, a flow rate of 500 ml/min, and an applied power of 20W.

[0107] As the emulsion passed by the transducer, the trapping lines of oil droplets were observed and characterized. The characterization involved the observation and pattern of the number of trapping lines across the fluid channel, as shown in Figure 11A, for seven of the ten resonance frequencies identified in Figure 10.

[0108] Figure 11 B shows an isometric view of the flow chamber and acoustic wave zone of separation in which the trapping line locations are being determined. Figure 11C is a view of the ultrasonic transducer volume as it appears when looking down the inlet, along arrow 251. Figure 11 D is a view of the ultrasonic transducer volume as it appears when looking directly at the transducer face, along arrow 253.

[0109] The effect of excitation frequency in this example clearly determines the number of trapping lines, which vary from a single trapping line at the excitation frequency of acoustic resonance 5 and 9, to nine trapping lines for acoustic resonance frequency 4. At other excitation frequencies four or five trapping lines are observed. Different displacement profiles of the transducer can produce different (more or less) trapping lines in the standing waves, with more gradients in the displacement profile generally creating greater trapping forces and more trapping lines.

[0110] Figure 12 is a lin-log graph (linear y-axis, logarithmic x-axis) that shows the calculated scaling of the acoustic radiation force, fluid drag force, and buoyancy force with particle radius. The buoyancy force is applicable to negative contrast factor particles, such as oil particles in this example. The calculated buoyancy force may include elements of gravity forces. In examples using positive contrast factor particles, which may be some types of cells, a line indicating gravity forces is used in a graph for such positive contrast factor particles showing acoustic radiation force and fluid drag force. In the present example illustrated in Figure 12, calculations are done for a typical SAE-30 oil droplet used in experiments. The buoyancy force is a particle volume dependent force, e.g., proportional to the radius cubed, and is relatively negligible for particle sizes on the order of a micron, but grows, and becomes significant for particle sizes on the order of hundreds of microns. The fluid drag force scales linearly with fluid velocity, e.g., proportional to the radius squared, and typically exceeds the buoyancy force for micron sized particles, but is less influential for larger sized particles on the order of hundreds of microns. The acoustic radiation force scaling acts differently than the fluid drag force or the buoyancy force. When the particle size is small, the acoustic trapping force scales with the cube of the particle radius (volume) of the particle at a close to linear rate. Eventually, as the particle size grows, the acoustic radiation force no longer increases linearly with the cube of the particle radius. As the particle size continues to increase, the acoustic radiation force rapidly diminishes and, at a certain critical particle size, is a local minimum. For further increases of particle size, the radiation force increases again in magnitude but with opposite phase (not shown in the graph). This pattern repeats for increasing particle sizes. The particle size to acoustic radiation force relationship is at least partially dependent on the wavelength or frequency of the acoustic standing wave. For example, as a particle increases to a half- wavelength size, the acoustic radiation force on the particle decreases. As a particle size increases to greater than a half-wavelength and less than a full wavelength, the acoustic radiation force on the particle increases.

