CN109069954B - Multi-station acoustophoresis device - Google Patents

Abstract

Multi-station acoustophoresis devices for continuous separation of a second fluid or particulate from a host fluid are disclosed. Methods of operating the multi-station acoustophoresis device are also disclosed. The system includes a plurality of acoustophoresis devices fluidly connected in series with one another, each acoustophoresis device including a flow chamber, an ultrasonic transducer capable of generating a multi-dimensional acoustic standing wave, and a reflector. The system can also include a pump and a flow meter.

Description

Multi-station acoustophoresis device

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application No. 62/322,262 filed on 14/4/2016 and U.S. provisional patent application No. 62/307,489 filed on 12/3/2016. The disclosures of these applications are hereby incorporated by reference herein in their entirety.

Background

The ability to separate a particle/fluid mixture into its individual components is desirable in many applications. Physical size exclusion filters can be used for this purpose, wherein particles are captured on the filter and 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, working with physical filters can be complex. For example, when the physical filter is full, the filtration capacity is reduced. Also, the use of such filters initiates periodic stops to remove the filter and/or to capture or clear particles trapped thereon.

Acoustophoresis (acoustophoresis) is the separation of particles with high intensity sound waves and does not use membranes or physical size exclusion filters. It is known that the high intensity standing wave of sound can exert pressure on the particles. The standing wave has a pressure curve which finally shows "rest". The pressure spectrum in a standing wave contains net zero pressure regions at its nodes and antinodes. Depending on the density and compressibility of the particles, they may become trapped at nodes or antinodes of the standing wave. However, conventional acoustophoresis devices have limited efficacy due to several factors, including heat generation, restriction of fluid flow, and inability to capture different types of materials. There remains a need for improved acoustophoretic devices with improved fluid dynamics.

Disclosure of Invention

The present disclosure relates to a multi-station acoustophoretic (acoustophoretic) system that can be used to effect separation of particles from a particle/fluid mixture. In certain embodiments, the multi-station acoustophoresis systems described herein are capable of reacting with a living beingThe reactors are used together, such as in a perfusion process, to produce biomolecules, such as recombinant proteins or monoclonal antibodies, and to isolate these desired products from the cell culture in the bioreactor. A new mixture of 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 can be produced. In more particular embodiments, the particle is a biological cell, such as a Chinese Hamster Ovary (CHO) cell, NS0 hybridoma cell, Baby Hamster Kidney (BHK) cell, or a human cell; lymphocytes such as T cells (e.g., regulatory T cells (tregs), Jurkat T cells), B cells, or NK cells; precursors thereof, such as Peripheral Blood Mononuclear Cells (PBMCs); algae or other plant cells, bacteria, viruses, or microcarriers. The acoustophoretic systems described herein may be expandable, typically for use at about 20 x 10⁶cells/mL to about 50X 10⁶Cell density of cells/mL. Several different types of acoustophoretic systems are described herein.

In particular, the present disclosure provides novel, scalable, potentially disposable techniques for clarifying cell cultures based on acoustophoresis. Acoustic Wave Separation (AWS) techniques involve the use of low frequency acoustic forces (acoustic force) to create a multi-dimensional acoustic standing wave across a flow channel. Fluid medium from the bioreactor enters the flow channels and is captured or suspended by acoustic forces as the cells pass through the multi-dimensional acoustic standing wave. The trapped cells migrate to the pressure node of the standing wave and begin to clump together, eventually forming clusters that are large enough to settle out of suspension by gravity. The permeate from the system showed a significant drop in turbidity and reduced zone specifications for secondary clarification using depth filtration and subsequent filtration for bioburden control.

Disclosed herein are multi-station acoustophoresis systems, such as two-station, three-station, and four-station acoustophoresis systems. These multi-station acoustophoresis systems can incorporate a filter "train" comprising a depth filter, sterile chlorine, a centrifuge, and an affinity column to purify the cell culture by separating the recombinant protein therefrom. In such systems, the frequency/power of the multi-dimensional acoustic standing waves generated in different stations (stages) of the system can vary the subsequent filtration step of feeding different concentrations or different sizes of material into the filter "train", thereby improving the efficiency of the clarification process by effectively managing the material processed in each step of the filter "train". In this way, the acoustic filters (i.e., the various stations of the system) can be used to manage the next stage in the "queue" of filters to receive material for subsequent processing therein.

An example multi-station acoustophoresis system of the present disclosure includes three or more acoustophoresis devices fluidly connected to one another. The acoustophoretic devices may be connected in series such that each acoustophoretic device is connected with at least one other acoustophoretic device. In some examples, the acoustophoretic devices may or may not have a common connection. The acoustophoretic device may typically be connected to a recovery channel to recover material after separation. Each acoustophoresis 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., on a wall thereof), the transducer comprising a piezoelectric material capable of being driven by a drive signal to generate a multi-dimensional standing wave in the flow chamber; and a reflector opposite the at least one ultrasonic transducer (e.g., on a wall opposite the transducer). In a particular embodiment, there are four acoustophoretic devices. In some embodiments, two or more acoustophoresis devices are used.

The acoustophoresis devices of the multi-station acoustophoresis system can be fluidly connected to one another by a conduit. The acoustophoretic devices of a multi-station acoustophoretic system can be physically connected directly to each other, one on top of the other, or side-by-side.

In particular embodiments, the acoustophoresis device is configured to produce multi-dimensional acoustic standing waves, all having frequencies within an order of magnitude of each other. In particular embodiments, the acoustophoresis device is configured to produce a multi-dimensional acoustic standing wave, all having frequencies that are different from one another. In some constructions, each multi-dimensional acoustic standing wave generates an acoustic radiation force having an axial component and a lateral component that are on the same order of magnitude.

The multi-station acoustophoresis system can further include a feed pump upstream of the most upstream of the at least three acoustophoresis devices and a pump (e.g., a peristaltic pump) downstream of each acoustophoresis device. That is, the multi-station acoustophoresis system can include a feed pump upstream of the first acoustophoresis device, a first pump between the first acoustophoresis device and the second acoustophoresis device, a second pump between the second acoustophoresis device and the third acoustophoresis device, a third pump downstream of the third acoustophoresis device and upstream of the fourth acoustophoresis device (when present), and a fourth pump downstream of the fourth acoustophoresis device.

The multi-station acoustophoresis system can further include a feed flow meter upstream of the most upstream of the acoustophoresis device, and a flow meter downstream of each acoustophoresis device. That is, the multi-station acoustophoresis system can include a feed flow meter upstream of the first acoustophoresis device, a first flow meter between the first acoustophoresis device and the second acoustophoresis device, a second flow meter between the second acoustophoresis device and the third acoustophoresis device, a third flow meter downstream of the third acoustophoresis device and upstream of the fourth acoustophoresis device, and a fourth flow meter downstream of the fourth acoustophoresis device.

Each acoustophoresis device may have at least one sudden-expansion diffuser located at the inlet into the flow chamber. Each acoustophoresis device can further include a port below its at least one ultrasonic transducer. This port can be used to recover separated material from the acoustophoresis device.

In certain embodiments, the inlet of the multi-station acoustophoresis system can be fluidly connected to the outlet of a bioreactor. The multi-station acoustophoresis system can further include at least one in-line filtration stage downstream from the most downstream of the three or more acoustophoresis devices. The serial filtration station can be selected from depth filters, sterile filters, centrifuges, affinity chromatography columns, or other filtration techniques known in the art of protein purification.

Methods of continuously separating a second fluid or particulate from a host fluid using a multi-station acoustophoresis system are also disclosed. All of the multi-dimensional standing waves generated in the acoustophoresis devices of the multi-station acoustophoresis system may have different frequencies or the same frequency, or may have frequencies within the same order of magnitude.

In certain embodiments of the methods disclosed herein, a drive signal is sent to drive the ultrasonic transducer of the first acoustophoresis device to produce 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 and the remaining mixture continues into the second portion of the acoustophoresis device. Sending another drive signal to drive the ultrasonic transducer of the second acoustophoresis device to generate 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 and the remaining mixture continues into the third portion of the acoustophoresis device. Sending another drive signal to drive the ultrasonic transducer of the third acoustophoresis device to generate 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 and the remaining mixture continues into the fourth portion of the acoustophoresis device. Sending another drive signal to drive the ultrasonic transducer of the fourth acoustophoresis device to produce a fourth multi-dimensional acoustic standing wave therein such that at least a portion of the second fluid or particulate is continuously trapped in the fourth standing wave.

The drive signals sent to each acoustophoresis device can be different from one another and can be implemented for a particular range of particle sizes to selectively filter particles in a mixture. In certain embodiments, the voltage signal to the at least one acoustophoresis device is at least 50V. In certain embodiments, the voltage signal (i.e., AC) to each acoustophoresis device is from 50V to about 60V (rms). In certain embodiments, the voltage signal most downstream of the acoustophoresis device is from 40V to about 60V.

The second fluid or particle may be a Chinese Hamster Ovary (CHO) cell, an NS0 hybridoma cell, a Baby Hamster Kidney (BHK) cell, or a human cell; t cells, B cells, or NK cells; peripheral Blood Mononuclear Cells (PBMCs); algae; plant cells, bacteria, viruses or microcarriers.

In certain embodiments of the methods disclosed herein, a voltage signal is sent to drive the ultrasonic transducer of each acoustophoresis device to generate a multi-dimensional acoustic standing wave within each acoustophoresis device such that at least a portion of the second fluid or particulate is continuously trapped at each standing wave, the frequency and power of each multi-dimensional acoustic standing wave being varied to selectively manage the size and/or concentration of particulates in the mixture that pass through a selected portion of the acoustophoresis device to a directly downstream portion of the acoustophoresis device.

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

Brief description of the drawings

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

Fig. 1 illustrates an exemplary embodiment of a multi-station acoustophoresis system described in this disclosure. The acoustophoretic system includes four acoustophoretic stations connected to one another by a conduit.

Fig. 2A illustrates an exemplary embodiment of four acoustophoretic devices/stations physically connected to one another for use in the multi-station acoustophoretic system of the present disclosure.

Figure 2B illustrates an isolated view of one of the acoustophoresis devices/stations of figure 2A.

Fig. 2C is a cross-sectional view of one of the acoustophoresis devices/stations of fig. 2A. The device includes opposing flow pump diffuser inlets that produce flow symmetry and more uniform velocity.

Figure 3 illustrates three of the four acoustophoresis devices of the four-station acoustophoresis system of figure 1. The acoustophoresis devices are connected to each other and pumped through a conduit therebetween. The figure also shows the fluid entrained in the cell culture medium continuously flowing into each device through its inlet, while the settled/aggregated cells slough/settle out of each device through its ports, and the remaining fluid flows out of the device through the outlet into the subsequent device.

FIG. 4 is an enlarged side cross-sectional view illustrating the cell culture medium in the fluid flowing vertically downward through the flow chamber and into the isolated sonic zone.

FIG. 5 is a continuation of FIG. 4, showing cells in the fluid being trapped in the acoustic standing wave at the pressure standing wave node, due to the lateral component of the acoustic radiation force.

