Acoustic perfusion devices

Description

BACKGROUND

One of the key components that is utilized in the manufacturing processes of new biologically based pharmaceuticals is the bioreactor and the ancillary processes associated therewith. An area of growth in the bioreactor field has been with the perfusion process. The perfusion process is distinguished from the fed-batch process by its lower capital cost and continuous (rather than batch) operation.

A perfusion bioreactor processes a continuous supply of fresh media that is fed into the bioreactor while growth-inhibiting byproducts are constantly removed. The nonproductive downtime can be reduced or eliminated with a perfusion bioreactor process. The cell densities achieved in perfusion culture (30-100 million cells/mL) are typically higher than for fed-batch modes (5-25 million cells/mL). These improvements have led to lower contamination in the harvest and better yields without significant increase in cost. A perfusion bioreactor may use a cell retention device to prevent escape of the culture when byproducts are being removed. These cell retention systems add a level of complexity to the perfusion process, which may be seen in the additional management, control, and/or maintenance activities that can lead to successful operation. Operational issues such as malfunction or failure of the cell retention equipment has previously been a problem with perfusion bioreactors, which has limited their attractiveness in the past.

BRIEF DESCRIPTION

The present disclosure relates, in various embodiments, to acoustic devices and processes which are used for perfusion biomanufacturing. More particularly, the devices are coupled to an associated bioreactor. Within the bioreactor, biomolecules, such as recombinant proteins or monoclonal antibodies, are produced from cells. The acoustic device is used for separating the biomolecules from the cells on a continuous basis, and the cells are continuously returned to the bioreactor. In some examples, a fluid mixture containing the cells and the desired products (e.g., biomolecules) are passed or flowed through the acoustic device and separated therein by multi-dimensional standing wave(s). The fluid mixture may contain other materials, such as cell debris and fines. The fluid mixture can be continuously flowed into the device, with desired products being continuously removed. The acoustic perfusion device returns healthy viable cells to the bioreactor while desired products are harvested and flowed downstream for further processing, e.g., additional filtering, chromatography, etc. Additionally, the cell culture media in the bioreactor is clarified as cell fragments are also allowed to pass into the harvest stream and thereby out of the fluid mixture being recycled to the bioreactor.

In some examples, smaller, less productive cells are selected out of the bioreactor return stream thus leaving larger more productive cells in the bioreactor. This retention of larger cells in the bioreactor system increases the specific cell productivity due to the size of the cells and the greater amount of ribosomal content for protein production. This increase in productivity results in lower overall cell culture media usage, corresponding to a predicted cost savings of up to $20,000 per day for large bioreactors.

Disclosed in various embodiments are acoustic perfusion devices, comprising: an acoustic chamber and an outlet flow path from the acoustic chamber. At least one ultrasonic transducer is coupled to the acoustic chamber to one side of the outlet flow path. The at least one ultrasonic transducer includes a piezoelectric material that can be excited to generate an acoustic standing wave across a portion of the acoustic chamber. The acoustic standing wave may be planar or multi-dimensional, or a combination of such waves may be present within the acoustic chamber. Multiple transducers may be used to generate the acoustic standing wave(s). The acoustic standing wave can be thought of as a “force field” that holds back whole cells but permits smaller materials such as the desired biomolecules (e.g. recombinant proteins and/or monoclonal antibodies) and cell fragments, to pass through and be removed from the fluid that is returned to the bioreactor.

In some examples, an inlet port, an inlet flow path leading from the inlet port to the acoustic chamber; an outlet port for recirculating fluid flowing through the device back to its source (e.g. a bioreactor) may be provided. The outlet port may be arranged below the inlet port, and may be located at a bottom end of the device.

As mentioned above, the device may have one or more collection or harvest ports at the top of the device. In some more specific embodiments, the device may have a total of two harvest ports spaced apart from each other on the top end of the device.

In particular embodiments, the inlet port is at a first end of the device at a first height, the at least one ultrasonic transducer is at a second height above the first height, and a bottom wall extends from the inlet port to the outlet port. The outlet port may be located at a second end of the device opposite the first end. The bottom wall may be concave, relative to a line between the inlet port and the outlet port. The device may include an upper wall above the inlet flow path. The inlet port, the outlet port, and the at least one harvest port are sometimes all located on a front wall of the device. The front wall itself may be planar (i.e. flat).

The device can further comprise a reflector located in the acoustic chamber opposite the at least one ultrasonic transducer. Alternatively, the device can have a total of two ultrasonic transducers located on opposite sides of the harvest flow path at the same height and facing each other, or additional ultrasonic transducers can be located on multiple sides of the collection/harvest flow path. A reflector may be located between the two ultrasonic transducers. There may also be a plurality of transducer/reflector pairs located as appropriate to form planar, multi-dimensional, or combinations of such acoustic standing wave(s).

In particular embodiments, the acoustic standing wave results in an acoustic radiation force having an axial force component and a lateral force component that are of the same order of magnitude.

In other embodiments of the device disclosed herein, the inlet flow path leads from the inlet port downwards towards a bottom end of the device and past the outlet port, and then upwards to the acoustic chamber. Sometimes, the inlet port and the at least one harvest port are both located on a top wall of the device, and the outlet port is located on a front wall of the device. The at least one ultrasonic transducer may be mounted in a rear wall or a front wall of the device. The bottom wall of this acoustic chamber can be a sloped planar surface. The reflector may be made of a transparent material.

The inlet flow path may be shaped to generate a tangential flow path below an acoustic field generated by the acoustic standing wave. In still additional versions seen herein, the inlet flow path enters the acoustic chamber on a first side of the device, and the outlet port is located (i) on the first side of the device or (ii) on a second opposite side. The inlet port can be located on a front side of the device, and the at least one harvest port can be located on a top wall of the device. The at least one transducer can be located on a front side or a rear side of the device. In more particular embodiments, there can be two transducers, one on the front side and one of the rear side. In yet other particular embodiments, there is an ultrasonic transducer on the front or rear side, and a reflector located on the respective rear or front side opposite the transducer.

In additional embodiments, the perfusion device further comprises a recirculation flow path between the inlet port and the outlet port that does not enter the acoustic chamber, and the recirculation flow path is located below the acoustic chamber. In some particular embodiments, the inlet flow path travels through a different passage than the outlet flow path. In yet other embodiments, the inlet flow path and the outlet flow path travel through a common passage.

The device may be attached to a mounting piece having holes for attachment.

Also disclosed are methods for separating cells from a fluid mixture containing the cells. The fluid mixture is flowed through an acoustic perfusion device of the structure described above, having at least one ultrasonic transducer. The at least one ultrasonic transducer is driven to create the acoustic standing wave. A fluid enriched in cells can be collected from the outlet port and a clarified fluid, depleted in cells, can be collected from the at least one harvest port.

In particular embodiments, the flow rate through the collection/harvest flow path is at least one order of magnitude smaller than a flow rate through the inlet flow path. In specific embodiments, a flow rate of the fluid mixture entering the device through the inlet port is about 1 liter per minute and a flow rate of the fluid depleted in cells exiting the device through the at least one collection/harvest port is about 10 milliliters per minute. Alternatively, the ratio of the flow rate entering through the inlet port to the flow rate exiting through the at least one collection/harvest port is such that the acoustic standing wave is not overcome by the main body of cells, or in other words so that a large volume of cells do not begin exiting the device through the collection/harvest port(s).

The methods may further comprise pulling the fluid mixture through the device using a first pump attached to the at least one harvest port of the device and a second pump attached to the outlet port of the device.

Also disclosed herein are flow devices adapted to (i) receive a flowing mixture containing a primary fluid and cells; and (ii) to use a first acoustic standing wave to continuously draw off a harvest fluid stream depleted in cells from the flowing mixture, thereby changing the cell concentration of the flowing mixture. A pressure rise may be generated on the upstream interface region of the acoustic standing wave, along with an acoustic radiation force acting on the incoming suspended particles. This “interface effect”, which may also be termed “edge effect”, can act as a barrier. In some examples the interface effect is located at the upstream bounding surface of the volume of fluid that is ensonified by the transducer. For example, the flow mixture crosses the interface region to enter the ensonified volume of fluid. The frequency of the acoustic standing wave may be modified such that different contrast factor materials may be held back by or allowed through the acoustic standing wave, or such that particles of one given size range are retained and particles of a second given range are allowed to flow through the standing wave. The acoustic standing waves that form the “edge effect” may also be modulated so as to let selective materials through at different times in the process.

The device may further comprise a secondary flow chamber in which the harvest fluid stream depleted in cells passes through a second acoustic standing wave having a frequency different from, or equal to the first acoustic standing wave. For example, the second acoustic standing wave may have a higher or lower frequency than the first acoustic standing wave. The ratio of the frequency of the two standing waves is, in some embodiments, at least 2:1 (i.e. one of the frequencies is at least twice the other frequency, e.g. 3 MHz and 6 MHz).

Also disclosed herein are flow devices that comprise: at least one inlet for receiving a flowing mixture of a primary fluid and cells, an ultrasonic transducer that produces a first ultrasonic acoustic standing wave and uses a pressure rise and an acoustic radiation force generated on an upstream interface region of the first ultrasonic acoustic standing wave to separate the flowing mixture into a primary high cell concentration fluid stream and a secondary harvest fluid stream; an outlet port for the primary high cell concentration fluid stream; and at least one collection port for the secondary harvest fluid stream. A bleed port can also be present for extracting a concentrated fluid/cell mixture. The fluid mixture may comprise particles such as mammalian cells, bacteria, cell debris, fines, proteins, exosomes, vesicles, viruses, and insect cells.

The device may further comprise a secondary flow chamber in which the secondary harvest fluid stream passes through a second acoustic standing wave having a frequency different from, or equal to, the first ultrasonic acoustic standing wave.

Disclosed in various embodiments herein are processes for separating biomolecules (e.g., therapeutic antibodies) from a fluid mixture. The process comprises flowing a fluid mixture containing biomolecules and cells through an acoustic perfusion device. The acoustic perfusion device includes an acoustic chamber through which the fluid mixture containing the biomolecules and cells flows, and an ultrasonic transducer and a reflector opposite the ultrasonic transducer, the ultrasonic transducer including a piezoelectric material driven to create a multi-dimensional acoustic standing wave in the acoustic chamber. The ultrasonic transducer is driven to create the multi-dimensional acoustic standing wave. In particular, the reflector is set up opposite the ultrasonic transducer, and the ultrasonic transducer is electronically driven to form a multi-dimensional acoustic standing wave in the flow chamber. Alternatively, two opposing ultrasonic transducers may be used to generate the multi-dimensional acoustic standing wave. An ultrasonic transducer may be used to generate an acoustic wave, as well as to reflect an acoustic wave, which can contribute to generating the multi-dimensional acoustic standing wave. Aggregates of the biomolecules, form higher molecular weight species of the monoclonal antibodies or recombinant proteins. The acoustic standing wave may be modified to hold back these aggregated proteins and not separate them from the monomer or pure monoclonal antibody that is the product of the bioreactor process. Aggregated proteins are typically less effective or ineffective at the therapeutic aspects of the monoclonal antibody that is designed to be produced and expressed by the Cho cells.

The separated biomolecules can be subjected to further processing downstream of the acoustic perfusion device. The further processing can include at least one of chromatography and additional filtration (e.g., depth filtration, crossflow filtration, tangential filtration, sterile filtration).

In particular embodiments, the biomolecules are proteins, such as therapeutic antibodies. The mechanism of action of the therapeutic antibodies can include antibody-dependent, cell-mediated cytotoxicity (ADCC). The fucosylation of the therapeutic antibodies can be decreased and the efficacy of the therapeutic antibodies can be increased. The therapeutic antibodies can be non-fucosylated.

The biomolecules can, in certain embodiments, be produced by culturing cells in a bioreactor (e.g., a perfusion bioreactor) prior to flowing the fluid mixture through the acoustic perfusion device. In particular embodiments, a pressure rise and an acoustic radiation force on cells are generated at an interface region of the multi-dimensional acoustic standing wave to clarify the fluid mixture as it passes through the multi-dimensional acoustic standing wave. The acoustic perfusion device can further comprise a recirculating fluid stream that transports away cells that are constantly held back at the interface region of the multi-dimensional acoustic standing wave. The multi-dimensional acoustic standing wave may result in an acoustic radiation force having an axial force component and a lateral force component that are of the same order of magnitude.

The process of the present disclosure are useful for producing desired biomolecules (e.g., monoclonal antibodies, recombinant proteins). In particular embodiments, the biomolecules can be glycoengineered to produce antibodies with predetermined glycoforms.

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

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG. 1 illustrates a single standing acoustic wave generated by an ultrasonic transducer and a reflector.

FIG. 2 is an illustration comparing a fed-batch bioreactor system with a perfusion bioreactor system.

FIG. 3 is a cross-sectional view that shows the various components of a stirred-tank bioreactor.

FIG. 4 is a schematic view illustrating a perfusion bioreactor coupled with an acoustic perfusion device of the present disclosure, and a recycle path.

FIG. 5 is a front cross-sectional view of an example implementation of an acoustic perfusion device of the present disclosure.

FIG. 6 is an exterior perspective view of the acoustic perfusion device of FIG. 5.

FIG. 7 is a front cross-sectional view of another example implementation of an acoustic perfusion device of the present disclosure.

FIG. 8 is a perspective view of the acoustic perfusion device of FIG. 7.

FIG. 9 is a cross-sectional diagram of a conventional ultrasonic transducer.

FIG. 10 is a cross-sectional diagram of an ultrasonic transducer of the present disclosure. An air gap is present within the transducer, and no backing layer or wear plate are present.

FIG. 11 is a cross-sectional diagram of an ultrasonic transducer of the present disclosure. An air gap is present within the transducer, and a backing layer and wear plate are present.

FIG. 12 is a graph of electrical impedance amplitude versus frequency for a square transducer driven at different frequencies.

FIG. 13 illustrates the trapping line configurations for seven of the resonance frequencies (minima of electrical impedance amplitudes) of FIG. 12 from the direction orthogonal to fluid flow.

FIG. 14 is a computer simulation of the acoustic pressure amplitude (right-hand scale in Pa) and transducer out of plane displacement (left-hand scale in meters). The text at the top of the left-hand scale reads “×10⁻⁷”. The text at the top of the left-hand scale by the upward-pointing triangle reads “1.473×10⁻⁶”. The text at the bottom of the left-hand scale by the downward-pointing triangle reads “1.4612×10⁻¹⁰”. The text at the top of the right-hand scale reads “×10⁶”. The text at the top of the right-hand scale by the upward-pointing triangle reads “1.1129×10⁶”. The text at the bottom of the right-hand scale by the downward-pointing triangle reads “7.357”. The triangles show the maximum and minimum values depicted in this figure for the given scale. The horizontal axis is the location within the chamber along the X-axis, in inches, and the vertical axis is the location within the chamber along the Y-axis, in inches.

FIG. 15 shows the In-Plane and Out-of-Plane displacement of a crystal where composite waves are present.

FIG. 16 is a view of an acoustic perfusion device, showing a reflector in the acoustic chamber between first and second transducers. The device is fluidly connected to an associated bioreactor. A fluid mixture is also present in the device and arrows are shown indicating the direction of flow in addition to waves indicating the acoustic field between the reflector and first and second transducers.

FIG. 17 is a front view of the device of FIG. 7, showing the flow paths, acoustic field, and acoustic interface effect.

FIG. 18 shows the geometry of a model simulation of the acoustic device used for cell retention. The model contains two fluids, one a clarified fluid within the acoustic field, the other a high cell density fluid to the left of the acoustic field, a piezoelectric transducer, a steel reflector, and an aluminum housing. The first fluid was water within the acoustic field and the second fluid was a 15% concentration of CHO cells in water solution outside (to the left) of the acoustic field. The blue solid line in the model indicates the separation line between the two fluids.

FIGS. 19A, 19B, and 19C are graphs showing the displacement of the piezoelectric material, the aluminum housing, and the steel reflector (left-side scale); and the acoustic pressure in the two fluids (right-side scale) of the model simulation of FIG. 18 at several frequencies of operation. FIG. 19A is at a frequency of 2.218 MHz. FIG. 19B is at a frequency of 2.2465 MHz. FIG. 19C is at a frequency of 2.3055 MHz. For all three graphs, the left-side scale is indicated with text at the top of the scale reading “×10⁻⁶” or “×10⁻⁷”, and is in units of inches. The right-side scale is indicated with text at the top of the scale reading “×10⁶”, and is in units of Pascals. The y-axis runs from −0.8 to 1.6 in intervals of 0.2. The x-axis runs from −0.5 to 1.5 in intervals of 0.5.

FIG. 20 is a graph showing the average lateral force (N) and the average lateral force normalized by power (N/W) acting on suspended CHO cells at several frequencies of operation.

FIG. 21 is a picture (top view) of an acoustic perfusion device of the present disclosure. Arrows indicate the flow into the inlet port; the flow out of the outlet port; the clarified fluid flow out the top of the device and the flow of concentrate out the bottom of the device.

FIG. 22 is a picture (side view) of the acoustic perfusion device of FIG. 21.

FIG. 23 is a graph of cell retention vs. perfusate flowrate for the device of FIG. 21.

FIG. 24 is a prior art illustration showing depth flow filtration (DFF) and tangential flow filtration (TFF).

FIG. 25 is a perspective view of an example implementation of an acoustic perfusion device of the present disclosure. This embodiment includes a direct recirculation flow path between the inlet port and the outlet port. An inflow passageway and an outflow passageway join the recirculation flow path to the acoustic chamber, and create a tangential sweeping flow underneath the acoustic field.

FIG. 26 is a front view picture of the device of FIG. 25. The inflow passageway and the outflow passageway are clearly visible, along with the recirculation pipe.

FIG. 27 is a diagrammatic side view of the device of FIG. 25.

FIG. 28 is a graph of viable cell density versus time for both a tangential flow filtration (TFF) process and an acoustic wave separation (AWS) process according to the present disclosure. The left y-axis represents VCD in units of cells/mL and runs from 0 to 120 in intervals of 10. The right y-axis represents viability (% via) and runs from 0% to 100% in intervals of 10%. The x-axis represents time (in culture days) and runs from 0 to 30 days in intervals of 10.

FIG. 29 is a graph of cell specific perfusion rate (CSPR) versus time for both a TFF process and an AWS process according to the present disclosure. The y-axis represents CSPR in units of pico liters per cell per day (pl/c/d) and runs from 0 to 30 in intervals of 5. The x-axis represents time (in culture days) and runs from 0 to 40 days in intervals of 10.

FIG. 30 is a graph of specific growth rate (SGR) in the bioreactor versus time for both a TFF process and an AWS process according to the present disclosure. The y-axis represents SGR in units of 1/day, and runs from −10% to 70% in intervals of 10%. The x-axis represents time (in culture days) and runs from 0 to 40 days in intervals of 10.

FIG. 31 is a graph of specific death rate (Kd) in the bioreactor versus time for both a TFF process and an AWS process according to the present disclosure. The y-axis represents Kd in units of 1/day, and runs from −2% to 12% in intervals of 2%. The x-axis represents time (in culture days) and runs from 0 to 40 days in intervals of 10.

FIG. 32 is a graph illustrating titer in the bioreactor (BRX) versus in the permeate or harvest stream for TFF and AWS. The y-axis represents the reversed-phase high-performance liquid chromatography (RP-HPLC) titers (expressed in mg/L) and runs from 0 to 2500 in intervals of 500. The x-axis represents time (in culture days) and runs from 0 to 40 days in intervals of 10.

FIG. 33 is a graph of volumetric productivity (VP) versus time for both a TFF process and an AWS process according to the present disclosure. The y-axis represents VP in units of grams/liter/day, and runs from 0 to 1.4 in intervals of 0.2. The x-axis represents time (in culture days) and runs from 0 to 40 days in intervals of 10.

FIG. 34 is a graph of specific productivity (Qp) versus time for both a TFF process and an AWS process according to the present disclosure. The y-axis represents Qp in units of pcd (protein/cell/day), and runs from 10 to 30 in intervals of 2. The x-axis represents time (in culture days) and runs from 0 to 40 days in intervals of 10.

FIGS. 35A-35D illustrate four graphs of metabolism using nutrient uptake markers for both a TFF process and an AWS process according to the present disclosure versus time. The x-axis of all four graphs represents time (in culture days) and runs from 0 to 40 days in intervals of 10. The y-axis of FIG. 35A represents dextrose uptake (expressed in pg/cell/day) and runs from 0 to 250 in intervals of 50. The y-axis of FIG. 35B represents lactate production rate (expressed in pg/cell/day) and runs from −10 to 80 in intervals of 10. The y-axis of FIG. 35C represents glutamine production rate (expressed in pg/cell/day) and runs from −1 to 5 in intervals of 1. The y-axis of FIG. 35D represents glutamate uptake (expressed in pg/cell/day) and runs from 0 to 16 in intervals of 2.

FIGS. 36A-36C illustrate three graphs of ion exchange chromatography (IEX) results of charge variants for both a TFF process and an AWS process according to the present disclosure versus time. The x-axis of all three graphs represents time (in culture days) and runs from 0 to 35 days in intervals of 5. The y-axis of FIG. 36A represents IEX acidic variants (percentage) and runs from 0 to 14 in intervals of 2. The y-axis of FIG. 36B represents IEX basic variants (percentage) and runs from 0 to 35 in intervals of 5. The y-axis of FIG. 36C represents IEX main peak (percentage) and runs from 62 to 76 in intervals of 2.

FIGS. 37A-37B illustrate two graphs of size exclusion chromatography (SEC) results for both a TFF process and an AWS process according to the present disclosure versus time. The x-axis of both graphs represents time (in culture days) and runs from 0 to 35 days in intervals of 5. The y-axis of FIG. 37A represents SEC HMW (high molecular weight) species (percentage) and runs from 0 to 3 in intervals of 0.5. The y-axis of FIG. 37B represents SEC monomer (percentage) and runs from 97 to 100 in intervals of 0.5.

FIGS. 38A-38B illustrate two graphs of glycosylation (N-glycan) results for both a TFF process and an AWS process according to the present disclosure versus time. The x-axis of both graphs represents time (in culture days) and runs from 0 to 35 days in intervals of 5. The y-axis of FIG. 38A represents N-glycan maturity (percentage) and runs from 90 to 100 in intervals of 1. The y-axis of FIG. 38B represents primary form (percentage of G0F glycoforms) and runs from 50 to 90 in intervals of 5.

FIGS. 39A-39C illustrate three additional graphs of glycosylation (N-glycan) results for both a TFF process and an AWS process according to the present disclosure versus time. The x-axis of all three graphs represents time (in culture days) and runs from 0 to 35 days in intervals of 5. The y-axis of FIG. 39A represents galactosylation (percentage) and runs from 0 to 18 in intervals of 2. The y-axis of FIG. 39B represents afucosylation (percentage) and runs from 0 to 7 in intervals of 1. The y-axis of FIG. 39C represents high mannose (percentage) and runs from 0 to 4.5 in intervals of 0.5.

