CN109069954A - Multi-drop sound swimsuit is set - Google Patents

Abstract

The multi-drop sound swimsuit for continuously separating second fluid or particle from main fluid is disclosed to set.Also disclose the method that the multi-drop sound swimsuit is set that operates.System is set comprising the multiple sound swimsuits fluidly connected that are one another in series, and each sound swimsuit sets the ultrasonic transducer and reflector that comprising flow chamber, can generate multidimensional sound standing wave.The system can also include pump and flowmeter.

Description

Multi-drop sound swimsuit is set

Cross reference to related applications

The U.S. Provisional Patent Application No. 62/322,262 submitted this application claims on April 14th, 2016 and 2016 3 The priority for the U.S. Provisional Patent Application Serial No. 62/307,489 that the moon is submitted on the 12nd.The disclosure of these applications is led to herein Incorporated is crossed to be included in herein.

Background technique

Need for particle/fluid mixture to be separated into the ability of its independent component in numerous applications.Physical size exclusion Filter can for this purpose, and wherein particle capture is on filter and fluid flow filter.The example of physics filter includes by cutting Those of operated to filter, deep stream filtering, hollow fibre filtering and centrifugation is flowed through.However, can be with the work of physics filter Complicated.For example, filter capacity reduces when physics filter is filled.Equally, caused using this kind of filter and periodically stopped removing The particle captured on filter and/or acquisition or removing.

Sound swimming (acoustophoresis) is with high sound intensity wavelength-division from particle, and does not use film or physical size exclusion Filter.The high-intensitive standing wave of known sound can apply pressure on particle.Standing wave has pressure curve, last to show " static " It is motionless.Pressure Spectrum in standing wave contains the net zero pressure region at its node and antinode.According to the density and compressibility of particle, The node or antinode of standing wave can be captured in.However, conventional sound swimsuit sets limited efficacy, this is attributed to a number of factors, including heat Occur, the limitation of fluid flow and different types of material can not be captured.There is still a need for fluid dynamic changed with improved It is set into sound swimsuit.

Summary of the invention

(acoustophoretic) system this disclosure relates to which multi-drop sound is swum, can be for realizing from particle/fluid mixture Middle separation particle.In certain embodiments, multi-drop sound swimming system as described herein can be used together with bioreactor, such as In instillation process, to generate biomolecule such as recombinant protein or monoclonal antibody, and from the cell culture in bioreactor Object separates these desired products.The increased new blend of granule density is obtained, or particle itself can be obtained, or can produce and contain There is the clear fluid of biomolecule such as recombinant protein or monoclonal antibody.In embodiment particularly, the particle is raw Object cell, such as Chinese hamster ovary (CHO) cell, NS0 hybridoma, baby hamster kidney (BHK) cell or people's cell；Lymph Cell such as T cell (such as regulatory T-cell (Treg), Jurkat T cell), B cell or NK cell；Its precursor, such as peripheral blood list A nucleus (PBMCs)；Algae or other plant cells, bacterium, virus or microcarrier.Sound swimming system described herein can be can Extension, it is generally used for about 20 × 10⁶Cell/mL- about 50 × 10⁶Cell/mL cell density.Several inhomogeneities are described herein The sound swimming system of type.

Specifically, the disclosure provide novel, expansible, potential disposable technology for based on sound swimming clarified cell Culture.Sound wave separation (AWS) technology is related to using all-bottom sound educational level (acoustic force) to generate across runner (flow Channel multidimensional sound standing wave).Fluid media (medium) from bioreactor enters runner, as cell passes through multidimensional sound standing wave, It is captured or is suspended by acoustic force.Pressure node and beginning together agglomerating, most end form of the cell migration being captured to standing wave At the cluster for being large enough to precipitate from suspension by gravity.Display turbidity is permeated from system to be remarkably decreased, and is reduced and be used for second level Clear region specification, the second level clarification are controlled using in-depth filtration and subsequent filter for biological load.

Disclosed herein is multi-drop sound swimming system, such as two standing postures, three-station type and four station type sound swimming system.These multi-drops Sound swimming system can be included in the filter " queue " comprising depth filter, sterile chlorine, centrifuge and affinity chromatographic column, thus by from Middle separation recombinant protein carrys out purifying cells culture.It is produced in the different stations (stage) of the system in this kind of system Multidimensional sound standing wave frequency/power it is changeable various concentration or various sizes of material are sent to it is subsequent in filter " queue " Filtration step, to improve the efficiency of clarification process, this is by effectively managing the material processed in filter " queue " each step It carries out.It receives in this way, acoustics filter (i.e. the different stations of system) can be used to manage the interior next stage of filter " queue " for it The material of middle following process.

The exemplary multi-drop sound swimming system of the disclosure includes that three or more the sound swimsuits being fluidly coupled to each other are set.Sound Swimsuit, which is set, to be connected in series, and connect so that each sound swimsuit is set to set at least another sound swimsuit.In some instances, the sound swimsuit Setting can be with or without common connection.Sound swimsuit, which is set, can connect recovery passage usually with salvage material after isolation.Each sound swimming Device may include flow chamber, have at least one entrance and at least one outlet；It is coupled flow chamber (as being located on its wall) extremely A few ultrasonic transducer, energy converter includes piezoelectric material, can be driven by driving signal to generate multidimensional in flow chamber and stay Wave；(such as it is located on the wall on energy converter opposite) in the reflector at least one ultrasonic transducer opposite.In specific embodiment party In case, there are four sound swimsuits to set.In some embodiments, it is set using two or more sound swimsuit.

The sound swimsuit of multi-drop sound swimming system, which is set, to flow connection each other by pipeline.The sound swimsuit of multi-drop sound swimming system Set can physical connection directly with one another, level-one is on another grade of top, or side by side.

In certain embodiments, the sound swimsuit, which is set, is configured to generate multidimensional sound standing wave, all has one each other Frequency in the order of magnitude.In certain embodiments, the sound swimsuit, which is set, is configured to generate multidimensional sound standing wave, all has that This different frequency.In certain buildings, each multidimensional sound standing wave generates acoustic radiation force, has the axial direction in the same order of magnitude Component and cross component force.

Multi-drop sound swimming system can further comprise the feed pump of the upstream for the most upstream set positioned at least three sound swimsuits The pump (such as peristaltic pump) in downstream is set with each sound swimsuit.That is, multi-drop sound swimming system can include the charging of the first sound swimming device upstream Pump, the first sound swimsuit set with rising tone swimsuit set between the first pump, rising tone swimsuit set with third sound swimsuit set between the Two pumps, third sound swimsuit sets downstream and falling tone swimsuit set (in the presence of) the third pump of upstream and falling tone swimsuit set downstream The 4th pump.

Multi-drop sound swimming system can also include feed flow meter and the swimming of each sound of the upstream for the most upstream set positioned at sound swimsuit The flowmeter in device downstream.That is, multi-drop sound swimming system can include the feed flow meter of the first sound swimming device upstream, the swimming of the first sound Device and rising tone swimsuit set between first flowmeter, rising tone swimsuit set with third sound swimsuit set between second flow Meter, third sound swimsuit sets the third flowmeter in downstream and falling tone swimming device upstream and falling tone swimsuit sets the 4th of downstream Flowmeter.

Each sound swimsuit, which is set, can have at least one to be located at the dump diffuser into the inlet of flow chamber.Each sound swimsuit is set It can further comprise the port below its at least one ultrasonic transducer.This port can be used to put back into from sound swimsuit contracture from Material.

In certain embodiments, the entrance of the multi-drop sound swimming system can connect with the outlet fluid of bioreactor It connects.Multi-drop sound swimming system can further comprise at least one that the downstream of most downstream is set positioned at three or more sound swimsuits Cascade filtration station (in-line filtration stage).Cascade filtration station can be selected from depth filter, sterile filters, centrifugation Other filtering techniques known to machine, affinity chromatographic column or protein purification art.

Also disclose the method for continuously separating second fluid or particle from main fluid with multi-drop sound swimming system.Multi-drop sound All multi-dimensional standing-waves that each sound swimsuit of swimming system sets middle generation can have different frequency or identical frequency, or can have same number Frequency in magnitude.

In certain embodiments of method disclosed herein, it is super to drive the first sound swimsuit to set to send driving signal Sonic transducer, so that the first multidimensional sound standing wave is generated wherein, in this way, at least partly second fluid or particle are continuously captured in One standing wave, remaining mixture go successively to sound swimsuit and set second part.Another driving signal is sent to drive rising tone swimsuit to set Ultrasonic transducer, so that the second multidimensional sound standing wave is generated wherein, in this way, at least part second fluid or particle are continuously captureed It obtains in the second standing wave, remaining mixture goes successively to sound swimsuit and sets Part III.Another driving signal is sent to drive third sound The ultrasonic transducer that swimsuit is set, so that third multidimensional sound standing wave is generated wherein, in this way, at least part second fluid or particle It continuously is captured in third standing wave, remaining mixture goes successively to sound swimsuit and sets Part IV.Another driving signal is sent to drive The ultrasonic transducer that falling tone swimsuit is set, so that the 4th multidimensional sound standing wave is generated wherein, in this way, at least part second fluid Or particle is continuously captured in the 4th standing wave.

Be sent to driving signal that each sound swimsuit is set can it is different from each other and can implement the particle size for particular range with Particle in selective filter mixture.In certain embodiments, the voltage signal set at least one sound swimsuit is At least 50V.In certain embodiments, the voltage signal (i.e. AC) set to each sound swimsuit is 50V- about 60V (rms).? In certain embodiments, the voltage signal for setting most downstream to sound swimsuit is 40V- about 60V.

Second fluid or particle can be Chinese hamster ovary (CHO) cell, NS0 hybridoma, baby hamster kidney (BHK) Cell or people's cell；T cell, B cell or NK cell；Peripheral blood mononuclear cells (PBMC)；Algae；Plant cell, bacterium, Virus or microcarrier.

In certain embodiments of presently disclosed method, sends voltage signal and changed with the ultrasound for driving each sound swimsuit to set Energy device, so that interior generation multidimensional sound standing wave is set in each sound swimsuit, in this way, at least partly second fluid or particle are continuously captured in respectively Standing wave, the frequency and power of each multidimensional sound standing wave change size and/or concentration that particle in mixture is managed with selectivity, described The direct downstream part that the selected part arrival sound swimsuit that particle is set by sound swimsuit is set.

These and other non-limiting feature particularly describes as follows.

Brief Description Of Drawings

It is being briefly described for attached drawing below, provides its purpose for being used to illustrate exemplary embodiment disclosed herein, and It is not used in the purpose for limiting it.

Fig. 1 illustrates the exemplary embodiment of the swimming system of multi-drop sound described in the disclosure.Sound swimming system includes passing through pipe Four sound that road is connected to each other, which are swum, stands.

Fig. 2A illustrates the physically connected to each other four sound swimming device/station for the swimming system of multi-drop sound described in the disclosure Exemplary embodiment.

Fig. 2 B illustrates the separate views of one of Fig. 2A sound swimming device/station.

Fig. 2 C is the cross-sectional view of one of Fig. 2A sound swimming device/station.Described device includes that opposite flow pumps diffuser enters Mouthful, it generates and flows symmetrical and more evenly speed.

Fig. 3 illustrate Fig. 1 four station type sound swimming system four sound swimsuits set in three.Sound swimsuit, which is set, to be connected to each other and passes through Pipeline pumping therebetween.The figure also shows the fluid that the cell culture medium entrainment of each device is continuously flowed into through its entrance, sinks simultaneously The cell of the cohesion/fall off through its port/formed sediment is settled out each device, and residual fluid, which is exported to flow out from the device, enters subsequent dress It sets.