[0111] Initially, when a suspension is flowing through the acoustic standing wave with primarily small micron sized particles, the acoustic radiation force balances the combined effect of fluid drag force and buoyancy force to trap a particle in the standing wave. In Figure 12, trapping occurs for a particle size of about 3.5 micron, labeled as Rci . In accordance with the graph in Figure 12, as the particle size continues to increase beyond Rci, larger particles are trapped, as the acoustic radiation force increases compared to the fluid drag force. As small particles are trapped in the standing wave, particle coalescence/clumping/aggregation/agglomeration takes place, resulting in continuous growth of effective particle size. Other, smaller particles continue to be driven to trapping sites in the standing wave as the larger particles are held and grow in size, contributing to continuous trapping. As the particle size grows, the acoustic radiation force on the particle increases, until a first region of particle size is reached. As the particle size increases beyond the first region, the acoustic radiation force on the particle begins to decrease. As particle size growth continues, the acoustic radiation force decreases rapidly, until the buoyancy force becomes dominant, which is indicated by a second critical particle size, R _C 2, at which size the particles rise or sink, depending on their relative density or acoustic contrast factor with respect to the host fluid. As the particles rise or sink and leave the antinode (in the case of negative contrast factor) or node (in the case of positive contrast factor) of the acoustic standing wave, the acoustic radiation force on the particles may diminish to a negligible amount. The acoustic radiation force continues to trap small and large particles, and drive the trapped particles to a trapping site, which is located at a pressure antinode in this example. The smaller particle sizes experience a reduced acoustic radiation force, which, for example, decreases to that indicated near point Rci. As other particles are trapped and coalesce, clump, aggregate, agglomerate and/or cluster together at the node or antinode of the acoustic standing wave, effectively increasing the particle size, the acoustic radiation force increases and the cycle repeats. All of the particles may not drop out of the acoustic standing wave, and those remaining particles may continue to grow in size. Thus, Figure 12 explains how small particles can be trapped continuously in a standing wave, grow into larger particles or clumps, and then eventually rise or settle out because of the relationship between buoyancy force, drag force and acoustic radiation force with respect to particle size.

[0112] Various coatings may be used on the internal flow chambers of the acoustophoretic devices. Such coatings can include epoxies, for example epichlorohydrin bisphenol crosslinked with an amine or a polyamide; or polyurethane coatings, for example a polyester polyol crosslinked with aliphatic isocyanates. Such coatings are useful for producing a smooth surface and/or reducing surface tension, permitting cells to slide better under the influence of gravity along the flow chamber surface and into desired locations (such as collection well modules).

[0113] The flow rate of the acoustophoretic device is controlled, for example, by a pump. The flow rate can be regulated so that gravity/buoyancy can act on particle aggregates. Particle/fluid mixture passing in/out of the flow chambers in the acoustophoretic devices through the inlets/outlets thereof can flow at rates of up to about 10 liters per hour (L/hr), including up to about 50 L/hr, but often at about 3.6 L/hr. By way of comparison, the flow rate out of the collection well modules through the ports is much less, from about 3 ml/min up to about 10 ml/min.

[0114] The acoustophoretic systems of the present disclosure can be used in a filter "train," in which multiple different filtration steps are used to clarify or purify an initial fluid/particle mixture to obtain the desired product and manage different materials from each filtration step. Each filtration step can be implemented to remove a particular material, improving the overall efficiency of the clarification process. An individual acoustophoretic device can operate as one or multiple filtration steps. For example, each individual ultrasonic transducer within a particular acoustophoretic device can be operated to trap materials within a given particle range. The acoustophoretic device can be used to remove large quantities of material, reducing the burden on subsequent downstream filtration steps / stages. Any of the various filtration steps / stages discussed herein can be placed upstream or downstream of the acoustophoretic device(s). Alternatively, or in addition, multiple acoustophoretic devices can be used. Desirable biomolecules or cells can be recovered / separated after such filtration / purification. [0115] The outlets of the acoustophoretic devices of the present disclosure (e.g. clarified fluid and concentrated cells) can be fluidly connected to any other filtration step or filtration stage. Such filtration steps can include various methods such as depth filtration, sterile filtration, size exclusion filtration, or tangential filtration. Depth filtration uses physical porous filtration mediums that can retain material through the entire depth of the filter. In sterile filtration, membrane filters with extremely small pore sizes are used to remove microorganisms and viruses, generally without heat or irradiation or exposure to chemicals. Size exclusion filtration separates materials by size and/or molecular weight using physical filters with pores of given size. In tangential filtration, the majority of fluid flow is across the surface of the filter, rather than into the filter.

[0116] Chromatography can also be used, including cationic chromatography columns, anionic chromatography columns, affinity chromatography columns and/or mixed bed chromatography columns. Other hydrophilic / hydrophobic processes can also be used for filtration purposes.