FIG. 40 is a graph of cell viability over time. The y-axis is cell viability in % of original cell count, and runs from 50% to 100% in intervals of 10%. The x-axis is culture days, and runs from 0 to 14 days in intervals of 1. Dark circles are for a TFF process with cell bleed. White circles are for an AWS process using a 2 L flow chamber. Dark inverted triangles are for an AWS process using a 10 L flow chamber. White triangles are for a TFF process without cell bleed.

DETAILED DESCRIPTION

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

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

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

The term “comprising” is used herein as requiring the presence of the named component and allowing the presence of other components. The term “comprising” should be construed to include the term “consisting of”, which allows the presence of only the named component, along with any impurities that might result from the manufacture of the named component.

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

All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 2 grams to 10 grams” is inclusive of the endpoints, 2 grams and 10 grams, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and/or values.

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

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

The terms “horizontal” and “vertical” are used to indicate direction relative to an absolute reference, i.e. ground level. However, these terms should not be construed to require structures to be absolutely parallel or absolutely perpendicular to each other. For example, a first vertical structure and a second vertical structure are not necessarily parallel to each other. The terms “top” and “bottom” or “base” are used to refer to surfaces where the top is always higher than the bottom/base relative to an absolute reference, i.e. the surface of the earth. The terms “upwards” and “downwards” are also relative to an absolute reference; upwards is always against the gravity of the earth.

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

The acoustic devices discussed herein may operate in a multimode or planar mode. Multimode refers to generation of acoustic waves by an acoustic transducer that create acoustic forces in three dimensions. The multimode acoustic waves, which may be ultrasonic, are generated by one or more acoustic transducers, and are sometimes referred to herein as multi-dimensional or three-dimensional acoustic standing waves. Planar mode refers to generation of acoustic waves by an acoustic transducer that create acoustic forces substantially in one dimension, e.g. along the direction of propagation. Such acoustic waves, which may be ultrasonic, that are generated in planar mode are sometimes referred to herein as one-dimensional acoustic standing waves.

The acoustic transducers may be composed of piezoelectric material. Such acoustic transducers can be electrically excited to generate planar or multimode acoustic waves. The three-dimensional acoustic forces generated by multimode acoustic waves include radial or lateral forces that are unaligned with a direction of acoustic wave propagation. The lateral forces may act in two dimensions. The lateral forces are in addition to the axial forces in multimode acoustic waves, which are substantially aligned with the direction of acoustic wave propagation. The lateral forces can be of the same order of magnitude as the axial forces for such multimode acoustic waves. The acoustic transducer excited in multimode operation may exhibit a standing wave on its surface, thereby generating a multimode acoustic wave. The standing wave on the surface of the transducer may be related to the mode of operation of the multimode acoustic wave. When an acoustic transducer is electrically excited to generate planar acoustic waves, the surface of the transducer may exhibit a piston-like action, thereby generating a one-dimensional acoustic standing wave. Compared to planar acoustic waves, multimode acoustic waves exhibit significantly greater particle trapping activity on a continuous basis with the same input power. One or more acoustic transducers may be used to generate combinations of planar and multi-dimensional acoustic standing waves. In some modes of operations, multimode acoustic waves generate an interface effect that can hold back or retain particles of a certain size, while smaller particles can flow through the multimode acoustic waves. In some modes of operation, planar waves can be used to deflect particles at certain angles that are characteristic of the particle size.

Bioreactors are useful for making biomolecules such as recombinant proteins or monoclonal antibodies. Very generally, cells are cultured in a bioreactor vessel with media in order to produce the desired product, and the desired product is then harvested by separation from the cells and media in an acoustic perfusion device, such as the device of the present disclosure. The acoustic filtering device permits the withdrawal of some desired product, a small portion of the media, and cellular fragments/debris smaller than the cells, with the remainder being recycled back to the bioreactor (particularly the cells). The use of mammalian cell cultures including Chinese hamster ovary (CHO), NS0 hybridoma cells, baby hamster kidney (BHK) cells, insect cells, and human cells (e.g. T-cells, B-cells, stem cells, red blood cells), and living/biological cells in general has proven to be a very efficacious way of producing/expressing the recombinant proteins and monoclonal antibodies used in various applications such as pharmaceuticals or vaccines. Two general types of bioreactor processes exist: fed-batch and perfusion.

A perfusion bioreactor may be utilized to generate cells that would be utilized in a cell therapy process. In this type of perfusion bioreactor, biological cells such as CAR T-cells, Jurkat T-cells and the like are cultured in a perfusion bioreactor. The acoustic standing wave used in the perfusion devices of the present disclosure can be used to separate viable and nonviable cells after the transfection process. This allows for improved efficacy of the inoculation of the patient with this T-cell therapy as only viable cells are utilized. The nonviable cells and cell fragments are separated out through the perfusion process, with these materials going into the secondary flow and exiting the bioreactor.

A perfusion bioreactor may also be used for production of exosomes, microvesicles, or vesicles by cells. The acoustic perfusion device can then be used to harvest the exosomes, or other desired cell products. In a similar fashion, a perfusion bioreactor can be used to produce viruses, such as lentivirus, which are used in cell and gene therapy to transfect cells. The acoustic perfusion device can then be used to harvest the virus. In all cases, the device is a cell retention device.

Recent developments in perfusion bioreactor technology also favor its use. Control technology and general support equipment is improving for perfusion bioreactors, increasing the robustness of perfusion processes. The perfusion process can now be scaled up to bioreactors having a volume up to 1000 liters (L). Better cell retention systems for perfusion bioreactors result in lower cell loss and greater cell densities than have been seen previously. Cell densities greater than 50 million cells/mL are now achievable, compared to fed-batch cell densities of around 20 million cells/mL. Lower contamination and infection rates have improved the output of perfusion bioreactors. Higher product concentrations in the harvest and better yields without significant increase in cost have thus resulted for perfusion processes.

Perfusion bioreactors are particularly attractive because of the continuous production of the biomolecules from the expressing cell culture, and shorter residence time of said biomolecules in the process prior to harvest. The target cells are held back by a filtration process, such as tangential flow filtration (TFF) or alternating tangential flow filtration (ATF) while the expressed biomolecules are extracted from the perfusion bioreactor. The cells are then returned to the bioreactor to ensure they receive the nutrition and oxygen to maintain the production of the overall cell culture. In the perfusion reactor process, the cells continue to multiply. Some of the cell culture population may be bled off via a portion or all of the perfusion production process.

The TFF and ATF processes of filtration have several issues, such as clogging/fouling and loss of biomolecule product (particularly at high cell densities), all directly related to the nature of the hollow fiber membranes used in the filtration. It is therefore desirable to find a new filtration process that does not clog and minimizes loss of the desired biomolecule product. In addition, TFF and ATF will retain all cellular debris and fines within the bioreactor, which is not desirable. A process capable of distinguishing between cell retention while allowing for the passing of cell debris and fines may therefore be favorable. Further yet, undesirable protein aggregation during monoclonal antibody production is known to occur in upstream and downstream processing, which is a major concern for therapeutic applications where aggregates influence drug performance and safety. Thus, processes that remove protein aggregates are desirable. Such processes are especially desirable for multiple reasons including that protein aggregates are typically non-functional and are a problem for the efficacy of therapeutic drugs, which makes their removal even more desirable.

Briefly, the present disclosure relates to acoustic perfusion devices capable of generating multi-dimensional acoustic standing wave(s) from one or more piezoelectric transducers, where the transducers are electrically excited such that they move in a multimode displacement pattern rather than a “piston” mode of vibration. Through this manner of acoustic standing wave generation, a higher lateral trapping force is generated than if the piezoelectric transducer is excited in a “piston” mode where only one large standing wave is generated. Thus, with the same input power to a piezoelectric transducer, the multi-dimensional acoustic standing waves can have a higher lateral trapping force compared to a planar acoustic standing wave. The input power is tunable for a controlled flow. This can be used to facilitate proteinaceous fluid purification of a fluid stream coming from a bioreactor. Alternatively, the acoustic standing wave may also be a planar standing wave where the piezoelectric transducer is excited in the piston mode, generating a planar wave. The acoustic standing wave(s) may also be a combination of planar and multi-dimensional acoustic standing waves. All of these standing waves generate an “interface effect” such that the cells from the bioreactor are held back and the biomolecule product expressed from the cells, cell fragments and small debris are allowed to pass through.

Acoustophoresis is a low-power, no-pressure-drop, no-clog, solid-state approach to particle separation from fluid dispersions (i.e., it is used to achieve separations that are more typically performed with porous filters, but it has none of the disadvantages of filters). In particular, the acoustic perfusion devices of the present disclosure are suitable for use with macro-scale bioreactors for separations in flowing systems with high flow rates. The acoustic perfusion device is designed to create a high intensity multi-dimensional ultrasonic standing wave that results in an acoustic radiation force that can overcome the combined effects of fluid drag and buoyancy or gravity at certain flow rates. As a result, the radiation force acts as a filter that prevents targeted particles (e.g., biological cells) from crossing through the standing wave. As explained above, the trapping capability of a standing wave may be varied as desired, for example by varying the flow rate of the fluid, the acoustic radiation force, and the shape of the acoustic filtering device to maximize cell retention through trapping and settling. This technology offers a green and sustainable alternative for separation of secondary phases with a significant reduction in cost of energy. Excellent particle separation efficiencies have been demonstrated for particle sizes as small as one micron.

Generally, the scattering of the acoustic field off the particles results in a three-dimensional acoustic radiation force, which acts as a three-dimensional trapping field. The acoustic radiation force is proportional to the particle volume (e.g., the cube of the radius) when the particle is small relative to the wavelength. It is proportional to frequency and the acoustic contrast factor. It also scales with acoustic energy (e.g., the square of the acoustic pressure amplitude). For harmonic excitation, the sinusoidal spatial variation of the force is what drives the particles to the stable positions within the standing waves. When the acoustic radiation force exerted on the particles is stronger than the combined effect of fluid drag force and buoyancy/gravitational force, the particle is trapped within the acoustic standing wave field. The action of the lateral and axial acoustic forces on the trapped particles results in formation of tightly packed clusters through concentration, clustering, clumping, agglomeration and/or coalescence of particles that, when reaching a critical size, settle continuously through enhanced gravity for particles heavier than the host fluid or rise out through enhanced buoyancy for particles lighter than the host fluid. Additionally, secondary inter-particle forces, such as Bjerkness forces, aid in particle agglomeration.

Most biological cell types present a higher density and lower compressibility than the medium in which they are suspended, so that the acoustic contrast factor between the cells and the medium has a positive value. As a result, the axial acoustic radiation force (ARF) drives the cells towards the standing wave pressure nodes. The axial component of the acoustic radiation force drives the cells, with a positive contrast factor, to the pressure nodes, whereas cells or other particles with a negative contrast factor are driven to the pressure anti-nodes. The radial or lateral component of the acoustic radiation force is the force that traps the cells. The radial or lateral component of the ARF is larger than the combined effect of fluid drag force and gravitational force. Desirably, the ultrasonic transducer(s) generates a multi-dimensional standing wave in the fluid that exerts a lateral force on the suspended particles to accompany the axial force. Typical results published in literature state that the lateral force is two orders of magnitude smaller than the axial force. In contrast, the technology disclosed in this application provides for a lateral force to be of the same order of magnitude as the axial force. However, in certain embodiments described further herein, the device use both transducers that produce multi-dimensional acoustic standing waves and transducers that produce planar acoustic standing waves. For purposes of this disclosure, a standing wave where the lateral force is not the same order of magnitude as the axial force is considered a “planar acoustic standing wave.” The lateral force component of the total acoustic radiation force (ARF) generated by the ultrasonic transducer(s) of the present disclosure is significant and is sufficient to overcome the fluid drag force at linear velocities of up to 1 cm/s, and to create tightly packed clusters, and is of the same order of magnitude as the axial force component of the total acoustic radiation force.

It may be helpful to contrast the technology of the present disclosure with that of prior filtration technology. FIG. 24 shows two prior art filtration methods. The left-hand side of FIG. 24 illustrates depth flow filtration (DFF). In DFF, the entire feed stream 4005 of fluid and particles is directed towards the filter. The filter 4010 holds back the particles 4020 that are larger than the filter's pore size, whereas smaller particles 4030 and the fluid pass through the filter. The right-hand side of FIG. 24 illustrates tangential flow filtration (TFF). In TFF, the feed stream is not directed towards the filter. Rather, the feed stream is directed tangentially to the filter, such that a majority of the feed stream passes tangentially over the filter surface. Typically, this feed stream is recirculated to pass by the filter more than once. A much smaller filtrate stream 4006 is pulled through the filter membrane containing the smaller particles 4030. One advantage of TFF over DFF is that the tangential stream reduces the clogging and fouling of the filter and the formation of a gel layer that sits on top of the filter.

In the devices of the present disclosure, during startup, the fluid ensonified by the acoustic standing wave is clarified by the process of trapping cells and growing them into tightly packed clusters, such that continuous gravitational separation of the clusters of cells takes place. Since there is a limited amount of new cells flowing into this volume, this results in a rapid clarification of the fluid subjected to the acoustic standing wave. When this state is reached, the system can be described as including two fluids: a first fluid containing the desired product and small cell fragments/debris (which have passed through the acoustic standing wave), and a second fluid containing the bioreactor fluid and all of the cells (which are held back by the acoustic standing wave). The two fluids may be of different effective acoustic properties, such as density and speed of sound, with a well-defined interface between these two fluids. The acoustic standing wave is a three-dimensional acoustic field, which, in the case of excitation by a rectangular transducer, can be described as occupying a roughly rectangular prism volume of fluid. Typically, two opposing faces are the transducer and reflector, an adjacent pair of opposing faces are the walls of the device, and the final opposing pair of faces, the upstream and downstream faces of the cube, extend through the fluid. The interface between the two fluids is generally located near the upstream face of the acoustic standing wave field, generating an “acoustic barrier or edge effect”. This location is also referred to as an upstream interface region. The first fluid (i.e., the fluid that has been clarified and contains the product, some cells, and cell fragments) is downstream of the interface and represents the harvest flow and occupies the volume of fluid ensonified by the acoustic standing wave field. The second fluid (i.e., the fluid containing the bioreactor fluid and most of the cells) is upstream of the interface. During operation at increased flow rates, the interface effect location may move downstream and is then located within the volume of fluid ensonified by the transducer.

The acoustic standing wave field exerts an acoustic radiation pressure (i.e. a pressure rise) and an acoustic radiation force on the cells at the interface region between the two fluids, thereby keeping the upstream cells from entering the acoustic field. The occurrence of the radiation pressure and the force on the interface allows for the first fluid containing the product to pass through the interface while retaining the cells in the upstream fluid. The cells that are held back by the effect of the acoustic radiation force at the interface between the two fluids can be continuously returned to the bioreactor to ensure they receive the nutrition and oxygen to maintain the production of the overall cell culture.

The circulating motion of the flow field underneath the interface transports the cells that are retained by the acoustic field back to the bioreactor. The circulating flow motion is driven by the primary recirculation stream and can be optimized with acoustic chamber geometry variations for maximum system efficiency.

During perfusion, the acoustic perfusion devices of the present disclosure have multiple possible modes of operation. One of these modes may be dominant in the device or they may occur concurrently depending on the distribution of cells and fluid within the device. In a first mode of operation (Mode 1), the fluid containing cells enters the acoustic standing wave field, which is produced between the transducer and the reflector. A multi-dimensional acoustic standing wave traps the cells at specific points, packs the cells into tightly packed clusters, and continuously separates the clusters through enhanced gravitational settling. The cell clusters settle out, enter the tangential flow path and are redirected to the bioreactor by the recirculation stream. Smaller particles 4030 are not trapped by, and pass through, the acoustic standing wave, to be harvested. The orientation of this device is significant due to the use of gravitational settling.

In the second mode of operation (Mode 2), the acoustophoretic system creates a strong barrier for cells at the interface between the two fluids and prevents cells from entering the acoustic field. Here, a barrier of cells is established between the two fluids through the interface effect of the acoustic standing wave. A first clarified fluid stream contains the smaller particles/desired byproducts within the acoustic standing wave field and the harvest stream. A second fluid stream contains the retained cells upstream of the acoustic standing wave field. In this mode of operation, an acoustic interface effect is realized in the interface region between the two fluids, clarified fluid downstream and flow mixture and cells on the upstream side. Very generally, the acoustic interface effect holds the cells back and prevents them from entering the acoustic field while a portion of the fluid stream containing the produced biomolecules and cell fragments is permitted to pass through this barrier. The tangential flow path underneath the acoustic interface (arrow) collects the retained cells and flows them back into the main recirculation stream and back to the bioreactor.

In perfusion applications, the setup of the acoustophoretic device is similar to that of TFF. A feed stream containing the cells, cell debris, fines, and product, i.e., protein, flows from the bioreactor into the perfusion system. A portion of the stream flows in a tangential fashion along the upstream/lower interface region of the acoustic standing wave and is recirculated back to the bioreactor. A smaller portion of the feed stream is harvested, i.e., diverted and flows through the acoustic standing wave. Here the acoustic standing wave functions very similarly to the filter in TFF, preventing the cells from entering the acoustic field. The harvest stream contains smaller particles such as cell debris and fines as well as the desired biomolecule product. The cells that are retained by the acoustic standing wave are transported by the recirculation stream back to the bioreactor. FIG. 17, discussed further herein, also illustrates a perfusion device that uses a tangential flow stream.

Perfusion applications typically entail high cell densities, e.g., >50 million cells/m L, and lower harvest velocities contrary to cell clarification or oil/water applications. The two fluid streams also have different effective acoustic properties, i.e., speed of sound and density of the media/cell mixture. As cell density increases, the difference in acoustic properties of the two fluid streams will be more pronounced as well. The acoustic standing wave field will now exert an acoustic radiation pressure, i.e., a pressure rise, on the second fluid stream, enriched with cells, as well as acoustic radiation forces on the cells suspended in the fluid. This radiation pressure and radiation force act at the interface between the two fluids which coincides with the upstream bounding surface of the acoustic field. When this “acoustic interface” effect of acoustic radiation force is sufficiently strong, it will prevent the cells from entering the acoustic field. Equally important is a tangential flow path to collect the retained cells and transport them back to the bioreactor.

The acoustic interface effect may also be referred to as an acoustic wall effect and results from the interface of the acoustic field exerting a strong lateral force (i.e., in the opposite direction to the harvest flow and perpendicular to the axis of the acoustic standing wave) on the suspended particles, thereby keeping the relatively larger sized particles from entering the acoustic field and allowing only clarified fluid (i.e., the fluid containing the smaller-sized product) to enter the acoustic field, thereby creating an acoustic perfusion cell retention device. In this way, only the clarified fluid can escape and the cells are held down by the radiation force. This force is never positive, meaning that it always holds the cells down at the interface, i.e., the force is acting in the upstream flow direction, not allowing the cells to pass through the acoustic interface. The multiple peaks in the power curve (see discussion of FIG. 20 below) show the existence of multiple modes of operation including planar resonance modes and multi-dimensional modes of operation, indicating that this type of operation can be generated through utilization of planar and multi-dimensional standing waves alike. In systems having 1″×1″ dimensions, there exists a planar resonance about every 30 kHz. FIG. 20 shows evidence of additional peaks indicating the existence of the multi-dimensional modes. Per unit power, these modes can be equally or even more effective as the planar resonance modes. As explained above, the cells that are held back by the acoustic radiation force may then picked up by the scrubbing motion of the fluid flow field (i.e., the recirculating flow underneath the interface), and be continuously returned to the bioreactor to ensure they receive the nutrition and oxygen to maintain the production of the overall cell culture.

The clarified fluid contains both the desired products and cell fragments, all of which are smaller than whole viable cells. In this way, the media that is returned to the bioreactor is clarified of cell fragments. Cell fragments absorb media without producing desired product, making the perfusion process less efficient. Thus, there is an efficiency gain and a cost savings obtained by removing these cell fragments using the acoustic perfusion devices of the present disclosure. Further clarification of the clarified fluid may be achieved downstream using a second device or a secondary flow chamber that contains another transducer-reflector pair that operates at a different frequency. This traps, clumps, clusters, or agglomerates particles having a size of about 10 microns or less that may have passed through the original acoustic standing wave, in the same manner as described before. A third transducer-reflector pair operating at another frequency, 3 MHz to 20 MHz, or higher, may be utilized to trap, clump, cluster, or agglomerate and drop out the small cell fragments and debris that passed through the initial acoustic standing wave and the “interface effect”. This triple-clarified fluid containing the desired biomolecules can then directly enter a sterile filter. For example, the original acoustic perfusion device may operate at frequencies up to about 4 MHz. The frequency of this second and third acoustic standing wave field may be from about 6 MHz to about 20 MHz, and possibly higher, to trap smaller sized cell fragments.

During startup of a bioreactor at low cell density, e.g., 2 million cells/mL, the first described mode of operation dominates (Mode 1). As cell density in the bioreactor increases over time, the mode of operation gradually switches from mode 1 to mode 2, and both modes may coexist at the same time.

When an acoustic standing wave is employed for perfusion in a bioreactor with an already high cell density, e.g., 50 million cells/mL, the device typically starts in the first mode of operation, until the volume of fluid within the acoustic standing wave is clarified, at which point the operation gradually switches to the second described mode of operation. At times, during operation, an instability, usually manifested as a perturbation or oscillation of the interface between the two fluids, may grow sufficiently strong such that cells enter the volume of fluid within the acoustic standing wave, at which point, for a short period of time, the device acts in a combined mode of operation, where both modes are active (i.e., the interface effect prevents cells from entering the acoustic field as explained above, while the acoustic field clarifies the cells that have entered the volume of fluid within the acoustic standing wave field). Once the tightly packed cell clusters have settled out (i.e., once the volume of fluid within the acoustic standing wave has been sufficiently clarified), the mode of operation is then again that of the second described mode of operation, namely, the acoustic interface effect. It is important to note that the device can operate in both/either of the modes of operation, as described above, without external switching. In other words, the properties of the fluid streams, e.g., cell concentrations in the streams, and acoustic field dictate which mode dominates.

The acoustic standing wave(s) perfusion devices of the present disclosure are operated differently compared to prior acoustic filter usages, previously described in literature. Previously, acoustophoresis was operated such that the protein-producing materials, such as Chinese hamster ovary cells (CHO cells), the most common host for the industrial production of recombinant protein therapeutics, were trapped within a planar ultrasonic standing wave (i.e., remain in a stationary position). Cells were retained in an acoustic field by causing individual cells to migrate towards the pressure nodal planes of the planar acoustic standing wave. There, as the cells were retained in the standing wave, there was also a physical scrubbing of the cell culture media flowing past, whereby more cells were trapped as they came in contact with the cells that were already held within the standing wave. The standing wave and harvest fluid flow were then intermittently shut off to allow the cells to drop out of the standing wave and return to the bioreactor.