Fig. 4 is the cross-sectional side view of amplification, illustrates the fluid for flowing through flow chamber vertically downward and entering separation sonic wave region In cell culture medium.

Fig. 5 is the continuation of Fig. 4, shows that the cell in fluid is captured in sound standing wave at Standing pressure waves node, this is because The cross component force of acoustic radiation force.

Fig. 6 is the continuation of Fig. 5, and the cell aggregation that display is captured in sound standing wave forms cell cluster, due to buoyancy reduction/weight Stress effect increases and is settled out suspension and falls on flow chamber bottom.

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

Fig. 8 is the cross-sectional view of disclosure ultrasonic transducer.There are air gaps in energy converter, and support layer or wear-resisting shield is not present Plate.

Fig. 9 is the cross-sectional view of disclosure ultrasonic transducer.There are air gaps in energy converter, and there are support layers and wear-resisting shield Plate.

Figure 10 is the figure of electrical impedance amplitude versus frequency (rectangular (square) energy converter for driving in different frequency).

Figure 11 A illustrates configuring for seven capture lines in Figure 10 peak amplitude for the direction orthogonal with fluid stream.

Figure 11 B is the perspective view for illustrating separator.Show direction of fluid flow and capture line.

Figure 11 C is along the view of the fluid inlet of Figure 11 B fluid flow direction (arrow 251), and display can capture particle Standing wave capture node.

Figure 11 D is to configure the view obtained along arrow 253 shown in Figure 11 B to capture line by energy converter planar.

Figure 12 shows the relationship of acoustic radiation force, buoyancy and Stokes drag and granular size.Trunnion axis is with micron (μ M) it counts and vertical axis is in terms of newton (N).

The performance figure of Figure 13 is shown in the pressure for having the system for the disclosure set there are three concatenated sound swimsuit in the first experiment Drop.Y-axis is the pressure drop in terms of psig, from 0 to 30, is divided into 5.X-axis be by rise/every square metre in terms of volume flux capacity (volumetric throughput capacity) is divided into 20 from 0 to 140.

Figure 14 is the group of two figures, this two figures show the performance of the second experimental system.The y-axis of bottom diagram is decline percentage Than being divided into 10% from 0% to 100%.X-axis is test time in minutes, from 0 to 80, is divided into 10.Top The y-axis of figure is decline percentage, and is logarithmic form, and being worth is 0,90% and 99.0%.

Figure 15 is the group of two figures, this two figures show the performance of third experimental system.The y-axis of bottom diagram is decline percentage Than being divided into 10% from 0% to 100%.X-axis is test time in minutes, from 0 to 80, is divided into 10.Top The y-axis of figure is decline percentage, and is logarithmic form, and being worth is 0,90% and 99.0%.

Figure 16 is the performance for showing the 5th experimental four station type sound swimming every station of system or the figure of volume flux (VT).Along figure The line at the top on the right represents DOHC filter pressure drop.The dotted line extended from DOHC pressure drop line represents DOHC filter turbidity.Along figure The line of the bottom on the right represents XOHC filter pressure drop.The dotted line extended from XOHC pressure drop line represents XOHC filter turbidity.

Figure 17 be show the 5th four station type sound swimming experimental system it is all 4 station performances or volume carrying capacity (VT) it is another Figure.Overall presure drop is represented along the line at the top on figure the right.DOHC filter pressure drop is represented along second line of slave few top on figure the right. The dotted line extended from DOHC pressure drop line represents DOHC filter turbidity.XOHC filter pressure drop is represented along the line of the bottom on figure the right. The dotted line extended from XOHC pressure drop line represents XOHC filter turbidity.

Figure 18 illustrates performance of the 6th experimental system compared to deep flow filtering (DFF).DFF performance is shown in the line at top simultaneously Usable floor area is 35m²And 11.6m²Filter to realize required performance.Along the line of bottom, it is combined three-station type AWS system and depth Layer filter makes filter area used be reduced to 10.2m², being combined four station type AWS system and depth filter subtracts filter area used It is few to arrive only 4.5m²。

Figure 19 illustrates the exemplary setting of the swimming system of the three-station type sound as experimental system, some performances for the disclosure Test.

Figure 20 illustrates the exemplary setting of the swimming system of the four station type sound as experimental system, many performances for the disclosure Test.

Figure 21 shows the turbidity reduction vs charging PCM of Figure 20 system.Y-axis is turbidity reduction percentage, from 0% to 100%, Between be divided into 10%.X-axis is that charging PCM percentage is divided into 5% from 0% to 15%.There is the biaxial generation of big circular data point Table uses [Q_F/(Q_S/Q_F)]=experimental run of [0.6x/10%].There is the dotted line of big triangular data points to represent and uses [Q_F/ (Q_S/Q_F)]=experimental run of [1.0x/10%].There is the line of box-X data point to represent and uses [Q_F/(Q_S/Q_F)]=[1.0x/ 20%] the first experimental run.There are three axis of big diamond data points to represent and uses [Q_F/(Q_S/Q_F)]=[1.0x/20%] Second experimental run.There is the dotted line of big square data points to represent and uses [Q_F/(Q_S/Q_F)] the experiment of=third of [1.0x/20%] Operating.Optimal (i.e. highest) turbidity reduction appears in~5-6% charging PCM.

Figure 22 shows the system (Q of Figure 20_S/Q_F=20%) decline for three different factors (TCD, turbidity, PCM) Percentage vs feed flow rate (Q_F).Y-axis is that decline percentage is divided into 10% from 0% to 100%.X-axis is feed flow rate (x Flowing), from 0 to 3.5, it is divided into 0.5.

Figure 23 shows the system (Q of Figure 20_S/Q_F=20%) turbidity reduction vs flow-rate ratio.Y-axis is turbidity reduction percentage, From 0% to 100%, it is divided into 10%.X-axis is flow-rate ratio (Q_S1/Q_S2), from 0 to 5, it is divided into 1.The line at top represents 0.6x stream Dynamic, the line of bottom represents 1.3x flowing.

Figure 24 shows the PCM vs solid velocity of the system of Figure 20.Y-axis be packed cell group (packed cell mass, PCM) percentage is divided into 5% from 0% to 50%.X-axis is flow velocity (Q_S1/Q_S2), from 0 to 5, it is divided into 1.Along figure the right The line at top represent solid station (solids stage) 2.Solid station 1 is represented along the medium line on figure the right.Along figure the right Bottom line represent penetrate object.

Figure 25 shows the turbidity reduction vs flow-rate ratio of the system of Figure 20.Y-axis is turbidity reduction percentage, from 0% to 100%, it is divided into 10%.X-axis is flow-rate ratio (Q_S1/Q_S2), from 0% to 25%, it is divided into 5%.

Figure 26 shows the decline %vs charging for three different factors (TCD, turbidity, PCM) of the system of Figure 20 PCM.Y-axis is that decline percentage is divided into 10% from 0% to 100%.X-axis is charging PCM percentage, from 0% to 12%, It is divided into 2%.Diamond data points represent standardized turbidity reduction.Square data points represent TCD reduction.Triangular data points generation Table PCM is reduced.

Figure 27 shows the decline %vs charging cell viability of the system of Figure 20.Y-axis be decline percentage, from 0% to 100%, it is divided into 10%.X-axis is that charging cell viability percentage is divided into 20% from 0% to 100%.Equally, diamond shape number Strong point represents standardized turbidity reduction, and square data points represent TCD reduction, and triangular data points represent PCM reduction.

Figure 28 is another figure for showing the performance of the 7th experimental system.The performance respectively stood with time showing illustrates performance not It degenerates.Y-axis is that TCD reduces percentage, from 0% to 100%, is divided into 10%.Along x-axis, the column of Far Left group is represented 2 hours The data obtained afterwards, the data that second group of column obtains after representing 4 hours from left to right, what the column of middle groups obtained after representing 6 hours Data, the data that second group of column obtains after representing 9 hours from right to left, the number that the column of rightmost group obtains after representing 12.5 hours According to.In each group column, the column of the leftmost side represents station 1, and the second column represents station 2 from left to right, and the second column represents station 3, the column of the rightmost side from right to left Represent station 4.

Figure 29 is another figure for showing the performance of the 7th experimental system.The figure shows the depth for independent depth filter (DFF) Layer filter area (left hand view) is to the comparison between the equivalent area (right part of flg) for the DFF for using sound swimming filter to obtain same capacity As a result.In both images, y-axis is with m²The specified area of the depth filter of meter is divided into 10 from 0 to 60.In two images In, along x-axis, the column of Far Left group represents the first user, and the column of middle groups represents second user, and the column of rightmost group represents Three users.In the column of each group, the column of the leftmost side represents station 1, and the second column represents station 2 from left to right, and the second column represents station 3 from right to left, most right Side column (in the presence of) represent station 4.

Figure 30 is another figure for showing the performance of the 7th experimental system.Y-axis be cell density decline percentage, from 0% to 100%, it is divided into 10%.X-axis is that the TCD for cell viability reduces drop percentage.Along x-axis, the column of the leftmost side is represented 22 × 10 when cell viability is 82%⁶Cell/mL cell density, from left to right second column represent 22 when cell viability is 80% × 10⁶Cell/mL cell density, the column of third represents 22 × 10 when cell viability is 64% from left to right⁶Cell/mL cell density, The column of third represents 40 × 10 when cell viability is 62% from right to left⁶Cell/mL cell density, second column represents cell from right to left 70 × 10 when vigor is 60%⁶Cell/mL cell density, the column of the rightmost side represent 100 × 10 when cell viability is 70%⁶Carefully Born of the same parents/mL cell density.

Detailed description of the invention

With reference to desired embodiment and wherein included exemplary detailed description below, can be more easily to understand The disclosure.It is described below in claims later, quotes some terms, should be defined as having following meanings.

Although having used specific term in the following description, for the sake of clarity, these terms are meant only to be related to selecting For illustrating the specific structure of the embodiment of attached drawing, and it is not intended to define or limit disclosure range.In following attached drawing and In being described below, it should be understood that same Numeral name refers to the component of identical function.Further, it should be understood that attached drawing does not weigh to scale Amount.

Singular " one (a) ", " one (an) " and " described " include plural referents, are clearly advised unless the context otherwise It is fixed.

Such as it is used for specification and claims, term "comprising" may include embodiment " consist of " and " basic By ... form ".The term as used herein "comprising", " comprising ", " with (having) ", " having (has) ", " energy ", " containing " Mean open conjunction, term or word with its variant, it is required that there is specified component/step and allow that there are other groups Point/step.However, this description should be interpreted that also describe composition or technique for " being made of the component/step enumerated " and " being made of substantially the component/step enumerated " allows there is only specified ingredients/step, and resulting possible What impurity, and exclude other component/steps.

Numerical value is understood to include the identical numerical value when being reduced to the effective digital of same number and is different from setting The numerical value of value, the difference are less than to determine described value, the experiment of the conventional measurement technology of the type of description described herein Error.

All ranges disclosed herein includes cited endpoint and independent can combine (for example, the model of " from 2 grams to 10 gram " It encloses comprising 2 grams and 10 grams of terminal and all medians).

By the value of one or more terms such as " about " and " generally " modification, specified exact value can be not limited to.Approximate language Speech can correspond to the instrument precision for measured value.Modifier " about " should also be regarded as what disclosure was defined by 2 terminal absolute values Range.For example, statement also discloses the range " from 2 to 4 " " from about 2 to about 4 ".