[0117] For example, the multi-stage acoustophoretic systems described herein can include or be used in conjunction with an in-line filtration stage. One or more in-line filtration stages may be located upstream or downstream of all or some of the acoustophoretic devices. The in-line filtration stages may be used to further purify the liquid and to recover and obtain desirable proteins therefrom. Suitable examples of inline filtration stages include depth filters, sterile filters, centrifuges, and affinity chromatography columns.

[0118] Secondary depth filtration product selection can be achieved with some screening of the material to be filtered. In a typical fed-batch culture of a CHO-S based cell line expressing a humanised lgG1 mAb, depth filters having total volumes of less than about 5L to less than about 25L and total areas of about 0.002 m ² to about 0.1 m ² can be used for secondary depth filtration. In this regard, suitable depth filters include the Supracap™ HP depth filter capsules available from Pall Corporation. Post clarification, the harvested cell culture fluid (HCCF) may be optionally stored, filtered to control the bioburden, and stored or filtered to control the bioburden and be processed chromatographically. In a typical fed-batch culture of a CHO-S based cell line expressing a humanised lgG1 mAb, the sterile filters (i.e., sterilizing-grade membrane filters) having total volumes of less than about 5L to less than about 25L and total areas of about 220 cm ² to about 375 cm ² can be used. In this regard, suitable sterile filters include the Kleencar® capsules and mini Kleenpak capsules available from Pall Corporation.

[0119] Tertiary depth filtration may optionally be omitted at small scale, but, when used, can prevent fouling of subsequent filters and allow for a reduction in size of the bioburden control filter. In a typical fed-batch culture of a CHO-S based cell line expressing a humanised lgG1 mAb, the same depth filters used for secondary depth filtration can be used for tertiary depth filtration. Post clarification, the same sterile filters as described above can be used.

[0120] In some example biological applications, all of the parts of the system (i.e., each stage, tubing fluidly connecting the same, etc.) can be separated from each other and be disposable. With regard to filtering cells in biological applications, centrifuges and conventional filters can impose undesirable or harmful conditions on the cells. The acoustophoretic devices discussed herein permit separation of cells from a host fluid without the undesirable or harmful effects of centrifuges and conventional filters. Accordingly, the use of acoustophoretic devices to avoid centrifuges and filters allows separation of cells without necessarily lowering the viability of the cells. Ultrasonic transducers may also be used to create rapid pressure changes to prevent or clear blockages due to agglomeration of cells. The frequency of the transducers may be controlled and/or varied to obtain an operating point to increase or maximize effectiveness for a given power.

[0121] The present disclosure will further be illustrated in the following non-limiting working examples, it being understood that these examples are intended to be illustrative only and that the disclosure is not intended to be limited to the modules, devices, conditions, process parameters and the like recited herein.

EXAMPLES

[0122] Various mixtures of CHO cells in cell culture media were filtered. Example 1

[0123] The acoustophoretic separation process was compared to depth flow filtration (DFF). First, a baseline of DFF capacity was obtained by performing two rounds of clarification, a primary clarification and a secondary clarification. The setup for this baseline is illustrated in Figure 13.

[0124] The pressure drop was measured during the two rounds. The separation apparatus was operated at 145 LMH (liters/m ² /hour). The pressure was measured at three different locations P1 , P2, and P3. Located between each set of sensors was a filter. The filter used during the primary clarification was a DOHC filter, and the filter used in the secondary clarification was a XOHC filter, both available from Millipore.

[0125] A mixture of CHO cells and culture media were flowed through the filters, and the permeate was then collected in a tank. The CHO cells were removed by the filters. The feed had a total cell density (TCD) of 6.34x10 ⁶ cells/mL and a turbidity of 815 NTU. The final permeate in the third tank had a turbidity of 1 .75 NTU.