In contrast, in the present disclosure, the ultrasonic standing waves are used as a blanket or selector or “force field”. Rather than just trapping and retaining the biological cells within the standing wave, fluid flows through the perfusion device in a manner such that gravity first operates on the biological cells, causing them to sink. The standing wave is created near the top of the filtering device and acts like a filter to prevent the cells from entering the acoustic field and exiting through the top of the filtering device (i.e., acting similar to a force field holding the cells back from entering the acoustic field). Thus, two output streams are created, one output stream retaining the cells and exiting through a port at the bottom of the device, and the other output stream being depleted in cells and exiting through a port at the top of the device (the cell concentration in the two output streams being compared to each other). In this mode of operation, there is almost no reliance on clustering, clumping, or agglomeration of the cells within the acoustic field. This mode may be particularly advantageous in certain applications as it does not necessarily rely on retention time of the cells in the acoustic filtering device.

Described another way, the acoustic perfusion device has two fluid streams flowing at different rates. The main fluid stream, carrying the expressing cell culture, culture media, product, and other bioreactor constituents, enters the device and is partially diverted into a secondary, lower volume, lower flow fluid stream. This secondary fluid stream passes through the multi-dimensional acoustic standing wave, where the multi-dimensional acoustic standing wave (or generally the interface effect created by the acoustic standing wave) holds back the main cell culture and allows the expressed biomolecules, the monoclonal antibodies and recombinant proteins, along with other small particles such as submicron and micron-sized cell debris, to pass through and be further collected and processed outside/downstream of the bioreactor. The main fluid stream, containing the main cell culture, is then recycled back to the bioreactor. The acoustic standing wave and its “interface effect” can be considered to act as a filter, preventing large cells, other particles or bodies, from exiting the bioreactor.

In another application, the acoustic perfusion devices can act as a retention device and cell washing device for cell therapy applications. In continuous cell-culture applications, such as autologous and allogeneic cell therapy, cells may be purified, isolated, and/or proliferated that are initially harvested at a very low cell-density. Relatively few cells can be used to seed a bioreactor, with the intention of increasing the number of cells. Further processing steps such as concentrating, washing, and/or media exchange may be used for various applications. These applications may include operations to continuously circulate, add, and/or remove media while retaining cells in a bioreactor (which may be traditional or single-use) with little or no effect to their viability. The acoustic cell retention systems described herein operate over a range of cell recirculation rates, efficiently retain cells over a range of perfusion (or media removal rates), and can be tuned to fully retain or selectively pass some percentage of cells through fluid flow rate, transducer power or frequency manipulation. Power and flow rates can all be monitored and used as feedback in an automated control system. Specialty flow paths may also be used such that a small volume of the main fluid flow is “sipped” off and the expressed biomolecules are separated from the main cell culture.

One advantage of acoustophoresis is that the acoustic radiation force does not harm or negatively affect the biological cells or the desired biomolecule product. Moreover, perfusion is continuous, such that the cell culture is kept viable and desired products can be continually recovered therefrom.

In a perfusion bioreactor system, it is desirable to be able to filter and separate the viable biological cells from the expressed materials that are in the fluid stream (i.e., cell culture media) and cellular debris. As previously mentioned, such biological cells may include Chinese hamster ovary (CHO) cells, whose cell genome is manipulated to express large biomolecules. Such biomolecules can include recombinant proteins or monoclonal antibodies, and are the desired product to be recovered.

The acoustic perfusion devices of the present disclosure are designed to maintain a high intensity multi-dimensional acoustic standing wave that can act as a filter, permitting smaller particles (such as recombinant proteins or cellular debris) to pass through while excluding larger particles (such as viable cells). In some examples, the device is driven by an oscillator and amplifier (not shown), and the device performance is monitored and controlled by a computer (not shown). Acoustic streaming can be controlled by modulating the frequency or amplitude of the standing wave. The modulation or control may be implemented by amplitude modulation, including voltage and/or current amplitude modulation, and/or by frequency modulation, which may include phase angle modulation. The duty cycle of the propagation of the standing wave may also be utilized to achieve certain results (i.e. the acoustic beam may be turned on and shut off at different time periods or rates).

FIG. 1 illustrates a single standing wave system 100 that is comprised of a reflector plate 101 and an ultrasonic transducer 103 that is set to resonate so as to form a standing wave 102. Excitation frequencies typically in the range from 100 kHz to 100 MHz are applied by the transducer 103. One or more multi-dimensional standing waves are created between the transducer 103 and the reflector 101. An ideal standing wave is the sum of two propagating waves that are equal in frequency and intensity and that are traveling in opposite directions, i.e. from the transducer to the reflector and back. The propagating waves constructively interfere with each other and thus generate the standing wave. A dotted line 105 is used to indicate the zero-amplitude of the wave. A node is a point where the wave has minimum amplitude, and is indicated with reference numeral 107. An anti-node is a point where the wave has maximum amplitude, and is indicated with reference numeral 109.

FIG. 2 is a schematic diagram that compares a fed-batch bioreactor system 201 (left side) with a perfusion bioreactor system 202 (right side). Beginning with the fed-batch bioreactor on the left, the bioreactor 210 includes a reaction vessel 220. The cell culture media is fed to the reaction vessel through a feed inlet 222. An agitator 225 is used to circulate the media throughout the cell culture. Here, the agitator is depicted as a set of rotating blades, though any type of system that causes circulation may be used. The bioreactor permits growth of a seed culture through a growth/production cycle, during which time debris, waste and unusable cells will accumulate in the bioreactor and the desired product (e.g. biomolecules such as monoclonal antibodies, recombinant proteins, hormones, etc.) will be produced as well. Due to this accumulation, the reaction vessel of a fed-batch process is typically much larger than that in a perfusion process. The desired product is then harvested at the end of the production cycle. The reaction vessel 220 also includes an outlet 224 for removing material.

Turning now to the perfusion bioreactor 202 on the right-hand side, again, the bioreactor includes a reaction vessel 220 with a feed inlet 222 for the cell culture media. An agitator 225 is used to circulate the media throughout the cell culture. An outlet 224 of the reaction vessel is fluidly connected to the inlet 232 of an acoustic perfusion device 230 of the present disclosure, and continuously feeds the bioreactor contents (containing cells and desired product) to the filtering device. The perfusion device is located downstream of the reaction vessel, and separates the desired product from the cells. The acoustic perfusion device 230 has two separate outlets, a product outlet 234 and a recycle outlet 236. The product outlet 234 fluidly connects the acoustic perfusion device 230 to a containment vessel 240 downstream of the perfusion device, which receives the flow of the desired product (plus media) from the perfusion device. From there, further processing/purification can occur to isolate/recover the desired product. For example, further downstream of this acoustic perfusion device may be additional filters such as an ATF, TFF, depth filter, centrifuge, etc. The recycle outlet 236 fluidly connects the acoustic perfusion device 230 back to a recycle inlet 226 of the reaction vessel 220, and is used to send the cells and cell culture media back into the reaction vessel for continued growth/production. Put another way, there is a fluid loop between the reaction vessel and the perfusion device. The reaction vessel 220 in the perfusion bioreactor system 202 has a continuous throughput of product and thus can be made smaller. The filtering process is critical to the throughput of the perfusion bioreactor. A poor filtering process will allow for only low throughput and result in low yields of the desired product.

FIG. 3 is a cross-sectional view of a generic bioreactor 300 that is useful for the systems of the present disclosure. As illustrated here, the bioreactor includes a reaction vessel 320 having an internal volume 323. A feed inlet 322 at the top of the vessel is used to feed cell culture media into the vessel. An agitator 325 is present. An outlet 324 is shown at the bottom of the vessel. A thermal jacket 310 surrounds the reaction vessel, and is used to regulate the temperature of the cells/media. An aerator 312 is located on the bottom of the vessel for providing gas to the internal volume. Sensors 314 are shown at the top right of the vessel. A pump 316 is illustrated for feeding the cell culture media into the vessel, as is another pump 318 for removing cell culture media from the vessel.

The perfusion systems described above use an acoustic perfusion device of the present disclosure. The contents of the bioreactor are continuously flowed through the acoustic perfusion device to capture the desired products.

In some embodiments, the acoustic perfusion device includes an inlet port, an outlet port, a first collection port, a bottom wall, and an acoustic chamber. The acoustic chamber can also be referred to as a fluid cell.

The inlet port is located at a first end of the device. Generally, the inlet port is fluidly connected to an associated bioreactor and serves as the inlet through which the fluid mixture with cells, fines, and product is introduced to the device. An inlet flow path leads from the inlet port to the acoustic chamber, which contains an internal volume. An upper wall can be present above the inlet flow path leading from the inlet port to the acoustic chamber, the upper wall having a substantially horizontal orientation. The inlet flow path has a cross-sectional area.

The inlet port is located at a first height above the outlet port, which defines a bottom end of the device. Put another way, the outlet port is located below the acoustic chamber or below the inlet port, or at the bottom end of the device. The placement of the outlet port below the inlet port ensures that fluid flow through the device is passively urged by gravity towards the outlet port, and that a hydraulic head is created within the device. The outlet port may also be referred to as a fluid recycle port because the host fluid is recycled or returned from the device to the associated bioreactor through the outlet port. The outlet port is also located at a second end of the device, opposite the first end. The first end and second end can be considered as being opposite ends of an x-axis, while the bottom end and top end can be considered as being opposite ends of a z-axis.

The first collection port is located above the acoustic chamber at the top end of the device, and is fluidly connected to the acoustic chamber. The device may include additional collection ports, such as second collection port, which is spaced apart from the first collection port. The first and second collection ports, are generally used to harvest and recover a portion of the desired biomolecule byproducts from the device. A collection or harvest flow path leads from the acoustic chamber to the collection ports. The collection flow path has a cross-sectional area. In some particular embodiments, the cross-sectional area of the collection flow path is greater than the cross-sectional area of the inlet flow path. This is one method by which the flow rate of fluid through the collection ports, can be made much lower than the incoming flow rate of fluid. When used in perfusion biomanufacturing, the collection ports can also be referred to as perfusion or harvest ports. Because fluid depleted in cells and enriched in desired biomolecule products, cell debris, and other fines is harvested, the collection ports can also be referred to as harvest ports, and the collection flow path can also be referred to as the harvest flow path.

In some embodiments, the bottom wall extends from the inlet port to the outlet port of the device. The exact shape of the bottom wall can vary to obtain the desired fluid flow. The bottom wall can curve in a concave fashion from the inlet port to the outlet port of the device. An outlet flow path leads from the acoustic chamber to the outlet port.

A first ultrasonic transducer can be located on a sidewall of the device at a second height that is above the first height (i.e. closer to the top end of the device) and below the collection ports. This volume above the acoustic chamber and below the collection ports can be identified as a harvest or collection zone. The first ultrasonic transducer includes a piezoelectric material that can be driven (e.g. by a voltage signal) to create a multi-dimensional standing wave in the acoustic chamber across the collection flow path. An acoustic radiation force field thus separates the acoustic chamber from the collection ports.

The acoustic perfusion device may include a reflector located on a wall opposite from the first ultrasonic transducer. The reflector is also located at the second height (i.e. the same height as the transducer). Together, the transducer and reflector generate a multi-dimensional acoustic standing wave, as illustrated in FIG. 1.

In some embodiments, the inlet port, outlet port, and the collection ports may all be located on a front wall of the device. The ports can face in any other direction, as desired. The front wall can have a flat or planar face, and has a constant thickness. However, the shape of the front wall may also vary if desired, for example to change the cross-sectional areas. Finally, the rear wall of the device is attached to a mounting piece, which contains holes for attaching the perfusion device to a surface for operation.

The flow rate through the collection or harvest flow path is, in various embodiments, at least one order of magnitude smaller than the flow rate through the inlet flow path. In more particular embodiments, the flow rate of the fluid mixture entering the device through the inlet port is about 1 liter per minute (L/min) and the flow rate of the fluid depleted in cells exiting the device through the collection port(s) is about 10 milliliters per minute (mL/min). In some tests, bioreactors having a size of 2 liters to 10 liters have been tested with solutions containing up to 10% yeast and up to 50 million cells/m L. The flow rate through the inlet port has been from about 0.75 L/min to about 3 L/min, with the flow rate through the collection flow path (i.e. all collection ports together) being about 1 mL/min to about 30 mL/min. A 95% cell recovery rate has been achieved.

The acoustic perfusion devices of the present disclosure can filter very high cell densities, around 100 million cells per mL and possibly in the range of about 20 million to about 120 million cells per mL, whereas other filtering technologies such as ATF can only filter at densities less than 80 million cells per mL. Unlike hollow fiber membranes, the acoustic standing wave(s) can also be tuned to allow passage of cells if desired, as well as allow the passage of fines/debris. This can act as a cleaning operation for the bioreactor. Continuous, steady-state operation is possible without pressure fluctuations, and the product stream does not accumulate in the bioreactor or the filtering device.

The acoustic perfusion device can be made of appropriate materials, such as, for example, high density polyethylene (HDPE), other plastics, metals and/or glasses. It has been found very convenient for the device to be transparent, so that fluid flow and ultrasonic transducer operation can be visually confirmed.

Turning now to FIG. 4, a processing system is shown including an associated bioreactor 610 and an acoustic perfusion device 630 of the present disclosure. The system is set up for use as a perfusion bioreactor. The bioreactor 610 includes a reaction vessel 620 having a feed inlet 622, an outlet 624, and a recycle inlet 626. Fresh media is added into the feed inlet 622 by an addition pipe 650. Some reactors will also include an outlet or bleed port (not shown here) to remove or “bleed” cells in order to maintain a constant cell density within a reactor. The contents of the reaction vessel (reference numeral 605) are mixed with an agitator 625. The desired product (e.g., recombinant proteins) is continuously produced by cells located within the vessel 620, and are present in the media of the bioreactor. The product and the cells in the perfusion bioreactor are drawn from the reaction vessel through pipe 652, and enter the acoustic perfusion device 630 through inlet port 632. Therein, a portion of the desired product is separated from the cells. The desired product can be drawn off through a first collection port 634 (which is a product recovery port) and pipe 654 into a containment vessel 640, or in the case of a truly continuous production system, some other downstream purification process. The cells are returned to the perfusion bioreactor after separation, passing from outlet port 636 (which is a fluid recycle port) of the acoustic perfusion device through pipe 656 to recycle inlet 626 of the reaction vessel, which form a recycle path. The multi-dimensional standing wave(s) of the acoustic perfusion device are used to create a separation barrier between the fluid cell of the device and the collection port, so that a highly reduced number of biological cells are collected in collection port 634.

FIG. 5 and FIG. 6 are views of another exemplary embodiment of an acoustic perfusion device. FIG. 5 is a front cross-sectional view, and FIG. 6 is an exterior perspective view. Notably, this embodiment is specifically designed such that it can be fabricated with clean machining techniques, using Class VI materials (medical device grade HDPE, for example), or even as single or welded injection molded part. In this manner, this embodiment is an example of a single-use device, which is gamma-stable. The devices are flushed to remove bioburden and then gamma-irradiated (generally from 25-40 kGy) to sterilize any potential contamination that could destroy a healthy cell culture, such as that present in a perfusion bioreactor.

Referring first to FIG. 5, in this device 700, the inlet port 710 and the collection port 770 are both located at the top end 718 of the device, or on the top wall 776 of the device. The outlet port 730 is located at a bottom end 716 of the device. Here, the inlet port 710 and the outlet port 730 are both on a first side 712 of the device. The inlet flow path 751 is in the form of a channel 755 that runs from the inlet port downwards towards the bottom end and past the outlet port, the channel being separated from the acoustic chamber 750 (here, the separation occurring by an internal wall 756). Fluid will flow downwards in the channel, then rise upwards into the acoustic chamber 750. The bottom wall 720 of the acoustic chamber is a sloped planar surface that slopes down towards the outlet port 730. The location of the ultrasonic transducers 760 are shown here as two squares, between the top end and the bottom end of the device. The collection flow path 753 is located above the transducers.

Referring now to FIG. 6, the device 700 is shown as being formed within a three-dimensional rectangular housing 706. It can be seen that the outlet port 730 at the bottom end 716 of the device is located on a front wall 775. Again, the collection port 770 and the inlet port 710 are located on a top wall 776. A viewing window 708 made of a transparent material is present in the front wall. Through that viewing window, it can be seen that the ultrasonic transducers are mounted in the rear wall 778 of the device housing. The viewing window acts as a reflector to generate the multi-dimensional acoustic standing waves.

FIG. 7 and FIG. 8 are views of yet another exemplary embodiment of an acoustic perfusion device. FIG. 7 is a front cross-sectional view, and FIG. 8 is a perspective view.

Referring first to FIG. 7, in this device 900, there is an inlet port 910 present on a front side 975 of the device along the first side 912 of the device. An outlet port 930 (best seen in FIG. 8) is located directly opposite and at the same height as the inlet port 910, and is also located on first side 912. In this embodiment, there is a main fluid stream that flows almost directly from the inlet port 910 to the outlet port 930, and the inlet flow path 951 diverts only a small side flow into the acoustic chamber 950 from the first side 912 of the device. The collection port 970 is located at the top end 918 of the device, or on the top wall 976 of the device. A secondary outlet port 980 is located on the first side 912 of the device as well, extending from first side wall 979, and located below the inlet port 910, and can act as a bleed port. The bottom wall 920 of the acoustic chamber is shaped in a pyramid-like fashion to taper downwards to a vertex. A drain line 981 runs from the bottom of the acoustic chamber 950 to the secondary outlet port 980. The secondary outlet port can be used to capture a small flow of highly concentrated cells, which can either be discarded (cell bleed) or can also be returned back to the bioreactor.

Referring now to FIG. 8, the front wall 975 of the device has a rectangular space 960, and the rear wall 978 of the device has a rectangular space 962. One transducer and one reflector can be placed in these two rectangular spaces 960/962 in either orientation, or that two transducers could be placed in the two rectangular spaces. The inlet port 910 and outlet port 930 are both visible in this view. The inlet port 910 is located on the front side of the device, and the outlet port 930 is located on the rear side of the device (though this could be reversed if desired). The clarification flow path 953 is located above the transducers. Although not depicted here, a mounting piece similar to that in FIG. 4 could be attached to the second side 914 of the device.

FIGS. 25-27 are views of yet another exemplary embodiment of an acoustic perfusion device. FIG. 25 is a perspective view, FIG. 26 is a picture showing a front view, and FIG. 27 is a side view.

Referring now to FIGS. 25-27, in this device 4300, the inlet port 4310 and the outlet port 4330 are both located at the bottom end 4316 of the device, and the collection port 4370 is located at the top end 4318 of the device. The inlet port 4310 is located on a first side 4312 of the device, and the outlet port 4330 is located on a second side 4314 of the device. In FIG. 25, the outlet port 4330 is attached to a pump 4305, which creates flow through the device 4300. A viewing window 4308 is present on the front wall 4375 of the device. The front wall 4375, top wall 4376, rear wall 4378, and first side wall 4379 are part of the housing 4306 that surrounds the interior of the device.

Referring now to FIG. 25 and FIG. 27, the ultrasonic transducer 4360 is located on the rear wall 4378 at the top end 4318 of the device. The viewing window 4308 acts as a reflector to generate the multi-dimensional acoustic standing waves.

In this embodiment, a recirculation pipe 4340 connects the inlet port 4310 directly to the outlet port 4330, and forms a recirculation flow path (arrow 4356) through which cell culture media containing cells and other materials can be continuously recirculated through the perfusion device without entering the acoustic chamber 4350. The recirculation pipe 4340 and the recirculation flow path 4356 are located below the acoustic chamber 4350.

An inflow passageway 4380 and an outflow passageway 4390 connect the acoustic chamber 4350 to the recirculation pipe 4340, and split off a portion of the flow of cell culture media from the recirculation pipe into the acoustic chamber. Arrow 4351 indicates the inlet flow path, and arrow 4355 indicates the outlet flow path. These two passageways are particularly visible in FIG. 26. Put another way, the inlet flow path travels through a different passage than the outlet flow path. This creates a secondary recirculating flow that is tangential to the acoustic interface, and allows for constant recirculation of cells beneath this acoustic interface, traveling in the same net direction as the recirculation flow path 4356.

All of the components of the acoustic perfusion devices of the present disclosure (i.e. housing, acoustic chamber, transducer, reflector) can be made of materials that are stable when exposed to gamma radiation (typically used for sterilization prior to usage). Examples of such gamma-stable materials can include plastics such as polyethylene, polypropylene, polycarbonates, and polysulfones, and potentially metals and glasses. The devices of the present disclosure can be operated as single-use, consumable, disposable devices. This prevents contamination of subsequent cell culture batches by previous cell culture batches.

It may be helpful now to describe the ultrasonic transducer(s) used in the acoustic filtering device in more detail. FIG. 9 is a cross-sectional diagram of a conventional ultrasonic transducer. This transducer has a wear plate 50 at a bottom end, epoxy layer 52, ceramic piezoelectric element 54 (made of, e.g. Lead Zirconate Titanate (PZT)), an epoxy layer 56, and a backing layer 58. On either side of the ceramic piezoelectric element, there is an electrode: a positive electrode 61 and a negative electrode 63. The epoxy layer 56 attaches backing layer 58 to the piezoelectric element 54. The entire assembly is contained in a housing 60 which may be made out of, for example, aluminum. The housing is used as the ground electrode. An electrical adapter 62 provides connection for wires to pass through the housing and connect to leads (not shown) which attach to the piezoelectric element 54. Typically, backing layers are designed to add damping and to create a broadband transducer with uniform displacement across a wide range of frequency and are designed to suppress excitation of particular vibrational eigen-modes of the piezoelectric element. Wear plates are usually designed as impedance transformers to better match the characteristic impedance of the medium into which the transducer radiates.

FIG. 10 is a cross-sectional view of an ultrasonic transducer 81 of the present disclosure, which is used in the acoustic filtering device of the present disclosure. Transducer 81 is shaped as a square, and has an aluminum housing 82. The aluminum housing has a top end and a bottom end. The transducer housing may also be composed of plastics, such as medical grade HDPE or other metals. The piezoelectric element is a mass of perovskite ceramic, each consisting of a small, tetravalent metal ion, usually titanium or zirconium, in a lattice of larger, divalent metal ions, usually lead or barium, and O²⁻ ions. As an example, a PZT (lead zirconate titanate) piezoelectric element 86 defines the bottom end of the transducer, and is exposed from the exterior of the bottom end of the housing. The piezoelectric element is supported on its perimeter by a small elastic layer 98, e.g. epoxy, silicone or similar material, located between the piezoelectric element and the housing. Put another way, no wear plate or backing material is present. However, in some embodiments, there is a layer of plastic or other material separating the piezoelectric element from the fluid in which the acoustic standing wave is being generated. The piezoelectric material/element/crystal has an exterior surface (which is exposed) and an interior surface as well.