It should be noted that many terms used herein are relative terms.For example, term " top " and " lower part " are relative to that This position, i.e., in assigned direction, upper element is located at height more higher than lower component, if but device overturning, these terms It can change.Term " entrance " and " outlet " are related to given structure, enter institute as fluid flows through entrance relative to its fluid is flowed through It states structure and flows through outlet and leave the structure.The direction of multiple components is flowed through in term " upstream " and " downstream " relative to fluid, I.e. fluid flows through upstream component, then flows through downstream components.It should be noted that first assembly can be described as the second component in ring Upstream and downstream.

Term "horizontal" and " vertical " are used to indicate the direction relative to absolute reference (i.e. ground level).Term " upward " and " downward " is also relative to absolute reference；It flows up always opposite with terrestrial gravitation.

This application involves " the same orders of magnitude ".If larger number is at least 1 and less than 10 divided by the quotient compared with decimal fractions Value, then 2 numbers are the same order of magnitude.

The application also relates to " sharp " angles.For disclosure purpose, term " sharp " refers to 0 ° -90 ° of angle, excludes 0 ° and 90 °.

The propulsion of fed-batch cell culture leads to up to 50 × 10⁶Cell/mL higher cell density and > 5g/L Product titre.Disposable technology is applied with turning in cell culture, the efficiency for seeking cell harvest and clarification stage improves To generate the cell culture fluid (HCCF) of harvest, for capturing chromatography and subsequent downstream processing.The progress of continuous processing (evolution) it is also possible to the factor of efficiency consideration, wherein preferential continuous feed HCCF, HCCF can be used for being loaded directly into even Continuous multicolumn captures chromatographic step.Existing cell culture clarification is carried out using centrifugation or in-depth filtration, usually in batch mode It operates and in the process using the charging of bulk storage or HCCF.

The sound swimming isolation technics of the disclosure uses ultrasonic standing wave to capture second level phase material in primary fluid stream, including fluid And/or particle, that is, keep its static.Capture second level phase material is the important difference of front method therewith, in method before, particle Track is only changed by acoustic radiation force effect.The sound field scattering of particle leads to three dimensional acoustic radiation power, is used as three-dimensionally capturing field. Combine the three dimensional acoustic radiation power generated with ultrasonic standing wave and is known as three-dimensional or multi-dimensional standing-wave in the disclosure.When particle is relative to wavelength Hour, acoustic radiation force is proportional to particle volume (cube of such as radius).Itself and frequency and acoustics coefficient of correlation (contrast Factor) proportional.It is also proportional to sound energy (squares of such as sound pressure amplitudes).Harmonic wave is excited, the sine space of power becomes Settling position in amount driving particle to standing wave.When the acoustic radiation force being applied on particle is better than fluid drag power and buoyancy and again When the joint effect of power, particle can be captured in sound standing wave field.This capture causes captured particulate condensation, aggregation and/or merging. In addition, second level intergranular power such as Bjerkness power assists particle aggregation.The particle for overweighting main fluid (i.e. than main fluid densification) passes through The gravitational settling of enhancing separates.

The specific application that sound swimsuit is set is processing bioreactor material.Need the expressed material from fluid stream Material filters as much as possible or all cells and cell fragment.Expressed material is made of biomolecule, such as recombinant protein or Monoclonal antibody, and be desired product to be recycled.Swum by using sound, separate cell and cell fragment it is highly effective and So that expression product loss is minimum.This isolation technics is one compared to current filter technique (in-depth filtration, tangential flow filtration, centrifugation) Kind is improved, and the current filter technique is shown in limited efficacy under high-cell density, wherein expressed material is in itself filter bed Interior loss may material caused by up to 5% bioreactor.Use mammalian cell cultures (including Chinese storehouse Mouse ovary (CHO) cell, NS0 hybridoma, baby hamster kidney (BHK) cell and people's cell), it is proved to be generation/expression and uses In the recombinant protein of current pharmacy and a kind of highly effective method of monoclonal antibody.By sound swim filtering mammalian cell and Mammalian cell fragment helps that bioreactor yield is significantly increased.The sound swimming skills art that is discussed herein allow cell and/or It expresses material recovery.

In the sound swimming skills art being discussed herein, coefficient of correlation is the difference between particle and fluid itself compressibility and density It is different.These properties are the features of particle Yu fluid itself.Most cells type is higher in the presence of culture medium therein is suspended from than it Density and lower compressibility, so that the acoustics coefficient of correlation between cell and culture medium is positive value.Axial acoustic radiation force (ARF) driving has the cell of positive coefficient of correlation to pressure pitch plane, and driving has the cell of negative coefficient of correlation or other particles to arrive Pressure antinode plane.Cell is captured in radial direction or the cross component help of acoustic radiation force.In some instances, the radial direction of ARF or side It is greater than the joint effect of fluid drag power and gravity to component.

As cell is assembled in standing wave node, the physics for cell culture medium also occur is cleaned, wherein more many cells are with connecing It touches the cell being maintained in standing wave and is captured.This phenomenon or phenomenon combination, help to separate cell and cell culture medium.Institute The biomolecule of expression is maintained in nutrition liquid stream (i.e. cell culture medium).

It is desirable that ultrasonic transducer generates three-dimensional or multidimensional sound standing wave in a fluid, to suspended particulate apply lateral force with Increase the particle capture ability of standing wave.Ultrasonic transducer publication in acoustics pertinent literature provides typical consequence, indicates plane Or the one-dimensional aborning lateral force of sound wave two orders of magnitude smaller than axial force.In contrast to this, technology disclosed in the present application mentions For the lateral force with the same order of magnitude of axial force.

Arbitrary phasing and/or difference or a variety of ultrasonic transducers of variable-frequency drive may also be used.A variety of energy converters can be with It works to separate the material in fluid stream, and is mutually out of phase and/or is operated with different or variable frequency.Alternatively or in addition, The single ultrasonic transducer for being divided into oldered array can be operated, so that some array components can be with other array component out-phase.

Three-dimensional (3-D) or multidimensional sound standing wave are produced from one or more piezoelectric transducers, and wherein energy converter is electricity or machine Tool excitation, so that it is migrated with more excitation modes.The type of the wave thus generated can be characterized as complex wave, and displacement spectra is similar In symmetrical (leaky symmetric) (also referred to as compressing or stretching, extension) Lamb wave of leakage.Wave is leaky wave, because its width is penetrated Enter water layer, this causes to generate sound standing wave in water layer.Neutral axis pair of the displacement spectra of symmetrical Lamb wave relative to piezoelectric element Claim, this causes multiple standing waves to generate in the space 3-D.By this wave producing method, if piezoelectric transducer is with " piston " mould Formula (wherein only generating single, plane standing wave) excitation, then generate higher lateral capture power.Therefore, to piezoelectric transducer with Same input power, 3-D or multidimensional sound standing wave can have higher lateral capture power, it may be possible to the single sound that piston mode generates Standing wave up to and more than 10 times.

Due to acoustic streaming, the frequency for needing to adjust standing wave or voltage amplitude there may come a time when.This kind of adjusting can be adjusted by amplitude And/or frequency modulation is completed.The duty ratio that standing wave is propagated can also be used for realizing certain results of trapping material.In other words, may be used not Same frequency opens and closes acoustic beam to realize required result.

In some applications, implementable multiple sound swimming cell filtering devices are to clarify bioreactor cell culture and incite somebody to action Biomolecule/albumen is separated with the cell for expressing them.This disclosure relates to be made of modular assembly sound swimming system and this The kit of generic module.Each module is collaboratively engaged with other modules, is then reversibly separated.Kit and module allow to use The different of sound swimming system needed for family obtains configure to provide improved particle precipitating and improved particle and separate with fluid.Letter speech It, the particle for being suspended from main fluid can be separated with inducing with fluid itself by generating multiple energy converters of a variety of standing waves.Also can make Improved fluid dynamics are provided with modular assembly, increases particle and is separated with fluid.

Multiple separating stations are considered using a variety of standing waves from multiple ultrasonic transducers.For example, super by four For sound station-reflector in the process at (i.e. four sound swimming stations), first stop (and its standing wave) collects a certain amount of particle, second station (and its standing wave) collects the particle by first stop, and third station (and its standing wave) collects the particle for passing through the first and second stations, the Collect the particle for passing through the first, second, and third station in four stations (and its standing wave).When particle/fluid is than high (i.e. large-volume particulate), This building can be separating capacity that is useful, and reaching any upstream transducer.This building can be used for having it is bimodal or The particle of larger size distribution, wherein each energy converter can be implemented for capturing the particle within the scope of certain size.

Fig. 1 illustrates the first exemplary embodiment of multi-drop sound swimming system 2000.System 2000 includes the first sound swimsuit Set 2010, rising tone swimsuit set 2020, third sound swimsuit set 2030 and falling tone swimsuit set 2040.Each device may be regarded as not in unison Swimming or filter station.In this respect, system 2000 is four station type sound swimming system, because each sound swimsuit, which is set, to be constructed as described herein, Including energy converter-reflector pair, to generate at least one multidimensional sound standing wave in each device.In certain embodiments, each dress The acoustical chamber set can have the area of 2 inches of 1 inch of x.It can be about 4L/ hours by the nominal flow rate of system, normally about 3.64L/ hours (i.e. about 60mL/ minutes).

In system 2000 shown in Fig. 1, device 2010,2020,2030,2040 is one another in series, and each device/station is logical The pipeline 2060 that runs therebetween is crossed to connect neighbor stations.With pipeline connection neighbor stations (with the device that is directly connected to each station and adjacent Device is opposite) more preferably fluid and separation of particles are provided.

Pump (such as peristaltic pump) can be provided between each device, can be provided in the upstream of first device and the downstream of final unit Extra pumps.In this respect, it should be noted that in four station type system 2000 shown in Fig. 1, there are 5 pumps to be used for 4 device/station: (1) First sound swimsuit sets the feed pump 2008 of 2010 upstreams；(2) first sound swimsuits set 2010 downstreams and rising tone swimsuit is set on 2020 First pump 2018 of trip；(3) rising tone swimsuit sets 2020 downstreams and third sound swimsuit sets the second pump 2028 of 2030 upstreams；(4) Third sound swimsuit sets 2030 downstreams and falling tone swimsuit sets the third pump 2038 of 2040 upstreams；(5) falling tone swimsuit sets 2040 4th pump 2048 in downstream.More concisely, the embodiment of system 2000 shown in FIG. 1 respectively includes the first sound swimsuit and sets 2010 The feed pump 2008 of upstream and each sound swimsuit set the pump 2018,2028,2038,2039 in 2010,2020,2030,2040 downstreams. Pump 2008,2018,2028,2038,2048 is fluidly connected between each neighboring devices/stand by pipeline 2060.

In addition to pump, the embodiment of system 2000 shown in FIG. 1 includes adjoining the flowmeter of each pump.As shown here, it deposits In 5 flowmeters: (1) the first sound swimsuit sets the first flowmeter 2009 of 2010 upstreams；(2) first sound swimsuits set 2010 downstreams and Rising tone swimsuit sets the second flowmeter 2019 of 2020 upstreams；(3) rising tone swimsuit sets 2020 downstreams and third sound swimsuit is set The third flowmeter 2029 of 2030 upstreams；(4) third sound swimsuit sets 2030 downstreams and falling tone swimsuit sets the 4th of 2040 upstreams the Flowmeter 2039；(5) falling tone swimsuit sets the 5th flowmeter 2049 in 2040 downstreams.More concisely, system shown in Figure 1 2000 Embodiment respectively include the first sound swimsuit set the feed flow meter 2009 of 2010 upstreams and each sound swimsuit set 2010, 2020, the flowmeter 2019,2029,2039,2049 in 2030,2040 downstreams.Using pump, flowmeter 2009,2019,2029, 2039, it 2049 is fluidly connected between each neighboring devices/stand by pipeline 2060.Therefore, fluid stream passes through feed pump, followed by Flowmeter is followed by pump followed by the first sound swimming/filter station, is flowmeter etc. later, and ending is last pump and flowmeter.