[0126] Figure 13 is a performance graph showing the pressure drop versus volumetric throughput capacity. The pressure drop in the primary filter was lowest at low throughput, then became greater than the pressure drop in the secondary filter above approximately 88 L/m ² capacity. The total pressure drop is the top line in the graph. A pressure drop of 15 psig was attained at a volumetric throughput of 88 L/m ² (indicated by dashed lines). This relationship indicates that if scaled up with a maximum pressure drop of 15 psig (Pmax=15 psig), the area of the filter for the primary clarification and for the secondary clarification would each be 1 1 .4 m ²

Example 2

[0127] Next, the two-step DFF described in Example 1 was compared to a two-step clarification process in which the primary clarification was performed by acoustic wave separation (AWS) and the secondary clarification was performed by DFF. This comparison setup is described in Figure 14.

[0128] As indicated in Figure 14, in the two-step DFF, each filter had an area of 1 1 m ² Each filter was operated with a pressure drop of 7.5 psig. The volumetric throughput (VT) at 7.5 psig (VT7.5) was 84 L/m ² for each filter. [0129] The acoustophoretic system used to perform the AWS was made up of three acoustophoretic devices linked in series. The transducer in each device was 1 inch by 1 inch. The system had a total acoustic volume of 49 cm ³ . The AWS system was paired with a DFF filter having a total area of 6 m ² . Because there is no pressure drop in the AWS system, the DFF filter could be operated at a pressure drop of 1 5 psig, resulting in a VT15 of 160 L/m ²

[0130] The feed had a total cell density (TCD) of 6.7x 1 0 ⁶ cells/mL and a turbidity of 835 NTU, and 77% cell viability. The feed rate to the acoustophoretic system was 4 kg at 2.5 liters per hour (LPH).

[0131] The results for the primary clarification using the AWS system are shown in Figure 14. The acoustophoretic system achieved 91 % TCD reduction, 90% turbidity reduction, and 91 .2% recovery of protein. The graph at the bottom left is percent reduction versus time, and shows that the AWS system operated consistently during the test.

[0132] In comparison to Example 1 , in which both the primary and secondary clarifications were performed by depth filtration, replacing the primary clarification with acoustic wave separation (AWS) achieves economic benefits by reducing the overall operating footprint, reducing the secondary depth filter area specification and the associated conditioning and flush buffers, as well as reduced storage and disposal costs. These become key process drivers as processes enter clinical manufacture. An example of the anticipated process specifications for a 1 000L CHO cell culture is summarized below.

Example 3

[0133] The same experiment as described in Example 2 was performed again, but with a higher cell density. The feed had a higher TCD of 1 5.6x 10 ⁶ cells/mL and a turbidity of 3608 NTU, and 68% cell viability. This comparison setup is described in Figure 15.

[0134] The two-step DFF process used filters of 38 m ² and 17 m ² , respectively. As indicated, the VT7.5 was 26 L/m ² for the primary clarification and 58 L/m ² for the secondary clarification. In the AWS-DFF process, the AWS system had two acoustophoretic devices in series (not three as in Example 2), with a total acoustic volume of 33 cm ³ . The DFF filter had a total area of 1 1 m ² , and a VT15 of 85 L/m ² . The feed rate to the acoustophoretic system was 8 kg at 2.5 liters per hour (LPH).

[0135] The results for the primary clarification using the AWS system are shown in Figure 15. The acoustophoretic system achieved 94% TCD reduction, 91 % turbidity reduction, and 92% recovery of protein. The graph at the bottom left is percent reduction versus time, and shows that the system operated consistently during the test. Higher cell densities were more difficult for the DFF device to process, as indicated by the lower VT in the primary clarification. However, the acoustophoretic device was able to handle the higher density with better VT.

Example 4

[0136] The feed had a TCD of 7.5 ^χ 10 ⁶ cells/mL and a turbidity of 819 NTU, and 88% cell viability. Clarification was performed using a three-stage acoustophoretic system as in Example 1 .

[0137] The first stage reduced the cell density by 62%. The second stage reduced the remaining cell density by 87% (cumulative 95%). The third stage reduced the remaining cell density by 63% (cumulative 98%). Two stages were used to attain greater than 90% cell density reduction.