Screws 88 attach an aluminum top plate 82a of the housing to the body 82b of the housing via threads. The top plate includes a connector 84 for powering the transducer. The top surface of the PZT piezoelectric element 86 is connected to a positive electrode 90 and a negative electrode 92, which are separated by an insulating material 94. The electrodes can be made from any conductive material, such as silver or nickel. Electrical power is provided to the PZT piezoelectric element 86 through the electrodes on the piezoelectric element. Note that the piezoelectric element 86 has no backing layer or epoxy layer. Put another way, there is an interior volume or an air gap 87 in the transducer between aluminum top plate 82a and the piezoelectric element 86 (i.e. the air gap is completely empty). A minimal backing 58 and/or wear plate 50 may be provided in some embodiments, as seen in FIG. 11.

The transducer design can affect performance of the system. A typical transducer is a layered structure with the ceramic piezoelectric element bonded to a backing layer and a wear plate. Because the transducer is loaded with the high mechanical impedance presented by the standing wave, the traditional design guidelines for wear plates, e.g., half wavelength thickness for standing wave applications or quarter wavelength thickness for radiation applications, and manufacturing methods may not be appropriate. Rather, in one embodiment of the present disclosure the transducers, there is no wear plate or backing, allowing the piezoelectric element to vibrate in one of its eigenmodes with a high Q-factor, or in a combination of several eigenmodes. The vibrating ceramic piezoelectric element/disk is directly exposed to the fluid flowing through the fluid cell.

Removing the backing (e.g. making the piezoelectric element air backed) also permits the ceramic piezoelectric element to vibrate at higher order modes of vibration with little damping (e.g. higher order modal displacement). In a transducer having a piezoelectric element with a backing, the piezoelectric element vibrates with a more uniform displacement, like a piston. Removing the backing allows the piezoelectric element to vibrate in a non-uniform displacement mode. The higher order the mode shape of the piezoelectric element, the more nodal lines the piezoelectric element has. The higher order modal displacement of the piezoelectric element creates more trapping lines, although the correlation of trapping line to node is not necessarily one to one, and driving the piezoelectric element at a higher frequency will not necessarily produce more trapping lines.

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

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

In some embodiments, for applications such as oil/water emulsion splitting and others such as perfusion, the ultrasonic transducer has a nominal 2 MHz resonance frequency. Each transducer can consume about 28 W of power for droplet trapping at a flow rate of 3 GPM. This translates to an energy cost of 0.25 kW hr/m³. This is an indication of the very low cost of energy of this technology. Desirably, each transducer is powered and controlled by its own amplifier. In other embodiments, the ultrasonic transducer uses a square piezoelectric element, for example with 1″×1″ dimensions. Alternatively, the ultrasonic transducer can use a rectangular piezoelectric element, for example with 1″×2.5″ dimensions. Power dissipation per transducer was 10 W per 1″×1″ transducer cross-sectional area and per inch of acoustic standing wave span in order to get sufficient acoustic trapping forces. For a 4″ span of an intermediate scale system, each 1″×1″ square transducer consumes 40 W. The larger 1″×2.5″ rectangular transducer uses 100 W in an intermediate scale system. The array of three 1″×1″ square transducers would consume a total of 120 W and the array of two 1″×2.5″ transducers would consume about 200 W. Arrays of closely spaced transducers represent alternate potential embodiments of the technology. Transducer size, shape, number, and location can be varied as desired to generate desired multi-dimensional acoustic standing wave patterns.

The size, shape, and thickness of the transducer determine the transducer displacement at different frequencies of excitation, which in turn affects separation efficiency. Typically, the transducer is operated at frequencies near the thickness resonance frequency (half wavelength). Gradients in transducer displacement typically result in more trapping locations for the cells/biomolecules. Higher order modal displacements generate three-dimensional acoustic standing waves with strong gradients in the acoustic field in all directions, thereby creating equally strong acoustic radiation forces in all directions, leading to multiple trapping lines, where the number of trapping lines correlate with the particular mode shape of the transducer.

To investigate the effect of the transducer displacement profile on acoustic trapping force and separation efficiencies, an experiment was repeated ten times using a 1″×1″ square transducer, with all conditions identical except for the excitation frequency. Ten consecutive acoustic resonance frequencies, indicated by circled numbers 1-9 and letter A on FIG. 12, were used as excitation frequencies. The conditions were experiment duration of 30 min, a 1000 ppm oil concentration of approximately 5-micron SAE-30 oil droplets, a flow rate of 500 ml/min, and an applied power of 20 W. Oil droplets were used because oil is less dense than water, and can be separated from water using acoustophoresis.

FIG. 12 shows the measured electrical impedance amplitude of a square transducer as a function of frequency in the vicinity of the 2.2 MHz transducer resonance. The minima in the transducer electrical impedance correspond to acoustic resonances of the water column and represent potential frequencies for operation. Additional resonances exist at other frequencies where multi-dimensional standing waves are excited. Numerical modeling has indicated that the transducer displacement profile varies significantly at these acoustic resonance frequencies, and thereby directly affects the acoustic standing wave and resulting trapping force. Since the transducer operates near its thickness resonance, the displacements of the electrode surfaces are essentially out of phase. The typical displacement of the transducer electrodes is not uniform and varies depending on frequency of excitation. As an example, at one frequency of excitation with a single line of trapped oil droplets, the displacement has a single maximum in the middle of the electrode and minima near the transducer edges. At another excitation frequency, the transducer profile has multiple maxima leading to multiple trapped lines of oil droplets. Higher order transducer displacement patterns result in higher trapping forces and multiple stable trapping lines for the captured oil droplets.

As the oil-water emulsion passed by the transducer, the trapping lines of oil droplets were observed and characterized. The characterization involved the observation and pattern of the number of trapping lines across the fluid channel, as shown in FIG. 13, for seven of the ten resonance frequencies identified in FIG. 12. Different displacement profiles of the transducer can produce different (more) trapping lines in the standing waves, with more gradients in displacement profile generally creating higher trapping forces and more trapping lines.

FIG. 14 is a numerical model showing a pressure field that matches the 9 trapping line pattern. The numerical model is a two-dimensional model; and therefore only three trapping lines are observed. Two more sets of three trapping lines exist in the third dimension perpendicular to the plane of the page.

The lateral force of the acoustic radiation force generated by the transducer can be increased by driving the transducer in higher order mode shapes, as opposed to a form of vibration where the piezoelectric material (e.g., a piezoelectric crystal) effectively moves as a piston having a uniform displacement. The acoustic pressure is proportional to the driving voltage of the transducer. The electrical power is proportional to the square of the voltage. The transducer is typically a thin piezoelectric plate, with electric field in the z-axis and primary displacement in the z-axis. The transducer is typically coupled on one side by air (i.e., the air gap within the transducer) and on the other side by the fluid mixture of the cell culture media. The types of waves generated in the plate are known as composite waves. A subset of composite waves in the piezoelectric plate is similar to leaky symmetric (also referred to as compressional or extensional) Lamb waves. The piezoelectric nature of the plate typically results in the excitation of symmetric Lamb waves. The waves are leaky because they radiate into the water layer, which result in the generation of the acoustic standing waves in the water layer. Lamb waves exist in thin plates of infinite extent with stress free conditions on its surfaces. Because the transducers of this embodiment are finite in nature, the actual modal displacements are more complicated.

FIG. 15 shows the typical variation of the in-plane displacement (x-displacement) and out-of-plane displacement (y-displacement) across the thickness of the plate, the in-plane displacement being an even function across the thickness of the plate and the out-of-plane displacement being an odd function. Because of the finite size of the plate, the displacement components vary across the width and length of the plate. In general, a (m,n) mode is a displacement mode of the transducer in which there are m undulations in transducer displacement in the width direction and n undulations in the length direction, and with the thickness variation as described in FIG. 15. The maximum number of m and n is a function of the dimension of the piezoelectric material (e.g., a piezoelectric crystal) and the frequency of excitation. Additional three-dimensional modes exist that are not of the form (m,n).

The transducers are driven so that the piezoelectric element vibrates in higher order modes of the general formula (m, n), where m and n are independently 1 or greater. Generally, the transducers will vibrate in higher order modes than (2,2). Higher order modes will produce more nodes and antinodes, result in three-dimensional standing waves in the water layer, characterized by strong gradients in the acoustic field in all directions, not only in the direction of the standing waves, but also in the lateral directions. As a consequence, the acoustic gradients result in stronger trapping forces in the lateral direction.

Generally, the ultrasonic transducer(s) may be driven by an electrical signal, which may be controlled based on voltage, current, phase angle, power, frequency or any other electrical signal characteristic. In particular, the driving signal for the transducer may be based on voltage, current, magnetism, electromagnetism, capacitive or any other type of signal to which the transducer is responsive. In embodiments, the signal driving the transducer can have a sinusoidal, square, sawtooth, pulsed, or triangle waveform; and have a frequency of 500 kHz to 10 MHz. The signal can be driven with pulse width modulation, which produces any desired waveform. The signal can also have amplitude or frequency modulation start/stop capability to eliminate streaming.

The transducers are used to create a pressure field that generates acoustic radiation forces of the same order of magnitude both orthogonal to the standing wave direction and in the standing wave direction. When the forces are roughly the same order of magnitude, particles of size 0.1 microns to 300 microns will be moved more effectively towards “trapping lines”, so that the particles will not pass through the pressure field and continue to exit through the collection ports of the filtering device. Instead, the particles will remain within the acoustic chamber to be recycled back to the bioreactor.

In biological applications, some or all of the parts of the system (i.e., the bioreactor, acoustic filtering device, tubing fluidly connecting the same, etc.) can be separated from each other and be disposable. Avoiding centrifuges and filters can permit better separation of the CHO cells without lowering the viability of the cells. The transducers may also be driven to create rapid pressure changes to prevent or clear blockages due to agglomeration of CHO cells. The frequency of the transducers may also be varied to obtain optimal effectiveness for a given power.

The acoustic perfusion/filtering devices of the present disclosure can be used in a filter “train,” in which multiple different filtration steps are used to clarify or purify an initial fluid/particle mixture to obtain the desired product and manage different materials from each filtration step. Each filtration step can be optimized to remove a particular material, improving the overall efficiency of the clarification process. An individual acoustophoretic device can operate as one or multiple filtration steps. For example, each individual ultrasonic transducer within a particular acoustophoretic device can be operated to trap materials within a given particle range. The acoustophoretic device can be used to remove large quantities of material, reducing the burden on subsequent downstream filtration steps/stages. Furthermore, additional filtration steps/stages can be placed upstream or downstream of the acoustophoretic device, such as physical filters or other filtration mechanisms, such as depth filters (e.g., polymeric morphology, matrix media adsorption), sterile filters, crossflow filters (e.g., hollow fiber filter cartridges), tangential flow filters (e.g., tangential flow filtration cassettes), adsorption columns, separation columns (e.g., chromatography columns), or centrifuges. Multiple acoustophoretic devices can be used as well. Desirable biomolecules or cells can be recovered/separated after such filtration/purification, as explained herein.

The outlets of the acoustophoretic separators/filtering devices of the present disclosure (e.g., product outlet, recycle outlet) can be fluidly connected to any other filtration step or filtration stage. Similarly, the inlets of the acoustophoretic separators/filtering devices of the present disclosure could also be fluidly connected to any other filtration step or filtration stage. The additional filtration steps/stages could be located either upstream (i.e., between the acoustophoretic separators(s) and the bioreactor), downstream, or both upstream and downstream of the acoustophoretic separators(s). In particular, it is to be understood that the acoustophoretic separators of the present disclosure can be used in a system with as few or as many filtration stages/steps located upstream or downstream thereof as is desired. The present systems can include a bioreactor, an acoustophoretic separator/filtering device, and multiple filtrations stages/steps located upstream and downstream of the acoustophoretic separator, with the filtrations stage(s) and acoustophoretic separator(s) generally serially arranged and fluidly connected to one another.

For example, when it is desired that the system include a filtration stage (e.g., a porous filter) located upstream of the acoustophoretic separator, the outlet of the bioreactor can lead to an inlet of the porous filter and the outlet of the porous filter can lead to an inlet of the acoustophoretic separator, with the porous filter pre-processing the fluid therein. As another example, when it is desired that the system include a filtration stage (e.g., a separation column) located downstream of the acoustophoretic separator, the outlet of the bioreactor can lead to an inlet of the acoustophoretic separator and the outlet of the acoustophoretic separator can lead to an inlet of the separation column, with the separation column further processing the fluid therein.

Filtration steps/stages can include or implement various methods such as an additional acoustophoretic separator/filtering device, or physical filtration, such as depth filtration, sterile filtration, size exclusion filtration, or tangential filtration. Depth filtration uses physical porous filtration mediums that can retain material through the entire depth of the filter. In sterile filtration, membrane filters with extremely small pore sizes are used to remove microorganisms and viruses, generally without heat or irradiation or exposure to chemicals. Size exclusion filtration separates materials by size and/or molecular weight using physical filters with pores of given size. In tangential filtration, the majority of fluid flow is across the surface of the filter, rather than into the filter.

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

The techniques and implementations described herein may be used for integrated continuous automated bioprocessing. As a non-limiting example, CHO mAb processing may be carried out using the techniques and apparatuses described herein. Control can be distributed to some or all units involved in the bioprocessing. Feedback from units can be provided to permit overview of the bioprocess, which may be in the form of screen displays, control feedbacks, reporting, status reports and other information conveyance. Distributed processing permits a high degree of flexibility in achieving a desired process control, for example by coordinating steps among units and providing a batch executive control.

The bioprocessing can be achieved with commercially available components, and obtain 100% cell retention. Cell density can be controlled via an external cell bleed based on a capacitance signal. The perfusion device utilizing an acoustic wave system can be implemented with biocompatible materials, and may include gamma sterilized and single use components. The processing system also permits ultrasonic flow measurement, which is noninvasive, and is capable of operating with high viscosity fluids. The system can be implemented with single use sterile the septic connectors and a simple graphical user interface (GUI) for control.

The acoustic wave system includes a sweeping flow that is induced below the acoustic chamber. An acoustic standing wave can act as a barrier for particulates in the fluid to permit a clarified stream to be passed and extracted. The recirculation loop can be implemented with high fluid velocity and with a low shear rate. The fluid velocity through the acoustic field can be lower than the fluid velocity through the recirculation loop, which may help to improve separation with low shear forces.

Cell SE is dominated by harvest to recirculation flow ratio. Particle size distribution is shown in the figures, where a parent size-based separation occurs. The system passes fines and smaller cells in the harvest, e.g. Greater than 90% cell separation can be achieved, with 30% of the power and a 1.25% harvest to recirculation ratio with the disclosed system compared to previous systems. In addition, the acoustic wave system separation does not appear to affect quality between the bioreactor and the harvest. The quality is comparable between the acoustic wave system and tangential flow filtering. The acoustic wave system may have negligibly higher HMW species, and a similar charge of variance profile compared to a 0.22 μm membrane tangential flow filter.

The disclosed acoustic wave separator is a viable perfusion technology capable of supporting greater than 50 MVC/m L. The productivity of the acoustic wave be significantly improved, and the acoustic wave separator is scalable. For example, a relatively small unit is capable of operation at 2 L to 50 L scale. The perfusion device can be used with depth filtration or other upstream or downstream filtering or processing components, including chromatography.

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

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

A statement that a value exceeds (or is more than) a first threshold value is equivalent to a statement that the value meets or exceeds a second threshold value that is slightly greater than the first threshold value, e.g., the second threshold value being one value higher than the first threshold value in the resolution of a relevant system. A statement that a value is less than (or is within) a first threshold value is equivalent to a statement that the value is less than or equal to a second threshold value that is slightly lower than the first threshold value, e.g., the second threshold value being one value lower than the first threshold value in the resolution of the relevant system. Also, configurations may be described as a process that is depicted as a flow diagram or block diagram. Although each may describe the operations as a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations may be rearranged. A process may have additional stages or functions not included in the figure.

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

The following examples are provided to illustrate the devices, components, and methods of the present disclosure. The examples are merely illustrative and are not intended to limit the disclosure to the materials, conditions, or process parameters set forth therein.

EXAMPLES
Example 1

FIG. 16 is a picture of another acoustic perfusion device of the present disclosure, having two ultrasonic transducers and a concave bottom wall leading from the inlet port to the outlet port at the bottom end of the device. A cell containing fluid mixture is also present in the device. In this picture, acoustic standing waves are created in the collection zone between the reflector and first and second transducers as described above. The acoustic field generated thereby is indicated by waves and reference numeral 1664. The flow pattern of the fluid mixture through the device from the inlet port to the outlet port is shown with an arrow (reference numeral 1610) indicating the direction of fluid flow into the device and arrows (reference numeral 1630) indicating the direction of fluid flow through the device towards the outlet port. Finally, the general flow pattern of the desired product out of the device through the first and second collection ports is shown with arrows (reference numeral 1670) indicating the direction of flow.

Acoustophoretic separation has been tested using the acoustic perfusion device of FIG. 16 and the methods of separation of the present disclosure on different lines of Chinese hamster ovary (CHO) cells. FIGS. 20-28 show various test results varying different parameters and measuring different values using a Beckman Coulter Cell Viability Analyzer.

The perfusion flow rates with the acoustic filtering device were from about 2 mL/min to about 10 mL/min, or the flow rates were about 1 VVD to about 5 VVD for a 2.7 L working volume bioreactor. The VVD refers to the “vessel volume per day”, or how many times the volume of the bioreactor vessel is cycled through the acoustic filtering device in one day. The perfusion flow rate (Qp) was collected through the perfusion ports. In contrast, the feed flow rates (Qf) were from about 40 mL/min to about 200 mL/min.

The feed solution had a starting CHO cell density of 50×10⁶cells/mL. The reactor size was 2.7 L and the feed volume of the host fluid was 1.5 L. In total, a series of seven tests (T1-T7) were performed to study the effect of varying the VVD and flow split in a 2.7 L volume reactor. The parameters for the tests are shown in Table 1 below.

TABLE 1

System results for a 2.7 L reactor and feed volume of 1.5 L

Flow Split
Qp

(Qp/Qf)
1 VVD
1.5 VVD
2 VVD
5.2 VVD

Qf
5.0%
T1
T2
T3
T7

2.5%
T4
T5
T6

The results included a cell clarification efficiency between 89-93% at a DC voltage of 45V, regardless of the flow rate. It is noted that the DC voltage for T1 was fixed at 60V, whereas for tests T2-T7 the DC voltage was reduced to a fixed amount of 45V. The transducer voltage amplitude is estimated to be about half of these values.

The results further included a perfusate turbidity reduction of 90-94% compared to the feed. The feed entered the inlet port, the recirculated fluid exited the outlet port and was recirculated, and the perfusate exited the perfusion port of the acoustic filtering device. It is noted that the turbidity measurements for tests T1 and T2 resulted in a hardware error, so only tests T3-T7 were used, which equated to a 6-10% turbidity in the perfusion stream relative to the feed stream, regardless of flow rate.

The tests revealed a cell viability for each flow rate that was within the error of instrument (i.e., ±6%), with the control ranging from 79-84% over all tests.

Further testing was performed using a solution designated “CHO Line A”. The solution had a starting cell density of 50×10⁶cells/mL, a turbidity of 2,400 NTU, and cell viability of roughly 80%. The solution was separated using a device of the present disclosure in a system having a reactor size of 2.7 L. The volume of the feed fluid was between 1.5 L and 2.0 L. The perfused flow rates were from 2 mL/min to 10 mL/min, or from 1 to 5 VVD. A series of six tests were performed to study the effect of varying the VVD and flow split on acoustic filtration performance for the 2.7 L volume reactor. The parameters for the tests are shown in Table 2 below.

TABLE 2

System results for a 2.7 L reactor and feed volume from 1.5 L-2.0 L

T1
T2
T3

VVD
1.5
VVD
2
VVD
1

Flow Split
5.00%
Flow Split
5.00%
Flow Split
2.50%

Perfused Flow
2.8
Perfused Flow
3.8
Perfused Flow
1.9

(ml/min)

(ml/min)

(ml/min)

Feed Flow (ml/min)
56
Feed Flow (ml/min)
75
Feed Flow (ml/min)
75

T4
T5
T6

VVD
1.5
VVD
2
VVD
5.2

Flow Split
2.50%
Flow Split
2.50%
Flow Split
5.00%

Perfused Flow
2.8
Perfused Flow
3.8
Perfused Flow
10

(ml/min)

(ml/min)

(ml/min)

Feed Flow (ml/min)
112.5
Feed Flow (ml/min)
150
Feed Flow (ml/min)
194.2

The bioreactor cell retention for the tests shows an approximately 90% perfusion efficiency. There was no significant change in measured cell viability across the tests.

Next, additional testing was performed using a solution designated “CHO Line B”. The solution had a starting cell density of 75×10⁶cells/mL, a turbidity of 2,300 NTU, and cell viability of roughly 80%. The solution was separated using a device of the present disclosure in a system having a reactor size of 2.7 L. Four tests were performed (T1-T4). Two of the tests (T1, T3) used a device having a single transducer. The other two tests (T2, T4) used a device having two transducers in series (such that the fluid ran through both standing waves). The parameters for the tests are shown in Table 3 below.

TABLE 3

System results for a 2.7 L reactor and feed volume from 1.5 L-2.0 L

T1
T2

Transducers
1
Transducers
2

VVD
1
VVD
1

Perfused Flow (mL/min)
1.9
Perfused Flow (mL/min)
1.9

Feed Flow (mL/min)
75
Feed Flow (mL/min)
75

T3
T4

Transducers
1
Transducers
2

VVD
2
VVD
2

Perfused Flow (mL/min)
3.8
Perfused Flow (mL/min)
3.8

Feed Flow (mL/min)
150
Feed Flow (mL/min)
150

The bioreactor cell retention for the tests shows a perfusion efficiency greater than 90%. The results further evidenced an approximately 3-5% greater efficiency when using two transducers rather than a single transducer. There was no significant change in measured cell viability across the tests. Practically speaking, operating at low VVD offers a number of advantages, such as media cost reduction.

Example 2

Another experimental setup included an acoustic perfusion device similar to that illustrated in FIG. 5. Tubes are connected to the inlet port, outlet port, and the collection port.

The device was tested at a transducer voltage of 40V peak to peak, a perfused flow rate of 15-30 mL/min, and a recirculation flow rate of 2 L/min. Samples were taken every 45-60 minutes, and the cell retention rate was determined. The cell retention efficiency remained above 95% for perfused flow rates over about 12 mL/min and up to 20 mL/min, and remained above 90% up to about 25 mL/min.

Next, experiments were performed to determine what factors would affect cell retention. The perfused flow rate was varied, as was the transducer voltage. When the perfused flow rate was varied, the transducer voltage was maintained at 40V peak to peak and the recirculation flow rate was maintained at 2 L/min. When the transducer voltage was varied, the perfused flow rate was maintained at 20 mL/min and the recirculation flow rate was maintained at 2 L/min. The results indicated that, for this particular embodiment, a perfused flow rate of about 15 mL/min to about 28 mL/min was optimum, and a transducer voltage of about 15V peak to peak to about 28V peak to peak was optimum.

A computational fluid dynamics (CFD) model was made of this device. As expected, the highest velocities were found in the channel leading downwards from the inlet port to the outlet port. The velocity was near zero in the fluid cell and out through the collection port. This is important for two reasons: the acoustic field is more effective in a flow with a lower, more uniform velocity, and because the cells used in biomanufacturing are sensitive to flow, and the induced shear rate.