FIG. 6 is a continuation of FIG. 5, showing the aggregation of cells trapped in the acoustic standing wave to form clusters of cells that settle out of the suspension and fall to the bottom of the flow chamber due to the reduced buoyancy/increased gravitational effect.

Fig. 7 is a cross-sectional view of a conventional ultrasonic transducer.

Fig. 8 is a cross-sectional view of an ultrasound transducer of the present disclosure. There is an air gap within the transducer and no backing layer or wear plate.

Fig. 9 is a cross-sectional view of an ultrasound transducer of the present disclosure. An air gap exists within the transducer, a backing layer and a wear plate are present.

Fig. 10 is a graph of electrical impedance amplitude versus frequency for a square (square) transducer driven at different frequencies.

FIG. 11A illustrates a capture line configuration for seven of the peak amplitudes of FIG. 10 in a direction orthogonal to fluid flow.

Fig. 11B is a perspective view illustrating the separator. Showing fluid flow direction and trapping lines.

FIG. 11C is a view of the fluid inlet along the direction of fluid flow (arrow 251) of FIG. 11B, showing a standing wave trapping node that would trap particles.

FIG. 11D is a view taken along arrow 253 shown in FIG. 11B with the transducer facing the trapping line configuration.

Fig. 12 shows the relationship of acoustic radiation force, buoyancy, and stokes drag force to particle size. The horizontal axis is in micrometers (μm) and the vertical axis is in newtons (N).

Figure 13 is a representation showing the pressure drop of the system of the present disclosure with three acoustophoresis devices in series in a first experiment. The y-axis is the pressure drop in psig from 0 to 30 with an interval of 5. The x-axis is the volumetric flux capacity (volumetric throughput capacity) in liters per square meter, from 0 to 140, at an interval of 20.

Fig. 14 is a set of two graphs showing the performance of the second experimental system. The bottom graph, the y-axis, is the percent reduction, from 0% to 100%, with a 10% interval. The x-axis is the test duration in minutes, from 0 to 80, at an interval of 10. The y-axis of the top graph is the percent reduction and is logarithmic with values of 0, 90% and 99.0%.

Fig. 15 is a set of two graphs showing the performance of the third experimental system. The bottom graph, the y-axis, is the percent reduction, from 0% to 100%, with a 10% interval. The x-axis is the test duration in minutes, from 0 to 80, at an interval of 10. The y-axis of the top graph is the percent reduction and is logarithmic with values of 0, 90% and 99.0%.

Fig. 16 is a graph showing the performance or volumetric flux (VT) per station for the fifth experimental four-station acoustophoresis system. The line along the top of the right side of the figure represents the DOHC filter pressure drop. The dashed line extending from the DOHC pressure drop line represents the DOHC filter turbidity. The line along the bottom of the right side of the figure represents the XOHC filter pressure drop. The dashed line extending from the XOHC pressure drop line represents the XOHC filter turbidity.

Fig. 17 is another graph showing the performance or volume loading (VT) of all 4 stations of the fifth four-station acoustophoresis experimental system. The line along the top of the right side of the figure represents the total pressure drop. The second line from the top along the right side of the figure represents the DOHC filter pressure drop. The dashed line extending from the DOHC pressure drop line represents the DOHC filter turbidity. The line along the bottom of the right side of the figure represents the XOHC filter pressure drop. The dashed line extending from the XOHC pressure drop line represents the XOHC filter turbidity.

Figure 18 illustrates the performance of the sixth experimental system compared to deep layer flow filtration (DFF). DFF showed the line shown at the top and used an area of 35m²And 11.6m²To achieve the desired performance. Along the bottom line, a combination of the three-station AWS system with a depth filter reduces the filtration area used to 10.2m²The combination of a four-station AWS system with a depth filter reduces the filter area to only 4.5m²。

Fig. 19 illustrates an exemplary setup of a three-station acoustophoresis system used as an experimental system for some performance tests of the present disclosure.

Fig. 20 illustrates an exemplary setup of a four-station acoustophoresis system used as an experimental system for many performance tests of the present disclosure.

FIG. 21 shows the turbidity reduction vs feed PCM of the FIG. 20 system. The y-axis is the percent reduction in haze, from 0% to 100%, with an interval of 10%. The x-axis is the percentage of PCM fed, from 0% to 15%, with a 5% interval. Biaxial representation with large circular data points using [ Q ]_F/(Q_S/Q_F)]＝[0.6x/10％]Experimental runs of (2). Dotted line with large triangle data points represents using [ Q ]_F/(Q_S/Q_F)]＝[1.0x/10％]Experimental runs of (2). Line representation with box-X data points employs [ Q ]_F/(Q_S/Q_F)]＝[1.0x/20％]The first experimental run of (1). Three-axis representation with large diamond data points is taken as [ Q ]_F/(Q_S/Q_F)]＝[1.0x/20％]The second experiment of (1). Dotted lines with large square data points represent the use of [ Q ]_F/(Q_S/Q_F)]＝[1.0x/20％]The third experiment of (1). Most preferablyThe best (i.e. highest) turbidity reduction occurs at-5-6% of the feed PCM.

FIG. 22 shows the system of FIG. 20 (Q)_S/Q _F20%) percentage reduction in terms of three different factors (TCD, turbidity, PCM) vs feed flow rate (Q)_F). The y-axis is the percent reduction, from 0% to 100%, with a 10% interval. The x-axis is the feed flow rate (x-flow) from 0 to 3.5 with an interval of 0.5.

FIG. 23 shows the system of FIG. 20 (Q)_S/Q _F20%) turbidity drop vs flow ratio. The y-axis is the percent reduction in haze, from 0% to 100%, with an interval of 10%. The x-axis is the flow ratio (Q)_S1/Q_S2) From 0 to 5, with an interval of 1. The top line represents 0.6x flow and the bottom line represents 1.3x flow.

FIG. 24 shows the PCM vs. solids flow rate for the system of FIG. 20. The y-axis is the percentage of Packed Cell Mass (PCM) from 0% to 50% with 5% spacing. The x-axis is the flow velocity (Q)_S1/Q_S2) From 0 to 5, with an interval of 1. The line along the top of the right side of the figure represents the solids station (solids stage) 2. The middle line along the right of the figure represents the solids station 1. The line along the bottom of the right side of the figure represents the permeate.

FIG. 25 shows the turbidity reduction vs. flow ratio for the system of FIG. 20. The y-axis is the percent reduction in haze, from 0% to 100%, with an interval of 10%. The x-axis is the flow ratio (Q)_S1/Q_S2) From 0% to 25% with an interval of 5%.

FIG. 26 shows the% reduction in vs feed PCM for the system of FIG. 20 for three different factors (TCD, turbidity, PCM). The y-axis is the percent reduction, from 0% to 100%, with an interval of 10%. The x-axis is the percentage of PCM fed, from 0% to 12%, with a 2% interval. The diamond data points represent normalized turbidity drops. The square data points represent the TCD reduction. The triangle data points represent PCM reduction.

Figure 27 shows the% vs feed cell viability reduction for the system of figure 20. The y-axis is the percent reduction, from 0% to 100%, with an interval of 10%. The x-axis is the percent viability of the feed cells, from 0% to 100%, with an interval of 20%. Likewise, the diamond data points represent normalized turbidity drop, the square data points represent TCD drop, and the triangle data points represent PCM drop.

Fig. 28 is another graph showing the performance of the seventh experimental system. The performance of each station is displayed over time, indicating that the performance has not degraded. The y-axis is the percentage reduction in TCD from 0% to 100% with a 10% interval. Along the x-axis, the leftmost set of bars represents data acquired after 2 hours, the second set of bars from the left represents data acquired after 4 hours, the middle set of bars represents data acquired after 6 hours, the second set of bars from the right represents data acquired after 9 hours, and the rightmost set of bars represents data acquired after 12.5 hours. Within each group of columns, the leftmost column represents station 1, the leftmost column represents station 2, the rightmost column represents station 3, and the rightmost column represents station 4.

Fig. 29 is another graph showing the performance of the seventh experimental system. The figure shows the results of a comparison between the area of the depth filter (left panel) for a single depth filter (DFF) versus the equivalent area of DFF (right panel) using an acoustophoretic filter to obtain the same volume. In both images, the y-axis is m²The depth filter was counted from 0 to 60 in a specified area with a spacing of 10. In both images, along the x-axis, the leftmost set of bars represents the first user, the middle set of bars represents the second user, and the rightmost set of bars represents the third user. Within each group of columns, the leftmost column represents station 1, the second column from the left represents station 2, the second column from the right represents station 3, and the rightmost column (when present) represents station 4.

Fig. 30 is another graph showing the performance of the seventh experimental system. The y-axis is the percentage decrease in cell density from 0% to 100% with a 10% interval. The x-axis is the percentage reduction in TCD in terms of cell viability. Along the x-axis, the leftmost bar represents 22X 10 at 82% cell viability⁶Cell density of cells/mL, the column second from the left represents 22X 10 at 80% cell viability⁶Cell density of cells/mL, the third bar from the left represents 22X 10 at 64% cell viability⁶Cell density of cells/mL, the third column from the right represents 40X 10 at 62% cell viability⁶Cell density of cells/mL, the second column from the right represents 70X 10 at 60% cell viability⁶Cell density of cells/mL, right-most bar represents 1 at 70% cell viability00×10⁶Cell density of cells/mL.

Detailed Description

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

Although specific terms are used in the following description, for purposes 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 following drawings and description, it is to be understood that like numeric designations refer to components of like function. Further, it should be understood that the drawings are not to scale.

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

As used in the specification and claims, the term "comprising" may include embodiments "consisting of and" consisting essentially of. As used herein, the terms "comprising," "including," "having," "has," "capable of," "containing," and variations thereof mean open-ended terms, or words that require the presence of the specified component/step and allow for 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 for the presence of only the specified components/steps, and any impurities that may result therefrom, and excludes other components/steps.

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

All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (e.g., ranges of "from 2 grams to 10 grams" are inclusive of the endpoints, 2 grams and 10 grams, and all intermediate values).

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 2 end points. For example, the expression "from about 2 to about 4" also discloses a range of "from 2 to 4".

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 position, i.e., the upper component is at a higher elevation than the lower component in a given orientation, but these terms can vary if the device is turned over. The terms "inlet" and "outlet" relate to a given structure relative to a fluid flowing therethrough, e.g., a fluid flowing through an inlet into the structure and flowing through an outlet out of the structure. The terms "upstream" and "downstream" are relative to the direction of fluid flow through the various components, i.e., fluid flows through an upstream component and then through a downstream component. It should be noted that in a ring, a first component can be described as being upstream and downstream of a second component.

The terms "horizontal" and "vertical" are used to indicate directions relative to an absolute reference (i.e., the ground plane). The terms "upward" and "downward" are also relative to absolute references; the upward flow is always against the earth's gravity.

The present application relates to "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, then 2 numbers are of the same order of magnitude.

The present application also relates to "sharp" angles. For the purposes of this disclosure, the term "acute" refers to an angle of 0 ° to 90 °, excluding 0 ° and 90 °.