[0138] The first stage reduced the turbidity by 68% from 819 NTU to 260 NTU. The second stage reduced the remaining turbidity down to 54 NTU (cumulative 94%). The third stage reduced the remaining turbidity to 42 NTU (cumulative 95%). Two stages were used to attain greater than 90% turbidity reduction. This result is important for secondary filtration processes further downstream.

[0139] The percent reduction for both cell density reduction and turbidity reduction was consistent over the entire time, meaning the device operated well on a continuous basis. The improved reduction of cell density and turbidity contributes to simplifying secondary filtration processes further downstream, and ultimately contributes to greater product recovery in the chromatographic separation of monoclonal antibodies or recombinant proteins from the clarified fluid.

Example 5

[0140] Five different lots were tested through the three-stage system of Example 1. Each lot had its own cell size and density characteristics. The feeds had a TCD of 7 to 8.5 x10 ⁶ cells/mL, a turbidity of 780 to 900 NTU, and 82% to 93% cell viability. This example tested the consistency of performance of the system across different batches.

[0141] Over the five different lots, the turbidity of the permeate was reduced in a range of 84% to 86%, with a standard deviation of 1 % after three passes. The cell density of the permeate was reduced in a range of 93% to 97%, with a standard deviation of 2% after three passes.

[0142] In other experiments not described here, it was found that the acoustic wave separation processes using a multi-dimensional acoustic standing wave did not affect the physical or chemical characteristics of protein or monoclonal antibodies recovered from the permeate.

Example 6

[0143] Next, the effect of voltage input on clarification performance was observed in a three-stage or a four-stage acoustophoretic system.

[0144] A mixture of CHO cells and culture media were flowed through the stages of the device, and the permeate was then collected in a tank. The CHO cells were removed by the filters. The feed had a total cell density (TCD) of 25x10 ⁶ cells/mL, a turbidity of 2048 NTU, and a cell viability of 72%. The feed flow rate was 30 mL/min and the solids draws were 2.34 mL/min for stage one, 1.41 mL/min for stage two, 0.94 mL/min for stage three, and 0 mL/min for stage four.

[0145] The TCD reduction after three and four stages of filtering are shown in the following table. T1 refers to the voltage in the first stage, T2 to the voltage in the second stage, T3 to the voltage in the third stage, and T4 to the voltage in the fourth stage. The results for five different test runs using different voltages in different stages are shown.

[0146] For all voltage conditions of 50V and 60V, the system achieved a greater than 90% cell density reduction. The addition of the fourth stage yielded a high 95% reduction, lowering the TCD from 2.9x10 ⁶ cells/mL after three stages to 1.3*10 ⁶ cells/mL after four stages. The following table shows the cumulative %TCD reduction per stage after 30 minutes for the test runs identified above.

[0147] As can be seen, voltages of 50V and 60V yielded the best performances through each stage. As can be seen in Test Run 5, adding a fourth stage at 50V increased the TCD reduction by 10% aggregate. Generally, a voltage of 50V to 60V should be used in the first and/or second stages to obtain high TCD reduction.

[0148] The total turbidity reduction after three and four stages of filtering are shown in the following table. For all voltage conditions of 50V and 60V, the system achieved a greater than 90% turbidity reduction. The addition of the fourth state yielded a 94% reduction, lowering permeate from 390 NTU after three stages to 177 NTU after four stages. Test T1 voltage T2 voltage T3 voltage T4 voltage %Turbidity Run (V) (V) (V) (V) reduction

1 40 40 40 - 81

2 50 50 50 - 90

3 60 60 60 - 91

4 60 50 40 - 91

5 50 50 50 50 94

[0149] In summary, the 50V and 60V operating conditions yielded higher and near- equivalent clarification over the 40V operating condition, and use of the 40V operating condition in the third stage after 50V / 60V in the first and second stages lowered the clarification efficiency.

Example 7

[0150] Next, similar to Example 1 , the acoustophoretic separation process was compared to depth flow filtration (DFF) to determine the effect of DFF performance on acoustic wave separation (AWS) performance.