In some embodiments, embodiment as shown in Figure 2 A, 2010,2020,2030,2040 object of isolated system station Position is side by side, and physically connected to each other in reason.However, it is not necessary to physical access --- standing can be separated from each other, and pass through pipeline It fluidly connects, as shown in figures 1 and 3.Fig. 2 B is the rearview that the first sound swimsuit sets 2010.

Fig. 2 C shows the cross-sectional view of exemplary sound swimming device/station 2100, can serve as multi-drop sound swimming described herein system Any device/station of system.This device can be used to alleviate some the problem of being in low particle Reynolds number about fluid, and formed and passed through The liquid stream (flow) more evenly of device.Device 2100 has the upward vertical liquid stream by acoustical chamber 2111.Acoustical chamber Also there are 2 opposite dump diffusers 2112 and collector design, provide flowing symmetrical vertical face or line.Generally, The device cross section of flow direction is round or rectangle.In this example, acoustical chamber is empty (without fluid), for example, in transducing There is no other structures in room between device and reflector, and fluid is allowed to flow through acoustical chamber.Have at least one in acoustical chamber upper end It is a to export 2114 through object.In acoustical chamber lower end, there are at least one concentrated solution outlets 2116.In acoustical chamber lower end, there are shallow walls 2118, and lead to concentrated solution outlet 2116.Shallow wall is with respect to the horizontal plane at an angle of, this can be described by acoustical chamber bottom.At least One ultrasonic transducer (not shown) is coupled acoustical chamber, for example, can be located on acoustical chamber side wall.At least one reflector (is not shown Show) or another ultrasonic transducer be located at ultrasonic transducer opposite, for example, can be located at ultrasonic transducer opposite side wall on.Multidimensional Standing wave can be generated with energy converter and opposite reflector, or can be generated with 2 opposite energy converters.

Device 2110 includes symmetrical, dual dump diffuser, booster cavity (plenum) import configuration.Here, 2 sudden expansions Diffuser 2112 is placed on the opposite side of device.Each dump diffuser has the pressurization with upper end 2120 and lower end 2122 Chamber/room.Charge volume provides flowing and spreads and significantly reduce incoming flow heterogeneity.Air inlet 2124 is located at the upper of lower end 2122 Side, at least one outflux 2126 are located at booster cavity lower end.Solid wall 2128 is present in booster cavity upper end.These dump diffusers Form of slots or a line hole, and its top for being placed on acoustical chamber bottom can be used in outflux.The offer of diffuser 2112 passes through sound The flow direction for learning room, the sound standing wave generated with ultrasonic transducer is axial at an angle, such as orthogonal.Also arrangement acoustical chamber enters Mouthful, so that it is in opposite position, so that horizontal velocity is reduced in acoustical chamber center is intended to zero.

Dump diffuser helps to reduce or eliminate the flowing that fluid mixture is downward in acoustical chamber.Fluid mixture is full of Booster cavity in dump diffuser, subsequent level stream enter acoustical chamber, and wherein mixture flows through sound standing wave vertically upward.Sudden expansion diffusion Device reduces/eliminates flow pulsation and flowing heterogeneity, is produced from pump, hose flushing and/or horizontal inlet stream, weight at this Stress effect is dominant.Dump diffuser enters mixture below ultrasonic transducer, and thus the node that is formed in ultrasonic standing wave Acoustical chamber below cluster or line.This arrangement helps to reduce or reduce as far as possible any interference of cluster, this may be in addition by flowing into Material causes.

Symmetrical vertical face or line are consistent with gravity direction (is aligned with).Also show streamline, need symmetrically with Reduce or reduce as far as possible heterogeneity, vortex interference, recycle and descend through concentrated solution outlet 2116 to be collected cluster it is dry It disturbs.The gravity symmetrically also contributed in air inlet flow distribution and particle collection process is uniformly distributed.Because it is overweighted at the top of device That leaves penetrates object, and the incoming mixture of (opposite) weight is extended to room bottom by gravity close to acoustical chamber bottom, and is provided certainly Lower and upper nearly consistent normal-moveout spectrum.As mixture is close to acoustical chamber center, the horizontal velocity of mixture can be reduced to it is close or Equivalent zero, this is attributed to the liquid stream of dual opposition import.This horizontal velocity decline facilitate reduce or as far as possible reduce room stream with Interference between the cluster of particle of sedimentation.Uniform speed improves and maximizing separates and collect result.The lateral sound of sound standing wave Educational level can overcome particle to pull and particle is allowed to be captured to form cluster and growth and be continuously removed from sound standing wave by falling into.Uniformly Speed can assist to avoid the non-uniform disturbance or interference with lateral acoustic force.Uniform speed allows import fluid flow distributor to be It is optionally present.

As cluster of particle is detached from, it is complete that axial direction acoustic force relevant to standing wave helps to maintain cluster.Keep cluster that can completely help Maintenance is helped to have the cluster rapid decrease of high terminal velocity, magnitude 1cm/sec.Compared to room flow velocity, amount in some instances Grade is 0.1cm/sec-0.3cm/sec, and the sinking speed of cluster may be exceedingly fast.Shallow wall angle means that cylindrical cluster of particle can be at it Pass through relatively short distance before leaving acoustical chamber, to hardly happen cluster dispersion.Preferably, system transducing per square inch 3-12 crystal vibration node operating of device.The symmetrical, flow disturbance of reduction and shallow collector wall are changed in centeral acquisition zone Kind collection, and allow baffle/lamellae optional.

Fig. 3 generally illustrates the fluid flow path by sound swimming station 2010.Fresh fluid/cell culture based mixtures are through entrance 2012 continuously introduce the station on its top, and cocurrent crosses flow chamber 2050.In flow chamber, cell aggregation simultaneously takes off from sound standing wave It falls/settles.Cell condensation object/aggregation of these sedimentations then falls on 2050 bottom of flow chamber, and can be 2016 times oral It receives.Remaining mixture flows out from flow chamber and proceeds to downstream unit on device top through outlet 2014, as described herein.

Fig. 4-6 illustrates the function at sound swimming station.It swims as shown in figure 4, fluid/cell culture based mixtures enter sound by pipeline The sonic wave region for standing and entering separation, i.e., wherein generate the region of at least one multidimensional sound standing wave.This sonic wave region is defined on ultrasound and changes It can be between device and reflector.As shown in figure 5, cell is captured in sound at node and stays due to the effect of acoustic radiation force cross stream component Wave.As shown in fig. 6, acoustic radiation force declines and/or buoyancy reduction/gravitational effect enhancing causes to gather as cell aggregation is in node The cell of collection settles from suspension and falls into flow chamber bottom.

As illustrated in brief explanation above and Fig. 3, it should be noted that in many implementations that multi-drop sound swimsuit as described herein is set In scheme, the ultrasonic transducer directly adjoins flow chamber, and is directly exposed to any fluid across flow chamber.Energy converter energy Separated by film and flow chamber, film by such as polyether-ether-ketone (PEEK) or entrant sound any suitable material or can serve as transducing Other suitable materials of barrier are constituted between fluid in device and flow chamber.Reflector be it is hard or flexible, can be by high acoustic resistance Anti- material is constituted, such as steel or tungsten, or any other suitable material of the good sound reflecting of offer.For multi-drop described herein Sound swim systematic reflection device it is a kind of special consideration should be given to material be borosilicate glass.Another energy converter can also be used as reflector.

The different piece such as flow chamber of multi-drop sound swimming system as described herein can be made of any suitable material, the material Material can dispose fluid mixture.This kind of suitable material and associated components for flow chamber includes medical grade plastic, such as poly- carbonic acid Ester or polymethyl methacrylate or other acrylate.For flow chamber/shell in multi-drop sound swimming system described herein One kind is polyphenylsulfone (PPS) special consideration should be given to material.The material can be configured at least some transparent conduct transparent windows to allow Inner flow passage and process are visible during sound swimming device/system operating.

Multidimensional sound standing wave for powder collection obtains as follows: not only to generate sound standing wave but also excite the piezoelectric material of energy converter Basic 3D vibration mode frequency drive ultrasonic transducer.Using disturbing for the piezoelectric material in the ultrasonic transducer of multimode form It is dynamic to allow to generate multidimensional sound standing wave.Piezoelectric material can be specifically designed to deform in the form of multimode by assigned frequency, allow to generate Multidimensional sound standing wave.Multidimensional sound standing wave can be generated by the different mode of piezoelectric material, such as 3x3 mode, can be generated multidimensional sound and be stayed Wave.By allowing piezoelectric material to vibrate through many different vibration shapes, it also can produce a large amount of multidimensional sound standing waves.Therefore, piezoelectricity can be excited Material arrives 1x1,2x2,1x3,3x1,3x3 and other higher order modes to generate various modes such as 0x0 mode (i.e. piston mode), And then it is recycled back to piezoelectric material low step mode (must not be using directly sequence).The conversion of the piezoelectric material between mode Or shake allows at the appointed time to generate different multi-dimensional waveforms and single piston mode shape.

Some other explanations of the ultrasonic transducer of devices, systems, and methods for the disclosure are also useful.In this respect, Energy converter uses piezoelectric material, and the material can be ceramic material, crystal or polycrystal such as PZT-8 (lead zirconate titanate).It is this kind of Crystal can have 1 inch diameter and the nominal resonant frequencies of 2MHz.Each ultrasonic transducer module can only have a kind of piezoelectric material, Or there can be a variety of piezoelectric materials.A variety of piezoelectric materials can respectively be used as independent ultrasonic transducer and can be controlled by one or more Device, driver or amplifier control.

Fig. 7 is the cross-sectional view of conventional ultrasonic transducer.This energy converter has wear plate 50, the epoxy layer in bottom end 52, piezo-electric crystal 54 (being made of such as PZT), epoxy layer 56 and support layer 58.In any surface of piezo-electric crystal, there is electrode: Anode 61 and cathode 63.Epoxy layer 56 makes support layer 58 invest crystal 54.Entire combination is contained in shell 60, which can be by example As aluminium is made.Electric adapter 62 provides power lug to pass through shell and connect and invest the conducting wire (not shown) of crystal 54.In general, Support layer is designed to that addition damps and generates the wide-band transducer for having unified displacement in extensive frequency, and is designed to inhibit in spy Determine the excitation of vibration performance-mode.It is therein more preferably to match energy converter radiation that wear plate is typically designed to impedance transformer Culture medium characteristic impedance.