Advances in fed-batch cell culture have led to advances of up to 50X 10⁶Higher cell density of cells/mL and>product titer of 5 g/L. With the switch to the application of disposable technology in cell culture, increased efficiency of the cell harvest and clarification stage is sought to produce Harvested Cell Culture Fluid (HCCF) for capture chromatography and subsequent downstream processing. Progress in continuous processes (evolution) may also be a factor in efficiency considerations, where preference is given to continuous feed of HCCF, which may be used directly loaded in a continuous multi-column capture chromatography step. ExistingCell culture clarification of (a) is performed using centrifugation or depth filtration, usually operating in batch mode and using bulk stored feed or HCCF in the process.

The acoustophoretic separation techniques of the present disclosure employ ultrasonic standing waves to capture, i.e., hold stationary, secondary phase materials, including fluids and/or particles, in a main fluid stream. Trapping the secondary phase material is an important distinction from previous methods, where particle trajectories were altered only by acoustic radiation force effects. The acoustic field scattering of the particles results in three-dimensional acoustic radiation forces, which act as three-dimensional trapping fields. The three-dimensional acoustic radiation force generated in conjunction with the ultrasonic standing wave is referred to in this disclosure as a three-dimensional or multi-dimensional standing wave. When the particles are small with respect to wavelength, the acoustic radiation force is proportional to the particle volume (e.g., the cube of the radius). Which is proportional to the frequency and the acoustic contrast factor (contrast factor). Which is also proportional to the acoustic energy (e.g., the square of the sound pressure amplitude). For harmonic excitation, the sinusoidal spatial variation of force drives the particles to stable positions within the standing wave. Particles can be trapped in an acoustic standing wave field when the acoustic radiation force exerted on the particles is stronger than the combined effect of fluid drag and buoyancy and gravity. This capture causes the captured particles to concentrate, aggregate, and/or coalesce. In addition, secondary interparticle forces such as Bjerkness forces assist in particle aggregation. Particles heavier than (i.e. denser than) the primary fluid are separated by enhanced gravitational settling.

One particular application of acoustophoretic devices is the treatment of bioreactor materials. It is desirable to filter as many or all of the cells and cell debris as possible from the expressed material in the fluid stream. The expressed material consists of biomolecules, such as recombinant proteins or monoclonal antibodies, and is the desired product to be recovered. By using acoustophoresis, the separation of cells and cell debris is very efficient and the loss of expression product is minimal. This separation technique is an improvement over current filtration processes (depth filtration, tangential flow filtration, centrifugation) which show limited efficiency at high cell densities, where the loss of expressed material within the own filter bed can be as much as 5% of the bioreactor-produced material. The use of mammalian cell cultures, including Chinese Hamster Ovary (CHO) cells, NS0 hybridoma cells, Baby Hamster Kidney (BHK) cells, and human cells, has proven to be a very efficient method for the production/expression of recombinant proteins and monoclonal antibodies for pharmaceutical use today. Filtration of mammalian cells and mammalian cell debris by acoustophoresis helps to greatly increase bioreactor yield. The acoustophoresis techniques discussed herein allow for the recovery of cells and/or their expressed material.

In the acoustophoresis technique discussed herein, the contrast ratio 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 itself. Most cell types present a higher density and lower compressibility than the medium in which they are suspended, so that the acoustic contrast coefficient between the cells and the medium is positive. Axial Acoustic Radiation Force (ARF) drives cells with positive contrast to the pressure node plane and cells or other particles with negative contrast to the pressure antinode plane. The radial or lateral component of the acoustic radiation force helps to trap the cells. In some examples, the radial or lateral component of the ARF is greater than the combined effect of the fluid drag force and gravity.

Physical scrubbing of the cell culture medium also occurs as cells accumulate at the nodes of the standing wave, where more cells are captured as they contact cells already held within the standing wave. This phenomenon, or combination of phenomena, facilitates the separation of cells from the cell culture medium. The expressed biomolecules remain in the nutrient stream (i.e., the cell culture medium).

Ideally, the ultrasonic transducer generates a three-dimensional or multi-dimensional acoustic standing wave in the fluid, exerting a lateral force on the suspended particles to increase the particle trapping capacity of the standing wave. The ultrasound transducer publications in the acoustic related literature provide typical results indicating that the lateral force in planar or one-dimensional acoustic wave generation is two orders of magnitude less than the axial force. In contrast, the disclosed technology provides lateral forces on the same order of magnitude as axial forces.

It is also possible to drive a variety of ultrasonic transducers with arbitrary phasing and/or different or variable frequencies. The various transducers may function to separate materials in a fluid stream to be out of phase with each other and/or to operate at different or variable frequencies. Alternatively or additionally, a single ultrasound transducer divided into an ordered array may be operated so that some array assemblies will be out of phase with other array assemblies.

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 so that they migrate in multiple excitation modes. The resulting wave type can be characterized as a complex wave with a displacement spectrum similar to a leaky symmetric (also called a compressional or extensional) lamb wave. The wave is a leaky wave because it radiates into the water layer, which results in an acoustic standing wave in the water layer. The displacement spectrum of the symmetric lamb wave is symmetric with respect to the neutral axis of the piezoelectric element, which results in multiple standing waves being generated in 3-D space. By this way of wave generation, higher lateral trapping forces are generated if the piezoelectric transducer is excited in a "piston" mode (where only a single, planar standing wave is generated). Thus, for the same input power to the piezoelectric transducer, a 3-D or multi-dimensional acoustic standing wave can have a higher lateral trapping force, possibly up to and over 10 times the single acoustic standing wave generated by the piston mode.

Due to acoustic streaming, it may sometimes be necessary to adjust the frequency or voltage amplitude of the standing wave. Such adjustment may be accomplished by amplitude adjustment and/or frequency modulation. The duty cycle of the standing wave propagation may also be used to achieve some of the results of the trapping material. In other words, the beams may be turned on and off at different frequencies to achieve the desired result.

In certain applications, multiple acoustophoretic cell filtration devices may be implemented to clarify bioreactor cell cultures and separate biomolecules/proteins from cells expressing them. The present disclosure relates to acoustophoresis systems constructed of modular components, and kits of such modules. Each module is cooperatively engaged with the other modules and then reversibly separable. The kits and modules allow a user to obtain different configurations of the acoustophoresis system as desired to provide improved particle precipitation and improved particle separation from a fluid. In short, particles suspended from a host fluid can pass through multiple transducers that generate multiple standing waves to induce separation from the fluid itself. The module assembly can also be used to provide improved fluid dynamics, increasing particle separation from the fluid.

The use of multiple standing waves from multiple ultrasonic transducers allows for multiple separation stations. For example, in a flow through four ultrasonic station-reflector pairs (i.e., four acoustophoresis stations), a first station (and its standing wave) collects a quantity of particles, a second station (and its standing wave) collects particles that pass through the first station, a third station (and its standing wave) collects particles that pass through the first and second stations, and a fourth station (and its standing wave) collects particles that pass through the first, second, and third stations. This configuration may be useful when the particle/fluid ratio is high (i.e., large volume of particles), and achieves the separation capability of any upstream transducer. This construction can also be used for particles having a bimodal or larger size distribution, where each transducer can be implemented to capture particles within a range of sizes.

Fig. 1 illustrates a first exemplary embodiment of a multi-station acoustophoresis system 2000. The system 2000 includes a first acoustophoresis device 2010, a second acoustophoresis device 2020, a third acoustophoresis device 2030, and a fourth acoustophoresis device 2040. Each device can be viewed as a different acoustophoresis or filtration station. In this regard, system 2000 is a four-station acoustophoresis system in that each acoustophoresis device can be constructed as described herein, including a transducer-reflector pair, to produce at least one multi-dimensional acoustic standing wave within each device. In certain embodiments, the acoustic chamber of each device may have an area of 1 inch x2 inches. The nominal flow rate through the system may be about 4L/hr, typically about 3.64L/hr (i.e., about 60 mL/min).

In the system 2000 shown in fig. 1, the devices 2010, 2020, 2030, 2040 are connected in series with each other, each device/station being connected to adjacent stations by a conduit 2060 running between them. Connecting adjacent stations with piping (as opposed to directly connecting the devices of each station to adjacent devices) provides for better separation of fluid from particulates.

Pumps (e.g., peristaltic pumps) may be provided between each device, and additional pumps may be provided upstream of the first device and downstream of the last device. In this regard, it should be noted that in the four-station system 2000 shown in fig. 1, there are 5 pumps for 4 devices/stations: (1) a feed pump 2008 upstream of the first acoustophoresis device 2010; (2) a first pump 2018 downstream of the first acoustophoresis device 2010 and upstream of the second acoustophoresis device 2020; (3) a second pump 2028 downstream of the second acoustophoresis device 2020 and upstream of the third acoustophoresis device 2030; (4) a third pump 2038 downstream of the third acoustophoresis device 2030 and upstream of the fourth acoustophoresis device 2040; and (5) a fourth pump 2048 downstream of the fourth acoustophoresis device 2040. More briefly, the embodiment of system 2000 shown in fig. 1 includes a feed pump 2008 upstream of first acoustophoresis device 2010 and pumps 2018, 2028, 2038, 2039 downstream of each acoustophoresis device 2010, 2020, 2030, 2040, respectively. The pumps 2008, 2018, 2028, 2038, 2048 are fluidly connected between each adjacent device/station by conduits 2060.

In addition to the pumps, the embodiment of the system 2000 shown in FIG. 1 includes flow meters adjacent to each pump. As shown here, there are 5 flow meters: (1) a first flow meter 2009 upstream of the first acoustophoresis device 2010; (2) a second flow meter 2019 downstream of the first acoustophoresis device 2010 and upstream of the second acoustophoresis device 2020; (3) a third flow meter 2029 downstream of the second acoustophoresis device 2020 and upstream of the third acoustophoresis device 2030; (4) a fourth flow meter 2039 downstream of third acoustophoresis device 2030 and upstream of fourth acoustophoresis device 2040; and (5) a fifth flow meter 2049 downstream of the fourth acoustophoresis device 2040. More briefly, the embodiment of the system 2000 shown in fig. 1 includes a feed flow meter 2009 upstream of the first acoustophoresis device 2010 and flow meters 2019, 2029, 2039, 2049 downstream of each acoustophoresis device 2010, 2020, 2030, 2040, respectively. With the pump, flow meters 2009, 2019, 2029, 2039, 2049 are fluidly connected between each adjacent device/station by conduits 2060. Thus, the fluid flow passes through the feed pump, then the flow meter, then the first acoustophoresis/filtration station, then the pump, then the flow meter, etc., ending with the last pump and flow meter.

In some embodiments, such as the embodiment shown in fig. 2A, the individual apparatus stations 2010, 2020, 2030, 2040 are physically located side-by-side and physically connected to each other. However, physical access is not required-the stations may be separated from each other and fluidly connected by conduits, as shown in fig. 1 and 3. Fig. 2B is a rear view of first acoustophoresis device 2010.