[0151] A mixture of CHO cells and culture media was flowed through two filters, a D0HC filter and a X0HC filter, both available from Millipore, and the permeate was then collected in a tank. The CHO cells were removed by the filters. The feed had a total cell density (TCD) of 24.7x10 ⁶ cells/mL and a turbidity of 2850 NTU. The final permeate in the tank had a turbidity of 4.9 NTU.

[0152] Figure 16 is a performance graph showing the pressure drop versus volumetric throughput capacity. The profiles in Figure 16 were plotted per stage based on the total filter area in each stage. The volumetric throughput capacity was calculated at 23 cm ² . The pressure drop in the D0HC filter was lowest at low throughput, then became greater than the pressure drop in the X0HC filter above approximately 16 L/m ² capacity. A pressure drop of 15 psig would be attained at a volumetric throughput of 47 L/m ² for the D0HC filter and approximately 1 10 L/m ² for the D0HC filter (estimated based on pressure-drop ratios— the D0HC filter accounting for 12.0 psig of the total 15.0 psig pressure drop and the X0HC filter accounting for the remaining 3.0 psig).

[0153] Figure 17 is a performance graph showing the pressure drop versus volumetric throughput capacity. The profiles in Figure 17 were plotted for the total filter area in series (i.e., across all stages). The volumetric throughput capacity was calculated at 46 cm ² . The pressure drop in the DOHC filter was lowest at low throughput, then became greater than the pressure drop in the XOHC filter above approximately 8 L/m ² capacity. The total pressure drop is the top line in the graph. A pressure drop of 15 psig would be attained at a volumetric throughput of 21 .5 L/m ² . This result indicates that if scaled up with a maximum pressure drop of 15 psig (Pmax=15 psig), the area of the filter for the primary clarification and secondary clarifications would be 46.5 m ² total with a D0HC:X0HC filter size ratio of 3: 1 (e.g., the DOHC filter would have an area of 34.9 m ² and the XOHC filter would have an area of 1 1 .6 m ² ).

Example 8

[0154] Next, the two-step DFF described in Examples 6 and 7 were compared to a two-step clarification process in which the primary clarification was performed by acoustic wave separation (AWS) and the secondary clarification was performed by DFF. This comparison setup is described in Figure 18.

[0155] As indicated there, in the two-step DFF, the DOHC filter had an area of 34.9 m ² and the XOHC filter had an area of 1 1 .6 m ² , for a total area of 46.5 m ² . Each filter was operated with a pressure drop of 15 psig. The volumetric throughput (VT) at 15 psig (VTi5) was 47 L/m ² for the DOHC filter and 1 10 L/m ² for the XOHC filter. The turbidity of the permeate was 4.9 NTU.

[0156] Two different acoustophoretic systems were used to perform the AWS. The first acoustophoretic system was a three-stage system (e.g., made up of three acoustophoretic devices) linked in series. The second acoustophoretic system was a four-stage system (e.g., made up of four acoustophoretic devices) as illustrated in Figure 1 and linked in series. The transducers used in each device of each system were 1 inch by 1 inch.

[0157] The first AWS system was paired with a DFF filter having a total area of 10.2 m ² The area by stage was 7.6 m ² (3x) and 2.6 m ² (1x). Because there is no pressure drop in the AWS system, the DFF filter could be operated at a pressure drop of 15 psig, resulting in a VT15 of 214 L/m ² . The feed had a total cell density (TCD) of 2.9x10 ⁶ cells/mL and a turbidity of 380 NTU. The turbidity of the permeate was 8.8 NTU.

[0158] The second AWS system was paired with a DFF filter having a total area of 4.5 m ² The area by stage was 3.4 m ² (3x) and 1 .1 m ² (1x). Again, because there is no pressure drop in the AWS system, the DFF filter could be operated at a pressure drop of 15 psig, resulting in a VT15 of 490 L/m ² . The feed had a total cell density (TCD) of 1 .3*10 ⁶ cells/mL and a turbidity of 176 NTU. The turbidity of the permeate was 1 1 .4 NTU.