Fig. 8 is the cross-sectional view of the ultrasonic transducer 81 of the disclosure.Energy converter 81 has aluminum enclosure 82 shaped like disk or dish. Aluminum enclosure has top and bottom end.Transducer housing can also be made of plastics (such as medical grade HDPE) or other metals.Piezoelectricity member Part is a large amount of perovskite ceramics, is respectively made of small quadrivalent metallic ion, usually titanium or zirconium, in biggish bivalent metal ion In lattice, usually lead or barium and O^2-Ion.For example, PZT (lead zirconate titanate) piezoelectric element 86 defines energy converter bottom end, and in shell Body bottom end outer exposed.Piezoelectric element passes through the small elastic layer 98 such as ring between piezoelectric element and shell on its circumference Oxygen, silicone or similar material are supported.Energy converter 81 does not include wear plate or base material.In some embodiments, exist Plastics or other materials layer (not shown) are provided on 86 outer surface of piezoelectric element.The material has separation piezoelectric element 86 and stream The characteristics of body, sound standing wave generate in the fluid.The material can be relatively thin, such as in 10 μm of -1mm ranges, and example Such as, useful binders are fixed on piezoelectric element 86.Material can be basic entrant sound, can be adhesive.Piezoelectric element 86 has Outer surface (exposure) and inner surface.

Screw makes the aluminium top plate 82a of shell invest the main body 82b of shell through screw thread 88.Top plate includes connector 84 with to changing It can device power supply.The top surface jointed anode 90 and cathode 92 of PZT piezoelectric element 86, anode and cathode are separated by insulating materials 94.Electricity The pole of pole 90,92 shows overturning, and electrode 90,92 can be located at 86 opposite face of piezoelectric element or the same face, and wherein the same face can be Inner surface or outer surface.Electrode can be made of any conductive material, such as silver or nickel.It is pressed by the electrode on piezoelectric element to PZT Electric device 86 provides electrical power.It should be noted that piezoelectric element 86 does not have support layer or epoxy layer.Energy converter 81 is in aluminium top plate 82a There is inner space or air gap 87 (such as shell is empty or comprising air) in energy converter between piezoelectric element 86.One In a little embodiments, it is possible to provide minimum support (backing) 58 (on inner surface) and/or wear plate 50 (on outer surface), As shown in Figure 9.

Transducer designs can influence system performance.Typical energy converter is layered structure, ceraminator combination support layer And wear plate.Since energy converter is loaded with standing wave bring (presented) high mechanical impedance, the routine for wear plate Design guidelines (for example, quarter-wave thickness for the half-wavelength thickness of standing wave application or for radiation application) and manufacture Method may be improper.In the example of energy converter 81, as shown in figure 8, allowing piezoelectric element without wear plate or support 86 have one of eigen mode of high Q factor or with it with the combination vibration of several eigen modes.Piezoelectric element 86, which has, to be directly exposed to The outer surface of fluid, the fluid flow through the flow chamber of installation energy converter 81.

Removal support (such as supporting piezoelectric element air) also allows ceraminator to damp small high frequent vibration Mode (such as high order mode displacement) vibration.In such as preceding energy converter with the piezoelectric element with support implemented, piezoelectricity Element is with more evenly displacement vibration, such as piston.Removal support allows piezoelectric element to be easier to vibrate with non-uniform bit mode shifter. The piezoelectric element vibration shape gets over high-order, and the nodal line that piezoelectric element can generate is more.The displacement of the higher order mode of piezoelectric element generates More capture lines, although capture line and the relationship of node must not be it is one-to-one, higher or lower frequency drive piezoelectric element Not must give operating frequency generate more or less capture line.

In some embodiments of the sound filter device of the disclosure, the piezoelectric element can have to piezoelectric element Q The support of Effects of Factors relatively small (such as less than 5%).Support can be made of basic acoustic window material, as cork wood, foam or Cork is allowed piezoelectric element to be vibrated with high order mode and maintain high Q factor, while still providing some machinery for piezoelectric element Support.Support layer can be solid, or can be the lattice with hole in layer, so that the lattice are using specific high frequent vibration mode Vibration piezoelectric element node after, provide support in node position, while allowing remaining piezoelectric element free vibration.Grid Or the purpose of acoustic window material is to provide support, the excitation of Q factor or the specific vibration shape of interference without reducing piezoelectric element.

By avoiding the damping and energy absorption of epoxy layer and wear plate, piezoelectric element is directly contacted to fluid and is put It sets and also contributes to high Q factor.Other embodiments of energy converter can have wear plate or wear-resistant surface to prevent the PZT comprising lead Contact main fluid.These layers between energy converter and main fluid can be suitble to such as biological applications, such as separate blood, bio-pharmaceuticals fills Note or fed-batch filter mammalian cell.Wearing layer such as chromium, electrolytic nickel or chemical nickel plating can be used in this kind of application.Chemical gas Mutually deposition can be used for using poly- (paraxylene) (such as parylene) layer or another polymeric layer.Organic and biocompatibility packet Wear-resistant surface is also served as by such as silicone or polyurethane.Film such as PEEK film can also serve as the covering of piezoelectric material outer surface, advantage It is as biocompatible materials.In one embodiment, the PEEK film invests piezoresistive material with contact adhesive (PSA) Expect surface.Could be used that with compared with low acoustic impedance and/or be biocompatibility other films.

In the implementation being discussed herein, particle capture remain stationary state in ultrasonic standing wave.Along clearly capture line Particle is collected, is separated by half-wavelength.In each pitch plane, particle capture is in minimum acoustic radiation gesture.The axial direction of acoustic radiation force point Power drive has the particle of positive coefficient of correlation to pressure pitch plane, and there have the particle of negative coefficient of correlation to be driven to pressure antinode to be flat Face.The radially or laterally component of acoustic radiation force facilitates lateral trap particles.The radially or laterally component and acoustically radiating of acoustic radiation force The axial thrust load for penetrating power has same order.As discussed above, cross force can increase as follows: drive the transducing of high order mode Device, it is opposite with the wherein crystal vibration mode effectively mobile as the piston with uniform position.The driving of acoustic pressure and energy converter Voltage is proportional.Electrical power is proportional to voltage squared.

Energy converter can be driven by driving signal, such as voltage signal (AC or DC), current signal, magnetic signal, electromagnetic signal, electricity Hold signal or energy converter responds it to generate any other signal type of multidimensional sound standing wave.In embodiments, the driving The voltage signal of energy converter can have pulse, sine, side, sawtooth or triangular waveform；With with 500kHz-10MHz frequency.Voltage Signal can be modulated with pulse width and be driven, and can be used for generating any required waveform.Voltage signal can carry out amplitude or frequency tune System.Driving signal is openable or closes and/or configured with starting/termination ability for example to eliminate crossfire (streaming).

In some instances, the transducer dimensions, shape and thickness can determine that the energy converter position under different stimulating frequencies It moves.Separative efficiency can be influenced by having the transducer displacement of different frequency.In some instances, the energy converter is to approach thickness resonance The frequency run of frequency (half-wavelength).There are gradients can produce more multiposition for particle to be captured in transducer displacement.It is high Rank modal displacement can generate three-dimensional sound standing wave, have strong gradient in the directive sound field of institute, to generate in all direction intensity Similar acoustic radiation force, such as the power can be at the same order of magnitude.High order mode displacement can cause a variety of capture lines.Capture line number Mesh is related to the special transducer vibration shape.

The influence for capturing power and separative efficiency to sound for research transducer displacement curve, is repeated with 1 " x1 " square transducer Experiment ten times, all conditions are identical, in addition to stimulating frequency.It is continuous by 10 of digital 1-9 and letter the A expression of zone circle on Figure 10 Acoustic resonance frequency is used as stimulating frequency.Condition is Therapy lasted 30 minutes, about 5 microns of SAE-30 oil droplets of 1000ppm oil concentration, 500ml/min flow velocity and 20W input power.Using oil droplet, because oil density is lower than water, and it can be swum and be separated from water with sound.

Figure 10 shows with the frequency function near 2.2MHz transducer resonant when operating in the water column containing oil droplet, transducing The measured electrical impedance amplitude of device.The anti-minimum value of transducer resistance corresponds to the acoustic resonance of water column and represents the potential of operating Frequency.There are additional resonants at other frequencies of excitation multi-dimensional standing-wave.Numerical simulation shows transducer displacement curve at these Acoustic resonance frequency significant changes, to directly affect sound standing wave and resulting capture power.Since energy converter can be in its thickness resonance Operating nearby, electrode surface displacement substantially out-phase.Transducer electrode displacement can unevenly and according to stimulating frequency change.Example Such as, under the stimulating frequency for having single capture oil droplet line, there is single largest value in centre and has at energy converter edge in the electrodes for displacement There is minimum value.In another stimulating frequency, energy converter curve has multiple maximum values, causes a plurality of oil droplet capture line.High-order energy converter Displacement model can generate higher capture power and a plurality of stable capture line for captured oil droplet.

The influence for capturing power and oily separative efficiency to sound for research transducer displacement curve, repeats experiment ten times, all Part is identical, in addition to stimulating frequency.It is used as by 10 continuous acoustic resonance frequencies that the digital 1-9 and letter A of zone circle on Figure 10 are indicated Stimulating frequency.Condition is Therapy lasted 30 minutes, about 5 microns of SAE-30 oil droplets of 1000ppm oil concentration, 500ml/min flow velocity With 20W input power.

As lotion is by energy converter, the capture line of oil droplet is observed and identified.As shown in Figure 11 A, identification is related to across runner The observation and mode of capture line number, 7 in 10 resonant frequencies identified for Figure 10.

Figure 11 B shows flow chamber and separates the isometric view of sonic wave region, wherein measurement capture line position.Figure 11 C is to work as edge Arrow 251 look down the view of the ultrasonic transducer volume presented when entrance.Figure 11 D is to work as directly to see along arrow 253 The view of the ultrasonic transducer volume presented when energy converter planar.

In this example, the influence of stimulating frequency clearly determines capture line number, the list under the stimulating frequency of acoustic resonance 5 and 9 One capture line changes to 9 capture lines of acoustic resonance frequency 4.Under other stimulating frequencies, 4 or 5 capture lines are observed.Transducing The different displacement curves of device can generate difference (more or less) capture line in standing wave, and more gradients in displacement curve generally produce Raw bigger capture power and more capture lines.

The lin-log figure (linear y-axis, logarithm x-axis) of Figure 12 shows the calculating ratio of acoustic radiation force, fluid drag power and buoyancy Example, using particle radius.Buoyancy can be applied to the particle of negative coefficient of correlation, the oil particles in such example.The buoyancy of calculating can Including gravity element.In using the example of positive coefficient of correlation particle that can be certain cell types, indicate that the line of gravity is used In the figure of the particle of this kind of positive coefficient of correlation, the figure shows acoustic radiation force and fluid drag power.In this example for being shown in Figure 12 In, just complete to calculate for the typical SAE-30 oil droplet of experiment.Buoyancy is particle volume dependence power, such as with radius cube at than Example, and it is relatively negligible for micron particles size, but buoyancy increases and becomes for hundreds of microns grade particles size Obviously.Fluid drag power linearly increases and decreases (scale) with fluid velocity, such as proportional to square radius, generally more than micron The buoyancy of grade particles, but the influence to hundreds of micron-sized larger size particles is less.The effect of acoustic radiation force increase and decrease is different from Fluid drag power or buoyancy.When granular size is smaller, it is vertical with the particle radius of particle to approach linear ratio that sound captures power Side's (volume) increase and decrease.Finally, as granular size increases, acoustic radiation force no longer linearly increases with particle radius cube.With Granular size continue growing, acoustic radiation force is reduced rapidly, and a certain key particles size be local minimum.It is further Increase granular size, radiant force increases in scale again, but is in reverse phase (not shown).This mode is repeated to increase Grain size.Granular size and the relationship of acoustic radiation force rely at least partially upon the wavelength or frequency of sound standing wave.For example, with particle Increase to half-wavelength size, the acoustic radiation force of particle is reduced.As granular size is increased to greater than half-wavelength and is less than all-wave It is long, the acoustic radiation force of particle is increased.