Fig. 2C shows a cross-sectional view of an exemplary acoustophoretic device/station 2100, which can be used as any device/station of the multi-station acoustophoretic system described herein. This device can be used to alleviate some of the problems associated with fluids at low particle reynolds numbers and to create a more uniform flow of liquid through the device. The device 2100 has an upward vertical flow of fluid through the acoustic chamber 2111. The acoustic chamber also has 2 opposing dump diffusers 2112 and collector designs that provide vertical planes or lines of flow symmetry. Typically, the cross-section of the device in the direction of flow is circular or rectangular. In this example, the acoustic chamber is empty (no fluid), e.g., there are no other structures in the chamber between the transducer and the reflector, and fluid is allowed to flow through the acoustic chamber. There is at least one permeate outlet 2114 at the upper end of the acoustic chamber. At the lower end of the acoustic chamber there is at least one concentrate outlet 2116. At the lower end of the acoustic chamber there is a shallow wall 2118 and leads to a concentrate outlet 2116. The shallow wall is angled relative to the horizontal, which can be described by the acoustic chamber bottom. At least one ultrasonic transducer (not shown) is coupled to the acoustic chamber, e.g., can be located on a sidewall of the acoustic chamber. At least one reflector (not shown) or another ultrasound transducer is located opposite the ultrasound transducer, e.g., may be located on a sidewall opposite the ultrasound transducer. The multi-dimensional standing wave may be generated with a transducer and opposing reflectors, or may be generated with 2 opposing transducers.

The device 2110 includes a symmetrical, double sudden-expansion diffuser, plenum chamber (plenum) inlet configuration. Here, 2 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 inlet volume provides flow diffusion and significantly reduces incoming flow non-uniformity. An air inlet 2124 is located above the lower end 2122 and at least one flow outlet 2126 is located at the plenum lower end. A solid wall 2128 exists at the upper end of the plenum. These sudden diffuser flow outlets may take the form of slots or a row of holes and are placed above the bottom of the acoustic chamber. The diffuser 2112 provides a flow direction through the acoustic chamber at an angle, such as orthogonal, to the axial direction of the acoustic standing wave generated by the ultrasonic transducer. The acoustic chamber inlets are also arranged so that they are in opposing positions so that the horizontal velocity decreases towards zero in the center of the acoustic chamber.

The dump diffuser helps to reduce or eliminate the downward flow of the fluid mixture in the acoustic chamber. The fluid mixture fills the plenum cavity in the dump diffuser and then flows horizontally into the acoustic chamber where the mixture flows vertically upward through the acoustic standing wave. The dump diffuser reduces/eliminates flow pulsations and flow non-uniformities that result from the pump, hose flush and/or horizontal inlet flow where gravitational effects dominate. The sudden diffuser causes the mixture to enter the acoustic chamber below the ultrasonic transducer and thus below the node clusters or lines formed in the ultrasonic standing wave. This arrangement helps to reduce or minimize any interference with the clusters that might otherwise be caused by the influent material.

The vertical plane or line of symmetry coincides with the direction of gravity (is aligned with). Also shown are flow lines which need to be symmetrical to reduce or minimize non-uniformity, vortex interference, circulation and interference of falling through the clusters of concentrate outlets 2116 to be collected. Symmetry also helps in the distribution of the incoming air flow and the even distribution of gravity during particle collection. Because it is heavier than the permeate exiting at the top of the apparatus, the (relatively) heavy feed mixture approaches the bottom of the acoustic chamber, stretches to the bottom of the chamber by gravity, and provides a near uniform velocity spectrum from bottom to top. As the mixture approaches the center of the acoustic chamber, the horizontal velocity of the mixture may decrease to near or equal zero due to the flow of the dual opposing inlets. This horizontal velocity drop helps to reduce or minimize interference between the chamber flow and the settled particle clusters. Uniform speed increases and separation and collection of results can be maximized. The lateral acoustic force of the acoustic standing wave can overcome particle drag and allow particles to be trapped to form clusters and grow and continuously move out of the acoustic standing wave. Uniform velocity can help avoid uneven disturbances or disturbances with lateral acoustic forces. The uniform velocity allows the inlet flow distributor to be optional.

As the particle clusters break away, the axial acoustic forces associated with the standing wave help keep the clusters intact. Keeping the clusters intact helps to maintain a rapid drop in clusters with high terminal velocity, on the order of 1 cm/sec. The settling velocity of the clusters can be extremely fast compared to the chamber flow rate, which in some examples is on the order of 0.1cm/sec to 0.3 cm/sec. A shallow wall angle means that the cylindrical particle cluster can travel a relatively short distance before it leaves the acoustic chamber, so that little cluster dispersion occurs. Preferably, the system operates with 3-12 crystal vibration nodes per square inch of transducer. Symmetric, reduced fluid turbulence and shallow collector walls in the central collection region achieve improved collection and may allow for baffle/lamella plate options.

Fig. 3 generally illustrates a fluid flow path through the acoustophoresis station 2010. Fresh fluid/cell culture medium mixture is introduced into the station through inlet 2012 continuously at its top end and flows through flow chamber 2050. Within the flow cell, cells aggregate and fall/settle out of the acoustic standing wave. These settled cell aggregates then fall to the bottom of the flow chamber 2050 and can be recovered via the ports 2016. The remaining mixture flows out of the flow chamber at the top of the device through outlet 2014 and continues to downstream devices as described herein.

Figures 4-6 illustrate the function of the acoustophoresis station. As shown in fig. 4, the fluid/cell culture medium mixture enters the acoustophoresis station through the conduit and into a separate acoustic wave zone, i.e., a region in which at least one multi-dimensional acoustic standing wave is generated. The acoustic wave region is defined between the ultrasonic transducer and the reflector. As shown in fig. 5, the cell is trapped in the acoustic standing wave at the node due to the lateral component of the acoustic radiation force. As shown in fig. 6, as the cells accumulate at the node, the drop in acoustic radiation force and/or the decrease in buoyancy/increase in gravitational effects causes the accumulated cells to settle out of the suspension and fall to the bottom of the flow chamber.

As briefly explained above and illustrated in fig. 3, it should be noted that in many embodiments of the multi-station acoustophoresis device described herein, the ultrasonic transducer 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 membrane composed of any suitable material, such as Polyetheretherketone (PEEK) or 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 rigid or flexible and can be constructed of a material with high acoustic impedance, such as steel or tungsten, or any other suitable material that provides good acoustic reflection. One particularly contemplated material for the reflectors of the multi-station acoustophoretic system described herein is borosilicate glass. Another transducer may also be used as a reflector.

The various portions of the multi-station acoustophoresis systems described herein, such as the flow chamber, can be made of any suitable material that is capable of housing a fluid mixture. Suitable materials and related components of this type for the flow cell include medical grade plastics such as polycarbonate or polymethylmethacrylate, or other acrylates. One particularly contemplated material for the flow chamber/housing in the multi-station acoustophoresis systems described herein is polyphenylsulfone (PPS). The material may be configured to be at least somewhat transparent as a transparent window to allow the internal flow passages and flow paths to be visible during operation of the acoustophoresis device/system.

A multi-dimensional acoustic standing wave for particle collection is obtained as follows: the ultrasonic transducer is driven at a frequency that both creates an acoustic standing wave and excites the fundamental 3D vibration mode of the piezoelectric material of the transducer. Perturbation of the piezoelectric material in an ultrasound transducer in multimode form allows for the generation of a multi-dimensional acoustic standing wave. Piezoelectric materials can be specifically designed to deform in multiple modes at a given frequency, allowing for the generation of multi-dimensional acoustic standing waves. The multi-dimensional acoustic standing wave may be generated by different modes of the piezoelectric material, such as a 3x3 mode, which produces a multi-dimensional acoustic standing wave. By allowing the piezoelectric material to vibrate through many different vibration modes, a large number of multi-dimensional acoustic standing waves can also be generated. Thus, the piezoelectric material can be excited to produce multiple modes such as 0x0 mode (i.e., piston mode) to 1x1, 2x2, 1x3, 3x1, 3x3, and other higher order modes, and then cycle back to the lower order modes of the piezoelectric material (not necessarily in direct sequence). The transition or dithering of the piezoelectric material between modes allows for the generation of different multi-dimensional waveforms and a single piston mode shape at a given time.

Some other explanations of the ultrasound transducers used in the devices, systems, and methods of the present disclosure are also useful. In this regard, the transducer uses a piezoelectric material, which may be a ceramic material, crystalline or polycrystalline such as PZT-8 (lead zirconate titanate). Such crystals may have a diameter of 1 inch and a nominal resonant frequency of 2 MHz. Each ultrasonic transducer module can have only one piezoelectric material, or can have multiple piezoelectric materials. The various piezoelectric materials may each be used as a separate ultrasound transducer and may be controlled by one or more controllers, drivers, or amplifiers.

Fig. 7 is a cross-sectional view of a conventional ultrasonic transducer. The transducer has a wear plate 50 at the bottom end, an epoxy layer 52, a piezoelectric crystal 54 (made of, for example, PZT), an epoxy layer 56 and a backing layer 58. On either side of the piezoelectric crystal, there are electrodes: an anode 61 and a cathode 63. Epoxy layer 56 attaches support layer 58 to crystal 54. The entire combination is contained in a housing 60, which may be made of, for example, aluminum. The electrical adapter 62 provides a wire connection to pass through the housing and connect to a wire (not shown) attached to the crystal 54. In general, the support layer is designed to add damping and produce a broadband transducer with uniform displacement over a wide range of frequencies, and to suppress excitation at specific vibration signature-modes. The wear shield is typically designed as an impedance transformer to better match the characteristic impedance of the medium into which the transducer radiates.

Fig. 8 is a cross-sectional view of an ultrasound transducer 81 of the present disclosure. The transducer 81 is shaped like a disk or dish, having an aluminum housing 82. The aluminum housing has a top end and a bottom end. The transducer housing may also be constructed of plastic (e.g., medical grade HDPE) or other metal. The piezoelectric elements are a large number of perovskite ceramics, each consisting of small tetravalent metal ions, usually titanium or zirconium, in a larger lattice of divalent metal ions, usually lead or barium and O^2-Ions. For example, a PZT (lead zirconate titanate) piezoelectric element 86 defines the transducer bottom end and is exposed outside the case bottom end. The piezoelectric element is supported on its perimeter by a low-elasticity layer 98, such as epoxy, silicone or similar material, located between the piezoelectric element and the housing. Transducer 81 does not include a wear-resistant shield or backing material. In some embodiments, a layer of plastic or other material (not shown) is provided on the outer surface of the piezoelectric element 86. The material has the characteristic of separating the piezoelectric element 86 from the fluid in which the acoustic standing wave is generated. The material may be relatively thin, such as in the range of 10 μm-1 mm, and may be secured to the piezoelectric element 86 with an adhesive, for example. The material may be substantially acoustically transparent and may be an adhesive. The piezoelectric element 86 has an outer surface (exposed) and an inner surface.