FIG. 17 is a diagram illustrating several aspects of this embodiment. Fluid flows into the device through the inlet port 710 (arrow 780) and into the acoustic chamber 790. The volume of fluid 750 below the acoustic chamber contains the tangential flow path, indicated by arrow 782. Fluid with a relatively high amount of viable cells will exit through the outlet port 730, as indicated by arrow 781. The acoustic interface effect/region created by the standing waves is marked with reference numeral 783, and is upstream of the acoustic standing wave field 784. The acoustic interface roughly coincides with an x-y plane in this diagram. This interface effect separates large cells from smaller cell fragments, particulate debris, desired biomolecules, etc., which can pass through the interface 783 and the acoustic standing wave field 784. By way of comparison, the cell aggregates that arise within the acoustic standing wave field 784 during the first mode of operation (see FIG. 41) can be described as being aligned in the y-z plane. In operation, the separation caused by the interface effect occurs at the interface region 783 as any large cells are held back by the “interface” or “barrier” effect. The harvest flow stream 785 containing the smaller fragments, particulate debris, desired biomolecules, etc. then exits through harvest port 770. The tangential flow path is part of the inlet flow path, and is located below the interface region 783 generated by the acoustic standing wave. The tangential flow path will transport away both the clusters of cells that drop from the acoustic standing wave field 784 due to gravity effects and the cells that are retained by the acoustic interface effect.

Example 3

Another way of explaining the operation of the acoustic perfusion device can be understood by looking at the results of a numerical study. In the numerical study, two fluids with differing effective acoustic properties (i.e., speed of sound and density), were modeled with an interface between them in COMSOL, a numerical simulation software. The acoustic field is calculated and therefrom the lateral radiation force acting on a particle in the direction of the fluid velocity is calculated using Gorkov's equation.

FIG. 18 shows the geometry of the simulation, utilizing a piezoelectric transducer, steel reflector, aluminum housing, and two fluids: the first fluid being water within the acoustic field, simulating the clarified fluid, and the second fluid being a 15% concentration of CHO cells in water solution outside of the acoustic field, the second fluid having a higher density and higher speed of sound than the water fluid and simulating the bioreactor fluid containing the cells.

The two fluids were separated as indicated by the solid line in the model of FIG. 18. In this setup, the fluid velocity through the system was in a horizontal direction from left to right. In such a configuration, the acoustic field is implemented to obtain a retention device. The acoustic field generates a force on the cells that acts in the negative x-direction (i.e, opposite the fluid velocity). Water was modeled with a fluid density of 1000 kg/m³and a speed of sound of 1500 m/s. CHO cells were modeled having a density of 1050 kg/m³and a speed of sound of 1550 m/s. A coupled multi-physics numerical simulation that included a full piezoelectric simulation of the piezoelectric material, an acoustic simulation of the two fluids, and a linear elastic simulation in the steel and aluminum bodies was performed at various frequencies of excitation. The transducer was driven at a peak voltage of 40 V.

FIGS. 19A-19C show the acoustic pressure in the two fluids and the displacement of the piezoelectric material, the aluminum housing, and the steel reflector of the model at frequencies of operation of 2.218 MHz, 2.2465 MHz, and 2.3055 MHz. The lateral radiation force (i.e., horizontally in the direction of the fluid flow), was calculated at the interface between the two fluids along with real electrical power consumed by the transducer. The structural displacement of the transducer and steel are shown, along with the acoustic pressure in the fluid.

FIG. 20 shows the lateral radiation force (N) and the lateral radiation force normalized by power (N/W) versus frequency acting on the suspended CHO cells. This graph shows that at the resonance frequencies (i.e., local maxima in power), the average lateral radiation force on the interface is negative, meaning that it is in the negative-x direction. The result is the creation of an acoustic barrier effect or an acoustic interface effect. That is, the acoustic field at the interface between the two fluids exerts a strong lateral force on the suspended particles in a direction opposite to the fluid flow, thereby keeping the larger particles from entering the acoustic field and allowing only the first fluid (i.e., fluid containing only smaller particles, such as the desired product, and excluding whole cells) to enter the acoustic field, thereby creating an acoustic perfusion cell retention device. In this way, only the clarified fluid can escape and the cells are held back by the radiation force. This force is never positive, meaning that it always holds the cells back at the interface, not allowing them to cross the interface. The multiple peaks in the power curve show the existence of multiple modes of operation including planar resonance modes and multi-dimensional modes of operation, indicating that this type of operation can be generated through utilization of planar and multi-dimensional standing waves alike. In systems having 1″×1″ dimensions, there exists a planar resonance about every 30 kHz. The graph shows evidence of additional peaks indicating the existence of the multi-dimensional modes. Per unit power, these modes can be equally or even more effective as the planar resonance modes. As explained above, the cells that are held back by the acoustic radiation force may then be picked up by the scrubbing motion of the flow field (i.e., the recirculating flow underneath the interface), and be continuously returned to the bioreactor to ensure they receive the nutrition and oxygen to maintain the production of the overall cell culture.

Example 4

FIG. 21 and FIG. 22 show another experimental setup for an acoustic perfusion device similar to that illustrated in FIG. 7. Tubes are connected to the inlet port, outlet port, the collection port, and the secondary outlet port (for a flow concentrated cells). Arrows are included to illustrate fluid flow. Arrows indicate the flow into the inlet port; the flow out of the outlet port; the perfusate flow out the top of the device and the flow of concentrate out the bottom of the device. The flow through the inlet port to the outlet port is the recirculation flowrate. The perfusate flow out the top of the device is the perfused flowrate containing clarified fluid depleted in cells and containing desired product. The flow of concentrate out the bottom of the device is the concentrated cell flow. The concentrated cell flow can be used for a cell bleed operation or if desired, the cells can be returned to the bioreactor.

The device was tested at a transducer voltage of 40V peak to peak, a perfused flow rate (out the top) of 1-10 mL/min, a recirculation flow rate of 0.75-1 L/min, and a concentrate flow rate (out the bottom) of 15 mL/min. The cell retention rate was determined for different perfused flowrates. FIG. 23 shows the results. The y-axis is the retention with 1.00 indicating 100% retention. The cell retention efficiency remained above 98% for perfused flow rates up to 7 mL/min, and was just below 90% at 10 mL/min.

Example 5

The device of FIG. 5 and FIG. 6 was tested at different flowrates (5 mL/min, 1.5 mL/min, 8 mL/min) on two different days. Higher values are more desirable, and most values were over 95% at flowrates ranging from 1.5 mL/min to 8 mL/min. The perfusate cell density (million cells/mL) versus time was also measured. Lower values are more desirable (indicating successful cell separation). As expected, results were better at lower flowrates.

Example 6

A computational fluid dynamics (CFD) model was made of the device with the internal structure of FIG. 26. As expected, the highest velocities were found in the inflow passageway into the acoustic chamber. The velocity is near zero in the acoustic chamber, and near the collection port at the top thereof. This is a desirable velocity profile.

Next, the device with the internal structure of FIG. 26 was tested at different flowrates (1.5 mL/min, 0.37 mL/min). Higher values are more desirable, and most values were over 97% at flowrates ranging from 0.37 mL/min to 1.5 mL/min. The perfusate cell density (million cells/mL) versus time was also measured. Here, lower values are more desirable (indicating successful cell separation). As expected, results were better at lower flowrates.

The device was then tested using two different operating frequencies for the ultrasonic transducer, 1 MHz or 2 MHz, and at different flowrates. The cell retention (%) versus time was measured. Higher values are more desirable. At 2 MHz, the values were close to 100% for flow rates of 1.5 mL/min to 3 mL/min. At 1 MHz, the values stayed over 90% for flow rates of 1.5 mL/min to 3 mL/min. The frequency of 2 MHz generally performed better. The perfusate cell density (million cells/mL) versus time was also measured. Again, results were better for 2 MHz operating frequency.

Example 7

A comparative evaluation between an AWS (acoustic wave separation) process according to the present disclosure and a TFF (tangential flow filtration) process (similar to that shown in FIG. 24) was performed using an acoustic perfusion device as described herein. FIGS. 28-39C include graphs illustrating this comparative evaluation for a variety of parameters. For both evaluations, a cell mixture was cultured in a bioreactor for 30 days. TFF or AWS was used for the separation of desired product from the cell culture, with the cells continuously returned to the bioreactor throughout the culture period. In the AWS process, the desired product was continuously obtained due to the nature of the AWS process. In both the TFF and AWS process, chromatography columns were used for further filtration/processing of the desired product downstream of the TFF/AWS process.

FIG. 28 graphically illustrates the viable cell density (VCD) (expressed as cells/mL) and viability (expressed as a percentage) versus time for both a tangential flow filtration (TFF) process and an acoustic wave separation (AWS) process according to the present disclosure. In FIG. 28, the line having diamond-shaped data points represents the VCD of the TFF process; the line having square-shaped data points represents the viability of the TFF process in the bioreactor; the line having triangle-shaped data points represents the VCD of the AWS process; the line having x-shaped data points represents the viability of the AWS process in the bioreactor; the line having circular-shaped data points represents the viability of the AWS process in the harvest stream (i.e., after acoustic wave separation); and the line having *-shaped data points represents the viability of the TFF process in the permeate stream (i.e., after tangential flow filtration). As can be seen in FIG. 28, the harvest % viability was near 100% after day 8, and generally remained at this high level.

FIG. 29 graphically illustrates the cell specific perfusion rate (CSPR) (expressed as picoliters per cell per day) for both a TFF process and an AWS process according to the present disclosure. In FIG. 29, the line having diamond-shaped data points represents the CSPR of the AWS process, and the line having square-shaped data points represents the CSPR of the TFF process. The offset between the lines is due to differences in the cell densities for each process. Even with this offset, it can be seen that the AWS CSPR is improved over the TFF CSPR.

FIG. 30 graphically illustrates the specific growth rate (SGR) (per day) in the bioreactor versus time for both a TFF process and an AWS process according to the present disclosure. In FIG. 30, the line having diamond-shaped data points represents the SGR of the TFF process, and the line having square-shaped data points represents the SGR of the AWS process. The specific growth rate accounts for both death and harvest losses. As can be seen in FIG. 30, there is no significant difference between the TFF and AWS processes, which indicates that AWS is a suitable addition to or substitution for TFF in conventional processes.

FIG. 31 graphically illustrates the specific death rate (Kd) (per day) in the bioreactor versus time for both a TFF process and an AWS process according to the present disclosure. In FIG. 31, the line having diamond-shaped data points represents the Kd of the TFF process, and the line having square-shaped data points represents the Kd of the AWS process. As can be seen in FIG. 31, there is no significant difference between the TFF and AWS processes, again indicating that AWS is a suitable addition to or substitution for TFF in conventional processes.

FIG. 32 graphically illustrates the titer (i.e., the concentration of protein available) in the bioreactor (BRX) for both a TFF process and an AWS process according to the present disclosure versus in the permeate (TFF) and harvest (AWS) streams of each process. In FIG. 32, the line having diamond-shaped data points represents the titer of the TFF process in the bioreactor; the line having square-shaped data points represents the titer of the TFF process in the permeate stream (i.e., after tangential flow filtration); the line having triangle-shaped data points represents the titer of the AWS process in the bioreactor; and the line having x-shaped data points represents the titer of the AWS process in the harvest stream (i.e., after acoustic wave separation). The large difference between the TFF permeate titer and the TFF bioreactor titer indicates that a low amount of the potential protein that could be harvested is actually harvested in the TFF process. In comparison, the relatively small offset between the AWS harvest titer and the AWS bioreactor titer is consistent with almost 100% efficiency in harvesting of the protein. In this way, FIG. 32 suggests that AWS is advantageous over TFF for capturing the desired protein and separating it from the bioreactor.

FIG. 33 graphically illustrates the volumetric productivity (VP) (expressed in grams/L/day) (i.e., the amount of protein produced per day) for both a TFF process and an AWS process according to the present disclosure. In FIG. 33, the line having diamond-shaped data points represents the VP of the TFF process, and the line having square-shaped data points represents the VP of the AWS process. As can be seen in FIG. 33, there is again no significant difference between the TFF and AWS processes, yet again indicating that AWS is a suitable addition to or substitution for TFF in conventional processes.

FIG. 34 graphically illustrates the specific productivity (Qp) (expressed in pico grams protein/cell/day) (i.e., the amount of product created by each cell in the bioreactor per day) for both a TFF process and an AWS process according to the present disclosure. In FIG. 34, the line having diamond-shaped data points represents the Qp of the TFF process, and the line having square-shaped data points represents the Qp of the AWS process. As can be seen in FIG. 34, the specific productivity of the AWS process is significantly greater than that for the TFF process, e.g. by thirty percent or more at different days in the culture process. The productivity increase with the AWS process is due to the selection of larger, more productive cells that are returned to the bioreactor. Over time, the cells in the bioreactor of the AWS process are thus selected to be more productive. The increase in productivity is believed to be due to the AWS process effectively selecting the cells that produce more protein. The selection of the more productive cells through the perfusion process tends to change the population of cells in the bioreactor over time, such that cells with greater ribosomal content, or larger cells, are retained over time, while nonproductive cells or lower productive cells are culled out of the system. In addition, or alternatively, cells that are in the process of mitosis, or reproduction, tend to be larger as well as more productive producers of biomolecules. Such cells tend to have greater ribosomal content as well. These cells are selected by the AWS process for return to the bioreactor, leading to a trend of cells that tend to be larger in the bioreactor population. Accordingly, over time, the AWS process tends to make the cells more productive, especially when compared to the TFF process, which does not posses these advantages, as illustrated in FIG. 34.

FIGS. 35A-35D graphically illustrate nutrient uptake markers for metabolism for both a TFF process and an AWS process according to the present disclosure versus time. In each graph, the line having diamond-shaped data points represents the AWS process, and the line having square-shaped data points represents the TFF process. As can be seen in FIGS. 35A-35D, there is no significant difference between the TFF and AWS processes, indicating that AWS is a suitable addition to or substitution for TFF in conventional processes.

FIGS. 36A-36C graphically illustrate ion exchange chromatography (IEX) results of charge variants for both a TFF process and an AWS process according to the present disclosure versus time. In FIGS. 36A-36C, the line having diamond-shaped data points represents the AWS process, and the line having square-shaped data points represents the TFF process. It is noted that while there was some increase in the basic variant (FIG. 36B), this increase is believed to have been caused by a discrepancy in the media used between the tests. Otherwise, there is no significant difference between the TFF and AWS processes as provided in FIGS. 36A-36C.

FIGS. 37A-37B graphically illustrate size exclusion chromatography (SEC) results for both a TFF process and an AWS process according to the present disclosure versus time. In FIGS. 37A-37B, the line having diamond-shaped data points represents the AWS process, and the line having square-shaped data points represents the TFF process. A decrease in HMW species is observed in FIG. 37A, and an increase in the monomer percent is observed in FIG. 37B for the AWS process versus the TFF process. Undesirable protein aggregation during monoclonal antibody production is known to occur in upstream and downstream processing, which is a major concern for therapeutic applications where aggregates influence drug performance and safety. These graphs suggest that the AWS process is efficient at separating out the protein aggregates in the system. The reduction in the amount of protein aggregates helps to lessen the burden on downstream filtration processes that could otherwise be overly subject to fouling and clogging due to the large aggregates. Moreover, protein aggregates are typically non-functional and are a problem for the efficacy of therapeutic drugs, which makes their removal even more desirable.

FIGS. 38A-38B graphically illustrate glycosylation (N-glycan) results for both a TFF process and an AWS process according to the present disclosure versus time. In each graph, the line having diamond-shaped data points represents the AWS process, and the line having square-shaped data points represents the TFF process. As can be seen from these two graphs, there is no significant difference between the TFF and AWS processes, once again indicating that AWS is a suitable addition to or substitution for TFF in conventional processes. This process refers particularly to the enzymatic process in which glycans are attached to the proteins of the biomolecules. Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. The biomolecules (e.g., antibodies) of the present disclosure can be glycoengineered to product antibodies with specific glycoforms. For example, specific glycoforms may be desired to achieve desired therapeutic effects, and glycoengineering can be used to achieve the same.

FIGS. 39A-39C graphically illustrate additional glycosylation (N-glycan) results for both a TFF process and an AWS process according to the present disclosure versus time. In each graph, the line having diamond-shaped data points represents the AWS process, and the line having square-shaped data points represents the TFF process. The majority of approved monoclonal antibodies have two N-linked biantennary complex-type oligosaccharides bound to the Fc region, making them of the IgG1 isotype type. The Fc region is important in that it exercises the effector function of the antibody-dependent cell-mediated cytotoxicity (ADCC) through its interaction with leukocyte receptors of the FcγR family. ADCC is important in the efficacy of cancer antibodies, but with many approved cancer antibodies there is less ADCC that could be desired due to nonspecific IgG competing with the drugs for binding to FcγIIIa on natural killer cells. As can be seen in FIG. 39B, the afucosylation percentage is decreased in the AWS process versus the TFF process.

Antibody glycosylation can be affected by the composition of growth and feed media, including the concentration of ammonia, glutamine, glucose, and metal ions. As a result, it is critical during media development and optimization to monitor and consider a culture medium's impact on glycosylation. For therapeutic antibodies whose mechanism of action includes antibody-dependent, cell-mediated cytotoxicity (ADCC), it is particularly important to measure N-glycan fucosylation, because the same is known to have a strong influence on ADCC activity. A decrease in fucosylation increases the antibodies' binding affinity on natural killer cells and ultimately increases ADCC potency (which has been identified as a crucial mechanism of anti-cancer therapeutic antibodies). Thus, there exists significant interest in lowering fucosylation to increase therapeutic antibody efficacy. Compared to fucosylated IgGs, these non-fucosylated forms have a much increased ADCC without any detectable chance in CDC or antigen binding capability.

FIG. 40 is a graph of cell viability over time. Dark circles are for a TFF process with cell bleed. White circles are for an AWS process using a 2 L flow chamber. Dark inverted triangles are for an AWS process using a 10 L flow chamber. White triangles are for a TFF process without cell bleed. As seen here, the AWS processes still maintain well over 90% viability after 14 days.

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

Claims

1. A process for separating biomolecules from a fluid mixture, the process comprising: flowing a fluid mixture containing biomolecules and cells through an acoustic perfusion device, the acoustic perfusion device comprising: an acoustic chamber that receives the fluid mixture containing the biomolecules and cells; andan ultrasonic transducer that includes a piezoelectric material that can be excited to generate a multi-dimensional acoustic standing wave in the acoustic chamber;exciting the ultrasonic transducer to create the multi-dimensional acoustic standing wave; andretaining cells with a larger size in the fluid mixture with the multi-dimensional acoustic standing wave.
2. The process of claim 1, wherein the cells with a larger size are trapped by the multi-dimensional acoustic standing wave and the biomolecules pass through the multi-dimensional acoustic standing wave.
3. The process of claim 2, wherein the trapped cells are recycled to a bioreactor upstream of the acoustic perfusion device.
4. The process of claim 1, wherein the biomolecules are therapeutic antibodies.
5. The process of claim 4, wherein a fucosylation of the therapeutic antibodies is increased and the efficacy of the therapeutic antibodies is changed.
6. The process of claim 1, wherein the biomolecules are produced by culturing cells in a bioreactor prior to flowing the fluid mixture through the acoustic perfusion device.
7. The process of claim 1, wherein a pressure rise and an acoustic radiation force on cells are generated at an interface region of the multi-dimensional acoustic standing wave to clarify the fluid mixture as it passes through the multi-dimensional acoustic standing wave.
8. The process of claim 1, wherein the acoustic perfusion device further comprises a recirculating fluid stream that transports away cells that are held back at the interface region of the multi-dimensional acoustic standing wave.
9. The process of claim 1, wherein the multi-dimensional acoustic standing wave results in an acoustic radiation force that includes an axial force component and a lateral force component that are of the same order of magnitude.
10. The process of claim 1, wherein biomolecule aggregates that pass through the multi-dimensional acoustic standing wave are collected outside and downstream of the acoustic perfusion device.
11. The process of claim 1, wherein biomolecules recovered from the acoustic perfusion device are subjected to further processing downstream of the acoustic perfusion device.
12. The process of claim 11, wherein the further processing includes at least one of chromatography and additional filtration.
13. The process of claim 12, wherein the additional filtration includes at least one of depth filtration, crossflow filtration, tangential filtration, and sterile filtration.
14. The process of claim 1, further comprising glycoengineering the biomolecules to produce antibodies with predetermined glycoforms.
15. A process for collecting biomolecules from a cell culture, the process comprising: flowing a nutrient fluid stream through the cell culture to collect the biomolecules;flowing the nutrient fluid stream through an acoustic perfusion device, the acoustic perfusion device including at least one ultrasonic transducer including a piezoelectric material configured to generate a multi-dimensional acoustic standing wave that holds the cell culture in the acoustic perfusion device; anddriving the ultrasonic transducer to generate the multi-dimensional acoustic standing wave and separate any biomolecule aggregates present in the nutrient fluid stream; andseparating the biomolecules from the nutrient fluid stream.
16. The process of claim 15, wherein the biomolecules are monoclonal antibodies.
17. The process of claim 15, wherein any cells in the nutrient fluid stream are recycled back to the cell culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Ser. No. 62/563,068, filed on Sep. 26, 2017, and to U.S. Provisional Patent Application Ser. No. 62/482,681, filed on Apr. 26, 2017. This application is also a continuation-in-part of U.S. patent application Ser. No. 15/696,176, filed Sep. 5, 2017, which is a continuation of U.S. patent application Ser. No. 15/139,187 filed Apr. 26, 2016, now U.S. Pat. No. 9,752,113, which is a continuation-in-part of U.S. patent application Ser. No. 14/975,307, filed Dec. 18, 2015, now U.S. Pat. No. 9,822,333, which claims priority to U.S. Provisional Patent Application Ser. No. 62/256,952, filed on Nov. 18, 2015, and to U.S. Provisional Patent Application Ser. No. 62/243,211, filed on Oct. 19, 2015, and to U.S. Provisional Patent Application Ser. No. 62/211,057, filed on Aug. 28, 2015, and to U.S. Provisional Patent Application Ser. No. 62/093,491, filed on Dec. 18, 2014. U.S. patent application Ser. No. 14/975,307 is also a continuation-in-part of U.S. patent application Ser. No. 14/175,766, filed on Feb. 7, 2014, now U.S. Pat. No. 9,416,344, which claims priority to U.S. Provisional Patent Application Ser. No. 61/761,717, filed on Feb. 3, 2013, and is also a continuation-in-part of U.S. patent application Ser. No. 14/026,413, filed on Sep. 13, 2013, now U.S. Pat. No. 9,458,450, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61/708,641, filed on Oct. 2, 2012. U.S. patent application Ser. No. 14/026,413 is also a continuation-in-part of U.S. Ser. No. 13/844,754, filed Mar. 15, 2013, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61/611,159, filed Mar. 15, 2012, and of U.S. Provisional Patent Application Ser. No. 61/611,240, filed Mar. 15, 2012, and of U.S. Provisional Patent Application Ser. No. 61/708,641, filed Oct. 2, 2012, and of U.S. Provisional Patent Application Ser. No. 61/754,792, filed Jan. 21, 2013. These applications are incorporated herein by reference in their entireties.