Initially, when suspension flows through the sound standing wave of mainly small micron particles, acoustic radiation force balanced fluid drag with The synergy of buoyancy is with the trap particles in standing wave.In Figure 12, just about 3.5 microns of granular size is captured, the particle Size is labeled as R_c1.According to Figure 12, as granular size is continued growing more than R_c1, larger particle is captured, because of acoustic radiation force Compare the increase of fluid drag power.As little particle is captured in standing wave, particle coalescence/agglutination/aggregation/reunion occurs, causes effectively Granular size sustainable growth.Continue the capture site for driving other little particles to standing wave, because larger particles are retained and ruler Very little increase helps continuously to capture.As granular size increases, the acoustic radiation force of particle is increased, until reaching the first particle Big cell.As granular size increases above the firstth area, the acoustic radiation force of particle is started to reduce.As granular size growth is held Continuous, acoustic radiation force declines rapidly, until buoyancy is dominant, this is by the second key particles size R_c2Instruction rises in the sized particles Or sink, this depends on its relative density or sound coefficient of correlation relative to main fluid.As particle rises or sinks and leaves The antinode (in negative coefficient of correlation) or node (in positive coefficient of correlation) of standing wave, can subtract the acoustic radiation force of particle Negligible quantity is arrived less.Acoustic radiation force continues to capture small and bulky grain, and drives by sunken particle to capture site, in this example In be located at pressure antinode.Lesser granular size undergoes reduced acoustic radiation force, such as point R shown in being reduced to_c1Neighbouring Acoustic radiation force.As other particles are captured in the node or antinode of sound standing wave and and are gathered, are aggregated, assemble, reunite and/or are concentrated Together, granular size is effectively increased, acoustic radiation force increases and circulating repetition.All particles may not be detached from from sound standing wave, Those remainder particulates can continue to expand size.Therefore, Figure 12 explains how little particle is continuously captured in standing wave, grows up to bigger Grain or agglomerate, then finally rise or be precipitated out, this be attributed to the buoyancy for being related to granular size, drag and acoustic radiation force it Between relationship.

A variety of coatings can be used for the internal flow room that sound swimsuit is set.This type coating can include epoxy resin, such as crosslinking amine Or the epoxychloropropane bis-phenol of polyamide；Or polyurethane coating, such as the polyester polyol of cross-linked aliphatic isocyanates.It is this kind of Coating allows cell more preferable along flowing chamber surface under the influence of gravity for generating smooth surface and/or reducing surface tension It slides and enters required position in ground (as collected hole module).

The flow velocity set for example, by pumping control sound swimsuit.It is adjustable flow velocity, so that gravity-buoyancy can act on particle aggregation Object.It can be every with up to about 10 liters by the particle/fluid mixture that the into/out sound swimsuit of its inlet/outlet sets middle flow chamber The speed flowing of hour (L/ hours), including up to about 50L/ hours, but typically about 3.6L/ hours.In comparison, through holding The flow velocity that mouth leaves collection hole module was small, from up to about 10ml/ minutes about 3ml/ minutes.

The sound swimming system of the disclosure can be used for filter " queue ", and plurality of difference filtration step is for clarifying or purifying just Beginning fluid/particulate mixture is to obtain the different materials of required product and management from each filtration step.Each filtration step can be real It is applied to removal certain material, improves the overall efficiency of clarifying process.Independent sound swimsuit, which is set, can be used as one or more filtering steps Rapid operating.For example, can operate specific sound swimsuit sets interior each independent ultrasonic transducer with the trapping material in given granulometric range. Sound swimsuit, which is set, can be used to remove lot of materials, reduce the burden to subsequent downstream filtration step/stand.A variety of filterings described herein Any one of step/stand can be placed in the upstream or downstream that sound swimsuit is set.Alternatively or in addition, a variety of sound swimsuits can be used to set. Required biomolecule or cell can be in the recovered/separateds of this kind of filtering/after purification.

The outlet (such as clear fluid and concentrating cells) that the sound swimsuit of the disclosure is set can fluidly connect any other filtering Step or filter station.This kind of filtration step may include a variety of methods such as in-depth filtration, aseptic filtration, size filtration or tangential Filtering.In-depth filtration using can in entire filter depth reserved materials physical holes filter medium.In aseptic filtration, aperture pole Small molecular filter is typically no to heat or radiate or be exposed among chemical substance for removing microorganism and virus.Size row Resistance is filtered through size and/or molecular weight separation material, uses the physics filter with intended size hole.In tangential flow filtration, big portion Liquid separation stream gets over filter surfaces, rather than enters filter.

It could be used that chromatography, including cation chromatographic column, anion chromatographic column, affinity chromatographic column and/or mixed bed chromatographic column. Other hydrophilic/hydrophobic processes can be used for filtering purpose.

For example, multi-drop sound swimming system as described herein may include cascade filtration station or be combined therewith.One or more string Connection filter station can be located at the upstream or downstream that all or some sound swimsuits are set.Cascade filtration station can be used for being further purified liquid simultaneously Albumen needed for therefrom recycling and obtain.The suitable example at cascade filtration station includes depth filter, sterile filters, centrifuge and affine Chromatographic column.

The selection of second level in-depth filtration product can be realized with certain material screening to be filtered.In expression humanization IgG1mAb The cell line based on CHO-S typical fed-batch culture object in, total volume be less than about 5L arrive less than about 25L and the gross area about 0.002m²About 0.1m²Depth filter can be used for second level in-depth filtration.In this respect, suitable depth filter includes Supracap^TMHP depth filter capsule, available from Solution (Pall Corporation).After clarification, optionally, save The cell culture fluid (HCCF) of harvest, filtering to control biological load, save or filter with control biological load and through chromatography at Reason.In the typical fed-batch culture object of the cell line based on CHO-S of expression humanization IgG1mAb, total volume can be used Less than about 5L to less than about 25L and the gross area about 220cm²About 375cm²Sterile filters (i.e. sterile grade molecular filter).In this side Face, suitable sterile filters includeCapsule and miniature Kleenpak capsule, available from Solution.

Optionally, three-level in-depth filtration can be being omitted on a small scale, but when in use, can prevent subsequent filter from polluting and allowing Reduce the size of biological load control filter.In the typical feed supplement point of the cell line based on CHO-S of expression humanization IgG1mAb It criticizes in culture, the same depth filter for second level in-depth filtration can be used for three-level in-depth filtration.After clarification, can be used with it is upper Identical sterile filters described in face.

In the application of some exemplary biologicals, all parts of system (i.e. each station fluidly connects the pipeline etc. at each station) It can be separated from each other and be disposable.About the filtration cell in biological applications, centrifuge and conventional filter can apply cell Add undesired or harmful condition.The sound swimsuit being discussed herein sets permission and separates cell from main fluid, without undesired Or harmful centrifuge and conventional filter influence.Therefore, it is set using sound swimsuit to avoid centrifuge and filter that from capable of separating cell, and Cell viability must not be reduced.Ultrasonic transducer can also be used for generate rapid pressure variation with prevent or scavenger-cell aggregation cause Blocking.The frequency of energy converter is controllable and/or variation is to obtain operation point, so that just given power increases effect or makes effect most Bigization.

The disclosure further illustrates in the following non-limiting examples, it should be understood that these embodiments are only intended to illustrate and this It is open to be not intended to be limited to module cited herein, device, condition, technological parameter etc..

Embodiment

A variety of Chinese hamster ovary celI mixtures in filtration cell culture medium.

Embodiment 1

Compare sound swimming separation process and deep layer flow filtration (DFF).Firstly, obtaining DFF capacity by carrying out two-wheeled clarification Baseline, i.e., primary clarification and second level clarification.In order to which the setting of this baseline is shown in Figure 13.

Pressure drop is measured during the two-wheeled.Separation equipment is in 145LMH (liter/m²/ hour) operating.Not at three Pressure is measured with position P1, P2 and P3.Filter is between every set sensor.The filter used during preliminary clarification is D0HC filter Device, the filter that second level uses during clarifying is X0HC filter, both available from Mi Libo (Millipore).

The mixture flow filter of Chinese hamster ovary celI and culture medium is then collected in slot and penetrates object.Chinese hamster ovary celI passes through filter It removes.Charging has 6.34 × 10⁶The turbidity of cell/mL total cell density (TCD) and 815NTU.Whole transmission in third slot Object has the turbidity of 1.75NTU.

The performance figure of Figure 13 shows pressure drop to volume flux.The pressure drop of primary filter is minimum under low pass excess, then exists Greater than about 88L/m²The pressure drop in second level filter that capacity becomes larger than.Overall presure drop is the line at the top in figure.In 88L/m²Body Product throughput obtains the pressure drop (being represented by the dotted line) of 15psig.This relationship shows that, if be scaling up, maximum pressure drop is 15psig (Pmax=15psig), then primary clarification and the clear filter area of second level are respectively 11.4m²。

Embodiment 2

Then, two-step DFF described in embodiment 1 is made comparisons with two-step clarification process, wherein primary clarification passes through Sound wave separates (AWS) progress and second level clarification passes through DFF and carries out.It is as shown in figure 14 that this compares setting.

As shown in figure 14, in two-step DFF, each filter has 11m²Area.Each filter is transported with the pressure drop of 7.5psig Turn.For each filter, 7.5psig (VT_7.5) under volume throughput (VT) be 84L/m²。

Sound swimming system for carrying out AWS is set by three sound swimsuits being connected in series to be constituted.Energy converter in each device is 1 Inch × 1 inch.The system has 49cm³Total sound volume.AWS system and the gross area are 6m²DFF filter pairing.Due to There is no pressure drop in AWS system, DFF filter can operate under 15psig pressure drop, generate 160L/m²VT₁₅。

Charging has 6.7 × 10⁶The turbidity and 77% cell viability of cell/mL total cell density (TCD) and 835NTU. The charging rate of sound swimming system is 4kg, 2.5 liter per hour (LPH).

It is as shown in figure 14 with the clear result of primary of AWS system.Sound swimming system realizes 91%TCD decline, 90% turbidity Decline and 91.2% protein salvage.The figure in the lower left corner is decline percentage comparison time, and display AWS system operates surely dduring test It is fixed.

Compared to the embodiment 1 that wherein clarification of primary and second level is all carried out by in-depth filtration, taken with sound wave separation (AWS) In generation, economic benefit can be achieved by the following procedure in primary clarification: reduce integrated operation footprint, reduce second level depth filter area specification and Correlated condition and dcq buffer liquid, and reduce storage and cost of disposal.As technique enters clinical production, these become key The process drive factor.The expection technological specification example of 1000L Chinese hamster ovary celI culture is summarized as follows.

Embodiment 3

Progress and identical experiment as described in example 2 again, but cell density is higher.Charging has 15.6 × 10⁶Carefully The turbidity of born of the same parents/mL total TCD and 3608NTU and 68% cell viability.It is as described in Figure 15 that this compares setting.

Two-step DFF technique uses 38m respectively²And 17m²Filter.As indicated, primary clear VT_7.5It is 26L/m², two Grade clarification is 58L/m².In AWS-DFF technique, there is AWS system concatenated two sound swimsuits to set (rather than in embodiment 2 Three), total acoustic volume is 33cm³.DFF filter has 11m²The gross area and 85L/m²VT₁₅.The charging speed of sound swimming system Degree is 8kg, 2.5 liter per hour (LPH).