The screws attach the aluminum top plate 82a of the housing to the body 82b of the housing via threads 88. The top plate includes a connector 84 to power the transducer. The top surface of the PZT piezoelectric element 86 is connected to an anode 90 and a cathode 92, which are separated by an insulating material 94. The poles of the electrodes 90, 92 appear inverted and the electrodes 90, 92 can be on opposite sides or the same side of the piezoelectric element 86, where the same side can be an inner or outer surface. The electrodes can be made of any conductive material, such as silver or nickel. Electrical power is provided to the PZT piezoelectric element 86 through electrodes on the piezoelectric element. Note that the piezoelectric element 86 does not have a support layer or an epoxy layer. The transducer 81 has an internal space or air gap 87 in the transducer between the aluminum top plate 82a and the piezoelectric element 86 (e.g., the housing is empty or contains air). In some embodiments, a minimal backing (backing)58 (on the inner surface) and/or a wear plate 50 (on the outer surface) may be provided, as shown in fig. 9.

The transducer design can affect system performance. A typical transducer is a layered structure with a ceramic piezoelectric element incorporating a support layer and a wear-resistant shield. Conventional design guidelines for wear shields (e.g., half-wavelength thickness for standing wave applications or quarter-wavelength thickness for radiation applications) and manufacturing methods may not be suitable due to the high mechanical impedance of the transducer loaded with standing waves. In the example of transducer 81, shown in FIG. 8, there is no wear plate or backing, allowing piezoelectric element 86 to vibrate in one of its eigenmodes, or in a combination of several eigenmodes, with a high Q factor. The piezoelectric element 86 has an outer surface directly exposed to the fluid flowing through the flow chamber in which the transducer 81 is mounted.

Removing the support (e.g., air supporting the piezoelectric element) also allows the ceramic piezoelectric element to vibrate in a damped small higher order vibration mode (e.g., higher order modal displacement). In transducers having a piezoelectric element with supports as previously implemented, the piezoelectric element vibrates with a more uniform displacement, such as a piston. Removing the support may allow the piezoelectric element to vibrate more easily in a non-uniform displacement mode. The higher the mode shape of the piezoelectric element, the more node lines the piezoelectric element can generate. Displacement of higher order modes of the piezoelectric element produces more trapping lines, although the trapping lines do not necessarily have to be one-to-one with nodes, driving the piezoelectric element at higher or lower frequencies does not necessarily produce more or less trapping lines for a given operating frequency.

In some embodiments of the acoustic filtering devices of the present disclosure, the piezoelectric element can have a support that has a relatively small effect (e.g., less than 5%) on the Q factor of the piezoelectric element. The support may be made of a substantially acoustically transparent material, such as balsa wood, foam, or cork, which allows the piezoelectric element to vibrate at high order mode shapes and maintain a high Q factor, while still providing some mechanical support for the piezoelectric element. The support layer may be a solid, or may be a lattice with holes in the layer, such that the lattice provides support at the node locations after the nodes of the vibrating piezoelectric element that adopt certain higher order modes of vibration, while allowing the remaining piezoelectric element to vibrate freely. The purpose of the mesh or acoustically transparent material is to provide support without reducing the Q factor of the piezoelectric element or interfering with the excitation of a particular mode shape.

Placing the piezoelectric element in direct contact with the fluid also contributes to a high Q factor by avoiding the damping and energy absorbing effects of the epoxy layer and the wear-resistant backplate. Other embodiments of the transducer may have a wear-resistant shield or surface to prevent PZT containing lead from contacting the primary fluid. These layers between the transducer and the main fluid may be suitable for e.g. biological applications such as separation of blood, biopharmaceutical perfusion or fed-batch filtration of mammalian cells. Such applications may use wear resistant layers such as chromium, electrolytic nickel or electroless nickel. Chemical vapor deposition can also be used to apply a layer of poly (p-xylylene) (e.g., parylene) or another polymer layer. Organic and biocompatible coatings such as silicone or polyurethane are also used as wear surfaces. Thin films such as PEEK films can also be used as a covering for the outer surface of the piezoelectric material, advantageously as a biocompatible material. In one embodiment, the PEEK film is attached to the piezoelectric material surface with a Pressure Sensitive Adhesive (PSA). Other films having lower acoustic impedance and/or being biocompatible can also be used.

In the implementations discussed herein, the particles are trapped in the ultrasonic standing wave, i.e., remain stationary. Particles were collected along a clear trapping line, separated by half a wavelength. Within each nodal plane, the particles are trapped at a minimum acoustic radiation potential. The axial component of the acoustic radiation force drives particles with a positive contrast coefficient to the pressure node plane, while particles with a negative contrast coefficient are driven to the pressure antinode plane. The radial or lateral component of the acoustic radiation force helps to trap the particles laterally. The radial or lateral component of the acoustic radiation force is of the same order of magnitude as the axial component of the acoustic radiation force. As discussed above, the lateral force can be increased as follows: a transducer of a higher order mode shape is driven, as opposed to a mode of vibration in which the crystal effectively moves as a piston with a uniform position. The sound pressure is proportional to the drive voltage of the transducer. The electrical power is proportional to the square of the voltage.

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 signal type to which the transducer responds to produce a multi-dimensional acoustic standing wave. In embodiments, the voltage signal driving the transducer can have a pulsed, sinusoidal, square, sawtooth or triangular waveform; and has a frequency of 500kHz-10 MHz. The voltage signal can be driven with pulse width modulation, which can be used to generate any desired waveform. The voltage signal may 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.

In some examples, the transducer size, shape, and thickness can determine transducer displacement at different excitation frequencies. Transducer displacements with different frequencies can affect separation efficiency. In some examples, the transducer operates at a frequency near the thickness resonance frequency (half wavelength). The presence of a gradient in transducer displacement may create more locations for particles to be captured. Higher-order modal displacements can produce three-dimensional acoustic standing waves with strong gradients in the acoustic field in all directions, thereby producing acoustic radiation forces of similar strength in all directions, which may be on the same order of magnitude, for example. Higher order modal displacements can cause multiple trapping lines. The number of trapping lines is related to the particular transducer mode shape.

To study the effect of transducer displacement curves on acoustic trapping force and separation efficiency, experiments were repeated ten times with a 1 "x 1" square transducer, all conditions being identical except for the excitation frequency. 10 consecutive acoustic resonance frequencies, indicated by circled numbers 1-9 and letter a on fig. 10, are used as excitation frequencies. Provided the experiment lasted 30 minutes, an oil concentration of 1000ppm of about 5 microns SAE-30 droplets, a flow rate of 500ml/min and 20W input power. Oil droplets are used because oil is less dense than water and can be separated from water using acoustophoresis.

FIG. 10 shows the measured electrical impedance amplitude of the transducer when operated in a water column containing oil droplets as a function of frequency near the 2.2MHz transducer resonance. The minimum value of the transducer electrical impedance corresponds to the acoustic resonance of the water column and represents the potential frequency of operation. Additional resonances exist at other frequencies that excite the multi-dimensional standing wave. Numerical simulations indicate that the transducer displacement curve varies significantly at these acoustic resonance frequencies, directly affecting the acoustic standing wave and the resulting trapping force. Since the transducer can be operated near its thickness resonance, the electrode surface displacements are essentially out of phase. The transducer electrode displacement may be non-uniform and vary according to the excitation frequency. For example, at an excitation frequency with a single trapped drop line, the displacement has a single maximum at the center of the electrode and a minimum at the transducer edge. At another excitation frequency, the transducer curve has multiple maxima, resulting in multiple drop capture lines. Higher order transducer displacement modes can produce higher trapping forces and multiple stable trapping lines for the trapped oil droplets.

To study the effect of the transducer displacement profile on acoustic trapping force and oil separation efficiency, the experiment was repeated ten times with all conditions being the same except for the excitation frequency. 10 consecutive acoustic resonance frequencies, indicated by circled numbers 1-9 and letter a on fig. 10, are used as excitation frequencies. Provided the experiment lasted 30 minutes, an oil concentration of 1000ppm of about 5 microns SAE-30 droplets, a flow rate of 500ml/min and 20W input power.

As the emulsion passed through the transducer, the capture line of the oil droplets was observed and identified. As shown in fig. 11A, the observations and patterns relating to the number of capture lines across the flow channel were identified for 7 of the 10 resonant frequencies identified in fig. 10.

FIG. 11B shows an isometric view of the flow cell and the isolated acoustic wave regions, where the trap line locations are determined. Fig. 11C is a view of the ultrasound transducer volume as it would appear when looking down at the inlet along arrow 251. Figure 11D is a view of the ultrasound transducer volume as it would appear when looking directly at the transducer face along arrow 253.

In this example, the influence of the excitation frequency clearly determines the number of trapping lines, varying from a single trapping line at the excitation frequencies of acoustic resonances 5 and 9 to 9 trapping lines at the excitation frequency of acoustic resonance 4. At other excitation frequencies, 4 or 5 trapping lines were observed. Different displacement curves of the transducer can generate different (more or less) trapping lines in the standing wave, with more gradients in the displacement curve generally producing greater trapping forces and more trapping lines.

The lin-log plot (linear y-axis, logarithmic x-axis) of fig. 12 shows the calculated ratio of acoustic radiation force, fluid drag force and buoyancy force, using the particle radius. Buoyancy may be applied to particles of negative contrast coefficient, such as the oil particles in this example. The calculated buoyancy may include a gravity element. In examples using positive contrast ratio particles, which may be of certain cell types, lines indicative of gravity are used for a map of such positive contrast ratio particles, the map showing acoustic radiation force and fluid drag force. In the present example shown in FIG. 12, the calculations were done for a typical SAE-30 oil drop used for the experiment. Buoyancy is a particle volume dependent force, such as proportional to the radius cube, and is relatively negligible in terms of micron particle size, but buoyancy increases and becomes significant in terms of hundreds of micron particle sizes. The fluid drag force increases or decreases linearly (scale) with the fluid velocity, as proportional to the square radius, generally exceeding the buoyancy of micron-sized particles, but having less effect on larger-sized particles of the order of hundreds of microns. The effect of the increase or decrease in acoustic radiation force is different from fluid drag or buoyancy. As the particle size is smaller, the acoustic trapping force increases and decreases at a nearly linear rate with the particle radius cubic (volume) of the particle. Finally, as the particle size increases, the acoustic radiation force no longer increases linearly with the particle radius cube. As the particle size continues to increase, the acoustic radiation force decreases rapidly and is a local minimum at some key particle size. To further increase the particle size, the radiation force is again increased in scale, but in phase opposition (not shown in the figure). This pattern is repeated to increase the particle size. The relationship of particle size to acoustic radiation force depends at least in part on the wavelength or frequency of the acoustic standing wave. For example, as the particles increase to a half wavelength size, the acoustic radiation force on the particles decreases. As the particle size increases to more than a half wavelength and less than a full wavelength, the acoustic radiation force on the particles increases.