US Referenced Citations (752)

Number	Name	Date	Kind
1016731	Bennis et al.	Feb 1912	A
1017524	Ferguson	Feb 1912	A
1035754	Shook	Aug 1912	A
2473971	Ross	Jun 1949	A
2667944	Crites	Feb 1954	A
3372370	Cyr	Mar 1968	A
3555311	Weber	Jan 1971	A
4055491	Porath-Furedi	Oct 1977	A
4065875	Srna	Jan 1978	A
4118649	Schwartzman et al.	Oct 1978	A
4125789	Van Schoiack	Nov 1978	A
4158629	Sawyer	Jun 1979	A
4165273	Azarov et al.	Aug 1979	A
4173725	Asai et al.	Nov 1979	A
4204096	Barcus et al.	May 1980	A
4211949	Brisken	Jul 1980	A
4254661	Kossoff et al.	Mar 1981	A
4320659	Lynnworth et al.	Mar 1982	A
4344448	Potts	Aug 1982	A
4398325	Piaget et al.	Aug 1983	A
4484907	Sheeran, Jr.	Nov 1984	A
4552669	Sekellick	Nov 1985	A
4666595	Graham	May 1987	A
4673512	Schram	Jun 1987	A
4699588	Zinn et al.	Oct 1987	A
4743361	Schram	May 1988	A
4759775	Peterson et al.	Jul 1988	A
4800316	Wang	Jan 1989	A
4821838	Chen	Apr 1989	A
4836684	Javorik et al.	Jun 1989	A
4860993	Goode	Aug 1989	A
4878210	Mitome	Oct 1989	A
4983189	Peterson et al.	Jan 1991	A
5059811	King et al.	Oct 1991	A
5062965	Bernou et al.	Nov 1991	A
5085783	Feke et al.	Feb 1992	A
5164094	Stuckart	Nov 1992	A
5225089	Benes et al.	Jul 1993	A
5371429	Manna	Dec 1994	A
5395592	Bolleman et al.	Mar 1995	A
5431817	Braatz et al.	Jul 1995	A
5443985	Lu et al.	Aug 1995	A
5452267	Spevak	Sep 1995	A
5475486	Paoli	Dec 1995	A
5484537	Whitworth	Jan 1996	A
5527460	Trampler et al.	Jun 1996	A
5560362	Sliwa, Jr. et al.	Oct 1996	A
5562823	Reeves	Oct 1996	A
5594165	Madanshetty	Jan 1997	A
5604301	Mountford et al.	Feb 1997	A
5626767	Trampler et al.	May 1997	A
5688405	Dickinson et al.	Nov 1997	A
5711888	Trampler et al.	Jan 1998	A
5831166	Kozuka et al.	Nov 1998	A
5834871	Puskas	Nov 1998	A
5844140	Seale	Dec 1998	A
5902489	Yasuda et al.	May 1999	A
5912182	Coakley et al.	Jun 1999	A
5947299	Vazquez et al.	Sep 1999	A
5951456	Scott	Sep 1999	A
6029518	Oeftering	Feb 2000	A
6090295	Raghavarao et al.	Jun 2000	A
6166231	Hoeksema	Dec 2000	A
6216538	Yasuda et al.	Apr 2001	B1
6205848	Faber et al.	Jun 2001	B1
6273262	Yasuda et al.	Aug 2001	B1
6332541	Coakley et al.	Dec 2001	B1
6391653	Letcher et al.	May 2002	B1
6475151	Koger et al.	Nov 2002	B2
6482327	Mori et al.	Nov 2002	B1
6487095	Malik et al.	Nov 2002	B1
6592821	Wada et al.	Jul 2003	B1
6641708	Becker et al.	Nov 2003	B1
6649069	DeAngelis	Nov 2003	B2
6699711	Hahn et al.	Mar 2004	B1
6727451	Fuhr et al.	Apr 2004	B1
6763722	Fjield et al.	Jul 2004	B2
6881314	Wang et al.	Apr 2005	B1
6929750	Laurell et al.	Aug 2005	B2
6936151	Lock et al.	Aug 2005	B1
7008540	Weavers et al.	Mar 2006	B1
7010979	Scott	Mar 2006	B2
7061163	Nagahara et al.	Jun 2006	B2
7081192	Wang et al.	Jul 2006	B1
7093482	Berndt	Aug 2006	B2
7108137	Lal et al.	Sep 2006	B2
7150779	Meegan, Jr.	Dec 2006	B2
7186502	Vesey	Mar 2007	B2
7191787	Redeker et al.	Mar 2007	B1
7235227	Lanza et al.	Jun 2007	B2
7322431	Ratcliff	Jan 2008	B2
7331233	Scott	Feb 2008	B2
7340957	Kaduchak et al.	Mar 2008	B2
7373805	Hawkes et al.	May 2008	B2
7541166	Belgrader et al.	Jun 2009	B2
7601267	Haake et al.	Oct 2009	B2
7673516	Janssen et al.	Mar 2010	B2
7674630	Siversson	Mar 2010	B2
7837040	Ward et al.	Nov 2010	B2
7846382	Strand et al.	Dec 2010	B2
7968049	Takahashi et al.	Jun 2011	B2
8075786	Bagajewicz	Dec 2011	B2
8080202	Takahashi et al.	Dec 2011	B2
8134705	Kaduchak et al.	Mar 2012	B2
8256076	Feller	Sep 2012	B1
8266950	Kaduchak et al.	Sep 2012	B2
8273253	Curran	Sep 2012	B2
8273302	Takahashi et al.	Sep 2012	B2
8309408	Ward et al.	Nov 2012	B2
8319398	Vivek et al.	Nov 2012	B2
8334133	Fedorov et al.	Dec 2012	B2
8387803	Thorslund et al.	Mar 2013	B2
8592204	Lipkens et al.	Nov 2013	B2
8679338	Rietman et al.	Mar 2014	B2
8691145	Dionne et al.	Apr 2014	B2
8865003	Yang	Oct 2014	B2
8873051	Kaduchak et al.	Oct 2014	B2
8889388	Wang et al.	Nov 2014	B2
9023658	Gauer et al.	May 2015	B2
9272234	Lipkens et al.	Mar 2016	B2
9357293	Claussen	May 2016	B2
9365815	Miyazaki et al.	Jun 2016	B2
9368110	Hershey et al.	Jun 2016	B1
9375662	Kambayashi et al.	Jun 2016	B2
9388363	Goodson et al.	Jul 2016	B2
9391542	Wischnewskiy	Jul 2016	B2
9403114	Kusuura	Aug 2016	B2
9410256	Dionne et al.	Aug 2016	B2
9416344	Lipkens et al.	Aug 2016	B2
9421553	Dionne et al.	Aug 2016	B2
9422328	Kennedy, III et al.	Aug 2016	B2
9457139	Ward et al.	Oct 2016	B2
9457302	Lipkens et al.	Oct 2016	B2
9458450	Lipkens et al.	Oct 2016	B2
9464303	Burke	Oct 2016	B2
9476855	Ward et al.	Oct 2016	B2
9480375	Marshall et al.	Nov 2016	B2
9480935	Mariella, Jr. et al.	Nov 2016	B2
9488621	Kaduchak et al.	Nov 2016	B2
9504780	Spain et al.	Nov 2016	B2
9512395	Lipkens et al.	Dec 2016	B2
9513205	Yu et al.	Dec 2016	B2
9514924	Morris et al.	Dec 2016	B2
9517474	Mao et al.	Dec 2016	B2
9532769	Dayton et al.	Jan 2017	B2
9533241	Presz, Jr. et al.	Jan 2017	B2
9550134	Lipkens et al.	Jan 2017	B2
9550998	Williams	Jan 2017	B2
9556271	Blumberg et al.	Jan 2017	B2
9556411	Lipkens et al.	Jan 2017	B2
9566352	Holmes et al.	Feb 2017	B2
9567559	Lipkens et al.	Feb 2017	B2
9567609	Paschon et al.	Feb 2017	B2
9572897	Bancel et al.	Feb 2017	B2
9573995	Schurpf et al.	Feb 2017	B2
9574014	Williams et al.	Feb 2017	B2
9580500	Schurpf et al.	Feb 2017	B2
9587003	Bancel et al.	Mar 2017	B2
9597357	Gregory et al.	Mar 2017	B2
9597380	Chakraborty et al.	Mar 2017	B2
9605074	Shah	Mar 2017	B2
9605266	Rossi et al.	Mar 2017	B2
9606086	Ding et al.	Mar 2017	B2
9608547	Ding et al.	Mar 2017	B2
9611465	Handa et al.	Apr 2017	B2
9616090	Conway et al.	Apr 2017	B2
9623348	McCarthy et al.	Apr 2017	B2
9629877	Cooper et al.	Apr 2017	B2
D787630	Lipkens et al.	May 2017	S
9644180	Kahvejian et al.	May 2017	B2
9645060	Fiering	May 2017	B2
9656263	Laurell et al.	May 2017	B2
9657290	Dimov et al.	May 2017	B2
9662375	Jensen et al.	May 2017	B2
9663756	Lipkens et al.	May 2017	B1
9670477	Lipkens et al.	Jun 2017	B2
9670938	Beliavsky	Jun 2017	B2
9675668	Bancel et al.	Jun 2017	B2
9675902	Lipkens et al.	Jun 2017	B2
9675906	Lipkens et al.	Jun 2017	B2
9677055	Jones et al.	Jun 2017	B2
9685155	Hershey et al.	Jun 2017	B2
9686096	Lipkens et al.	Jun 2017	B2
9688958	Kennedy, III et al.	Jun 2017	B2
9689234	Gregory et al.	Jun 2017	B2
9689802	Caseres et al.	Jun 2017	B2
9695063	Rietman et al.	Jul 2017	B2
9695442	Guschin et al.	Jul 2017	B2
9810665	Fernald et al.	Nov 2017	B2
9833763	Fernald et al.	Dec 2017	B2
9869618	Hoyos	Jan 2018	B2
9869659	Buckland et al.	Jan 2018	B2
9872900	Ciaramella et al.	Jan 2018	B2
9873126	Mao et al.	Jan 2018	B2
9873894	Conway et al.	Jan 2018	B2
9878056	Bancel et al.	Jan 2018	B2
9878536	Foresti et al.	Jan 2018	B2
9879087	DeSander et al.	Jan 2018	B2
9990297	Conway et al.	Jan 2018	B2
9907846	Morein et al.	Mar 2018	B2
9908288	Harkness	Mar 2018	B2
9909117	Kaduchak	Mar 2018	B2
9909313	Grubbs	Mar 2018	B1
9913656	Stulen	Mar 2018	B2
9913866	O'Shea et al.	Mar 2018	B2
9925277	Almarsson et al.	Mar 2018	B2
9926382	Fischer et al.	Mar 2018	B2
9937207	Gregory et al.	Apr 2018	B2
9938390	Storti et al.	Apr 2018	B2
9943599	Gehl et al.	Apr 2018	B2
9944702	Galetto	Apr 2018	B2
9944709	Galetto	Apr 2018	B2
9947431	El-Zahab et al.	Apr 2018	B2
9974898	Spain et al.	May 2018	B2
9983459	Arnold	May 2018	B2
10006052	Jarjour	Jun 2018	B2
10045913	Warner	Aug 2018	B2
10046028	Gregory	Aug 2018	B2
10046037	Weinschenk et al.	Aug 2018	B2
10047116	Morein	Aug 2018	B2
10047123	Weinschenk et al.	Aug 2018	B2
10047124	Weinschenk et al.	Aug 2018	B2
10047144	Elson et al.	Aug 2018	B2
10047365	Williams	Aug 2018	B2
10047451	Gaben	Aug 2018	B2
10047650	Abram	Aug 2018	B2
10052427	Flieg	Aug 2018	B2
10052431	Dreschel	Aug 2018	B2
10052631	Ben-Yakar et al.	Aug 2018	B2
10071148	Weinschenk	Sep 2018	B2
10071383	Dionne	Sep 2018	B2
10072062	Collingwood	Sep 2018	B2
10073098	Wong	Sep 2018	B2
10076574	Wang	Sep 2018	B2
10160786	Weinschenk et al.	Dec 2018	B1
10166255	Moriarity et al.	Jan 2019	B2
10166323	Fiering et al.	Jan 2019	B2
10167474	Rossi et al.	Jan 2019	B2
10167478	Williams	Jan 2019	B2
10190113	Forsyth	Jan 2019	B2
10190137	Zhang et al.	Jan 2019	B2
10195605	Reinbigler	Feb 2019	B2
10196608	Poirot	Feb 2019	B2
10196651	Conway et al.	Feb 2019	B2
10196652	Conway et al.	Feb 2019	B2
10201365	Boudreaux et al.	Feb 2019	B2
10201652	Dutra et al.	Feb 2019	B2
10202457	Ruiz-Opazo et al.	Feb 2019	B2
10202762	Sollohub	Feb 2019	B2
10208300	Messina et al.	Feb 2019	B2
10214013	Foresti et al.	Feb 2019	B2
10214718	Berteau et al.	Feb 2019	B2
10215760	Grove	Feb 2019	B2
10221843	Locker	Mar 2019	B2
10224015	Hsu	Mar 2019	B2
10236797	Wischnewskiy	Mar 2019	B2
10238365	Shiraishi	Mar 2019	B2
10238741	Creusot	Mar 2019	B2
10239058	Lavieu et al.	Mar 2019	B2
10239948	Jullerat et al.	Mar 2019	B2
10245064	Rhee et al.	Apr 2019	B2
10251664	Shelton et al.	Apr 2019	B2
10253296	Kahvejian et al.	Apr 2019	B2
10254212	Ward	Apr 2019	B2
10254401	Suyama	Apr 2019	B2
10258698	Hoge et al.	Apr 2019	B2
10261078	Branch	Apr 2019	B2
10272163	Laterza	Apr 2019	B2
10272412	Rubio Martinez et al.	Apr 2019	B2
10273283	Springer et al.	Apr 2019	B2
10286007	Galetto et al.	May 2019	B2
10308928	Lipkens et al.	Jun 2019	B2
10316063	Weinschenk et al.	Jun 2019	B1
10316101	Galetto et al.	Jun 2019	B2
10322949	Lipkens et al.	Jun 2019	B2
10323065	Weinschenk et al.	Jun 2019	B1
10323076	Ellsworth et al.	Jun 2019	B2
10324082	Taylor et al.	Jun 2019	B2
10326383	Stiebel et al.	Jun 2019	B2
10329531	Kahvejian et al.	Jun 2019	B2
10334390	Bakish	Jun 2019	B2
10342829	Smith et al.	Jul 2019	B2
10343187	Doyle et al.	Jul 2019	B2
10344051	Bracewell et al.	Jul 2019	B2
10344263	Kahvejian et al.	Jul 2019	B2
10350514	Lipkens et al.	Jul 2019	B2
10357766	Raghen et al.	Jul 2019	B2
20020038662	Schuler et al.	Apr 2002	A1
20020134734	Campbell et al.	Sep 2002	A1
20030015035	Kaduchak et al.	Jan 2003	A1
20030028108	Miller et al.	Feb 2003	A1
20030195496	Maguire	Oct 2003	A1
20030209500	Kock et al.	Nov 2003	A1
20030230535	Affeld et al.	Dec 2003	A1
20040016699	Bayevsky	Jan 2004	A1
20040035208	Diaz et al.	Feb 2004	A1
20040057886	Jona Zumeris et al.	Mar 2004	A1
20040112841	Scott	Jun 2004	A1
20040124155	Meegan, Jr.	Jul 2004	A1
20040149039	Cardelius	Aug 2004	A1
20050031499	Meier	Feb 2005	A1
20050055136	Hoffman	Mar 2005	A1
20050121269	Namduri	Jun 2005	A1
20050145567	Quintel et al.	Jul 2005	A1
20050196725	Fu	Sep 2005	A1
20050239198	Kunas	Oct 2005	A1
20060037915	Strand et al.	Feb 2006	A1
20060037916	Trampler	Feb 2006	A1
20060050615	Swisher	Mar 2006	A1
20070053795	Laugharn, Jr. et al.	Mar 2007	A1
20070138108	Hadfield et al.	Jun 2007	A1
20070224676	Haq	Sep 2007	A1
20070267351	Roach et al.	Nov 2007	A1
20070272618	Gou et al.	Nov 2007	A1
20070284299	Xu et al.	Dec 2007	A1
20080011693	Li et al.	Jan 2008	A1
20080035568	Huang	Feb 2008	A1
20080067128	Hoyos et al.	Mar 2008	A1
20080105625	Rosenberg et al.	May 2008	A1
20080181838	Kluck	Jul 2008	A1
20080217259	Siversson	Sep 2008	A1
20080245709	Kaduchak et al.	Oct 2008	A1
20080245745	Ward et al.	Oct 2008	A1
20080264716	Kuiper et al.	Oct 2008	A1
20080272034	Ferren et al.	Nov 2008	A1
20080272065	Johnson	Nov 2008	A1
20080316866	Goodemote et al.	Dec 2008	A1
20090029870	Ward et al.	Jan 2009	A1
20090042253	Hiller et al.	Feb 2009	A1
20090048805	Kaduchak et al.	Feb 2009	A1
20090053686	Ward et al.	Feb 2009	A1
20090087492	Johnson et al.	Apr 2009	A1
20090098027	Tabata et al.	Apr 2009	A1
20090104594	Webb	Apr 2009	A1
20090126481	Burris	May 2009	A1
20090178716	Kaduchak et al.	Jul 2009	A1
20090194420	Mariella, Jr. et al.	Aug 2009	A1
20090226994	Lemor et al.	Sep 2009	A1
20090227042	Gauer et al.	Sep 2009	A1
20090045107	Ward et al.	Dec 2009	A1
20090295505	Mohammadi et al.	Dec 2009	A1
20100000945	Gavalas	Jan 2010	A1
20100078323	Takahashi et al.	Apr 2010	A1
20100078384	Yang	Apr 2010	A1
20100124142	Laugharn et al.	May 2010	A1
20100139377	Huang et al.	Jun 2010	A1
20100192693	Mudge et al.	Aug 2010	A1
20100193407	Steinberg et al.	Aug 2010	A1
20100206818	Leong et al.	Aug 2010	A1
20100255573	Bond et al.	Oct 2010	A1
20100261918	Chianelli et al.	Oct 2010	A1
20100317088	Radaelli et al.	Dec 2010	A1
20100323342	Gonzalez Gomez et al.	Dec 2010	A1
20100330633	Walther et al.	Dec 2010	A1
20110003350	Schafran et al.	Jan 2011	A1
20110024335	Ward et al.	Feb 2011	A1
20110092726	Clarke	Apr 2011	A1
20110095225	Eckelberry et al.	Apr 2011	A1
20110123392	Dionne et al.	May 2011	A1
20110125024	Mueller	May 2011	A1
20110146678	Ruecroft et al.	Jun 2011	A1
20110154890	Holm et al.	Jun 2011	A1
20110166551	Schafer	Jul 2011	A1
20110189732	Weinand et al.	Aug 2011	A1
20110207225	Mehta et al.	Aug 2011	A1
20110245750	Lynch et al.	Oct 2011	A1
20110262990	Wang et al.	Oct 2011	A1
20110278218	Dionne et al.	Nov 2011	A1
20110281319	Swayze et al.	Nov 2011	A1
20110309020	Rietman et al.	Dec 2011	A1
20120088295	Yasuda et al.	Apr 2012	A1
20120145633	Polizzotti et al.	Jun 2012	A1
20120161903	Thomas et al.	Jun 2012	A1
20120163126	Campbell et al.	Jun 2012	A1
20120175012	Goodwin et al.	Jul 2012	A1
20120231504	Niazi	Sep 2012	A1
20120267288	Chen et al.	Oct 2012	A1
20120325727	Dionne et al.	Dec 2012	A1
20120325747	Reitman et al.	Dec 2012	A1
20120328477	Dionne et al.	Dec 2012	A1
20120329122	Lipkens et al.	Dec 2012	A1
20130017577	Arunakumari et al.	Jan 2013	A1
20130115664	Khanna et al.	May 2013	A1
20130175226	Coussios et al.	Jul 2013	A1
20130206688	El-Naas	Aug 2013	A1
20130217113	Srinivasan et al.	Aug 2013	A1
20130277316	Dutra et al.	Oct 2013	A1
20130277317	LoRicco et al.	Oct 2013	A1
20130284271	Lipkens et al.	Oct 2013	A1
20130309757	Sung-Chun Kim	Nov 2013	A1
20130316412	Schultz	Nov 2013	A1
20140011240	Lipkens et al.	Jan 2014	A1
20140017758	Kniep et al.	Jan 2014	A1