It is as described in Figure 15 with the clear result of primary of AWS system.Sound swimming system realizes 94%TCD decline, 91% turbidity Decline and 92% protein salvage.The figure in the lower left corner is decline percentage comparison time, shows that the system operates stabilization dduring test. More high-cell density is more difficult to handle for DFF device, as shown in the lower VT in primary clarification.However, sound swimsuit sets energy It is enough that higher density is handled with more preferably VT.

Embodiment 4

Charging has 7.5 × 10⁶The turbidity of cell/mL TCD and 819NTU and 88% cell viability.With three-station type sound Swimming system is clarified, such as embodiment 1.

First stop makes cell density reduce by 62%.Second station reduces cell density by 87% (accumulation 95%).Third station makes Cell density reduces by 63% (accumulation 98%).Obtaining the cell density greater than 90% using two stations reduces.

First stop makes haze reduction 68%, from 819NTU to 260NTU.Second station makes residual turbidity be down to 54NTU (accumulation 94%).Third station makes residual turbidity be down to 42NTU (accumulation 95%).The haze reduction greater than 90% is obtained using two stations.This As a result the secondary filtration process in downstream again is important.

Cell density reduces and the decline percentage of haze reduction is constant in whole time, it is meant that device is continuous On the basis of run well.The decline of cell density and turbidity improves the secondary filtration technique for helping to simplify again downstream, and final The product recycling for helping to separate the chromatography of monoclonal antibody or recombinant protein from clear fluid is higher.

Embodiment 5

Pass through 5 different batches of three-station type system testing of embodiment 1.Each batch has the cell size of their own and close Spend feature.Charging has 7-8.5 × 10⁶The turbidity of cell/mL TCD and 780-900NTU and the cell viability of 82%-93%. This embodiment tests performance consistency of the system in different batches.

In 5 different batches, after passing through three times, the turbidity reduction range through object is 84%-86%, and standard deviation is 1%.After passing through three times, the cell density decline range through object is 93%-97%, and standard deviation is 2%.

In other experiments not described herein, the sound wave separating technology of discovery multidimensional sound standing wave does not influence to be recovered from Cross the protein of object or the physically or chemically feature of monoclonal antibody.

Embodiment 6

Then, influence of the voltage input to clarification performance is observed in three-station type or four station type sound swimming system.

The mixture of Chinese hamster ovary celI and culture medium flows through each station of device, is then collected in slot through object.It is removed by filter Chinese hamster ovary celI.Charging has 25 × 10⁶The turbidity of cell/mL total cell density (TCD) and 2048NTU and 72% cell it is living Power.Feed flow rate is that draw (solid draw) be 2.34mL/ minutes to the solid of 30mL/ minutes and first stop, and second station is 1.41mL/ minutes, third station was 0.94mL/ minutes, and the 4th station is 0mL/ minutes.

TCD behind three stations and four station Filtration Filtrations reduces as shown in the table.T1 refers to that the voltage in first stop, T2 refer to second It stands, T3 refers to third station and T4 refers to the 4th station.Show 5 different test runs as a result, using different voltages in different stations.

For all voltage conditions of 50V and 60V, which realizes that the cell density greater than 90% declines.It is added the Four stations can produce higher 95% decline, make TCD from 2.9 × 10 behind three stations⁶Cell/mL be down to four station after 1.3 × 10⁶Cell/mL.Following table shows that for the operation of institute's characterization test above, the accumulation %TCD at every station declines after 30 minutes.

As can be seen that the voltage of 50V and 60V generates optimal representation in each station.Such as test run 5 as it can be seen that being added in 50V 4th station can make TCD decline increase by 10% aggregation.Generally, 50V-60V voltage is applied to first and/or second station to obtain height TCD decline.

Total turbidity reduction after three and four filter stations is shown in following table.It, should for all voltage conditions of 50V and 60V System realizes the turbidity reduction greater than 90%.The 4th station, which is added, can produce 94% decline, make through object behind three stations 390NTU is down to the 177NTU behind four stations.

Test run	T1 voltage (V)	T2 voltage (V)	T3 voltage (V)	T4 voltage (V)	% turbidity reduction
						1	40	40	40	-	81
2	50	50	50	-	90
						3	60	60	60	-	91
4	60	50	40	-	91
						5	50	50	50	50	94

In short, 50V and 60V operating condition ratio 40V operating condition generates higher and almost equivalent clarification, first and the Clarifying efficiency is reduced using 40V operating condition in third station after the 50V/60V at two stations.

Embodiment 7

Then, similar embodiment 1 makes comparisons sound swimming separating technology to determine that DFF is showed with deep layer flow filtration (DFF) Influence to sound wave separation (AWS) performance.

The mixture of Chinese hamster ovary celI and culture medium flows through two filters, i.e. D0HC filter and X0HC filter, both available from Mi Libo, and slot is then collected in through object.Chinese hamster ovary celI is removed by filter.Charging has 24.7 × 10⁶Cell/mL is total The turbidity of cell density (TCD) and 2850NTU and 72% cell viability.End in slot has the turbidity of 4.9NTU through object.

The performance figure of Figure 16 shows pressure drop to volume flux capacity.According to total filter region at each station, Figure 16 is drawn at every station In spectrum.In 23cm²Calculate volume flux capacity.Pressure drop in D0HC filter is minimum under low pass excess, then in about 16L/m² The pressure drop in X0HC filter become larger than on capacity.In the 47L/m of D0HC filter²The pact of volume flux and D0HC filter 110L/m²Under volume flux, the pressure drop for obtaining 15psig (causes total 15.0psig to press based on pressure drop ratio estimation-D0HC filter 12.0psig the and X0HC filter of drop causes remaining 3.0psig).

The performance figure of Figure 17 shows pressure drop to volume flux.Figure is drawn with regard to concatenated total filter area (crossing over all stations) 17 curve.In 46cm²Calculate volume flux capacity.Pressure drop in D0HC filter is minimum under low pass excess, is then being higher than About 8L/m²Capacity becomes larger than the pressure drop of X0HC filter.Overall presure drop is the line at the top in figure.In 21.5L/m²Volume flux Obtain 15psig pressure drop.This result shows that, if be scaling up, maximum pressure drop be 15psig (Pmax=15psig), then just It is 46.5m that grade clarification and the clear filter area of second level, which are always met together,², D0HC:X0HC filter size ratio is 3:1 (for example, D0HC is filtered Device can have 34.9m²Area, and X0HC filter can have 11.6m²Area).

Embodiment 8

Then, two-step DFF described in embodiment 6 and 7 is made comparisons with two-step clarification process, in the clarification process In, primary clarification is carried out by sound wave separation (AWS) and second level clarification is carried out by DFF.It is as shown in figure 18 that this compares setting.

As indicated, D0HC filter has 34.9m in two-step DFF²Area and X0HC filter have 11.6m²Area, always Area is 46.5m².Each filter is operated with the pressure drop of 7.5psig.For D0HC filter, in 15psig (VT₁₅) volume pass through Measuring (VT) is 47L/m², it is 110L/m for X0HC filter².Turbidity through object is 4.9NTU.

2 kinds swimming system is not used to carry out AWS in unison.First sound swimming system is the three-station type system of series connection (such as by three A sound swimsuit sets composition).Rising tone swimming system is the four station type system of series connection as shown in Figure 1 (such as by four sound swimsuits Set composition).Energy converter for each device in each system is 1 inch × 1 inch.

First AWS system and the gross area are 10.2m²DFF filter pairing.Step area is 7.6m²(3x) and 2.6m² (1x).Due to not having pressure drop in AWS system, DFF filter can operate under 15psig pressure drop, lead to 214L/m²VT₁₅.Charging With 2.9 × 10⁶The turbidity of cell/mL total cell density (TCD) and 380NTU.Turbidity through object is 8.8NTU.

2nd AWS system and the gross area are 4.5m²DFF filter pairing.Step area is 3.4m²(3x) and 1.1m² (1x).Once again, DFF filter can be operated in 15psig pressure drop due to not having pressure drop in AWS system, lead to 490L/m²VT₁₅。 Charging has 1.3 × 10⁶The turbidity of cell/mL total cell density (TCD) and 176NTU.Turbidity through object is 11.4NTU.

First AWS system (three-station type system) makes total filter area reduce by 78%, and second level clarification is clear compared to primary DFF reduces by 12% (centrifuge is active cell operation).

2nd AWS system (four station type system) makes total filter area reduce by 90%, and DFF-DFF reduction is compared in second level clarification 60%.Sound swimming system realizes 91%TCD decline, 90% turbidity reduction and 91.2% Protein Recovery.

Cutting generates 21.5 liters/m to DFF-DFF (primary and second level)²Extremely low volume throughput, to handle The cutting of 1000L will use almost 50m²Filter area.The volume flux of dual station formula DFF step behind third station is with addition 4th station and increase by 100%, from 98.5L/m²To 222L/m².TCD is from 2.9 × 10⁶Cell/mL is reduced to 1.4 × 10⁶Cell/ ML (~50% reduces).Turbidity is reduced to 220NTU from 338NTU (42% reduces).In order to compare, when centrifugation is used as primary clarification When step, the centrifugal filtrate from centrifuge has 0.07 × 10 through measurement⁶TCD, 450NTU turbidity and 6% living cells Density (VCD).High shear force leads to clasmatosis, to increase turbidity and reduce the VCD of remaining cell.

To make comparisons with the filtered dual station formula DFF (C0HC+X0HC) of sound, single phase is also carried out after four station type AWS DFF, using three different DFF filters (PDD1, X0HC, 90Z).The volume flux of PDD1 is 90L/m², X0HC is 21L/m², 90Z is 110L/m².These value ratios 222L/m²Dual station co-volume flux low 50-90%.

Embodiment 9

Then, feed flow rate (Q is observed in multi-drop sound swimming system_F), total flow ratio (Q_S/Q_F) and solid flow ratio (Q_S#/Q_S#) to clarification performance influence.In order to which the setting of this observation is shown in Figure 19.In Figure 19, Q_F#Indicate entry into given sound swimming station Feed flow rate, Q_P#Indicate the transmission logistics speed from given sound swimming station, Q_S#Indicate the solid velocity from given sound swimming station. Upstream device is feed flow rate (such as Q to stand into the swimming of subsequent sound through logistics speed_P1=Q_F2)。

Using 10 kinds of different cell lines, 135 isolated operations, the mixture tool of Chinese hamster ovary celI used and culture medium are executed Have 27-98% cell viability, 2.8 × 10⁶-56×10⁶Cell/mL cell density, 1.4-16.5% packed cell group (PCM) and the charging turbidity of 30-9000NTU.

Figure 20 illustrates exemplary four station type setting.Figure 21 shows turbidity reduction with the function for feeding PCM.Figure 22 is with feeding flow The function of speed shows turbidity reduction, and total flow is than 20%.Figure 23 shows turbidity reduction, total flow ratio with the function of solid flow ratio 20%.Figure 24 shows PCM with the function of solid flow ratio.Figure 25 shows turbidity reduction with the function of solid flow ratio.As solid The solid PCM of the function of body flow-rate ratio is listed in the table below.

Flow-rate ratio (QS/QF)	PCM (%)
		2.5%	61
5%	42
		10%	30
20%	17

It was found that feed flow rate largely effects on clarification.It was found that total flow influences yield than main, and influenced generally on clear. It was found that the ratio for providing optimum is (charging PCM/50%PCM efflux of solids).It was found that reduce total flow ratio can be improved yield and Increase solids package.It was found that solid flow, which compares overall clarification, not to be influenced, but discovery can control the solids distribution between station.Hair Now charging PCM influences clarification and station efficiency.It was found that the optimum of charging PCM is 5-6%, it is single to stand, and higher %PCM is not more The problem of standing posture system.