Initially, as the suspension flows through the acoustic standing wave, which is primarily small micron-sized particles, the acoustic radiation force balances the combined effect of the fluid drag force and the buoyancy force to trap the particles in the standing wave. In FIG. 12, trapping occurs for a particle size of about 3.5 microns, which is labeled R_c1. According to FIG. 12, as the particle size continues to increase beyond R_c1Larger particles are captured because the acoustic radiation force is increased compared to the fluid drag force. As small particles become trapped in the standing wave, particle coalescence/coagulation occursAggregation/agglomeration, causing the effective particle size to continue to grow. The other small particles continue to be driven to the trapping sites of the standing wave as the larger particles are retained and grow in size, facilitating continuous trapping. As the particle size increases, the acoustic radiation force on the particles increases until the first particle size zone is reached. As the particle size increases beyond the first zone, the acoustic radiation force on the particles begins to decrease. As the particle size growth continues, the acoustic radiation force falls rapidly until buoyancy dominates, which is dominated by the second key particle size R_c2Indicating that at this size the particles rise or sink depending on their relative density or acoustic contrast ratio with respect to the host fluid. As the particles rise or fall and leave the antinodes (in the case of negative contrast) or nodes (in the case of positive contrast) of the standing wave, the acoustic radiation force on the particles can be reduced to a negligible amount. The acoustic radiation force continues to capture small and large particles and drives the trapped particles to the capture site, which in this example is located at a pressure antinode. The smaller particle size experiences a reduced acoustic radiation force, which decreases, for example, to the point R shown_c1Nearby acoustic radiation forces. As other particles become trapped and coalesced, agglutinated, aggregated, agglomerated, and/or concentrated together at nodes or antinodes of the acoustic standing wave, effectively increasing particle size, the acoustic radiation force increases and the cycle repeats. All particles may not escape from the acoustic standing wave and those remaining particles may continue to expand in size. Thus, fig. 12 explains how small particles can be continuously trapped in a standing wave, grow into larger particles or clumps, and then eventually rise or settle out due to the relationship between buoyancy, drag force, and acoustic radiation force related to particle size.

A variety of coatings can be used for the internal flow chamber of an acoustophoretic device. Such coatings can include epoxy resins such as epichlorohydrin bisphenols of crosslinked amines or polyamides; or a polyurethane coating such as polyester polyols that crosslink the aliphatic isocyanate. Such coatings are used to create a smooth surface and/or reduce surface tension, allowing cells to better slide along the flow cell surface and into a desired location (e.g., collection well module) under the influence of gravity.

The flow rate of the acoustophoretic device is controlled by, for example, a pump. The flow rate can be adjusted so that gravity/buoyancy can act on the particle aggregate. The particle/fluid mixture entering/leaving the flow chamber in the acoustophoresis device through its inlet/outlet can flow at a rate of up to about 10 liters per hour (L/hr), including up to about 50L/hr, but typically about 3.6L/hr. In comparison, the flow rate exiting the collection well module via the port is much smaller, from about 3 ml/min up to about 10 ml/min.

The acoustophoretic system 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 a desired product and to manage different materials from each filtration step. Each filtration step can be performed to remove specific materials, increasing the overall efficiency of the clarification process. The individual acoustophoresis devices may operate as one or more filtration steps. For example, each individual ultrasonic transducer within a particular acoustophoretic device may be operated to capture material within a given particle range. The acoustophoresis device can be used to remove large amounts of material, reducing the burden on subsequent downstream filtration steps/stations. Any of the various filtering steps/stations discussed herein can be placed upstream or downstream of the acoustophoresis device. Alternatively or additionally, a variety of acoustophoretic devices may be used. The desired biomolecules or cells can be recovered/isolated after such filtration/purification.

The outlets (e.g., clarified fluid and concentrated cells) of the acoustophoresis devices of the present disclosure can be fluidly connected to any other filtration step or filtration station. Such filtration steps may include various methods such as depth filtration, sterile filtration, size exclusion filtration or tangential filtration. Depth filtration uses a physical pore filter media that retains material throughout the depth of the filter. In sterile filtration, very small pore size membrane filters are used to remove microorganisms and viruses, typically without heating or irradiation or exposure to chemicals. Size exclusion filtration through size and/or molecular weight separation materials, using a physical filter with pores of a given size. In tangential filtration, the majority of the liquid flow passes over the surface of the filter, rather than entering the filter.

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

For example, the multi-station acoustophoresis systems described herein may include or be used with in-line filtration stations. One or more in-line filtration stations may be located upstream or downstream of all or some of the acoustophoretic devices. The in-line filtration stations can be used to further purify the liquid and recover and obtain the desired protein therefrom. Suitable examples of serial filtration stations include depth filters, sterile filters, centrifuges, and affinity chromatography columns.

Secondary depth filtration product selection can be achieved by screening with certain materials to be filtered. In a typical fed-batch culture of CHO-S based cell lines expressing humanized IgG1 mAbs, the total volume was less than about 5L to less than about 25L and the total area was about 0.002m²About 0.1m²The depth filter of (2) can be used for secondary depth filtration. In this regard, suitable depth filters include Supracap^TMHP depth filter capsules, available from Pall Corporation. After clarification, the Harvested Cell Culture Fluid (HCCF) is optionally stored, filtered to control bioburden, stored or filtered to control bioburden and chromatographed. In a typical fed-batch culture of CHO-S based cell lines expressing humanized IgG1 mAbs, total volumes of less than about 5L to less than about 25L and total areas of about 220cm can be used²About 375cm²A sterile filter (i.e., a sterile grade membrane filter). In this regard, suitable sterile filters include

Capsules and mini-Kleenpak capsules available from pall corporation.

Optionally, the tertiary depth filtration can be omitted on a small scale, but when used, prevents subsequent filter contamination and allows for a reduction in the size of the bioburden control filter. In a typical fed batch culture of a CHO-S based cell line expressing humanized IgG1mAb, the same depth filter used for secondary depth filtration can be used for tertiary depth filtration. After clarification, the same sterile filters as described above can be used.

In some exemplary biological applications, all portions of the system (i.e., the stations, the conduits fluidly connecting the stations, etc.) can be separate from one another and disposable. With respect to filtering cells in biological applications, centrifuges and conventional filters can impose unwanted or deleterious conditions on the cells. The acoustophoresis device discussed herein allows for the separation of cells from a host fluid without the unwanted or deleterious effects of centrifuges and conventional filters. Thus, the use of an acoustophoretic device to avoid a centrifuge and filter can separate cells without necessarily reducing cell viability. Ultrasonic transducers may also be used to generate rapid pressure changes to prevent or clear blockages caused by cell aggregation. The frequency of the transducer may be controlled and/or varied to achieve an operating point to increase or maximize effectiveness for a given power.

The present disclosure is further illustrated in the following non-limiting examples, with the understanding that these examples are intended to be illustrative only and the present disclosure is not intended to be limited to the modules, devices, conditions, process parameters, etc. recited herein.

Examples

The mixture of multiple CHO cells in the cell culture medium was filtered.

Example 1

The acoustophoretic separation process was compared to Deep Flow Filtration (DFF). First, the DFF capacity baseline, i.e., primary and secondary clarification, was obtained by performing two rounds of clarification. The setting for this baseline is shown in fig. 13.

The pressure drop was measured during the two runs. The separation apparatus is at 145LMH (liters per m)²Per hour) was run. Pressure was measured at three different positions, P1, P2, and P3. A filter is located between each set of sensors. The filter used during primary clarification was a D0HC filter and the filter used during secondary clarification was an X0HC filter, both available from Millipore (Millipore).

The mixture of CHO cells and culture medium was passed through a filter and the permeate was subsequently collected in a tank. CHO cells were removed through the filter. The feed had a mass of 6.34X 10⁶Total Cell Density (TCD) of cells/mL and turbidity of 815 NTU. The final permeate in the third tank had a turbidity of 1.75 NTU.

The representation of fig. 13 shows pressure drop versus volumetric flux. The pressure drop across the primary filter is lowest at low throughputs and then above about 88L/m²The capacity becomes greater than the pressure drop in the secondary filter. The total pressure drop is the top line in the figure. At 88L/m²By volume ofA pressure drop of 15psig (indicated by the dashed line) is obtained. This relationship indicates that if scaled up, the maximum pressure drop is 15psig (Pmax ═ 15psig), then the filter areas for primary and secondary clarification are each 11.4m²。

Example 2

Next, the two-step DFF described in example 1 was compared to a two-step clarification process, where primary clarification was performed by sonic separation (AWS) and secondary clarification was performed by DFF. This comparison setup is shown in fig. 14.

As shown in FIG. 14, in the two-step DFF, each filter had 11m²The area of (a). Each filter was operated with a pressure drop of 7.5 psig. For each filter, 7.5psig (VT)_7.5) The Volume Throughput (VT) at bottom is 84L/m²。

The acoustophoretic system for performing AWS consists of three acoustophoretic devices connected in series. The transducer in each device was 1 inch by 1 inch. The system has a height of 49cm³Total acoustic volume of (c). AWS System and Total area 6m²The DFF filter pair of (1). Since there is no pressure drop in the AWS system, the DFF filter can be run at 15psig pressure drop, yielding 160L/m²VT of₁₅。

The feed had a mass of 6.7X 10⁶Total Cell Density (TCD) of cells/mL and turbidity of 835NTU and 77% cell viability. The feed rate of the acoustophoretic system was 4kg, 2.5 Liters Per Hour (LPH).

The results of the primary clarification with the AWS system are shown in fig. 14. The acoustophoretic system achieved a 91% TCD reduction, a 90% turbidity reduction, and 91.2% protein recovery. The lower left graph is percent drop versus time showing the AWS system operating steadily during the test.

Compared to example 1, where both primary and secondary clarification were performed by depth filtration, replacing primary clarification with sonic separation (AWS) could achieve economic benefits by: reduce the overall operating footprint, reduce the secondary depth filter area specification and associated conditions and wash buffers, and reduce storage and disposal costs. As the process enters clinical production, these become key process drivers. An example of expected process specifications for 1000L CHO cell culture is summarized below.

Example 3

The same experiment as described in example 2 was performed again, but with a higher cell density. The feed had a particle size of 15.6X 10⁶Turbidity of total TCD and 3608NTU per mL and cell viability of 68%. This comparison setup is as described in fig. 15.

The two-step DFF process uses 38m each²And 17m²The filter of (1). As shown, the primary clarified VT_7.5Is 26L/m²The secondary clarification was 58L/m². In the AWS-DFF process, the AWS system has two acoustophoretic devices in series (instead of three as in example 2), and the total acoustic volume is 33cm³. The DFF filter had a thickness of 11m²Total area of (2) and 85L/m²VT of₁₅. The feed rate of the acoustophoretic system was 8kg, 2.5 Liters Per Hour (LPH).

The results of the primary clarification with the AWS system are depicted in fig. 15. The acoustophoresis system achieved 94% TCD reduction, 91% turbidity reduction and 92% protein recovery. The lower left graph is percent decrease versus time, showing that the system is operating steadily during the test. Higher cell densities are more difficult to handle with DFF devices, as indicated by lower VT in primary clarification. However, the acoustophoresis device is able to handle higher densities with better VT.

Example 4

The feed had a 7.5X 10⁶Turbidity of TCD and 819NTU per mL and cell viability of 88%. Clarification was performed with a three-station acoustophoresis system as in example 1.