[0159] The first AWS system (the three-stage system) reduced the overall filter area by 78%, with a decrease of 12% in secondary clarification as compared to DFF for primary clarification (centrifuge is the current unit operation).

[0160] The second AWS system (the four-stage system) reduced the overall filter area by 90%, with a decrease of 60% in secondary clarification as compared to DFF- DFF. The acoustophoretic system achieved 91 % TCD reduction, 90% turbidity reduction, and 91 .2% recovery of protein.

[0161] Harvest to DFF to DFF (primary and secondary) produced a very low volumetric throughput of 21 .5 liters/m ² , so low that to process 1000L would use almost 50 m ² of filter area. The volumetric throughput of the dual-stage DFF step after the third stage was increased by 100% with the addition of a fourth stage, from 98.5 L/m ² to 222 L/m ² . TCD was decreased from 2.9*10 ⁶ cells/mL to 1 .4*10 ⁶ cells/mL (-50% reduction). Turbidity was decreased from 338 NTU to 220 NTU (42% reduction). For comparison, when centrifugation was used as the primary clarification step, centrate from the centrifuge was measured to have a TCD of 0.07x10 ⁶ , a turbidity of 450 NTU, and a viable cell density (VCD) of 6%. High shear forces caused cell disruption, thereby increasing turbidity and lowering the VCD of remaining cells.

[0162] For comparison to the dual-stage DFF (C0HC + X0HC) after acoustic filtering, single-stage DFF was also performed after four-stage AWS using three different DFF filters (PDD1 , X0HC, 90Z). The volumetric throughput for PDD1 was 90 L/m ² , for X0HC was 21 L/m ² , and for 90Z was 1 10 L/m ² . These values were 50-90% below the dual stage volumetric throughput of 222 L/m ² . Example 9

[0163] Next, the effect of feed flow rates (QF), total flow ratios (QS/QF), and solids flow ratios (Qs#/Qs#) on clarification performance was observed in a multi-stage acoustophoretic system. The setup for this observation is shown in Figure 19. In Figure 19, the QF# indicates the feed flow rate into a given acoustophoretic stage, the Qp# indicates the permeate flow rate from the given acoustophoretic stage, and the Qs# indicates the solids flow rate from the given acoustophoretic stage. The permeate flow rate of an upstream device is the feed flow rate into the subsequent acoustophoretic stage (e.g., QPI = QF2).

[0164] Using ten different cell lines, 135 individual runs were performed using a mixture of CHO cells and culture media having 27-98% cell viabilities, 2.8x10 ⁶ to 56x10 ⁶ cells/mL cell densities, 1 .4 to 16.5% packed cell mass (PCM), and 30 to 9000 NTU feed turbidity.

[0165] Figure 20 illustrates an exemplary four-stage setup. Figure 21 shows the turbidity reduction as a function of the feed PCM. Figure 22 shows the turbidity reduction as a function of the feed flow rate for a total flow ratio of 20%. Figure 23 shows the turbidity reduction as a function of the solids flow ratio for a total flow ratio of 20%. Figure 24 shows the PCM as a function of the solids flow ratio. Figure 25 shows the turbidity reduction as a function of the solids flow ratio. The solids PCM as a function of the solids flow ratio is listed in the following table.

[0166] The feed flow rate was found to greatly impact clarification. The total flow ratio was found to primarily impact yield, while impacting clarification somewhat. The ratio that provided the best results was found to be (feed PCM / 50% PCM solids stream). Decreasing the total flow ratio was found to improve yield and increase solids packing. The solids flow ratio was found to not have an impact on overall clarification, but was found to allow control over solids distribution between stages. The feed PCM was found to impact clarification and stage efficiency. The best results for feed PCM was found to be 5-6%, single stage, and higher %PCM is not an issue with multi-stage systems.

Example 10

[0167] Next, the effect of feed flow rates (QF) on clarification performance was observed in three- and four-stage acoustophoretic systems.