20140033808	Ding et al.	Feb 2014	A1
20140046181	Benchimol et al.	Feb 2014	A1
20140102947	Baym et al.	Apr 2014	A1
20140141413	Laugham, Jr. et al.	May 2014	A1
20140154795	Lipkens et al.	Jun 2014	A1
20140193381	Warner et al.	Jul 2014	A1
20140230912	Aider et al.	Aug 2014	A1
20140319077	Lipkens et al.	Oct 2014	A1
20140329997	Kennedy, III et al.	Nov 2014	A1
20140377834	Presz, Jr. et al.	Dec 2014	A1
20150053561	Ward et al.	Feb 2015	A1
20150060581	Santos et al.	Mar 2015	A1
20150252317	Lipkens et al.	Sep 2015	A1
20150274550	Lipkens et al.	Oct 2015	A1
20150321129	Lipkens et al.	Nov 2015	A1
20160060615	Walther et al.	Mar 2016	A1
20160089620	Lipkens et al.	Mar 2016	A1
20160102284	Lipkens et al.	Apr 2016	A1
20160121331	Kapur et al.	May 2016	A1
20160123858	Kapur et al.	May 2016	A1
20160145563	Berteau et al.	May 2016	A1
20160153249	Mitri	Jun 2016	A1
20160175198	Warner et al.	Jun 2016	A1
20160184790	Sinha et al.	Jun 2016	A1
20160202237	Zeng et al.	Jul 2016	A1
20160208213	Doyle et al.	Jul 2016	A1
20160230168	Kaduchak et al.	Aug 2016	A1
20160237110	Gilmanshin et al.	Aug 2016	A1
20160237394	Lipkens et al.	Aug 2016	A1
20160237395	Lipkens et al.	Aug 2016	A1
20160252445	Yu et al.	Sep 2016	A1
20160279540	Presz, Jr. et al.	Sep 2016	A1
20160279551	Foucault	Sep 2016	A1
20160287778	Leach et al.	Oct 2016	A1
20160312168	Pizzi	Oct 2016	A1
20160314868	El-Zahab et al.	Oct 2016	A1
20160319270	Lipkens et al.	Nov 2016	A1
20160325039	Leach et al.	Nov 2016	A1
20160325206	Presz, Jr. et al.	Nov 2016	A1
20160332159	Dual et al.	Nov 2016	A1
20160339360	Lipkens et al.	Nov 2016	A1
20160347628	Dionne et al.	Dec 2016	A1
20160355776	Lipkens et al.	Dec 2016	A1
20160361670	Lipkens et al.	Dec 2016	A1
20160363579	Lipkens et al.	Dec 2016	A1
20160368000	Dionne et al.	Dec 2016	A1
20160369236	Kennedy, III et al.	Dec 2016	A1
20160370326	Kaduchak et al.	Dec 2016	A9
20170000413	Clymer et al.	Jan 2017	A1
20170002060	Bolen et al.	Jan 2017	A1
20170002839	Burkland et al.	Jan 2017	A1
20170007679	Maeder et al.	Jan 2017	A1
20170008029	Lipkens et al.	Jan 2017	A1
20170016025	Poirot et al.	Jan 2017	A1
20170016027	Lee et al.	Jan 2017	A1
20170020926	Mata-Fink et al.	Jan 2017	A1
20170029802	Lipkens et al.	Feb 2017	A1
20170035866	Poirot et al.	Feb 2017	A1
20170037386	Jones et al.	Feb 2017	A1
20170038288	Ward et al.	Feb 2017	A1
20170042770	Warner et al.	Feb 2017	A1
20170044517	Lipkens et al.	Feb 2017	A1
20170049949	Gilmanshin et al.	Feb 2017	A1
20170056448	Glick et al.	Mar 2017	A1
20170058036	Ruiz-Opazo et al.	Mar 2017	A1
20170065636	Moriarty et al.	Mar 2017	A1
20170066015	Lipkens et al.	Mar 2017	A1
20170067021	Moriarty et al.	Mar 2017	A1
20170067022	Poirot et al.	Mar 2017	A1
20170072405	Mao et al.	Mar 2017	A1
20170073406	Schurpf et al.	Mar 2017	A1
20170073423	Juillerat et al.	Mar 2017	A1
20170073638	Campana et al.	Mar 2017	A1
20170073684	Rossi et al.	Mar 2017	A1
20170073685	Maeder et al.	Mar 2017	A1
20170080070	Weinschenk et al.	Mar 2017	A1
20170080423	Dauson et al.	Mar 2017	A1
20170081629	Lipkens et al.	Mar 2017	A1
20170088809	Lipkens et al.	Mar 2017	A1
20170088844	Williams	Mar 2017	A1
20170089826	Lin	Mar 2017	A1
20170096455	Baric et al.	Apr 2017	A1
20170107536	Zhang et al.	Apr 2017	A1
20170107539	Yu et al.	Apr 2017	A1
20170119820	Moriarty et al.	May 2017	A1
20170128523	Ghatnekar et al.	May 2017	A1
20170128857	Lipkens et al.	May 2017	A1
20170130200	Moriarty et al.	May 2017	A1
20170136168	Spain et al.	May 2017	A1
20170137491	Matheson et al.	May 2017	A1
20170137774	Lipkens et al.	May 2017	A1
20170137775	Lipkens et al.	May 2017	A1
20170137802	Lipkens et al.	May 2017	A1
20170145094	Galetto	May 2017	A1
20170151345	Shah	Jun 2017	A1
20170152502	Scharenberg et al.	Jun 2017	A1
20170152503	Scharenberg et al.	Jun 2017	A1
20170152504	Scharenberg et al.	Jun 2017	A1
20170152505	Scharenberg et al.	Jun 2017	A1
20170152527	Paschon et al.	Jun 2017	A1
20170152528	Zhang et al.	Jun 2017	A1
20170158749	Cooper	Jun 2017	A1
20170159005	Lipkens et al.	Jun 2017	A1
20170159007	Lipkens et al.	Jun 2017	A1
20170166860	Presz, Jr. et al.	Jun 2017	A1
20170166877	Bayle et al.	Jun 2017	A1
20170166878	Thanos et al.	Jun 2017	A9
20170166903	Zhang et al.	Jun 2017	A1
20170173080	Lee et al.	Jun 2017	A1
20170173128	Hoge et al.	Jun 2017	A1
20170173498	Lipkens et al.	Jun 2017	A9
20170175073	Lipkens et al.	Jun 2017	A1
20170175125	Welstead et al.	Jun 2017	A1
20170175139	Wu et al.	Jun 2017	A1
20170175144	Zhang et al.	Jun 2017	A1
20170175509	Abdel-Fattah et al.	Jun 2017	A1
20170175720	Tang et al.	Jun 2017	A1
20170183390	Springer et al.	Jun 2017	A1
20170183413	Galetto	Jun 2017	A1
20170183418	Galetto	Jun 2017	A1
20170183420	Gregory et al.	Jun 2017	A1
20170184486	Mach et al.	Jun 2017	A1
20170189450	Conway et al.	Jul 2017	A1
20170190767	Schurpf et al.	Jul 2017	A1
20170191022	Lipkens et al.	Jul 2017	A1
20170232439	Suresh et al.	Aug 2017	A1
20170291122	Lipkens et al.	Oct 2017	A1
20170298316	Kennedy, III	Oct 2017	A1
20170369865	Lipkens	Dec 2017	A1
20170374730	Flores	Dec 2017	A1
20180000311	Lipkens et al.	Jan 2018	A1
20180000870	Britt	Jan 2018	A1
20180000910	Chakraborty et al.	Jan 2018	A1
20180001011	Paschon et al.	Jan 2018	A1
20180008707	Bussmer et al.	Jan 2018	A1
20180009158	Harkness et al.	Jan 2018	A1
20180009888	Baumeister et al.	Jan 2018	A9
20180009895	Smith et al.	Jan 2018	A1
20180010085	Lipkens et al.	Jan 2018	A1
20180010758	Mochizuki	Jan 2018	A1
20180014846	Rhee	Jan 2018	A1
20180015128	Britt	Jan 2018	A1
20180015392	Lipkens et al.	Jan 2018	A1
20180016570	Lipkens et al.	Jan 2018	A1
20180016572	Tang	Jan 2018	A1
20180020295	Pander et al.	Jan 2018	A1
20180021379	Galetto et al.	Jan 2018	A1
20180022798	Shurpf et al.	Jan 2018	A1
20180028683	Wong et al.	Feb 2018	A1
20180043473	Helvajian et al.	Feb 2018	A1
20180049767	Gee et al.	Feb 2018	A1
20180051089	Galettto et al.	Feb 2018	A1
20180051265	Cooper	Feb 2018	A1
20180052095	Cumbo et al.	Feb 2018	A1
20180052147	Zeng	Feb 2018	A1
20180055529	Messerly et al.	Mar 2018	A1
20180055530	Messerly et al.	Mar 2018	A1
20180055531	Messerly et al.	Mar 2018	A1
20180055532	Messerly et al.	Mar 2018	A1
20180055997	Cabrera et al.	Mar 2018	A1
20180056095	Messerly et al.	Mar 2018	A1
20180057810	Zhang et al.	Mar 2018	A1
20180058439	Locke et al.	Mar 2018	A1
20180066223	Lim	Mar 2018	A1
20180066224	Lipkens et al.	Mar 2018	A1
20180066242	Zhang	Mar 2018	A1
20180067044	Kaduchak et al.	Mar 2018	A1
20180071363	Ghatnekar et al.	Mar 2018	A1
20180071981	Collino et al.	Mar 2018	A1
20180078268	Messerly	Mar 2018	A1
20180080026	Rossi et al.	Mar 2018	A1
20180085743	Yavorsky et al.	Mar 2018	A1
20180087044	Lipkens et al.	Mar 2018	A1
20180088083	Sinha	Mar 2018	A1
20180092338	Hering et al.	Apr 2018	A1
20180092660	Ethicon	Apr 2018	A1
20180094022	Bracewell et al.	Apr 2018	A1
20180095067	Huff et al.	Apr 2018	A1
20180098785	Price et al.	Apr 2018	A1
20180100134	Lim	Apr 2018	A1
20180100204	O'Shea et al.	Apr 2018	A1
20180119174	Scharenberg et al.	May 2018	A1
20180130491	Mathur	May 2018	A1
20180136167	Smith et al.	May 2018	A1
20180140758	Vincent et al.	May 2018	A1
20180143138	Shreve et al.	May 2018	A1
20180147245	O'Shea et al.	May 2018	A1
20180147576	Lavieu et al.	May 2018	A1
20180148740	Conway	May 2018	A1
20180148763	Shimada et al.	May 2018	A1
20180153946	Alemany et al.	Jun 2018	A1
20180155716	Zhang et al.	Jun 2018	A1
20180157107	Koyama	Jun 2018	A1
20180161775	Kapur et al.	Jun 2018	A1
20180177490	Shiraishi	Jun 2018	A1
20180178184	Holland	Jun 2018	A1
20180180610	Taha	Jun 2018	A1
20180223256	Kim	Aug 2018	A1
20180223273	Lipkens	Aug 2018	A1
20180223439	Lipkens	Aug 2018	A1
20180230433	Kokkaliaris	Aug 2018	A1
20180231555	Davis	Aug 2018	A1
20180236103	Friedland	Aug 2018	A1
20180236280	Lipkens Bart et al.	Aug 2018	A1
20180237533	Juillerat et al.	Aug 2018	A1
20180237768	Reik	Aug 2018	A1
20180237798	Duchateau et al.	Aug 2018	A1
20180243382	Wang	Aug 2018	A1
20180243665	Lacki	Aug 2018	A1
20180244722	Stickel	Aug 2018	A1
20180246103	Lipkens	Aug 2018	A1
20180249688	Ayares	Sep 2018	A1
20180250424	Cotta-Ramusino	Sep 2018	A1
20180251723	Murthy	Sep 2018	A1
20180251770	Friedland	Sep 2018	A1
20180255751	Regev	Sep 2018	A1
20180256922	Mittelstein	Sep 2018	A1
20180257042	Hester	Sep 2018	A1
20180257076	Weitz	Sep 2018	A1
20180258160	Lai	Sep 2018	A1
20180258955	Levasseur	Sep 2018	A1
20180258957	Levasseur	Sep 2018	A1
20180296954	Trampler	Oct 2018	A1
20180353614	Peters	Dec 2018	A1
20180361053	Fiering et al.	Dec 2018	A1
20180361383	Kapur et al.	Dec 2018	A1
20180361384	Kapur et al.	Dec 2018	A1
20180369816	Ai	Dec 2018	A1
20180371418	Yang et al.	Dec 2018	A1
20190000932	Martini	Jan 2019	A1
20190000933	Martini	Jan 2019	A1
20190000947	Weinschenk et al.	Jan 2019	A1
20190000959	Ciaramella et al.	Jan 2019	A1
20190000982	Wang et al.	Jan 2019	A1
20190002497	Stickel et al.	Jan 2019	A1
20190002504	Weinschenk et al.	Jan 2019	A1
20190002561	Galetto	Jan 2019	A1
20190002573	Galetto	Jan 2019	A1
20190002578	Brayshaw et al.	Jan 2019	A1
20190002589	Bardroff et al.	Jan 2019	A1
20190002890	Martini et al.	Jan 2019	A1
20190004052	Herd et al.	Jan 2019	A1
20190008943	Poolman et al.	Jan 2019	A1
20190008948	Ciaramella et al.	Jan 2019	A1
20190010190	Weinschenk et al.	Jan 2019	A1
20190010192	Binder et al.	Jan 2019	A1
20190010471	Zhang et al.	Jan 2019	A1
20190010495	Boitano et al.	Jan 2019	A1
20190010514	Poirot et al.	Jan 2019	A1
20190011407	Lipkens et al.	Jan 2019	A9
20190015501	Ciaramella et al.	Jan 2019	A1
20190016753	Jang et al.	Jan 2019	A1
20190016767	Shah	Jan 2019	A1
20190016781	Bolen	Jan 2019	A1
20190021729	Smith et al.	Jan 2019	A1
20190022019	Martini	Jan 2019	A1
20190023577	Feng	Jan 2019	A1
20190024114	Bauer	Jan 2019	A1
20190030073	Kalayoglu	Jan 2019	A1
20190030151	Jones et al.	Jan 2019	A1
20190030533	Shachar et al.	Jan 2019	A1
20190031780	Eavarone et al.	Jan 2019	A1
20190031999	Suresh et al.	Jan 2019	A1
20190032036	Zhang	Jan 2019	A1
20190032052	Zhang	Jan 2019	A1
20190036152	Gaben et al.	Jan 2019	A1
20190036172	Gaben et al.	Jan 2019	A1
20190006036	Moriarty et al.	Feb 2019	A1
20190038671	Fan et al.	Feb 2019	A1
20190039060	Chien et al.	Feb 2019	A1
20190040099	Brellisford et al.	Feb 2019	A1
20190040117	Elson et al.	Feb 2019	A1
20190040414	Wu	Feb 2019	A1
20190046986	Yuan et al.	Feb 2019	A1
20190048060	Conway et al.	Feb 2019	A1
20190048061	Smeland et al.	Feb 2019	A1
20190054112	Gregoire	Feb 2019	A1
20190054119	Alma et al.	Feb 2019	A1
20190054122	Moriarity et al.	Feb 2019	A1
20190055286	Walz et al.	Feb 2019	A1
20190055509	Meacham et al.	Feb 2019	A1
20190056302	Berezin et al.	Feb 2019	A1
20190056399	Wong et al.	Feb 2019	A1
20190060363	Moriarity et al.	Feb 2019	A1
20190062185	Amouzadeh et al.	Feb 2019	A1
20190062690	Tostoes et al.	Feb 2019	A1
20190062735	Welstead et al.	Feb 2019	A1
20190064146	Glick	Feb 2019	A1
20190067554	Karrai et al.	Feb 2019	A1
20190070233	Yeung	Mar 2019	A1
20190070528	Luthe	Mar 2019	A1
20190071695	Wagner	Mar 2019	A1
20190071717	Zhang et al.	Mar 2019	A1
20190076473	Nguyen	Mar 2019	A1
20190076769	Meacham et al.	Mar 2019	A1
20190078133	Cavanagh et al.	Mar 2019	A1
20190079070	Shiffman et al.	Mar 2019	A1
20190083533	Soon-Shiong et al.	Mar 2019	A1
20190085067	Schurpf et al.	Mar 2019	A1
20190085082	Bicknell	Mar 2019	A1
20190085381	Neely et al.	Mar 2019	A1
20190090900	Rhee et al.	Mar 2019	A1
20190091683	Baudoin et al.	Mar 2019	A1
20190092794	Rubio Martinez et al.	Mar 2019	A1
20190092865	Ruiz-Opazo	Mar 2019	A1
20190093097	Madison et al.	Mar 2019	A1
20190094185	Athanassiadis	Mar 2019	A1
20190101541	Wandall et al.	Apr 2019	A1
20190105043	Jaworek et al.	Apr 2019	A1
20190106039	Winton et al.	Apr 2019	A1
20190106710	Zhang et al.	Apr 2019	A1
20190107420	Kincel	Apr 2019	A1
20190111480	Barbati et al.	Apr 2019	A1
20190119387	Brett	Apr 2019	A1
20190119701	Liang et al.	Apr 2019	A1
20190125839	Frederick et al.	May 2019	A1
20190127685	Fattah et al.	May 2019	A1
20190133633	Neurohr et al.	May 2019	A1
20190135942	Duthe et al.	May 2019	A1
20190136261	Conway	May 2019	A1
20190143013	Vincent et al.	May 2019	A1
20190153027	Natarajan et al.	May 2019	A1
20190153106	Ruiz-Opazo et al.	May 2019	A1
20190160463	Ai et al.	May 2019	A1
20190161540	Gearing et al.	May 2019	A1
20190167722	Soon-Shiong et al.	Jun 2019	A1
20190169233	Weinschenk et al.	Jun 2019	A1
20190169597	Astrakhan et al.	Jun 2019	A1
20190169639	Zhang et al.	Jun 2019	A1
20190170745	Hu et al.	Jun 2019	A1
20190173129	Gaben et al.	Jun 2019	A1
20190175517	Martini et al.	Jun 2019	A1
20190175651	Lee et al.	Jun 2019	A1
20190176150	Kapur et al.	Jun 2019	A1
20190177368	Weinschenk et al.	Jun 2019	A1
20190177369	Weinschenk et al.	Jun 2019	A1
20190183931	Alice et al.	Jun 2019	A1
20190184035	Jarjour et al.	Jun 2019	A1
20190184312	Liu et al.	Jun 2019	A1
20190184326	Davis et al.	Jun 2019	A1
20190185860	Kim et al.	Jun 2019	A1
20190191252	Lipkens et al.	Jun 2019	A1
20190192653	Hoge	Jun 2019	A1
20190194049	Lindemann	Jun 2019	A1
20190194087	Larsen	Jun 2019	A1
20190194340	Emtage et al.	Jun 2019	A1
20190194617	Emtage et al.	Jun 2019	A1
20190199312	Dasgupta et al.	Jun 2019	A1
20190199322	Dasgupta et al.	Jun 2019	A1
20190201041	Kimball et al.	Jul 2019	A1
20190201047	Yates et al.	Jul 2019	A1
20190201048	Nguyen et al.	Jul 2019	A1
20190209616	Galetto et al.	Jul 2019	A1
20190211109	Peshwa et al.	Jul 2019	A1
20190218254	Weinschenk et al.	Jul 2019	A1
20190218602	Zhang et al.	Jul 2019	A1
20190225694	Zien et al.	Jul 2019	A1
20190225990	Adbudl-Manan et al.	Jul 2019	A1
20190290201	Boudreaux et al.	Jul 2019	A1