The first stage reduced the cell density by 62%. The second stage reduced the cell density by 87% (95% cumulative). The third stage reduced cell density by 63% (98% cumulative). A reduction in cell density of greater than 90% was obtained using two stations.

The first stage reduced the turbidity by 68% from 819NTU to 260 NTU. The second station reduced the remaining turbidity to 54NTU (94% cumulative). The third station reduced the residual turbidity to 42NTU (95% cumulative). A turbidity reduction of more than 90% was obtained using two stations. This result is important for the secondary filtration process further downstream.

The percentage decrease in cell density and turbidity was constant over time, meaning that the device worked well on a continuous basis. The improved reduction in cell density and turbidity helps to simplify the downstream secondary filtration process and ultimately helps to achieve higher product recovery from chromatography for separation of monoclonal antibodies or recombinant proteins from the clarified fluid.

Example 5

5 different batches were tested by the three-station system of example 1. Each batch had its own cell size and density characteristics. The feed has a composition of 7-8.5X 10⁶TCD and 780-900NTU turbidity per mL and 82% -93% cell viability. This example tests the system for consistency of performance across batches.

In 5 different batches, the turbidity of the permeate decreased in the range of 84% to 86% after three passes with a standard deviation of 1%. After three passes, the cell density of the permeate decreased in the range of 93% to 97%, with a standard deviation of 2%.

In other experiments not described herein, it was found that the process of sonic separation with a multi-dimensional acoustic standing wave did not affect the physical or chemical characteristics of the protein or monoclonal antibody recovered from the permeate.

Example 6

Then, the effect of the voltage input on the clarification performance was observed in the three-or four-station acoustophoresis system.

The mixture of CHO cells and culture medium flows through the various stations of the device and the permeate is then collected in a tank. CHO cells were removed by filter. The feed had a 25X 10⁶Total Cell Density (TCD) of cells/mL and turbidity of 2048NTU and 72% cell viability. The feed flow rate was 30 mL/min and the solids draw (solid draw) at the first station was 2.34 mL/min, 1.41 mL/min at the second station, 0.94 mL/min at the third station, and 0mL/min at the fourth station.

The TCD reduction after three and four stage filtration is shown in the table below. T1 denotes a voltage in the first station, T2 denotes the second station, T3 denotes the third station and T4 denotes the fourth station. The results of 5 different test runs are shown, with different voltages being used in different stations.

For all voltage conditions of 50V and 60V, the system achieved a cell density drop of greater than 90%. The addition of the fourth station produced a higher 95% reduction in TCD from 2.9X 10 after the three stations⁶cell/mL 1.3X 10 after four stages⁶cells/mL. The table below shows the cumulative% TCD decrease per station after 30 minutes for the test runs identified above.

It can be seen that voltages of 50V and 60V produce the best performance in each station. As seen in test run 5, the addition of the fourth station at 50V increased the TCD reduction by 10% aggregation. Typically, a voltage of 50V-60V is applied to the first and/or second station to obtain a high TCD drop.

The total turbidity reduction after three and four filtration stations is shown in the table below. For all voltage conditions of 50V and 60V, the system achieved a turbidity drop of more than 90%. Addition of the fourth station produced a 94% reduction, allowing the permeate to drop from 390NTU after three stations to 177NTU after four stations.

Test run	T1 Voltage (V)	T2 Voltage (V)	T3 Voltage (V)	T4 Voltage (V)	% reduction in turbidity
						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

In summary, the 50V and 60V operating conditions produce higher and nearly equivalent clarification than the 40V operating conditions, and using 40V operating conditions at the third station after 50V/60V at the first and second stations reduces clarification efficiency.

Example 7

Next, similarly to example 1, the acoustophoretic separation process was compared to Deep Flow Filtration (DFF) to determine the effect of DFF performance on sonic separation (AWS) performance.

The mixture of CHO cells and culture medium was passed through two filters, a D0HC filter and an X0HC filter, both of which were obtained from millipore, and the permeate was subsequently collected in a tank. CHO cells were removed through the filter. The feed had a composition of 24.7X 10⁶Total Cell Density (TCD) and turbidity of 2850NTU and 72% cell viability per mL. The final permeate in the tank had a turbidity of 4.9 NTU.

The representation of fig. 16 shows pressure drop versus volumetric flux capacity. The spectra in fig. 16 are plotted for each station according to the total filter area of the station. At 23cm²The volumetric flux capacity was calculated. The pressure drop in the D0HC filter was lowest at low throughput, followed by about 16L/m²The pressure drop in the X0HC filter becomes greater above capacity. 47L/m on D0HC filter²Volume flux and about 110L/m for D0HC filter²At volumetric throughput, a pressure drop of 15psig was obtained (based on the pressure drop ratio estimate-D0 HC filter caused 12.0psig for the total 15.0psig pressure drop and X0HC filter caused the remaining 3.0 psig).

The plot of fig. 17 shows pressure drop versus volumetric flux. The curve of fig. 17 is plotted against the total filter area in series (i.e. across all stations). At 46cm²The volumetric flux capacity was calculated. The pressure drop across the D0HC filter was lowest at low throughput and then above about 8L/m²The capacity becomes greater than the pressure drop of the X0HC filter. The total pressure drop is the top line in the figure. At 21.5L/m²A 15psig pressure drop is obtained. This result shows that if scaled up to a maximum pressure drop of 15psig (Pmax ═ 15psig), the total filter area for primary and secondary clarification would be 46.5m total²The D0HC: X0HC filter size ratio is 3:1 (e.g., the D0HC filter would have a 34.9m filter²Area, and the X0HC filter would have a 11.6m²Area).

Example 8

Next, the two-step DFF described in examples 6 and 7 was compared to a two-step clarification process in which primary clarification was performed by sonic separation (AWS) and secondary clarification was performed by DFF. This comparison setup is shown in fig. 18.

As shown, in the two-step DFF, the D0HC filter had a depth of 34.9m²Area and X0HC Filter with 11.6m²Area, total area 46.5m². Each filter was operated with a pressure drop of 7.5 psig. For D0HC filters, at 15psig (VT)₁₅) Has a Volume Throughput (VT) of 47L/m²110L/m for an X0HC filter². The turbidity of the permeate was 4.9 NTU.

2 different acoustophoresis systems were used to perform the AWS. The first acoustophoretic system is a three-station system (e.g., consisting of three acoustophoretic devices) connected in series. The second acoustophoresis system is a series-connected four-station system (e.g. consisting of four acoustophoresis devices) as shown in fig. 1. The transducer used for each device in each system was 1 inch by 1 inch.

First AWS System and Total area 10.2m²The pair of DFF filters (1). The segmented area is 7.6m²(3x) and 2.6m²(1 x). Since there is no pressure drop in the AWS system, the DFF filter can be run at 15psig pressure drop, resulting in 214L/m²VT of₁₅. The feed had a 2.9X 10⁶Total Cell Density (TCD) of cells/mL and turbidity of 380 NTU. The turbidity of the permeate was 8.8 NTU.

Second AWS System with a Total area of 4.5m²The DFF filter pair of (1). The segmented area is 3.4m²(3x) and 1.1m²(1 x). Again, since there is no pressure drop in the AWS system, the DFF filter can be run at a 15psig pressure drop, resulting in 490L/m²VT of₁₅. The feed had a 1.3X 10⁶Total Cell Density (TCD) of cells/mL and turbidity of 176 NTU. The turbidity of the permeate was 11.4 NTU.

The first AWS system (three-station system) reduced the total filter area by 78%, and the secondary clarification by 12% compared to the DFF of the primary clarification (centrifuge is the current unit operation).

The second AWS system (four-station system) reduced the total filter area by 90% and the secondary clarification by 60% compared to DFF-DFF. The acoustophoretic system achieved a 91% TCD reduction, a 90% turbidity reduction, and a 91.2% protein recovery.

Harvest to DFF-DFF (primary and secondary) yield 21.5 liters/m²So that processing 1000L of harvest would use almost 50m²Area of filter. The volumetric flux of the two-station DFF step after the third station increased by 100% with the addition of the fourth station, from 98.5L/m²To 222L/m². TCD is from 2.9X 10⁶cell/mL reduction to 1.4X 10⁶cells/mL (. about.50% reduction). Turbidity decreased from 338NTU to 220NTU (42% decrease). For comparison, when centrifugation is used as the primary clarification step, the centrate from the centrifuge was measured to have a 0.07 x 10⁶TCD of 450NTU and Viable Cell Density (VCD) of 6%. The high shear forces cause cell disruption, thereby increasing turbidity and decreasing VCD of the remaining cells.

For comparison with the two-stage DFF after acoustic filtering (C0HC + X0HC), a single stage DFF was also performed after four-stage AWS, using three different DFF filters (PDD1, X0HC, 90Z). The volume flux of PDD1 was 90L/m²X0HC is 21L/m²90Z is 110L/m². These values are compared with 222L/m²The two-station volumetric flux of (a) is 50-90% lower.

Example 9

The feed flow rate (Q) was then observed in a multi-station acoustophoresis system_F) Total flow ratio (Q)_S/Q_F) And solid flow ratio (Q)_S#/Q_S#) The effect on clarification performance. The setup for this observation is shown in fig. 19. In FIG. 19, Q_F#Representing the feed flow rate, Q, into a given acoustophoresis station_P#Representing the flow rate of permeate from a given acoustophoretic station, Q_S#Representing the flow rate of solids from a given acoustophoretic station. The permeate flow rate of the upstream device is the feed flow rate (e.g., Q) to the subsequent acoustophoresis station_P1＝Q_F2)。

Using 10 different cell lines, 135 individual runs were performed, using a mixture of CHO cells and medium with 27-98% cellsVitality, 2.8X 10⁶-56×10⁶Cell density of 1.4-16.5% packed cell Pellet (PCM) and feed turbidity of 30-9000 NTU.

Fig. 20 illustrates an exemplary four-station setup. FIG. 21 shows the turbidity drop as a function of feed PCM. FIG. 22 shows the turbidity drop as a function of feed flow rate with a total flow rate of 20%. FIG. 23 shows the turbidity drop as a function of the solids flow ratio, total flow ratio 20%. FIG. 24 shows PCM as a function of solids flow ratio. FIG. 25 shows the turbidity reduction as a function of the solids flow ratio. The solid PCM as a function of the solid flow ratio is listed in the table below.

Flow ratio (Q)S/QF)	PCM(％)
		2.5％	61
5％	42
		10％	30
20％	17

The feed flow rate was found to greatly affect clarification. The total flow ratio was found to affect mainly the yield, but generally the clarification. The ratio found to provide the best results was (feed PCM/50% PCM solids stream). It was found that decreasing the total flow ratio can improve the yield and increase the solids packing. The solids flow rate was found to have no effect on overall clarification, but was found to control solids distribution between stations. Feed PCM was found to affect clarity and station efficiency. It was found that the best results for feed PCM were 5-6%, single station, and higher% PCM not a problem for multi-station systems.

Example 10

Next, the feed flow rate (Q) was observed in three and four station acoustophoresis systems_F) Effect on clarification performance.