[0168] A mixture of CHO cells and culture media were flowed through both systems as an AWS "train" that was then processed using a DFF filter. The CHO cells were removed by the filters. The feed had a total cell density (TCD) of 35.8x 1 0 ⁶ cells/mL, a turbidity of 4457 NTU, a PCM of 9.1 %, and a viability of 91 .9%.

[0169] The three-stage system used two runs with feed flow rates (QF) of 1.0x and 0.66x. The four-stage system used a single run with a feed flow rate (QF) of 1 .Ox. The 0.6x three-stage system performed comparably to the 1 .0x four-stage system, though all runs achieved > 85% yield and > ~90% turbidity reduction. Each permeate has a separate filtration chain. The full data recorded is provided below.

[0170] Again, the AWS performed much better than DFF to DFF and achieved a ~ 4x increase in volumetric throughput. The overall filtration is summarized below.

Example 11

[0171] Next, 1 1 individual runs were performed on a four-stage acoustophoretic system. A mixture of CHO cells and culture media were flowed through the four stages of the system in series in five different CHO cell lines. The feed had a total cell density (TCD) of 9.78x 10 ⁶ to 34.3x 1 0 ⁶ cells/mL, a turbidity of 920 to 4670 NTU, a PCM of 6.5 to 1 1 %, and a viability of 31 -91 %.

[0172] The AWS permeate had a TCD of 0.8x 1 0 ⁶ to 5.2x 1 0 ⁶ cells/mL (average = 2.9x 1 0 ⁶ cells/mL), a turbidity of 121 to 453 NTU ((average = 236 NTU), and a PCM of 1 .1 to 2.3% (average = 1 .6%). The reduction performance was a TCD reduction of 76% to 95% (average = 88%), a turbidity reduction of 86% to 97% (average = 94%), and a PCM reduction of 72% to 88% (average = 79%). The yield was 84% to 89% (average = 86%). Figure 26 shows the reduction performance as a function of the feed PCM, and Figure 27 shows the reduction performance as a function of the feed cell viability.

Example 12

[0173] Two CHO cell lines with a multi-specific antibody (MM-1 31 ) was flowed through a three-stage acoustophoretic system with stages having a 1 inch by 1 inch ultrasonic transducer. The feed had a total cell density (TCD) of 20x 1 0 ⁶ to 24x 1 0 ⁶ cells/mL. The first feed line had an 80% cell viability and the second feed line had a 65% cell viability. The total cell reduction, turbidity reduction, and protein recovery for the two feed lines are listed in the table below. Feed Line Total Cell Reduction Turbidity Reduction Protein Recovery

80% viable 90% 84% 82%

65% viable 89% 85% 78%

Example 13

[0174] In a four-stage system, the system performance was also measured over time for each stage. Figure 28 shows the TCD reduction at each stage and at intervals of 2 hours, 4 hours, 6 hours, 9 hours, and 12.5 hours. The leftmost bar in each series represents the first stage, the second bar from the left in each series represents the second stage, the second bar from the right in each series represents the third stage, and the rightmost bar in each series represents the fourth stage. Addition of the fourth stage showed an increased TCD reduction at each time interval. This graph shows that each acoustophoretic stage maintains its performance and its separation ability does not decrease over time, as a physical filter would.

Example 14

[0175] For three discrete customers, the reduction in depth filter area was compared between using DFF and using a four-stage AWS system fed into a DFF filter. Figure 29 shows that use of a four-stage AWS system reduced the depth filter area used between 3- and 10-fold.

Example 15

[0176] Figure 30 is a graph showing the cell density reduction for five different runs through a three-stage system having transducers of size 1 inch by 1 inch. The x-axis near the bottom of each bar indicates the total cell density (cells/mL) and the cell viability. The y-axis is the percent reduction of cells from the feed to the permeate, and a higher value is better. These results show good separation even for high total cell density.

[0177] The present disclosure has been described with reference to exemplary embodiments. Modifications and alterations may occur to others upon reading and understanding the preceding detailed description. It is intended that the present disclosure be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.