Foreign Referenced Citations (403)

Number	Date	Country
2002236405	Sep 2002	AU
105 087 788	Nov 2015	CN
104722106	Apr 2016	CN
30 27 433	Feb 1982	DE
32 18 488	Nov 1983	DE
196 48 519	Jun 1998	DE
103 19 467	Jul 2004	DE
10 2008 006 501	Sep 2008	DE
10 2014 206 823	Oct 2015	DE
0 292 470	Nov 1988	EP
0 167 406	Jul 1991	EP
0 641 606	Mar 1995	EP
1 175 931	Jan 2002	EP
1 254 669	Nov 2002	EP
1 308 724	May 2003	EP
2 209 545	Jul 2010	EP
270152	Jan 2018	EP
2419511	Jan 2018	EP
3068888	Jan 2018	EP
3257600	Jan 2018	EP
3274453	Jan 2018	EP
3274454	Jan 2018	EP
3275894	Jan 2018	EP
278108	Feb 2018	EP
3279315	Feb 2018	EP
3286214	Feb 2018	EP
2289535	Mar 2018	EP
2545068	Mar 2018	EP
2675540	Mar 2018	EP
2750683	Mar 2018	EP
2796102	Mar 2018	EP
3066201	Mar 2018	EP
3066998	Mar 2018	EP
3107552	Mar 2018	EP
3288660	Mar 2018	EP
3288683	Mar 2018	EP
3289362	Mar 2018	EP
3291842	Mar 2018	EP
3291852	Mar 2018	EP
3292142	Mar 2018	EP
3292195	Mar 2018	EP
3292515	Mar 2018	EP
3294343	Mar 2018	EP
3294764	Mar 2018	EP
3294857	Mar 2018	EP
3294871	Mar 2018	EP
3294888	Mar 2018	EP
3294896	Mar 2018	EP
3296302	Mar 2018	EP
3297740	Mar 2018	EP
3298046	Mar 2018	EP
3164488	Apr 2018	EP
3301115	Apr 2018	EP
3302783	Apr 2018	EP
3302789	Apr 2018	EP
3303558	Apr 2018	EP
3306310	Apr 2018	EP
2675901	May 2018	EP
2956772	May 2018	EP
3323444	May 2018	EP
3324996	May 2018	EP
3327127	May 2018	EP
3337819	Jun 2018	EP
2772196	Aug 2018	EP
2882091	Aug 2018	EP
2910568	Aug 2018	EP
3265805	Aug 2018	EP
3359676	Aug 2018	EP
3360955	Aug 2018	EP
3361252	Aug 2018	EP
3362102	Aug 2018	EP
3363456	Aug 2018	EP
3363813	Aug 2018	EP
3365062	Aug 2018	EP
3365095	Aug 2018	EP
3365441	Aug 2018	EP
3365447	Aug 2018	EP
3366696	Aug 2018	EP
3367118	Aug 2018	EP
2931892	Sep 2018	EP
3019606	Sep 2018	EP
3089800	Sep 2018	EP
3123534	Sep 2018	EP
3368528	Sep 2018	EP
3368670	Sep 2018	EP
3371295	Sep 2018	EP
3372813	Sep 2018	EP
3372814	Sep 2018	EP
2535355	Jan 2019	EP
2922902	Jan 2019	EP
3004338	Jan 2019	EP
3421975	Jan 2019	EP
3423092	Jan 2019	EP
3423580	Jan 2019	EP
3425386	Jan 2019	EP
3426271	Jan 2019	EP
3426372	Jan 2019	EP
3426375	Jan 2019	EP
3426690	Jan 2019	EP
3427815	Jan 2019	EP
3429753	Jan 2019	EP
3430050	Jan 2019	EP
3430134	Jan 2019	EP
3430146	Jan 2019	EP
3430463	Jan 2019	EP
3433363	Jan 2019	EP
3433366	Jan 2019	EP
3434774	Jan 2019	EP
3434776	Jan 2019	EP
2598533	Feb 2019	EP
2691422	Feb 2019	EP
2925431	Feb 2019	EP
3170185	Feb 2019	EP
3436030	Feb 2019	EP
3436196	Feb 2019	EP
3436575	Feb 2019	EP
3436579	Feb 2019	EP
3437740	Feb 2019	EP
3439698	Feb 2019	EP
3440191	Feb 2019	EP
3441468	Feb 2019	EP
3442598	Feb 2019	EP
3443002	Feb 2019	EP
3443084	Feb 2019	EP
3445407	Feb 2019	EP
3445848	Feb 2019	EP
3445853	Feb 2019	EP
3445856	Feb 2019	EP
2694091	Mar 2019	EP
3080260	Mar 2019	EP
3448291	Mar 2019	EP
3448995	Mar 2019	EP
3449850	Mar 2019	EP
3452133	Mar 2019	EP
3452499	Mar 2019	EP
3453406	Mar 2019	EP
3456339	Mar 2019	EP
3458081	Mar 2019	EP
3458083	Mar 2019	EP
3458104	Mar 2019	EP
3458105	Mar 2019	EP
3458107	Mar 2019	EP
3458108	Mar 2019	EP
3458590	Mar 2019	EP
3066115	Apr 2019	EP
3119807	Apr 2019	EP
3186281	Apr 2019	EP
3361252	Apr 2019	EP
3463433	Apr 2019	EP
3463660	Apr 2019	EP
3464198	Apr 2019	EP
3464594	Apr 2019	EP
3467276	Apr 2019	EP
3467491	Apr 2019	EP
3468225	Apr 2019	EP
3468351	Apr 2019	EP
3468594	Apr 2019	EP
3470089	Apr 2019	EP
3470519	Apr 2019	EP
3471621	Apr 2019	EP
3473707	Apr 2019	EP
2546144	May 2019	EP
3311588	May 2019	EP
3474904	May 2019	EP
3475307	May 2019	EP
3481361	May 2019	EP
3481867	May 2019	EP
2412817	Jun 2019	EP
3490562	Jun 2019	EP
3490574	Jun 2019	EP
3490694	Jun 2019	EP
3490712	Jun 2019	EP
3490801	Jun 2019	EP
3491124	Jun 2019	EP
3491126	Jun 2019	EP
3493836	Jun 2019	EP
3493907	Jun 2019	EP
3495376	Jun 2019	EP
3495811	Jun 2019	EP
3498846	Jun 2019	EP
3500244	Jun 2019	EP
3500271	Jun 2019	EP
3500297	Jun 2019	EP
3500659	Jun 2019	EP
3500696	Jun 2019	EP
3501619	Jun 2019	EP
3502137	Jun 2019	EP
3502253	Jun 2019	EP
3EP510161	Jul 2019	EP
2680877	Jul 2019	EP
2996789	Jul 2019	EP
3068535	Jul 2019	EP
3140319	Jul 2019	EP
3277333	Jul 2019	EP
3505098	Jul 2019	EP
3511342	Jul 2019	EP
3511420	Jul 2019	EP
3512540	Jul 2019	EP
2 420 510	May 2006	GB
HO2-290266	Nov 1990	JP
9-136090	May 1997	JP
H11-090110	Apr 1999	JP
2005-249267	Dec 2005	JP
1442486	Sep 2014	KR
2037327	Jun 1995	RU
94015846	Jun 1996	RU
2067079	Sep 1996	RU
2085933	Jul 1997	RU
629496	Oct 1978	SU
WO 198707178	Dec 1987	WO
WO 8911899	Dec 1989	WO
WO 9005008	Mar 1990	WO
WO 9501214	Jan 1995	WO
WO 9734643	Sep 1997	WO
WO 1998017373	Apr 1998	WO
WO 9850133	Nov 1998	WO
WO 0041794	Jul 2000	WO
WO 02072234	Sep 2002	WO
WO 02072236	Sep 2002	WO
WO 03089567	Oct 2003	WO
WO 2004079716	Sep 2004	WO
WO 2009063198	May 2009	WO
WO 2009111276	Sep 2009	WO
WO 2009144709	Dec 2009	WO
WO 2010024753	Apr 2010	WO
WO 2010040394	Apr 2010	WO
WO 2011023949	Mar 2011	WO
WO 2011025890	Mar 2011	WO
WO 2011027146	Mar 2011	WO
WO 2011130321	Oct 2011	WO
WO 2011131947	Oct 2011	WO
WO 2011161463	Dec 2011	WO
WO 2013043044	Mar 2013	WO
WO 2013043046	Mar 2013	WO
WO 2013043297	Mar 2013	WO
WO 2013030691	Mar 2013	WO
WO 2013049623	Apr 2013	WO
WO 2013055517	Apr 2013	WO
WO 2013138797	Sep 2013	WO
WO 2013148376	Oct 2013	WO
WO 2013159014	Oct 2013	WO
WO 2014014941	Jan 2014	WO
WO 2014029505	Feb 2014	WO
WO 2014035457	Mar 2014	WO
WO 2014046605	Mar 2014	WO
WO 2014055219	Apr 2014	WO
WO 2014124306	Aug 2014	WO
WO 2014153651	Oct 2014	WO
WO 2014165177	Oct 2014	WO
WO 2015006730	Jan 2015	WO
WO 2015102528	Jul 2015	WO
WO 2016004398	Jan 2016	WO
WO 2016124542	Aug 2016	WO
WO 2016176663	Nov 2016	WO
WO 2016209082	Dec 2016	WO
WO 2017011519	Jan 2017	WO
WO 2017021543	Feb 2017	WO
WO 2017041102	Mar 2017	WO
WO 20174201349	Nov 2017	WO
WO 2017218714	Dec 2017	WO
WO 2018009894	Jan 2018	WO
WO 2018002036	Jan 2018	WO
WO 2018005873	Jan 2018	WO
WO 2018013558	Jan 2018	WO
WO 2018013629	Jan 2018	WO
WO 2018013840	Jan 2018	WO
WO2018014174	Jan 2018	WO
WO2018015561	Jan 2018	WO
WO 20180011600	Jan 2018	WO
WO2018018958	Feb 2018	WO
WO2018021920	Feb 2018	WO
WO2018022158	Feb 2018	WO
WO 2018022513	Feb 2018	WO
WO2018022619	Feb 2018	WO
WO2018022651	Feb 2018	WO
WO2018022930	Feb 2018	WO
WO2018023114	Feb 2018	WO
WO2018024639	Feb 2018	WO
WO2018026644	Feb 2018	WO
WO2018026941	Feb 2018	WO
WO2018028647	Feb 2018	WO
WO 2018034343	Feb 2018	WO
WO2018034885	Feb 2018	WO
WO 2018035141	Feb 2018	WO
WO 2018035423	Feb 2018	WO
WO20180202691	Feb 2018	WO
WO2018034655	Mar 2018	WO
WO 2018038711	Mar 2018	WO
WO 2018039119	Mar 2018	WO
WO 2018039407	Mar 2018	WO
WO 2018039408	Mar 2018	WO
WO 2018039410	Mar 2018	WO
WO 2018039412	Mar 2018	WO
WO 2018039515	Mar 2018	WO
WO 2018045284	Mar 2018	WO
WO 2018049226	Mar 2018	WO
WO 2018050738	Mar 2018	WO
WO 2018057825	Mar 2018	WO
WO 2018063291	Apr 2018	WO
WO 2018058275	May 2018	WO
WO 2018081476	May 2018	WO
WO 2018091879	May 2018	WO
WO2018094244	May 2018	WO
WO 20180814701	May 2018	WO
WO 2018098671	Jun 2018	WO
WO 2018102752	Jun 2018	WO
WO 2018106163	Jun 2018	WO
WO 2018112145	Jun 2018	WO
WO 2018112335	Jun 2018	WO
WO 2018138385	Aug 2018	WO
WO 2018140573	Aug 2018	WO
WO 2018140845	Aug 2018	WO
WO 2018142364	Aug 2018	WO
WO 2018151811	Aug 2018	WO
WO 2018151823	Aug 2018	WO
WO 2018153772	Aug 2018	WO
WO 2018160548	Sep 2018	WO
WO 2018160909	Sep 2018	WO
WO 2018160993	Sep 2018	WO
WO 2018161017	Sep 2018	WO
WO 2018161026	Sep 2018	WO
WO 2018161038	Sep 2018	WO
WO 2018161905	Sep 2018	WO
WO 2018163183	Sep 2018	WO
WO2018227286	Dec 2018	WO
WO2018229612	Dec 2018	WO
WO2018231990	Dec 2018	WO
WO2018232045	Dec 2018	WO
WO2018232131	Dec 2018	WO
WO2018234421	Dec 2018	WO
WO2018235228	Dec 2018	WO
WO2018236708	Dec 2018	WO
WO2018237201	Dec 2018	WO
WO2018237239	Dec 2018	WO
WO2018183966	Jan 2019	WO
WO2019002551	Jan 2019	WO
WO2019002633	Jan 2019	WO
WO2019005155	Jan 2019	WO
WO2019005866	Jan 2019	WO
WO2019005871	Jan 2019	WO
WO2019006418	Jan 2019	WO
WO2019007869	Jan 2019	WO
WO2019008335	Jan 2019	WO
WO2019010422	Jan 2019	WO
WO2019018423	Jan 2019	WO
WO2019018491	Jan 2019	WO
WO2019018796	Jan 2019	WO
WO2019022671	Jan 2019	WO
WO2019023523	Jan 2019	WO
WO2019025661	Feb 2019	WO
WO2019025984	Feb 2019	WO
WO2019028172	Feb 2019	WO
WO2019032675	Feb 2019	WO
WO2019036382	Feb 2019	WO
WO209048639	Mar 2019	WO
WO2019041344	Mar 2019	WO
WO2019046450	Mar 2019	WO
WO2019048666	Mar 2019	WO
WO2019051106	Mar 2019	WO
WO2019051255	Mar 2019	WO
WO2019051278	Mar 2019	WO
WO2019051316	Mar 2019	WO
WO2019051355	Mar 2019	WO
WO2019055697	Mar 2019	WO
WO2019055817	Mar 2019	WO
WO2019055896	Mar 2019	WO
WO2019056015	Mar 2019	WO
WO2019057774	Mar 2019	WO
WO2019058321	Mar 2019	WO
WO2019058326	Mar 2019	WO
WO2019060253	Mar 2019	WO
WO2019060425	Mar 2019	WO
WO2019060779	Mar 2019	WO
WO2019067015	Apr 2019	WO
WO2019069101	Apr 2019	WO
WO2019070541	Apr 2019	WO
WO2019070974	Apr 2019	WO
WO2019072889	Apr 2019	WO
WO2019075409	Apr 2019	WO
WO2019079497	Apr 2019	WO
WO2019079819	Apr 2019	WO
WO2019080898	May 2019	WO
WO2019081521	May 2019	WO
WO2019094360	May 2019	WO
WO2019098839	May 2019	WO
WO2019099619	May 2019	WO
WO2019099736	May 2019	WO
WO2019099949	May 2019	WO
WO2019101691	May 2019	WO
WO2019101956	May 2019	WO
WO201912655	Jun 2019	WO
WO2018215686	Jun 2019	WO
WO2019111250	Jun 2019	WO
WO2019113310	Jun 2019	WO
WO2019118475	Jun 2019	WO
WO2019118885	Jun 2019	WO
WO2019126329	Jun 2019	WO
WO2019126558	Jun 2019	WO
WO2019126724	Jun 2019	WO
WO2019134007	Jul 2019	WO
WO2019135843	Jul 2019	WO
WO2019136288	Jul 2019	WO
WO 2018231759	Dec 2019	WO

Non-Patent Literature Citations (102)

Entry
Alvarez et al.; Shock Waves, vol. 17, No. 6, pp. 441-447, 2008.
Augustsson et al., Acoustophoretic microfluidic chip for sequential elution of surface bound molecules from beads or cells, Biomicrofluidics, Sep. 2012, 6(3):34115.
Benes et al.; Ultrasonic Separation of Suspended Particles, 2001 IEEE Ultrasonics Symposium; Oct. 7-10, 2001; pp. 649-659; Atlanta, Georgia.
Castilho et al.; Animal Cell Technology: From Biopharmaceuticals to Gene Therapy; 11—Animal Cell Separation; 2008.
Castro; Tunable gap and quantum quench dynamics in bilayer graphene; Jul. 13, 2010; Mathematica Summer School.
Chitale et al.; Understanding the Fluid Dynamics Associated with Macro Scale Ultrasonic Separators; Proceedings of Meetings on Acoustics, May 2015.
Cravotto et al.; Ultrasonics Sonochemistry, vol. 15, No. 5, pp. 898-902, 2008.
Ding, X et al., “Cell Separation Using Tilted-Angle Standing Surface Acoustic Waves”, Proceedings of the National Academy of Sciences, Sep. 9, 2014, vol. 111, No. 36, pp. 12992-12997, See abstract; p. 12994, left col. p. 12995, left col. figure 1-3 and 6.
Ensminger et al; Ultrasonics Fundamentals, Technologies, and Applications; 2011.
Evander et al; Acoustofluidics 20: Applications in acoustic trapping, Lab Chip, 2012,12,4667-4676.
Evander et al; Acoustiofluidics 5: Building microfluidic acoustic resonators, Lab Chip, 2012, 12, 684.
Gallego-Juarez et al; “Piezoelectric ceramic and ultrasonic transducers”; Journal of Physics E: Scientific Instruments. 1989.
Ganguly et al; Essential Physics for Radiology and Imaging; Academic Publishers, Jan. 2016.
Garcia-Lopez, et al; Enhanced Acoustic Separation of Oil-Water Emulsion in Resonant Cavities. The Open Acoustics Journal. 2008, vol. 1, pp. 66-71.
Greenhall et al; Dynamic behavior of microscale particles controlled by standing bulk acoustic waves; Applied Physics Letters, 105, 144105 (2014).
Grenvall et al.; Concurrent Isolation of Lymphocytes and Granulocytes Using Prefocused Free Flow Acoustophoresis; Analytical Chemistry; vol. 87; pp. 5596-5604; 2015.
Gorenflo et al; Characterization and Optimization of Acoustic Filter Performance by Experimental DesignMethodology (whole document).
Gor'Koy et al; On the Forces Acting on a Small Particle in an Acoustical Field in an Ideal Fluid; Soviet Physics Doklady, vol. 6, p. 773.
Higginson et al.; Tunable optics derived from nonlinear acoustic effects; Journal of Applied Physics; vol. 95; No. 10; pp. 5896-5904; 2004.
Hill et al.; Ultrasonic Particle Manipulation; Microfluidic Technologies for Miniaturized Analysis Systems, Jan. 2007, pp. 359-378.
Ilinskii et al.; Acoustic Radiation Force on a Sphere in Tissue; AIP Conference Proceedings; 2012.
Jin et al; Pharmaceutical Engineering; Jan. 2015; vol. 35 No. 1.
Kuznetsova et al.; Microparticle concentration in short path length ultrasonic resonators: Roles of radiation pressure and acoustic streaming; Journal of the Acoustical Society of America, American Institute of Physics for the Acoustical Society of America, vol. 116, No. 4, Oct. 1, 2004, pp. 1956-1966, DOI: 1.1121/1.1785831.
Latt et al.; Ultrasound-membrane hybrid processes for enhancement of filtration properties; Ultrasonics sonochemistry 13.4 (2006): 321-328.
Li et al.; Electromechanical behavior of PZT-brass unimorphs; J. Am. Ceram. Soc. vol. 82; No. 7; pp. 1733-1740, 1999.
Lipkens et al.; The effect of frequency sweeping and fluid flow on particle trajectories in ultrasonic standing waves; IEEE Sensors Journal, vol. 8, No. 6, pp. 667-677, 2008.
Lipkens et al.; Frequency sweeping and fluid flow effects on particle trajectories in ultrasonic standing waves; Acoustics 08, Paris, Jun. 29-Jul. 4, 2008.
Lipkens et al.; Prediction and measurement of particle velocities in ultrasonic standing waves; J. Acoust. Soc. Am., 124 No. 4, pp. 2492 (A) 2008.
Lipkens et al.; Separation of micron-sized particles in macro-scale cavities by ultrasonic standing waves; Presented at the International Congress on Ultrasonics, Santiago; Jan. 11-17, 2009.
Lipkens et al.; Separation of bacterial spores from flowering water in macro-scale cavities by ultrasonic standing waves; submitted/uploaded to http://arxiv.org/abs/1006.5467 on Jun. 28, 2010.
Lipkens et al., Macro-scale acoustophoretic separation of lipid particles from red blood cells, The Journal of the Acoustical Society of America, vol. 133, Jun. 2, 2013, p. 045017, XP055162509, New York, NY.
Meribout et al.; An Industrial-Prototype Acoustic Array for Real-Time Emulsion Layer Detection in Oil Storage Tanks; IEEE Sensors Journal, vol. 9, No. 12, Dec. 2009.
Musiak et al.; Design of a Control System for Acoustophoretic Separation, 2013 IEEE 56th International Midwest Symposium on Circuits and Systems (MWSCAS), Aug. 2013, pp. 1120-1123.
National Science Foundation, “Catalyzing Commercialization: putting sound to work for challenging separations”, CEP, Sep. 2015, p. 14.
Nilsson et al.; Review of cell and particle trapping in microfluidic systems; Department of Measurement Technology and Industrial Electrical Engineering, Div. of Nanobiotechnology, Lund University, P.O. Box 118. Lund, Sweden, Analytica Chimica Acta 649, Jul. 14, 2009, pp. 141-157.
Nienow et al.; A potentially scalable method for the harvesting of hMSCs from microcarriers; Biochemical Engineering Journal 85 (2014) 79-88.
Pangu et al.; Droplet transport and coalescence kinetics in emulsions subjected to acoustic fields; Ultrasonics 46, pp. 289-302 (2007).
Phys. Org. “Engineers develop revolutionary nanotech water desalination membrane.” Nov. 6, 2006. http://phys.org/news82047372.html.
Ponomarenko et al.; Density of states and zero Landau level probed through capacitance of graphene; Nature Nanotechnology Letters, Jul. 5, 2009; DOI: 10.1038/NNANO.2009.177.
“Proceedings of the Acoustics 2012 Nantes Conference,” Apr. 23-27, 2012, Nantes, France, pp. 278-282.
Ryll et al.; Performance of Small-Scale CHO Perfusion Cultures Using an Acoustic Cell Filtration Device for Cell Retention: Characterization of Separation Efficiency and Impact of Perfusion on Product Quality; Biotechnology and Bioengineering; vol. 69; Iss. 4; pp. 440-449; Aug. 2000.
Seymour et al, J. Chem. Edu., 1990, 67(9), p. 763, published Sep. 1990.
Shitizu et al; “A Tutorial Review on Bioprocessing Systems Engineering” (whole document).
Volpin et al.; Mesh simplification with smooth surface reconstruction; Computer-Aided Design; vol. 30; No. 11; 1998.
Wang et al.; Retention and Viability Characteristics of Mammalian Cells in an Acoustically Driven Polymer Mesh; Biotechnol. Prog. 2004, pp. 384-387 (2004).
Wicklund et al.; Ultrasonic Manipulation of Single Cells; Methods in Molecular Biology; vol. 853; pp. 1777-1196; 2012.
Woodside et al; Acoustic Force Distribution in Resonators for Ultrasonic Particle Separation; Biotechnology Laboratory and Dept of Chemical and Bio-Resource Engineering, University of British Columbia, Sep. 1998, vol. 44, No. 9.
Zhanqiu et al ;Culture Conditions and Types of Growth Media for Mammalian Cells (whole document).
Annex to Form PCT/ISA/206—Communication Relating to the Results of the Partial International Search Report dated Jul. 18, 2013.
European Search Report of European Application No. 11769474.5 dated Sep. 5, 2013.
European Search Report of European Application No. 11796470.0 dated Jan. 5, 2016.
European Search Report of European Application No. 13760840.2, dated Feb. 4, 2016.
European Search Report of European Application No. 13721179.3 dated Mar. 23, 2016.
European Search Report for European Application No. 14749278.9 dated Jan. 13, 2017.
Extended European Search Report for European Application No. EP 12833859.7 dated Mar. 20, 2015.
Extended European Search Report for European Application No. EP 14787587.6 dated Jan. 2, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2011/032181 dated Dec. 20, 2011.
International Search Report and Written Opinion for International Application No. PCT/US2011/040787 dated Feb. 27, 2012.
International Search Report and Written Opinion for International Application No. PCT/US2012/051804 dated Nov. 16, 2012.
International Search Report and Written Opinion for International Application No. PCT/US2013/037404 dated Jun. 21, 2013.
International Search Report and Written Opinion for International Application No. PCT/US2013/032705 dated Jul. 26, 2013.
International Search Report and Written Opinion for International Application No. PCT/US2013/050729 dated Sep. 25, 2013.
International Search Report and Written Opinion for International Application No. PCT/US2013/059640 dated Feb. 18, 2014.
International Search Report and Written Opinion for International Application No. PCT/US2014/015382 dated May 6, 2014.
International Search Report and Written Opinion for International Application No. PCT/US2014/035557 dated Aug. 27, 2014.
International Search Report and Written Opinion for International Application No. PCT/US2014/043930 dated Oct. 22, 2014.
International Search Report and Written Opinion for International Application No. PCT/US2014/046412 dated Oct. 27, 2014.
International Search Report and Written Opinion for International Application No. PCT/US2014/064088 dated Jan. 30, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/010595 dated Apr. 15, 2015.
European Search Report of European Application No. 12825592.4 dated Apr. 28, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/019755 dated May 4, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/030009 dated Jul. 30, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/039125 dated Sep. 30, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/053200 dated Dec. 28, 2015.
International Search Report and Written Opinion for International Application No. PCT/US2015/066884, dated Mar. 22, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/024082 dated Jun. 27, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/031357 dated Jul. 26, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/038233 dated Sep. 26, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2015/024365 dated Oct. 13, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/041664 dated Oct. 18, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/044586 dated Oct. 21, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/049088 dated Nov. 28, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/050415 dated Nov. 28, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2016/037104 dated Dec. 16, 2016.
International Search Report and Written Opinion for International Application No. PCT/US2017/015197 dated Apr. 3, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2017/015450 dated Apr. 10, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2016/047217 dated Apr. 11, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2016/048243 dated Apr. 20, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2017/017788 dated May 8, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2017/030903 dated Jul. 19, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2017/025108 dated Jul. 20, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2017/031425 dated Aug. 30, 2017.
Sony New Release: <http://www.sony.net/SonyInfo/News/Press/201010/10-137E/index.html>.
International Search Report and Written Opinion for International Application No. PCT/US2017/031425 dated Oct. 23, 2017.
International Search Report and Written Opinion for International Application No. PCT/US2018/026617, dated Jul. 4, 2018.
International Search Report and Written Opinion for International Application No. PCT/US2018/31267, dated Aug. 1, 2018.
European Search Report of European Application No. 15847217.5 dated Oct. 15, 2018.
International Search Report and Written Opinion for International Application No. PCT/US2017/057485, dated Apr. 23, 2019.
International Search Report and Written Opinion for International Application No. PCT/US18/65839, dated May 16, 2019.
International Search Report and Written Opinion for International Application No. PCT/US19/12950, dated May 24, 2019.
International Search Report and Written Opinion for International Application No. PCT/US18/63698, dated May 27, 2019.
International Search Report and Written Opinion for International Application No. PCT/US19/21492, dated Jun. 25, 2019.

Related Publications (1)

	Number	Date	Country
	20180298323 A1	Oct 2018	US

Provisional Applications (11)

Number	Date	Country
62563068	Sep 2017	US
62482681	Apr 2017	US
62256952	Nov 2015	US
62243211	Oct 2015	US
62211057	Aug 2015	US
62093491	Dec 2014	US
61611159	Mar 2012	US
61611240	Mar 2012	US
61754792	Jan 2013	US
61708641	Oct 2012	US
61761717	Feb 2013	US

Continuations (1)

	Number	Date	Country
Parent	15139187	Apr 2016	US
Child	15696176		US

Continuation in Parts (5)

	Number	Date	Country
Parent	15696176	Sep 2017	US
Child	15947770		US
Parent	14975307	Dec 2015	US
Child	15139187		US
Parent	14175766	Feb 2014	US
Child	14975307		US
Parent	14026413	Sep 2013	US
Child	14175766		US
Parent	13844754	Mar 2013	US
Child	14026413		US

Acoustic perfusion devices

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