The mixture of CHO cells and media was flowed through both systems as AWS "cohort" which was subsequently treated with DFF filters. CHO cells were removed by filter. The feed had a particle size of 35.8X 10⁶Total Cell Density (TCD) and turbidity of 4457NTU, 9.1% PCM and 91.9% viability per mL.

Three-station acoustophoresis System Using 2 runs, feed flow Rate (Q)_F) Are 1.0x and 0.66 x. Four-station acoustophoresis System Using Single-run, feed flow Rate (Q)_F) Is 1.0 x. The 0.6x three-station system performs equivalently to the 1.0x four-station acoustophoresis system, although all operations are performed>Yield of 85% and>haze decreases by 90%. Each permeate has a separate filter chain. All data recorded are provided below.

Again, AWS performs much better than DFF-DFF and achieves a volumetric flux increase of-4 x. The overall filtration is summarized below.

Example 11

Subsequently, 11 separate runs were performed in a four-station acoustophoresis system. The mixture of CHO cells and culture medium flowed through four stations of the system in series, 5 different CHO cell lines. The feed had a composition of 9.78X 10⁶-34.3×10⁶Total Cell Density (TCD) of cells/mL, turbidity of 920-4670NTU, PCM of 6.5-11% and viability of 31-91%.

AWS permeate has a density of 0.8X 10⁶-5.2×10⁶TCD of cells/mL (average 2.9X 10)⁶cells/mL), turbidity at 121-. The reductions were expressed as 76% -95% reduction in TCD (average 88%), 86% -97% reduction in turbidity (average 94%) and 72% -88% reduction in PCM (average 79%). The yield was 84% to 89% (average 86%). Fig. 26 shows the decrease performance as a function of feed PCM and fig. 27 shows the decrease performance as a function of feed cell viability.

Example 12

2 CHO cell lines with multispecific antibodies (MM-131) flowed through a three-station acoustophoresis system, each station having a 1 inch by 1 inch ultrasound transducer. The feed has a size of 20X 10⁶-24×10⁶Total Cell Density (TCD) of cells/mL. The first feed line had a cell viability of 80% and the second feed line had a cell viability of 65%. The total cell reduction, turbidity reduction and protein recovery for the 2 feed lines are listed in the table below.

Feed line	Decrease in total cells	Turbidity reduction	Protein recovery
					80% alive	90％	84％	82％
65% alive	89％	85％	78％

Example 13

In a four-station system, each station measures system performance over time. Fig. 28 shows TCD decline for each station with intervals of 2 hours, 4 hours, 6 hours, 9 hours, and 12.5 hours. The leftmost column of each series represents the first station, the left-most second column of each series represents the second station, the right-most second column of each series represents the third station, and the rightmost column of each series represents the fourth station. The addition of a fourth station showed an increase in the decrease in TCD at each time interval. This figure shows that each acoustophoresis station maintains its performance and its separation capacity does not decrease over time, while the physical filter decreases.

Example 14

For three different users, the depth filtration area was reduced using DFF and a four-station AWS system incorporating DFF filters. FIG. 29 shows that the use of a four-station AWS system reduces the depth filtration area used by a factor of 3-10.

Example 15

Fig. 30 shows the cell density drop over 5 different runs of a three station system with a 1 inch by 1 inch transducer. The x-axis near the bottom of each column indicates total cell density (cells/mL) and cell viability. The y-axis is the percentage decrease of cells from the feed to the permeate, with higher values being better. These results show good separation even for higher total cell densities.

The present disclosure is described with reference to exemplary embodiments. Modifications and alterations will occur to others upon reading and understanding the preceding detailed description. It is intended that the 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.

Claims (21)

1. A multi-station acoustophoresis system for use with a bioreactor, the system comprising:

at least a first acoustophoretic device, a second acoustophoretic device, and a third acoustophoretic device fluidly connected in series in sequence, each acoustophoretic device comprising:

a flow chamber comprising at least two inlets on opposite sides of the flow chamber, each inlet comprising a sudden-expansion diffuser, the flow chamber comprising at least one outlet;

at least one ultrasonic transducer coupled to the flow chamber, comprising a piezoelectric material, and configured to be driven to generate a multi-dimensional acoustic standing wave in the flow chamber;

a reflector opposite the at least one ultrasonic transducer; and

a recovery port below the at least one ultrasonic transducer, the recovery port in fluid connection with the bioreactor; and

a feed pump upstream of the first acoustophoresis device for controlling a flow rate of the first acoustophoresis device, a first pump downstream of the first acoustophoresis device for controlling a flow rate of the second acoustophoresis device, a second pump downstream of the second acoustophoresis device for controlling a flow rate of the third acoustophoresis device, and a third pump downstream of the third acoustophoresis device.

2. The multi-station acoustophoresis system of claim 1, wherein the first, second, and third acoustophoresis devices are fluidly connected by a conduit.

3. The multi-station acoustophoresis system of claim 1, wherein the first, second, and third acoustophoresis devices are configured to produce a multi-dimensional acoustic standing wave, within an order of magnitude of one another in frequency.

4. The multi-station acoustophoresis system of claim 1, further comprising a fourth acoustophoresis device downstream of and in series fluid connection with the third acoustophoresis device, and a fourth pump downstream of the fourth acoustophoresis device.

5. The multi-station acoustophoresis system of claim 1, further comprising a feed flow meter upstream of the first acoustophoresis device, a first flow meter downstream of the first acoustophoresis device, a second flow meter downstream of the second acoustophoresis device, and a third flow meter downstream of the third acoustophoresis device.

6. The multi-station acoustophoresis system of claim 5, further comprising a fourth acoustophoresis device downstream of and in series fluid connection with the third acoustophoresis device, and a fourth flow meter downstream of the fourth acoustophoresis device.

7. The multi-station acoustophoresis system of claim 1, wherein each multi-dimensional acoustic standing wave produces an acoustic radiation force having an axial component and a lateral component that are on the same order of magnitude.

8. The multi-station acoustophoresis system of claim 1, further comprising at least one in-line filtration station upstream of the first acoustophoresis device or downstream of the third acoustophoresis device, the in-line filtration station being one or more depth filters, sterile filters, centrifuges, or affinity chromatography columns.

9. The multi-station acoustophoresis system of claim 1, further comprising:

a fourth acoustophoresis device downstream of and in series fluid connection with the third acoustophoresis device;

a feed pump and a feed flow meter fluidly connected upstream of and in series with the first acoustophoresis device;

a first pump and a first flow meter fluidly connected in series with the first acoustophoresis device and the second acoustophoresis device and located between the first acoustophoresis device and the second acoustophoresis device;

a second pump and a second flow meter fluidly connected in series with and located between the second acoustophoresis device and the third acoustophoresis device;

a third pump and a third flow meter fluidly connected in series downstream of the third acoustophoresis device and the fourth acoustophoresis device and positioned between downstream of the third acoustophoresis device and the fourth acoustophoresis device; and

a fourth pump and a fourth flow meter downstream of and in series fluid connection with the fourth acoustophoresis device.

10. A method for continuously separating a second fluid or particulate from a main fluid mixture obtained from a bioreactor, the method comprising:

flowing the mixture of the primary fluid and the second fluid or particulate through a multi-station acoustophoresis system comprising at least a first acoustophoresis device, a second acoustophoresis device, and a third acoustophoresis device fluidly connected in series in sequence, a feed pump upstream of at least the first acoustophoresis device for controlling a flow rate of the first acoustophoresis device, a first pump downstream of the first acoustophoresis device for controlling a flow rate of the second acoustophoresis device, a second pump downstream of the second acoustophoresis device for controlling a flow rate of the third acoustophoresis device, and a third pump downstream of the third acoustophoresis device, each acoustophoresis device comprising:

a flow chamber comprising at least two inlets on opposite sides of the flow chamber, each inlet comprising a sudden-expansion diffuser, the flow chamber comprising at least one outlet;

at least one ultrasonic transducer coupled to the flow chamber, comprising a piezoelectric material, and configured to be driven to generate a multi-dimensional acoustic standing wave in the flow chamber;

a reflector opposite the at least one ultrasonic transducer; and

a recovery port below the at least one ultrasonic transducer, the recovery port in fluid connection with the bioreactor;

driving the at least one ultrasonic transducer of the first acoustophoretic device to generate a first multi-dimensional acoustic standing wave therein such that at least a first portion of the second fluid or particulate is continuously trapped in the first standing wave and the remaining mixture continues into the second acoustophoretic device;

driving the at least one ultrasonic transducer of the second acoustophoresis device to produce a second multi-dimensional acoustic standing wave therein such that at least a second portion of the second fluid or particulate is continuously trapped in the second standing wave and the remaining mixture continues into the third acoustophoresis device; and

driving the at least one ultrasonic transducer of the third acoustophoresis device to produce a third multi-dimensional acoustic standing wave therein such that at least a third portion of the second fluid or particulate is continuously trapped in the third standing wave.

11. The method of claim 10, wherein the first, second, and third acoustic standing waves are driven at different frequencies from one another.

12. The method of claim 10, wherein the frequencies of the first, second, and third acoustic standing waves are within an order of magnitude of one another.

13. The method of claim 10, wherein the second fluid or particulate is chinese hamster ovary cells, NS0 hybridoma cells, baby hamster kidney cells, or human cells; t cells, B cells, or NK cells; peripheral blood mononuclear cells; plant cells, bacteria, viruses or microcarriers.

14. The method of claim 10, wherein the second fluid or particulate is algae.

15. The method of claim 10, wherein the first, second, and third acoustophoresis devices are fluidly connected by a conduit.

16. The method of claim 10, wherein the first, second, and third acoustophoresis devices are each driven by a voltage signal of at least 50V.

17. The method of claim 10, wherein the first, second, and third acoustophoresis devices are each driven by a voltage signal of 50V-60V.

18. The method of claim 10, wherein the voltage signal driving the downstream-most acoustophoresis device in the multi-station acoustophoresis system is 40-60V.

19. The method of claim 10, wherein the multi-station acoustophoresis system further comprises at least one in-line filtration station downstream of a most downstream acoustophoresis device in the multi-station acoustophoresis system, the in-line filtration station being one or more depth filters, sterile filters, centrifuges, or affinity chromatography columns.

20. A system for collecting biomolecules from a cell culture, the system comprising:

a bioreactor housing a cell culture;

a plurality of acoustophoresis devices fluidly connected in series, each acoustophoresis device comprising:

a flow chamber having at least two inlets on opposite sides of the flow chamber, each inlet including a sudden-expansion diffuser;

an ultrasonic transducer coupled to the flow chamber and driven to produce a multi-dimensional acoustic standing wave in the flow chamber, an

A recovery port below the ultrasonic transducer, the recovery port in fluid connection with the bioreactor;

a pump upstream of each acoustophoresis device for controlling a flow rate of each acoustophoresis device;

at least one of the plurality of acoustophoresis devices is in direct fluid connection with the bioreactor; and

a filter fluidly connected to at least another one of the plurality of acoustophoretic devices.

21. The system of claim 20, wherein there are at least two acoustophoresis devices fluidly connected in series between the bioreactor and the filter.

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