Embodiment 10

Then, feed flow rate (Q is observed in three and four station type sound swimming system_F) to clarification performance influence.

The mixture of Chinese hamster ovary celI and culture medium flows through the two systems as AWS " queue ", then uses at DFF filter Reason.Chinese hamster ovary celI is removed by filter.Charging has 35.8 × 10⁶Cell/mL total cell density (TCD) and 4457NTU's is turbid It spends, the vigor of 9.1% PCM and 91.9%.

Three-station type sound swimming system uses 2 operations, feed flow rate (Q_F) it is 1.0x and 0.66x.Four station type sound swimming system makes With single operation, feed flow rate (Q_F) it is 1.0x.0.6x three-station type system is suitable with the swimming system performance of 1.0x four station type sound, although institute There is operation to realize > 85% yield and >~90% turbidity reduction.Respectively there is separated filtering chain through object.Note is provided below All data of record.

Once again, AWS ratio DFF-DFF execution is much better, and realize volume flux increase~4x.The following institute of overall filtration is general It includes.

Embodiment 11

Then, 11 isolated operations in four station type sound swimming system.The mixture of Chinese hamster ovary celI and culture medium flows through system Concatenated four stations, 5 different CHO cell lines.Charging has 9.78 × 10⁶-34.3×10⁶Cell/mL total cell density (TCD), the vigor of the turbidity of 920-4670NTU, the PCM of 6.5-11% and 31-91%.

AWS has 0.8 × 10 through object⁶-5.2×10⁶Cell/mL TCD (average=2.9 × 10⁶Cell/mL), 121- The turbidity (average=236NTU) of 453NTU and the PCM (average=1.6%) of 1.1-2.3%.Reducing performance is 76%-95% The PCM decline of TCD decline (average=88%), the turbidity reduction (average=94%) of 86%-97% and 72%-88% is (average =79%).Yield is 84%-89% (average=86%).Figure 26 display reduce performance with charging PCM and change, Figure 27 with The function display for feeding cell viability reduces performance.

Embodiment 12

There are 2 CHO cell lines of multi-specific antibody (MM-131) to flow through three-station type sound swimming system, each station has 1 inch × 1 Inch ultrasonic transducer.Charging has 20 × 10⁶-24×10⁶Cell/mL total cell density (TCD).First feeding line has 80% cell viability and the second feeding line have 65% cell viability.The total cell decline of 2 feeding lines, turbidity reduction and Protein salvage is listed in the table below.

Feeding line	Total cell decline	Turbidity reduction	Protein salvage
				80% is living	90%	84%	82%
65% is living	89%	85%	78%

Embodiment 13

It is also just each to stand as time measurement system shows in four station type system.Figure 28 shows the TCD decline at each station, and Between be divided into 2 hours, 4 hours, 6 hours, 9 hours and 12.5 hours.Each leftmost column of series represents first stop, and each series is from left to right Second column represents second station, and the second column represents third station to each series from right to left, and the column of each series rightmost represents the 4th station.It is added the Four stations show that the TCD at each time interval declines and increase.This figure show each sound swimming station maintain its performance and its separating capacity not with The time reduce, and physics filter can be reduced.

Embodiment 14

For three different users, dropped with DFF and the four station type AWS systematic comparison in-depth filtration area that DFF filter is added It is low.Figure 29 is shown makes in-depth filtration area used reduce 3-10 times using four station type AWS system.

Embodiment 15

Figure 30 shows the cell densities decline by 5 times of three-station type system different operatings, the system have 1 inch × 1 inch of energy converter.X-axis instruction total cell density (cell/mL) and cell viability near each column bottom.Y-axis is from charging Decline percentage to the cell through object, the higher the better for value.These are as the result is shown even for higher total cell Density Separation Well.

The disclosure is described with reference to exemplary embodiment.It can be modified after reading and understanding are described in detail above And change.The disclosure is intended to be construed to include all such modifications and change, as long as they are in appended claims or it is equivalent In the range of object.

Claims (26)

1. The system 1. a kind of multi-drop sound is swum, the system comprises:

   At least the first sound swimsuit of sequentially serial fluid connection is set, and rising tone swimsuit is set to be set with third sound swimsuit, and each sound swimsuit is set Include:

   Flow chamber, including at least one entrance and at least one outlet；

   At least one ultrasonic transducer, the ultrasonic transducer and the flow chamber are coupled, including piezoelectric material, and be configured to by Driving is with the sound standing wave of the generation multidimensional in flow chamber；With

   Reflector, at least one ultrasonic transducer opposite.
2. The system 2. multi-drop sound as described in claim 1 is swum, wherein the first, second, and third sound swimsuit is set and passes through pipeline It fluidly connects.
3. The system 3. multi-drop sound as described in claim 1 is swum, wherein the first, second, and third sound swimsuit is set and is configured to produce Raw multidimensional sound standing wave, frequency is in an order of magnitude each other.
4. The system 4. multi-drop sound as described in claim 1 is swum, the also feed pump comprising the first sound swimming device upstream, the swimming of the first sound First pump in device downstream, the third that rising tone swimsuit sets second pump in downstream and third sound swimsuit sets downstream pump.
5. The system 5. multi-drop sound as claimed in claim 4 is swum also sets downstream comprising third sound swimsuit and series connection with it fluid connects The falling tone swimsuit connect is set and falling tone swimsuit is set the 4th of downstream and pumped.
6. The system 6. multi-drop sound as described in claim 1 is swum, the also feed flow meter comprising the first sound swimming device upstream, first Sound swimsuit sets the first flowmeter in downstream, and rising tone swimsuit sets the second flowmeter in downstream and third sound swimsuit sets the of downstream Three flowmeters.
7. The system 7. multi-drop sound as claimed in claim 6 is swum also sets downstream comprising third sound swimsuit and series connection with it fluid connects The falling tone swimsuit connect is set and falling tone swimsuit sets the 4th flowmeter in downstream.
8. The system 8. multi-drop sound as described in claim 1 is swum, wherein each sound swimsuit, which is set to have to be located at, enters the flow chamber At least one dump diffuser of inlet.
9. The system 9. multi-drop sound as described in claim 1 is swum has wherein each multidimensional sound standing wave generates acoustic radiation force in same The axial thrust load and cross component force of one order of magnitude.
10. The system 10. multi-drop sound as described in claim 1 is swum, wherein each sound swimsuit is set also under at least one ultrasonic transducer Side includes port.
11. The system 11. multi-drop sound as described in claim 1 is swum, is also included in the first sound swimming device upstream or third sound swimsuit is set At least one cascade filtration station in downstream, the cascade filtration station be one or more depth filters, sterile filters, centrifuge or Affinity chromatographic column.
12. The system 12. multi-drop sound as described in claim 1 is swum also includes:

    The falling tone swimsuit that third sound swimsuit sets downstream and series connection with it fluidly connects is set；

    The feed pump and feed flow meter that first sound swimming device upstream and series connection with it fluidly connect；

    It sets to set serial fluid with rising tone swimsuit and connect and be located at the first sound swimsuit with the first sound swimsuit and sets and set with rising tone swimsuit Between first pump and first flowmeter；

    It sets to set serial fluid with third sound swimsuit and connect and be located at rising tone swimsuit with rising tone swimsuit and sets and set with third sound swimsuit Between second pump and second flowmeter；

    Downstream and falling tone swimsuit are set with third sound swimsuit set serial fluid and connect and be located at third sound swimsuit set downstream and the 4th Sound swimsuit set between third pump and third flowmeter；With

    Downstream is set positioned at falling tone swimsuit and series connection with it fluidly connects the 4th pump and the 4th flowmeter.
13. 13. a kind of method for continuously separating second fluid or particle from main fluid, which comprises

    The mixture flow of the main fluid and the second fluid or particle is set to cross multi-drop sound swimming system, the multi-drop sound swimming System is set comprising at least the first sound swimsuit that series connection sequentially fluidly connects, and rising tone swimsuit is set to be set with third sound swimsuit, each sound swimming Device includes:

    Flow chamber includes at least one entrance and at least one outlet；

    At least one ultrasonic transducer, the energy converter and flow chamber are coupled, and include piezoelectric material, and be configured to be actuated to Multidimensional sound standing wave is generated in flow chamber；With

    Reflector, at least one described ultrasonic transducer opposite；With

    At least one ultrasonic transducer for driving the first sound swimsuit to set to generate the first multidimensional sound standing wave wherein, thus at least The second fluid or particle of a part are continuously captured in the first standing wave, and remaining mixture goes successively to rising tone swimsuit and sets；

    At least one ultrasonic transducer for setting of driving rising tone swimsuit to generate the second multidimensional sound standing wave wherein, thus at least the The second fluid or particle of two parts are continuously captured in the second standing wave, and remaining mixture goes successively to third sound swimsuit and sets；With

    At least one ultrasonic transducer for setting of driving third sound swimsuit to generate third multidimensional sound standing wave wherein, thus at least the The second fluid or particle of three parts are continuously captured in third standing wave.
14. 14. method as claimed in claim 13, wherein first, second, and third sound standing wave is driven with different frequency It is dynamic.
15. 15. method as claimed in claim 13, wherein the frequency of first, second, and third sound standing wave is each other in a number In magnitude.
16. 16. method as claimed in claim 13, wherein the second fluid or particle be Chinese hamster ovary (CHO) cell, NS0 hybridoma, baby hamster kidney (BHK) cell or people's cell；T cell, B cell or NK cell；Peripheral blood mononuclear cells (PBMCs)；Algae；Plant cell, bacterium, virus or microcarrier.
17. 17. method as claimed in claim 13, wherein the first, second, and third sound swimsuit is set and connected by pipeline fluid It connects.
18. 18. method as claimed in claim 13, wherein the first, second, and third sound swimsuit is set respectively through at least 50V's Voltage signal driving.
19. 19. method as claimed in claim 13, wherein the first, second, and third sound swimsuit, which is set, respectively passes through about 50V- about The voltage signal of 60V drives.
20. 20. method as claimed in claim 13, wherein the sound swimsuit of most downstream in the multi-drop sound swimming system is driven to set Voltage signal is about 40V- about 60V.
21. 21. method as claimed in claim 13 is also included under at least one ultrasonic transducer wherein each sound swimsuit is set The port of side, the mouth, which is configured to put back into from sound swimsuit, receives second fluid or particle.
22. 22. method as claimed in claim 13, wherein multi-drop sound swimming system also includes the multi-drop sound swimming system The sound swimsuit of middle most downstream sets at least one cascade filtration station in downstream, and the cascade filtration station is one or more deep layer filters Device, sterile filters, centrifuge or affinity chromatographic column.
23. 23. a kind of system for collecting biomolecule from cell culture, the system comprises:

    Dispose the bioreactor of cell culture；

    Multiple sound swimsuits of serial fluid connection are set；

    The multiple sound swimsuit set at least one directly fluidly connected with the bioreactor；With

    At least another filter fluidly connected set with the multiple sound swimsuit.
24. 24. system as claimed in claim 23, wherein there is at least two serial fluids company between bioreactor and filter The sound swimsuit connect is set.
25. 25. system as claimed in claim 23, the recovery port at least first also set comprising the multiple sound swimsuit is used In the cell for recycling at least first cell culture separation from setting by the multiple sound swimsuit.
26. 26. system as claimed in claim 25, wherein the recovery port is connect with the bioreactor fluids.